Alginate based biomaterials for hemostatic applications: Innovations and developments.

Development of the efficient hemostatic materials is an essential requirement for the management of hemorrhage caused by the emergency situations to avert most of the casualties. Such injuries require the use of external hemostats to facilitate the immediate blood clotting. A variety of commercially available hemostats are present in the market but most of them are associated with limitations such as exothermic reactions, low biocompatibility, and painful removal. Thus, fabrication of an ideal hemostatic composition for rapid blood clot formation, biocompatibility, and antimicrobial nature presents a real challenge to the bioengineers. Benefiting from their tunable fabrication properties, alginate-based hemostats are gaining importance due to their excellent biocompatibility, with >85 % cell viability, high absorption capacity exceeding 500 %, and cost-effectiveness. Furthermore, studies have estimated that wounds treated with sodium alginate exhibited a blood loss of 0.40 ± 0.05 mL, compared to the control group with 1.15 ± 0.13 mL, indicating its inherent hemostatic activity. This serves as a solid foundation for designing future hemostatic materials. Nevertheless, various combinations have been explored to further enhance the hemostatic potential of sodium alginate. In this review, we have discussed the possible role of alginate based composite hemostats incorporated with different hemostatic agents, such as inorganic materials, polymers, biological agents, herbal agents, and synthetic drugs. This article outlines the challenges which need to be addressed before the clinical trials and give an overview of the future research directions.

Ultrasensitive Detection of Lipid-Induced Misfolding of the Prion Protein at the Aqueous-Liquid Crystal Interface.

The misfolding of the α-helical cellular prion protein into a self-propagating ß-rich aggregated form is a key pathogenic event in fatal and transmissible neurodegenerative diseases collectively known as prion diseases. Herein, we utilize the interfacial properties of liquid crystals (LCs) to monitor the lipid-membrane-induced conformational switching of prion protein (PrP) into ß-rich amyloid fibrils. The lipid-induced conformational switching resulting in aggregation occurs at the nanomolar protein concentration and is primarily mediated by electrostatic interactions between PrP and lipid headgroups. Our LC-based methodology offers a potent and sensitive tool to detect and delineate molecular mechanisms of PrP misfolding mediated by lipid-protein interactions at the aqueous interface under physiological conditions.

Single-molecule FRET unmasks structural subpopulations and crucial molecular events during FUS low-complexity domain phase separation.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies permitting us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

Enhanced oil-water emulsion separation through coalescence filtration utilizing milkweed fiber: a sustainable paradigm.

Over the past few years, the environment and public safety have suffered due to the detrimental effects of oily industrial effluents. Natural fibers have gained popularity for their affordability, reusability, and effectiveness in separating oil from oily wastewater. Milkweed fibers were characterized using FTIR (Fourier transform infrared spectroscopy), SEM (scanning electron microscopy), and contact angle techniques. With four porosities (0.90, 0.92, 0.95, and 0.98), deep bed coalescence filters were built at three different filter bed heights (10 mm, 20 mm, and 30 mm). Using milkweed coalescence filtering technology, a novel oil separation method is described along with a method to calculate oil film thickness following emulsified oily water saturation. By combining a bed height of 30 mm and a porosity of 0.98, a maximum oil separation of 99.73% and an optimized D50 droplet ratio were achieved. Throughout a prolonged operational period lasting 250 min, the filter bed, possessing a depth of 30 mm and a porosity of 98%, exhibited no discernible fouling indications. Following five cycles, the milkweed filter bed measuring 30 mm in depth and featuring a porosity of 98% displayed an impressive oil separation efficiency of 91.5%. This study found that using a milkweed deep bed filter, coalescence filtering effectively removes oil from oily effluent. Furthermore, milkweed is a natural and biodegradable fiber that is easy to dispose of after use and does not harm the environment.

Development of κ-carrageenan-PEG/lecithin bioactive hydrogel membranes for antibacterial adhesion and painless detachment.

The issue of wound dressing adherence poses a substantial challenge in the field of wound care, with implications both clinically and economically. Overcoming this challenge requires the development of a hydrogel dressing that enables painless removal without causing any secondary damage. However, addressing this issue still remains a significant challenge that requires attention and further exploration. The present study is focused on the synthesis of hydrogel membranes based on κ-carrageenan (CG), polyethylene glycol (PEG), and soy lecithin (LC), which can provide superior antioxidant and antibacterial attachment properties with a tissue anti adhesion activity for allowing an easy removability without causing secondary damage. The (CG-PEG)/LC mass ratio was varied to fabricate hydrogel membranes via a facile approach of physical blending and solution casting. The physicochemical properties of (CG-PEG)/LC hydrogel membranes were studied by scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and mechanical analyses. The membranes showed significantly enhanced mechanical properties with excellent flexibility and had high swelling capacity (˃1000 %), which would provide a moist condition for wound healing. The membranes also exhibited excellent free radical scavenging ability (>60 %). In addition, the (CG-PEG)/LC hydrogel membranes showed reduced peel strength 26.5 N/m as a result of weakening the hydrogel-gelatin interface during an in vitro gelatin peeling test. Moreover, the membrane showed superior antibacterial adhesion activity (>90 %) against both S. aureus and E. coli due to the presence of both PEG and LC. The results also suggested that the hydrogel membranes exhibit NIH3T3 cell antiadhesion property, making them promising material for easy detachment from the healed tissue without causing secondary damage. Thus, this novel combination of (CG-PEG)/LC hydrogel membranes have immense application potential as a biomaterial in the healthcare sector.

Hydrogen-Deuterium Exchange Vibrational Raman Spectroscopy Distinguishes Distinct Amyloid Polymorphs Comprising Altered Core Architecture.

Amyloid fibrils are ordered protein aggregates comprising a hydrogen-bonded central cross-ß core displaying a structural diversity in their supramolecular packing arrangements within the core. Such an altered packing results in amyloid polymorphism that gives rise to morphological and biological strain diversities. Here, we show that vibrational Raman spectroscopy coupled with hydrogen/deuterium (H/D) exchange discerns the key structural features that are responsible for yielding diverse amyloid polymorphs. Such a noninvasive and label-free methodology allows us to structurally distinguish distinct amyloid polymorphs displaying altered hydrogen bonding and supramolecular packing within the cross-ß structural motif. By using quantitative molecular fingerprinting and multivariate statistical analysis, we analyze key Raman bands for the protein backbone and side chains that allow us to capture the conformational heterogeneity and structural distributions within distinct amyloid polymorphs. Our results delineate the key molecular factors governing the structural diversity in amyloid polymorphs and can potentially simplify studying amyloid remodeling by small molecules.

Sustainable Photocatalytic Desizing Process for the Starch-Based Size.

Textile wet processing highly impacts the environment due to its massive water and energy consumption. High consumption of water also results in the generation of a considerable volume of effluents. In this regard, an ultraviolet C (UVC)-assisted desizing method of starch-sized cotton fabric has been developed to lower the utility consumption in textile pretreatment. A UVC cabinet is designed to control exposing temperature and energy of exposure on the starch-sized cotton fabric. The UVC exposure time is optimized concerning the desizing efficiency. The UVC-exposed-sized fabric is washed with different washing times and washing temperatures to optimize the process. The alkali consumption in washing is reduced by 75% and desizing efficiency is improved to 95%. The application of oxidizing agents like NaNO2, K2S2O8, and NaBO3·4H2O during sizing further reduced the washing temperature and washing time for desizing to obtain 100% desizing efficiency. The UVC-assisted desized fabric is characterized by the whiteness index, water absorbency, tensile strength, Fourier transform infrared (FTIR), and wide-angle X-ray diffraction and compared with the control. The UVC-assisted desizing process has the potential to save approximately 60% water, 90% energy, and more than 70% of the time. Life cycle analysis has also been done. The photocatalytic desizing process can reduce the impact on human health by more than 85% and save approximately 69% of mineral resources than the conventional technique. The textile industry can quickly adopt a novel approach for sustainable desizing.

Nontoxic Cross-Linked Graphitic Carbon Nitride-Alginate-Based Biodegradable Transparent Films for UV and High-Energy Blue Light Shielding.

Flexible, transparent, and biodegradable films that can shield dangerous UV and high-energy blue light (HEBL) are high in demand to satisfy the ever-increasing expectations for environmental sustainability. To achieve this goal, biopolymer alginate is an excellent choice that has an outstanding film-forming ability. However, alginate has the limitation of poor UV and HEBL blocking ability. Thus, in this study, UV and HEBL blocker graphitic carbon nitride (g-C3N4) was incorporated in alginate films to enhance the compatibility, applicability, and durability. The ATR-FTIR, TGA, DTG, and FE-SEM results indicated that the composite film formation was due to hydrogen bonding, and the composite films revealed synergistic properties of alginate and g-C3N4. Though the incorporation of g-C3N4 in films enhanced the mechanical and thermal stabilities of the films, the films were still flexible. The UV-visible transmittance characterization confirmed that the prepared films could block both UV and HEBL radiation while maintaining transparency in visible regions. In experiments involving only 2 wt % g-C3N4, nearly 90% of UV (200-400 nm) and 95% of HEBL (400-450 nm) irradiation were blocked. Additionally, the inclusion of g-C3N4 also facilitated the biodegradation process of composite films. Moreover, after 6 months, the composite films exhibited excellent UV and HEBL shielding with excellent mechanical durability.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates.

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.

Preparation of functional and reactive nanosilver nanogels using oxidized carboxymethyl cellulose.

The designing of functional and reactive nanosilver has been carried out by in-situ reduction of silver nitrate using oxidized carboxymethyl cellulose (OCMC). The reduction process is also accompanied by the stabilization of nanoparticles using the OCMC polymer chain, leading to the formation of a structure where nanosilver is entrapped within OCMC gel. The silver nanogels characterized using transmission electron microscopy (TEM) are found to be ∼22 nm. By virtue of the presence of dialdehyde functionality around the silver nanogels, they have the ability to react with a polymer having a complementary functional group. The nanogels have exhibited prominent antimicrobial activity against both gram-negative and gram-positive bacteria. It has been observed that a 0.3 mM concentration of silver nanogel is active in inhibiting bacterial growth. The antibacterial activity of the synthesized Ag nanogels was dose-dependent, with 99.9 % of E. coli and S. aureus destroyed within 5 h at a concentration of 0.4 mM Ag nanogels. The nanogels disrupted the bacterial cell wall and generated reactive oxygen species inside the cell, which resulted in cell death. This investigation provides a very interesting application as a coating for biomedical implants and devices.

Biophysics of biomolecular condensates.

The formation of biomolecular condensates has emerged as a new biophysical principle for subcellular compartmentalization within cells to facilitate the spatiotemporal regulation of a multitude of complex biomolecular reactions. In this Research Highlight, we summarize the findings that were published in Biophysical Journal during the past two years (2021 and 2022). These papers provided biophysical insights into the formation of biomolecular condensates via phase separation of proteins with or without nucleic acids.

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

Oil separation from oil in water emulsion by coalescence filtration using kapok fibre.

In this study, the stable emulsion of engine oil in water of concentration 10% was prepared using a non-ionic surfactant. Kapok fibres were used as filter beds to separate oil from the oil-water emulsion. The surface morphology of fibres was investigated using Scanning Electron Microscope (SEM) analysis and chemical bond analysis of fibres done using Fourier transform infrared (FTIR). Kapok filter beds were prepared with three different bed heights 10, 20 and 30 mm each with four different porosities 0.90, 0.92, 0.95 and 0.98 for preparing the coalescence filter. The oil-water emulsion (influent) was pumped into the filtration column and the coalesced oil droplets, water, as well as un-coalesced oil droplets, especially the finer oil droplets, were collected as effluent. Oil separation efficiency was evaluated in terms of change in droplet size (D50) and oil concentration from influent to effluent. With increasing porosity and bed height, apart from porosity of 0.92, the separation efficiency increases. Increasing the bed heights at lower porosities does not improve the efficiency of the process. A combination of 0.98 porosity and a bed height of 30 mm provided the highest filtration performance in terms of oil separation efficiency and D50 droplet ratio. At 0.98 porosity, increasing the bed height from 10 mm to 30 mm resulted in a D50 droplet ratio of 0.25-0.14, representing a significant decrease in droplet size in the effluent and therefore an increase in oil separation efficiency from 91.3% to 99.63%.

Shapeshifting proteins: the role of structural disorder and conformational plasticity in physiology and disease.

Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm and do not autonomously fold up into unique 3D structures for carrying out functions. They exist as rapidly interconverting conformational ensembles and are thought to expand the functional repertoire of proteins. Such shapeshifting proteins are associated with a multitude of biological functions and a wide range of human diseases. The thematic issue on 'Shapeshifting Proteins' in Essays in Biochemistry includes some exciting and emerging aspects of this class of proteins. Articles in this issue provide current trends and contemporary views on various intriguing features of these proteins involving their unique structural and dynamical characteristics, misfolding and aggregation behavior, and their phase transitions into biomolecular condensates. I hope that this thematic issue will be of considerable interest to the practitioners in protein biochemistry and biophysics as well as to the researchers in other allied areas involving cell and molecular biology, neuroscience, virology, pathophysiology, and so forth.

Molecular Origin of Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are controlled by an interplay of thermal and viscosity effects. The effect of viscous drag through the solvent molecules is described by the classic Kramers theory in the high friction limit, which considers the dampening of the reactant molecules in the solution and quantifies the dependence of the reaction rate on the frictional drag. In addition to the external energy dissipation originating from the surrounding solvent molecules, there is an additional mode of internal energy dissipative force operative within the polypeptide chain reflecting the internal resistance of the chain to its conformational alterations. This dry, solvent-independent intrinsic frictional drag is termed internal friction. In the case of natively folded proteins, the physical origin of internal friction is primarily attributed to the intrachain interactions or other nonnative interactions in their compact states. However, the molecular origin of internal friction in intrinsically disordered proteins (IDPs) remains elusive.In this Account, we address this fundamental issue: what are the principal drivers of viscosity-independent (dry) friction in highly solvated, expanded, conformationally flexible, rapidly fluctuating IDPs that do not possess persistent intrachain interactions? IDPs exhibit diffusive conformational dynamics that is predominantly dominated by the segmental motion of the backbone arising due to the dihedral rotations in the Ramachandran Φ-Ψ space. The physical origin of friction in a complex biopolymeric system such as IDPs can be described by classic polymer models, namely, Rouse/Zimm models with internal friction. These one-dimensional models do not invoke torsional fluctuation components. Kuhn's classic description includes the connection between internal friction and microscopic dihedral hopping. Based on our time-resolved fluorescence anisotropy results, we describe that the sequence-dependent, collective, short-range backbone dihedral rotations govern localized internal friction in an archetypal IDP, namely, α-synuclein. The highly sensitive, residue-specific fluorescence depolarization kinetics offers a potent methodology to characterize and quantify the directional decorrelation engendered due to the short-range dihedral relaxation of the polypeptide backbone in the dihedral space. We utilized this characteristic relaxation time scale as our dynamic readout to quantify the site-specific frictional component. Our linear viscosity-dependent model of torsional relaxation time scale furnished a finite nonzero time constant at the zero solvent viscosity representing the solvent-independent internal friction. These results unveil the effect of the degree of dihedral restraining parameter on the internal friction component by showing that a restrained proline residue imparts higher torsional stiffness in the chain segments and, therefore, exhibits higher internal friction. This Account sheds light on the molecular underpinning of the sequence-specific internal friction in IDPs and will be of interest to unmask the role of internal friction in a diverse range of biomolecular processes involving binding-induced folding, allosteric interaction, protein misfolding and aggregation, and biomolecular condensation via phase separation.

Single-droplet surface-enhanced Raman scattering decodes the molecular determinants of liquid-liquid phase separation.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are involved in a myriad of critical cellular functions and debilitating neurodegenerative diseases. Elucidating the role of intrinsic disorder and conformational heterogeneity of intrinsically disordered proteins/regions (IDPs/IDRs) in these phase-separated membrane-less organelles is crucial to understanding the mechanism of formation and regulation of biomolecular condensates. Here we introduce a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that utilizes surface-engineered, plasmonic, metal nanoparticles to unveil the inner workings of mesoscopic liquid droplets of Fused in Sarcoma (FUS) in the absence and presence of RNA. These highly sensitive measurements offer unprecedented sensitivity to capture the crucial interactions, conformational heterogeneity, and structural distributions within the condensed phase in a droplet-by-droplet manner. Such an ultra-sensitive single-droplet vibrational methodology can serve as a potent tool to decipher the key molecular drivers of biological phase transitions of a wide range of biomolecular condensates involved in physiology and disease.