Synthesis of Amino Acid-Based Cationic Lipids and Study of the Role of the Cationic Head Group for Enhanced Drug and Nucleic Acid Delivery.

Leveraging liposomes for drug and nucleic acid delivery, though promising due to reduced toxicity and ease of preparation, faces challenges in stability and efficiency. To address this, we synthesized cationic amphiphiles from amino acids (arginine, lysine, and histidine). Histidine emerged as the superior candidate, leading to the development of three histidine-rich cationic amphiphiles for liposomes. Using the hydration method, we have prepared the liposomes and determined the optimal N/P ratios for lipoplex formation via gel electrophoresis. In vitro transfection assays compared the efficacy of our lipids to Fugene, while MTT assays gauged biocompatibility across cancer cell lines (MDA-MB 231 and MCF-7). The histidine-based lipid demonstrated marked potential in enhancing drug and nucleic acid delivery. This improvement stemmed from increased zeta potential, enhancing electrostatic interactions with nucleic acids and cellular uptake. Our findings underscore histidine's crucial role over lysine and arginine for effective delivery, revealing a significant correlation between histidine abundance and optimal performance. This study paves the way for histidine-enriched lipids as promising candidates for efficient drug and nucleic acid delivery, addressing key challenges in the field.

Cationic and amphiphilic peptide-based hydrogels with dual activities as anticancer and antibacterial agents.

The emergence of peptide-based functional biomaterials is on the rise. To fulfil this purpose, a series of amphiphilic peptides, such as H2N-X-Met-Phe-C12H25, where X = L-lysine (CP1), X = L-histidine (CP2), and X = L-leucine (CP3), have been designed, synthesised, purified and fully characterised. Herein, we reported peptide-based supramolecular hydrogels with antibacterial and anticancer activities. An attempt has been made to investigate the antibacterial properties of these peptide-based hydrogels against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacteria. Investigations show that the L-lysine containing gelator, CP1, is active against both Gram-positive and Gram-negative bacteria and the L-histidine containing gelator, CP2, selectively inhibits the growth of Gram-negative bacteria. Interestingly, the L-leucine containing gelator, CP3, does not show any antibacterial properties. Moreover, the L-lysine containing gelator exhibits the best potency. Generation of reactive oxygen species (ROS) is a probable way to damage the bacterial membrane. To explore the cytotoxic properties and to determine the efficacy of the synthesized compounds in inhibiting cell viability, a comprehensive investigation was performed using three distinct cell lines: MDA-MB-231 (human triple-negative breast cancer), MDA-MB-468 (human triple-negative breast cancer) and HEK 293 (human embryonic kidney). Remarkably, the results of our study revealed a substantial cytotoxic impact of these peptide gelators on the MDA-MB-231 and MDA-MB-468 cell lines in comparison to the HEK 293 cells. Caspase 3/7 activity is the possible mechanistic path to determine the apoptotic rates of the cell lines. This finding emphasizes the promising potential of these peptide-based gelators in targeting and suppressing the growth of human triple negative breast cancer cells, while showing non-cytotoxicity towards non-cancerous HEK 293 cells. In a nutshell, these peptide-based materials are coming to light as next generation biomaterials.

Elucidation of the signalling pathways for enhanced exosome release from Mycobacterium-infected macrophages and subsequent induction of differentiation.

Exosomes are extracellular vesicles released by all cell types; perform several important functions such as cell-to-cell communication, growth, differentiation and so on. Exosomes elicit several signalling mechanisms as they carry information in the form of DNA, RNA or protein docked on them. We show that exosomes released from Mycobacterium tuberculosis (Mtb)-infected macrophages not only induce differentiation of naïve monocytes but also generate functionally active macrophages via MAPK-dependent signalling mechanism through MK-2 and NF-κß activation which is completely different from the differentiation induced by exosomes from uninfected macrophages. Further, we elucidate unequivocally the signalling mechanism behind the enhanced release of exosome generation from infected macrophages driven by AKT phosphorylation involving Rab7a and Rab11a. Genes of both ESCRT-dependent and -independent pathways are found to be involved in enhanced exosomes release and are modulated by AKT. However, interestingly, the genes of the ESCRT-independent pathway are dependent on NF-κß activation while the genes of the dependent pathway are not, suggesting two parallel signalling cascades operating in tandem.

Advances in Microscale Droplet Generation and Manipulation.

Microscale droplet generation and manipulation have widespread applications in numerous fields, from biochemical assays to printing and additive manufacturing. There are several techniques for droplet handling. Most techniques, however, can generate and work with only a limited range of droplet sizes. Furthermore, there are constraints regarding the workable variety of fluid properties (e.g., viscosity, surface tension, mass loading, etc.). Recent works have focused on developing techniques to overcome these limitations. This feature article discusses advances in this area that cover a wide range of droplet sizes from subpicoliter to microliter.

Histidine-Containing Amphiphilic Peptide-Based Non-Cytotoxic Hydrogelator with Antibacterial Activity and Sustainable Drug Release.

A histidine-based amphiphilic peptide (P) has been found to form an injectable transparent hydrogel in phosphate buffer solution over a pH range from 7.0 to 8.5 with an inherent antibacterial property. It also formed a hydrogel in water at pH = 6.7. The peptide self-assembles into a nanofibrillar network structure which is characterized by high-resolution transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy, small-angle X-ray scattering, Fourier-transform infrared spectroscopy, and wide-angle powder X-ray diffraction. The hydrogel exhibits efficient antibacterial activity against both Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). The minimum inhibitory concentration of the hydrogel ranges from 20 to 100 µg/mL. The hydrogel is capable of encapsulation of the drugs naproxen (a non-steroidal anti-inflammatory drug), amoxicillin (an antibiotic), and doxorubicin, (an anticancer drug), but, selectively and sustainably, the gel releases naproxen, 84% being released in 84 h and amoxicillin was released more or less in same manner with that of the naproxen. The hydrogel is biocompatible with HEK 293T cells as well as NIH (mouse fibroblast cell line) cells and thus has potential as a potent antibacterial and drug releasing agent. Another remarkable feature of this hydrogel is its magnification property like a convex lens.

Yellow-Emitting Carbon Dots for Selective Fluorescence Imaging of Lipid Droplets in Living Cells.

This study shows a one-pot preparation of carbon dots by a solvothermal method in ethylene glycol. The carbon dots show yellow-colored fluorescence emission in water. The carbon dots showed distinct preference to be present in the hydrophobic environment which was evident from their efficient transfer from aqueous phase to organic phase. They were also found to locate themselves in the vesicle bilayer and micelle core. This inherent lipophilic character of these carbon dots has been successfully utilized for the selective imaging of lipid droplets inside the living cells. The selective imaging of lipid droplets was confirmed by similar staining patterns with other staining dyes and the starvation study.

Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition.

The triple-negative phenotype is the most prevalent form of human breast cancer worldwide and is characterized by poor survival, high aggressiveness, and recurrence. Microvesicles (MV) are shredded plasma membrane components and critically mediate cell-cell communication, but can also induce cancer proliferation and metastasis. Previous studies have revealed that protease-activated receptor 2 (PAR2) contributes significantly to human triple-negative breast cancer (TNBC) progression by releasing nano-size MV and promoting cell proliferation, migration, and invasion. MV isolated from highly aggressive human TNBC cells impart metastatic potential to nonmetastatic cells. Over-expression of microRNA221 (miR221) has also been reported to enhance the metastatic potential of human TNBC, but miR221's relationship to PAR2-induced MV is unclear. Here, using isolated MV, immunoblotting, quantitative RT-PCR, FACS analysis, and enzymatic assays, we show that miR221 is translocated via human TNBC-derived MV, which upon fusion with recipient cells, enhance their proliferation, survival, and metastasis both in vitro and in vivo by inducing the epithelial-to-mesenchymal transition (EMT). Administration of anti-miR221 significantly impaired MV-induced expression of the mesenchymal markers Snail, Slug, N-cadherin, and vimentin in the recipient cells, whereas restoring expression of the epithelial marker E-cadherin. We also demonstrate that MV-associated miR221 targets phosphatase and tensin homolog (PTEN) in the recipient cells, followed by AKT Ser/Thr kinase (AKT)/NF-κB activation, which promotes EMT. Moreover, elevated miR221 levels in MV derived from human TNBC patients' blood could induce cell proliferation and metastasis in recipient cells. In summary, miR221 transfer from TNBC cells via PAR2-derived MV induces EMT and enhances the malignant potential of recipient cells.

Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to ß-catenin accumulation via the AKT/GSK3ß pathway and contributes to breast cancer progression.

Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3ß inactivation is involved in these processes and that ß-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of ß-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. ß-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent ß-catenin accumulation may represent a potential therapeutic approach to control breast cancer.

Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer.

Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter-cellular signaling, mediated predominantly by protease-activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease-mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor-FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK followed by MLC phosphorylation to alter myosin organization whereas, P38 reorganizes actin dynamics by the sequential activation of MK2 and HSP27. RhoA-dependent ROCK-II activation again contributes to remodeling myosin II activity. Further, both our in vitro and in vivo analyses showed that these MVs potentiate invasive and migratory property to the recipient cells. Breast cancer patients blood show an elevation of TF-bearing, pro-metastatic MVs than normal. These findings give an insight into the detailed signaling mechanism involved in the production of MVs with transforming ability from PAR2-activated human breast cancer cells. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in MVs-associated human breast cancer metastasis.

Impact of Bioinspired Nanotopography on the Antibacterial and Antibiofilm Efficacy of Chitosan.

Chitosan derived from chitin is one of the most abundant naturally occurring biocompatible polymers obtained from fungi and arthropods. In this work, we report the enhancement in the bactericidal efficacy of CHI in the presence of a sharp nanotopography. High-aspect ratio nanostructured surface (NSS) was fabricated using a single-step deep reactive ion etching technique (DRIE). Post fabrication, CHI coating was carried out using a layer-by-layer (LBL) dip coating process on the flat and nanostructured surfaces. Antibacterial efficacy of the flat silicon surface coated with CHI (Si_CHI) and NSS coated with CHI (NSS_CHI) was tested against both Gram-negative (G-ve) bacteria E. coli and Gram-positive (G+ve) bacteria S. aureus. NSS_CHI exhibited superior antibacterial property against G-ve and G+ve microbes as compared with Si_CHI and NSS substrates. Scanning electron microscopy (SEM) and fluorescence microscopy were used to study the morphology and viability of the bacteria on all the surfaces. Also, biofilm quantification was carried out on all the engineered surfaces for both E. coli and S. aureus using crystal violet (CV) staining. NSS_CHI was found to have the minimum biofilm formation on its surface exhibiting its superior antibacterial property. This study shows that the antibacterial and antibiofilm efficiency of CHI can be augmented by combining it with a sharp nanotopography.

Effect of geometrical parameters on rebound of impacting droplets on leaky superhydrophobic meshes.

When a droplet impacts a superhydrophobic sieve, a part of the droplet penetrates through it when the dynamic pressure (ρU2) of the impinging droplet exceeds the breakthrough pressure (γΓ/A). At higher impact velocities, the ejected-jet breaks and separates from the main droplet. The remaining part of the droplet bounces off the surface showing different modes (normal bouncing as a vertically elongated drop or pancake bouncing). In this work, we have studied the effect of different geometrical parameters of superhydrophobic copper meshes on different modes of droplet rebound. We observe three different effects in our study. Firstly, we observe pancake like bouncing, which is attributed to the capillary energy of the rebounding interface formed after the breaking of the ejected-jet. Secondly, we observe leakage of the droplet volume and kinetic energy due to the breaking of the ejected-jet, which leads to reduction in the contact times. Finally, we observe that for flexible meshes, the transition to pancake type bouncing is induced at lower Weber numbers. Flexibility also leads to a reduction in the volume loss from the ejected-jet. This study will be helpful in the design of superhydrophobic meshes for use under impact scenarios.

Anisotropic drop spreading on superhydrophobic grates during drop impact.

We study the influence of geometric anisotropy of micro-grate structures on the spreading dynamics of water drops after impact. It is found that the maximal spreading diameter along the parallel direction to grates becomes larger than that along the transverse direction beyond a certain Weber number, while the extent of such an asymmetric spreading increases with the structural pitch of grates and Weber number. By employing grates covered with nanostructures, we exclude the possible influences coming from the Cassie-to-Wenzel transition and the circumferential contact angle variation on the spreading diameter. Then, based on a simplified energy balance model incorporating slip length, we propose that slip length selectively enhances the spreading diameter along the parallel direction, being responsible for the asymmetric drop spreading. We believe that our work will help better understand the role of microstructures in controlling the drop dynamics during impact, which has relevance to various engineering applications.

Water Penetration through a Superhydrophobic Mesh During a Drop Impact.

When a water drop impacts a mesh having submillimeter pores, a part of the drop penetrates through the mesh if the impact velocity is sufficiently large. Here we show that different surface wettability, i.e., hydrophobicity and superhydrophobicity, leads to different water penetration dynamics on a mesh during drop impact. We show, despite the water repellence of a superhydrophobic surface, that water can penetrate a superhydrophobic mesh more easily (i.e., at a lower impact velocity) over a hydrophobic mesh via a penetration mechanism unique to a superhydrophobic mesh. On a superhydrophobic mesh, the water penetration can occur during the drop recoil stage, which appears at a lower impact velocity than the critical impact velocity for water penetration right upon impact. We propose that this unique water penetration on a superhydrophobic mesh can be attributed to the combination of the hydrodynamic focusing and the momentum transfer from the water drop when it is about to bounce off the surface, at which point the water drop retrieves most of its kinetic energy due to the negligible friction on superhydrophobic surfaces.

Axisymmetric and Nonaxisymmetric Oscillations of Sessile Compound Droplets in an Open Digital Microfluidic Platform.

Manipulating droplets of biological fluids in an electrowetting on dielectric (EWOD)-based digital microfluidic platform is a significant challenge because of biofouling and surface contamination. This problem is often addressed by operating in an oil environment. We study an alternate configuration of sessile compound droplets having an aqueous core surrounded by a smaller oil shell. In contrast to the conventional EWOD platform, an open digital microfluidic platform enabled by the core-shell configuration will allow electrical, mechanical, or optical probes to get unrestricted access to the droplet, thus enabling highly flexible and dynamically reconfigurable lab-on-chip systems. Understanding droplet oscillations is essential as they are known to enhance mixing. To our knowledge, this is the first study of axisymmetric and nonaxisymmetric oscillations of compound droplets actuated using EWOD platforms. Mode shapes for both axisymmetric and nonaxisymmetric oscillations were studied and explained. Enhancement in the axisymmetric oscillation of the core by decreasing the shell volume was obtained experimentally and modeled theoretically. Smaller shell volumes reduce the damping losses, allowing the appearance of nonaxisymmetric modes over a larger range of operating parameters. The oscillation frequency regime for obtaining prominent nonaxisymmetric oscillations for different shell volumes was identified. Compound droplets provide a mechanism to reduce biofouling, sample contamination, and evaporation. We demonstrate axisymmetric and nonaxisymmetric oscillations of compound droplets with the biological core of red blood cells, providing crucial first steps for promoting applications such as rapid efficient assays, mixing of biological fluids, and fluidic photonics on hysteresis-free surfaces.

Enhancing the Bactericidal Efficacy of Nanostructured Multifunctional Surface Using an Ultrathin Metal Coating.

Insects and plants exhibit bactericidal behavior through nanostructures, which leads to physical contact killing that does not require antibiotics or chemicals. Also, certain metallic ions (e.g., Ag+ and Cu2+) are well-known to kill bacteria by disrupting their cellular functionalities. The aim of this study is to explore the improvement in bactericidal activity by combining extreme physical structure with surface chemistry. We have fabricated tall (8-9 µm high) nanostructures on silicon surfaces (NSS) having sharp tips (35-110 nm) using a single-step, maskless deep reactive ion etching technique inspired by dragonfly wing. Bactericidal efficacy of the nanostructured surfaces coated with a thin layer of silver (NSS_Ag) or copper (NSS_Cu) was measured quantitatively using standard viability plate-count method and flow cytometry. NSS_Cu surfaces kill bacteria very efficiently (killing 97% within 30 min) when compared to the uncoated NSS. This can be attributed to the addition of a surface chemistry to the nanostructures. The antibacterial activity of NSS_Cu is further indicated by the morphological differences of the dying/dead bacteria observed in the SEM images. The nanostructured surfaces demonstrate excellent superhydrophobic behavior, even with an ultrathin layer of metal (Ag/Cu) coating. The nanostructured surfaces exhibit static contact angle greater than 150° and contact hysteresis less than 10°. Moreover, reflectance is found to be <1% (for NSS_Cu < 0.5%) for all the nanostructured surfaces in the wavelength range 250-800 nm. The results obtained suggest that the fabricated nanostructured surfaces are multifunctional and can be used in various practical applications.

Molecular determinants involved in differential behaviour between soluble tissue factor and full-length tissue factor towards factor VIIa.

During blood-coagulation, the transmembrane protein tissue factor (TF) binds to its ligand, factor (F)VII, activating and allosterically modifying it to form a mature active binary complex (TF-FVIIa). Although the extracellular domain of TF (sTF) can bind to FVII, it fails to activate it. Binding of TF with FVIIa only partially enhances FVIIa proteolytic activity. Our previous kinetic study revealed that sTF has a lower binding capacity with FVIIa compared to membrane bound full-length (fl)TF. The reason behind this incapability of FVII activation and reduced catalytic activity remains unexplored due to the lack of an flTF crystal structure. Here we employed a comparative dynamic study between sTF-FVIIa in solution and flTF-FVIIa in a membrane system to give probable explanations for the differential behaviour of these complexes. Based on potential of mean force and interaction energy calculations, the binding affinities between sTF and FVIIa are weaker than those of the flTF-FVIIa complex. We further observed domain-wise less stability, reduced height, and thus less inter and intra-domain interaction between the sTF and FVIIa complexes. We detected higher fluctuation among the inter-atomic distances of the catalytic triad (CT) residues in sTF-FVIIa over the flTF-FVIIa complex. The flTF-FVIIa complex forms two major interactions between EGF2 and TF. We showed the enhanced activity of the flTF-FVIIa complex over the sTF-FVIIa complex, which is guided by mainly two interactions between EGF2 and TF. Due to the lack of these interactions, sTF-FVIIa somehow forms a less stable binary complex and could not react upon binding its substrates (FIX, FX). Our study, for the first time, provides a possible explanation of the distinct behaviour of the two forms of TF (sTF and flTF) towards its only ligand FVII/FVIIa.

Identification of potent anti-fibrinolytic compounds against plasminogen and tissue-type plasminogen activator employing in silico approaches.

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibers) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) used for treating severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: the kringle-2 domain of tissue plasminogen activator, the kringle-1 domain of plasminogen, and the serine protease domain of plasminogen. In the present study, one million molecules were screened from the ZINC database. These ligands were docked to their respective protein targets using Autodock Vina, Schrödinger Glide, and ParDOCK/BAPPL+. Thereafter, the drug-likeness properties of the ligands were evaluated using Discovery Studio 3.5. Subsequently, we subjected the protein-ligand complexes to molecular dynamics simulation of 200 ns in GROMACS. The identified ligands P76(ZINC09970930), C97(ZINC14888376), and U97(ZINC11839443) for each protein target are found to impart higher stability and greater compactness to the protein-ligand complexes. Principal component analysis (PCA) implicates, that the identified ligands occupy smaller phase space, form stable clusters, and provide greater rigidity to the protein-ligand complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis reveals that P76, C97, and U97 exhibit better binding free energy (ΔG) when compared to that of the standard ligands. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents.Communicated by Ramaswamy H. Sarma.

Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection.

Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.

A molecular dynamics simulation study to understand the effect of cholesterol and tissue factor palmitoylation on tissue factor-factor VIIa-factor Xa ternary complex in different lipid environments.

BACKGROUND: Tissue factor (TF), a transmembrane glycoprotein, plays a profound role in the formation of the tissue factor-factor VIIa (TF-FVIIa) complex that initiates factor Xa (FXa) generation followed by thrombin activation and clot formation. Previous reports suggest that TF-FVIIa coagulant activity at the cell surface may be affected by various processes, including changes in cholesterol content and posttranslational modifications of TF. Numerous studies were conducted but yielded inconclusive results about the effect of cholesterol on TF expression. OBJECTIVE: The present study aimed to understand how cholesterol affects structural modulations on the tissue factor-factor VIIa-factor Xa ternary complex (TF-FVIIa-FXa). Additionally, we aimed to illustrate the effect of palmitoylation on the Cys245 residue of TF and understand its structural implications on the TF-FVIIa-FXa. METHODS: We set up the following 4 systems in different lipid environments: TF-FVIIa-FXa in POPC:POPS (CS), TF-FVIIa-FXa in POPC:POPS:CHOL (CSL), Palmitoylated TF-FVIIa-FXa in POPC:POPS:CHOL (CSLP), and Palmitoylated TF-FVIIa-FXa in POPC:CHOL (CLP), respectively, and subjected them to molecular dynamics simulation. RESULTS: Hydrogen-bond and contact probability analysis were performed between various important domains of TF-FVIIa-FXa and notable novel interactions: Asn93FVIIa:L-Lys48TF, Arg178FVIIa:H-Asp95FXa:B, Lys20FVIIa:H-Glu193FXa:A, Arg178FVIIa:H-Asp97FXa:B, and Arg153FVIIa:H-Gln135FXa:B have been reported. The protein stability study implies that the CS and CLP systems are thermodynamically less stable than CSL and CSLP systems. CONCLUSION: Analysis of molecular dynamic simulation data suggests that the presence of cholesterol and palmitoylation may contribute to structural rigidity, stability, and compactness of key domains of TF-FVIIa-FXa by augmenting protein-protein and protein-lipid interactions.

Suppression of droplet pinch-off by early onset of interfacial instability.

HYPOTHESIS: Interfacial instabilities cause undesirable droplet breakage during impact. Such breakage affects many applications, such as printing, spraying, etc. Particle coating over a droplet can significantly change the impact process and stabilize it against breakage. This work investigates the impact dynamics of particle-coated droplets, which mostly remains unexplored. EXPERIMENTS: Particle-coated droplets of different mass loading were formed using volume addition. The prepared droplets were impacted on superhydrophobic surfaces, and their dynamics were recorded using a high-speed camera. FINDINGS: We report an intriguing phenomenon where an interfacial fingering instability helps suppress pinch-off in particle-coated droplets. This island of breakage suppression, where the droplet maintains its intactness upon impact, appears in a regime of Weber numbers where bare droplet breakage is inevitable. The onset of fingering instability in particle-coated droplets is observed at much lower impact energy, around two times less than the bare droplet. The instability is characterized and explained using the rim Bond number. The instability suppresses pinch-off because of the higher losses associated with the formation of stable fingers. Such instability can also be seen in dust/pollen-covered surfaces, making it useful in many applications related to cooling, self-cleaning, anti-icing etc.