BSA-ICG-Cu( ii ) complex as an NIR responsive therapeutic agent exhibits bacteria-killing through photothermal and photodynamic properties, promotes angiogenesis, and supports fibroblast activity as required for chronic wound healing.

Viruses employ diverse strategies to gain entry into cells to reach their replication sites. Influenza A virus (IAV), a respiratory pathogen, takes advantage of clathrin-dependent endocytosis or macropinocytosis for its internalization. Here, we report that TRIM62, a member of the tripartite motif (TRIM) protein family, is required for IAV entry, independent of its E3 ubiquitin ligase activity. We find that TRIM62 specifically functions in a clathrin-independent, lipid raft-mediated pathway, which IAV exploits to enter the endocytic network. Additionally, we reveal the involvement of the WASH complex and the retromer component VPS35 in this pathway beyond their canonical functions in endosomal sorting. We observe that a pool of WASH and VPS35 localize to the plasma membrane and associate with lipid rafts, in addition to their typical endosomal presence. While WASH subunits except FAM21 play a proviral role in IAV endocytosis and intracellular trafficking, VPS35 acts antagonistically. We show that TRIM62 counteracts the antiviral function of VPS35 by limiting FAM21-VPS35 interaction. By directly binding WASH in the cytosol, TRIM62 restricts its VPS35-mediated endosomal recruitment and thereby enhances its surface availability to facilitate IAV entry. Together, this study uncovers previously unrecognized roles of TRIM62 and endosomal sorting machinery in IAV entry, offering new antiviral targets. ### Competing Interest Statement The authors have declared no competing interest. Department of Biotechnology, https://ror.org/03tjsyq23, BT/PR38441/MED/29/1498/2020

Influenza A virus (IAV) is a major threat to global human health and is a topic of intense research. With the continuous problem of seasonal influenza and the threat of potential pandemics due to frequent emergence of new viral strains, development of new, broad-spectrum antivirals is an urgent priority. In antiviral development against influenza, the process of host cell entry of IAV is of particular interest as inhibiting the virus at the entry step should stop infection early on, blocking the downstream infection processes including viral replication and transcription. Therefore, a detailed understanding of the IAV entry processes is essential to illuminate virus-assisting host factors that can serve as potentially valuable targets for therapeutic interventions. To accelerate the identification of novel antivirals or host-directed targets that play essential role in IAV entry, quantitative assays that can be used to monitor the virus at sequential entry steps would be important for performing high-content genetic or inhibitor screens. In this chapter, we describe how IAV entry can be monitored at the sequential entry steps, spanning from the initial attachment of the virus particle to the cell surface to the transmission of the viral genome to the nucleus, by fluorescence microscopy. Further, we provide the methods to quantify the images acquired with high-content microscope for each of the major IAV entry steps. The fluorescence microscopy-based IAV entry assays and the image quantification methods described here can be used to boost our understanding of the virus-host cell interactions and can lead to the discovery of novel host-directed prophylactic or therapeutic interventions.

The present study introduces carboxymethyl chitosan (CMC) delaminated Ti3C2 MXene (MX_CMC) as a novel therapeutic nanoplatform for chronic wound treatment. MXene was delaminated with CMC in water. Electron microscopy confirmed the 2D layered structure of MX_CMC, which demonstrated 60% higher dispersibility in aqueous media compared to MX alone. The nanoplatform showed high cytocompatibility with human adult dermal fibroblasts (HADF) and positively impacted cell migration, fibroblast differentiation, and VEGF expression. In vitro tube formation study with human umbilical vascular endothelial cells (HUVEC) further confirmed the angiogenic nature of MX_CMC. A low expression of proinflammatory cytokines (IL-6 and TNF-α) in the U937 cell line upon treatment with MX_CMC implied the non-immunogenic nature of the material. MX_CMC showed significant antimicrobial activity against S. aureus and B. subtilis, including biofilm prevention. In vivo testing in a rat model of infected diabetic chronic wounds yielded promising therapeutic outcomes. In this study, we have successfully prepared delaminated 2D Ti3C2 MXene in an environment-friendly way, which not only exhibits long-term dispersibility in biologically relevant aqueous media, but also possesses excellent antimicrobial, angiogenic, anti-inflammatory, and wound healing properties. In conclusion, MX_CMC could be a potential multifunctional nanoplatform for biomedical applications, especially for wound healing.

Glioblastoma Multiforme is an aggressive and complex cancer affecting mostly elderly patients above the age of 60 years. Originally classified as the fourth stage of glioma, it has an abysmal prognosis along with limited therapeutic options. Surgical removal of tumors, radiotherapy, and chemotherapy are prevalent treatment strategies with numerous therapeutic obstacles, including undefined boundary of tumor mass leaving traces even after excision, chances of secondary cancer formation, and presence of blood–brain barrier. These blood–brain and blood–brain tumor barriers actively restrict the permeability of many molecules from blood circulation to enter the central nervous system. Therefore, many conventional antineoplastic drugs fail to reach the tumor periphery except temozolomide. Meanwhile, active stem cells in the tumor microenvironment, genetic mutation inducing tumor growth, and epigenetic pattern alteration make this cancer chemoresistant. Our review delineates the recent approaches to resensitize the existing clinical drugs through specifically designed nanoformulations. Nanoparticles with modified physiological characteristics and modified through technological parameters can reduce the tumor's stemness, which increases tumor cells' apoptosis rate. Moreover, these nanoparticles can efficiently traverse the blood–brain barrier and escape from endosomal degradation with minimum toxicological impact. Overall, this review discusses the cancer chemoresistance phenomena and related pathways and highlights the potential of nanoformulation in reversing chemoresistance. Also, the existing limitations of this unique approach and suggestions are discussed at the end of the article, which may facilitate the identification of new directions for advancement of the nanoparticle-mediated reversal of chemoresistance.

The efficiency of oleogel as an oral delivery vehicle of probiotics depends on the chemical composition and gelator used. However, the gelators, which are surfactant in nature often interact with the probiotics and alter the therapeutic outcome. Keeping this perspective in mind, here we have developed oleogel of sunflower oil containing 5% (w/w) of sunflower wax and different emulsifiers, namely Span80 (S), Tween 80(T), stearyl alcohol (SA), and Span60 (SP), and checked their influence on probiotics in-vitro. Using confocal laser scanning microscopy, it was found that adding different emulsifiers changed the length and arrangement of the gelator network. SA and SP-modified oleogels, used at a concentration of 0.05% (w/v), demonstrated enhanced growth and metabolic activity of Lactiplantibacillus pentosus, which was employed as a model probiotic. Furthermore, the mucin adhesion test and scanning electron microscopy confirmed the negligible effect of those oleogels on the activity and morphology of the probiotic, respectively. When the secretome of such probiotics was applied to the colonic cell line, no negative effects were seen. This study implied that sunflower oil-sunflower wax oleogels modified using different emulsifiers can modulate probiotic growth.

The complex neoplasm of central nervous system malignancies, glioblastoma multiforme (GBM), is often challenging to manage due to its location complexity and anatomical barriers. The majority of conventional anticancer drugs are restricted by the blood-brain barrier, except Temozolomide, which also hampers patients' quality of life. Therefore, a potential carrier system is needed to enhance drug delivery efficiency. In this study, we have prepared the nanoformulation G4PFCP, a G4 PAMAM_OH dendrimer functionalized with folic acid and conjugated with carboplatin, to evaluate its efficacy in drug delivery to the GBM tumor microenvironment. Both the drug loaded (G4PFCP) and drug free (G4PF) nanoparticles have been characterized by spectroscopic and microscopic techniques. They have exhibited properties that are consistent with pH-specific sustained drug release. Both in vivo and in vitro studies have been performed. G4PFCP exhibited enhanced therapeutic efficacy compared to free carboplatin and G4PF in LN18 and LN229 GBM cell lines, which has been evaluated based on induced nuclear fragmentation and chromatin condensation. Furthermore, treatment with G4PFCP led to significant modulation of apoptotic markers, including Cleaved Caspase-9, XIAP, Bax, and DSB protein γ-H2AX. In the rat model, G4PFCP treatment resulted in substantial tumor size reduction and notable antiproliferative and antiangiogenic effects without showing any organ toxicity. The findings suggest that the G4PFCP nanoformulation represents a promising and effective drug delivery system for targeting GBM malignancies.

Nanoemulsions are isotropic systems made up of nanoscale droplets (about 200 nm in size) created by combining two immiscible liquids with the aid of emulsifiers. They are often regarded as harmless excipients and are made to enhance the release of active medicinal compounds. Improving drug distribution to specific areas is the main goal of employing nanoemulsions in cancer treatment. In addition to increasing bioavailability, nanoemulsions reduce adverse effects on healthy cells by encasing medications in a closed structure. This is especially crucial because, in the absence of such formulations, different medications fall short of their intended targets. The study shows that by increasing the solubility and bioavailability of anticancer medications, nanoemulsions can greatly improve their delivery. This is important because a lot of anticancer medications have low solubility, which reduces their ability to effectively target cancer cells. Nanoemulsions have been shown to effectively target tumor cells while minimizing the impact on healthy tissues. This targeted approach helps overcome the common issue of multidrug resistance seen in cancer treatments, as the nanoemulsions can be modified with specific ligands to focus on tumor cells. Targeting tumor cells and preventing multidrug resistance are two benefits of using nanoemulsions. Besides, hydrophilic and hydrophobic compounds can be encapsulated in nanoemulsions to satisfy a range of needs. Therefore, nanoemulsions are a promising new approach to cancer treatment. This review provides an overview of nanoemulsion in cancer therapeutics, aiming to highlight the current status of this technology.

In the life cycle of a virus, host cell entry represents the first step that a virus needs to undertake to gain access to the cell interior for replication. Once a virus attaches itself to its target cell receptor, it activates endogenous cellular responses and exploits host cell factors for its internalization, fusion, and genome release. Among the host factors that critically contribute to the viral entry processes are cathepsins, which are the most abundant endo/lysosomal proteases with diverse physiological functions. This review summarizes previous findings on how different cathepsins contribute to the host cell entry of human pathogenic viruses, focusing on their specific roles in the entry processes of both enveloped and non-enveloped RNA viruses. A comprehensive knowledge of the functions of different cathepsins in viral entry will provide valuable insights into the molecular mechanisms underlying viral infections and can be useful in the development of new antiviral strategies.

Influenza A virus (IAV) is a highly contagious respiratory pathogen that significantly threatens global health by causing seasonal epidemics and occasional, unpredictable pandemics. To identify new compounds with therapeutic potential against IAV, we designed and synthesized a series of 4'-morpholinodiazenyl chalcones using the molecular hybridization method, performed a high-content screen against IAV, and found that (E)-1-{4-[(E)-morpholinodiazenyl]phenyl}-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (MC-22) completely neutralized IAV infection. While MC-22 allowed IAV to successfully internalize into the cell and fuse at the acidic late endosomes, it prevented viral capsid uncoating and genome release. Since IAV majorly utilizes clathrin-mediated endocytosis (CME) for cellular entry, we examined whether MC-22 had any effect on CME, using nonviral cargoes that enter cells via clathrin-dependent or -independent pathways. Although MC-22 showed no effect on the uptake of choleratoxin B, a cargo that enters cells majorly via the clathrin-independent pathway, it significantly attenuated the clathrin-dependent internalization of both epidermal growth factor and transferrin. Cell biological analyses revealed a marked increase in the size of early endosomes upon MC-22 treatment, indicating an endosomal trafficking/maturation defect. This study reports the identification of MC-22 as a novel CME-targeting, highly potent IAV entry inhibitor, which is expected to neutralize a broad spectrum of viruses that enter the host cells via CME.

Physical properties of biomolecular condensates formed via phase separation of proteins and nucleic acids are associated with cell physiology and disease. Condensate properties can be regulated by several cellular factors including post-translational modifications. Here, we introduce an application of intermolecular energy migration via homo-FRET (F & ouml;rster resonance energy transfer), a nanometric proximity ruler, to study the modulation in short- and long-range protein-protein interactions leading to the changes in the physical properties of condensates of fluorescently-tagged FUS (Fused in Sarcoma) that is associated with the formation of cytoplasmic and nuclear membraneless organelles. We show that homoFRET captures modulations in condensate properties of FUS by RNA, ATP, and post-translational arginine methylation. We also extend the homoFRET methodology to study the in-situ formation of cytoplasmic stress granules in mammalian cells. Our studies highlight the broad applicability of homoFRET as a potent generic tool for studying intracellular phase transitions involved in function and disease. The properties of biomolecular condensates can be regulated by multiple factors, including intermolecular dynamics. Here, the authors use fluorescence anisotropy-based homoFRET imaging to monitor the intermolecular interactions and supramolecular packing that underlie the modulation of biomolecular condensate properties.

Oleogelation is an efficient oil-structuring technique commonly utilized to form oleogels using vegetable oil. This chapter aims to summarize current developments in the production of oleogels by structuring liquid oil with the help of lipids. The structuration occurs through oleogelators that are classified as high-molecular- and low-molecular-weight oleogelators (LMOGs). LMOGs, importantly waxes, can form a three-dimensional network within the oil at a low concentration (1%–3% w/w). Wax-based oleogels form through the direct dispersion method, giving rise to crystallite formation. The process of oleogel crystallization is similar to fat crystallization, which occurs in three stages, including nucleation, crystal growth, and polymorphic transition. Emulsifiers are amphiphilic molecules that often affect the stages of crystallization in oleogels. The effect of emulsifiers comes from the organization of crystals during the crystallization stages. Majorly the formulated oleogels are characterized through different microscopic studies, molecular characterization, and thermal studies. The field of oleogels has evolved quite progressively since 1960, which has been highlighted in this review through the bibliometric study. The study also confirmed the potential applications of the oleogels highlighting the field of novel delivery systems for drug and nutrient delivery along with their use in replacing saturated solid fat without compromising the organoleptic properties.

The performance of polyether-ether-ketone (PEEK) as an orthopedic biomaterial can be improved by bulk modification of PEEK through hydroxyapatite (HA) incorporation. In this context, we have studied the size effect of HA particles (from micro to nano) on the high-temperature extrusion, physicochemical and biological properties of the extruded PEEK-HA filaments. Our study showed that incorporation of HA into PEEK up to 5% w/w allows filament formation through single screw extrusion. However, a more significant temperature gradient between the hopper end and nozzle was necessary for the extrusion of nano-HA incorporated PEEK compared to micro-HA incorporated PEEK. The micro-CT revealed a homogeneous dispersion of HA particles within the extruded filaments. The inclusion of nano HA powder (<200 nm) in PEEK (5%w/w) did not alter the mechanical properties of PEEK. When checked in vitro using MG-63 cells, PnHA exhibited better cytocompatibility, as evidenced by calcein-AM staining and MTT assay. Cellular expression of vascular endothelial growth factor and alkaline phosphatase was also found to be 2-3 fold higher for PnHA. Further, PnHA was found to be a promoter of angiogenesis when checked by tube formation assay. The results together implied that nano-HA is more suitable than micro-HA for improving the essential qualities of PEEK for orthopedic applications.

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals.

Sunflower oil (SO) oleogel was initially prepared using 5 % (w/w) sunflower wax, hydrophobic (Span 80), or hydrophilic (Tween 80) emulsifiers. This study involved the physicochemical characterization of the batter and cake prepared through partial and complete replacement of butter with the oleogels. Batter and cake properties were improvised in T80, prepared with oleogel containing 0.015 % (w/w) of Tween 80. The polarized micrograph of the T80 batter displayed a large number of air bubbles stabilized by the wax crystals. Starch gelatinization was found highest in T80 batter and could be related to the ability of hydrophilic emulsifiers to form a complex with starch. The FTIR spectra in the T80 batter and cake displayed a reduced peak for gluten content. T80 cake crumb showed a homogenous distribution of smaller air cells supporting its softness. A reduction in the firmness and hardness of T80 was obtained from the texture studies.

The present approach of creating vasculature inside the scaffold or engineered tissue majorly relies on the use of cues from angiogenesis or vasculogenesis. In the recent years, artificial intelligence (AI) and machine learning (ML) have emerged as very powerful tool to analyze and predict various complex biological processes. In tissue engineering also, people have started using AIML extensively for material discovery and selection, scaffold designing, material composition, and process parameter optimization and for predicting the performance of the engineered tissue. This chapter provides a comprehensive picture of the potential application of AI in creating customized vasculature through angiogenesis and vasculogenesis. We also discussed the different models of AI that are used for the designing and developing of engineered tissue for analyzing vasculature and angiogenesis. Subsequently, we presented the concepts of designer vasculature and technical advances in this area with a conclusion on future directives for the research.

In this study, nanocomposite film was fabricated using cellulose nanocrystals (CNCs) as nanofiller in a polymer matrix of polyvinyl alcohol (PVA) and gum tragacanth (GT) via solution casting. CNCs were extracted from sugarcane bagasse using a steam explosion technique followed by acid hydrolysis. Initial analysis of CNCs by transmission electron microscopy (TEM) showed nanosized particles of 104 nm in length and 7 nm in width. Physical and chemical characteristics of neat PVA, PVA/GT, and PVA/GT/CNC films with varying concentrations of CNCs (from 2% to 10%) were analyzed by the scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrometry, mechanical test, and swelling test. The SEM analysis showed cluster formation of CNCs in the polymer matrix at high concentration. The developed films were transparent. FTIR spectrometry analysis confirmed the chemical functional groups of the various components in the film. The presence of GT and CNCs in the polymer matrix improved the characteristics of films as evident in the prolonged stability for 7 days and increased mechanical properties. The highest elastic modulus of 1526.11 ± 31.86 MPa and tensile strength of 80.39 MPa were recorded in PVA/GT/CNC2 film. The swelling ability, however, decreased from 260% to 230%. Cytotoxicity analysis of the PVA/GT/CNC film showed that it is nontoxic to mouse fibroblast cells L929 with 95% cell viability. Films loaded with betel leaf extract exhibited excellent antibacterial activities against Staphylococcus aureus DMST 8840 and Pseudomonas aeruginosa TISTR 781 with 28.20 ± 0.84 mm and 23.60 ± 0.55 mm inhibition zones, respectively. These results demonstrate that PVA/GT/CNC loaded with the betel leaf extract could act as promising and versatile wound dressings to protect the wound surface from infection and dehydration.

The frequent emergence of pathogenic viruses with pandemic potential has posed a significant threat to human health and economy, despite enormous advances in our understanding of infection mechanisms and devising countermeasures through developing various prophylactic and therapeutic strategies. The recent coronavirus disease (COVID-19) pandemic has re-emphasised the importance of rigorous research on virus infection mechanisms and highlighted the need for our preparedness for potential pandemics. Although viruses cannot self-replicate, they tap into host cell factors and processes for their entry, propagation and dissemination. Upon entering the host cells, viruses ingeniously utilise the innate biological functions of the host cell to replicate themselves and maintain their existence in the hosts. Influenza A virus (IAV), which has a negative-sense, single-stranded RNA as its genome, is no exception. IAVs are enveloped viruses with a lipid bilayer derived from the host cell membrane and have a surface covered with the spike glycoprotein haemagglutinin (HA) and neuraminidase (NA). Viral genome is surrounded by an M1 shell, forming a "capsid" in the virus particle. IAV particles use HA to recognise sialic acids on the cell surface of lung epithelial cells for their attachment. After attachment to the cell surface, IAV particles are endocytosed and sorted into the early endosomes. Subsequently, as the early endosomes mature into late endosomes, the endosomal lumen becomes acidified, and the low pH of the late endosomes induces conformational reaggangements in the HA to initiate fusion between the endosomal and viral membranes. Upon fusion, the viral capsid disintegrates and the viral ribonucleoprotein (vRNP) complexes containing the viral genome are released into the cytosol. The process of viral capsid disintegration is called "uncoating". After successful uncoating, the vRNPs are imported into the nucleus by importin α/β (IMP α/β), where viral replication and transcription take place and the new vRNPs are assembled. Recently, we have biochemically elucidated the molecular mechanisms of the processes of viral capsid uncoating subsequent viral genome dissociation. In this chapter, we present the molecular details of the viral uncoating process.

Regenerative medicine (RM) is a field of study that helps repair or restore native tissue function which has lost its functionality due to chronic diseases and trauma. The regeneration process can be promoted by constructing biomimetic systems, which can support cellular growth and proliferation. In this regard, the development of injectable hydrogels has gained enormous attention in recent times. An arrangement of cells and bioactive molecules in the three-dimensional extracellular matrix created by injectable gels is favorable for the regeneration of damaged tissues. Ideally, the injectable hydrogel remains in the solution form before injection and rapidly undergoes gelation at the physiological condition. A high water content, mechanical strength, scope of improved functionalization, injectability, and ease of implantation make the injectable hydrogel an ideal candidate for tissue-specific repair. This chapter aims to concisely summarize the mechanism and recent fabrication advancement of the injectable hydrogel that is being used in RM applications. A vast number of injectable hydrogels have been discovered for bone, cartilage, skin, and cardiovascular tissue regeneration, which are discussed in detail in the chapter. In gist, it is expected that injectable hydrogels will become a promising tool for a variety of tissue repair applications shortly.