Protonation-State Dependent Modulation of Hen Egg-White Lysozyme Fibrillation under the Influence of a Short Synthetic Peptide.

Under the influence of various conditions, misfolding of soluble proteins occurs, leading to the formation of toxic insoluble amyloids. The formation and deposition of such amyloids within the body are associated with detrimental biological consequences such as the onset of several amyloid-related diseases. Previously, we established a strategy for the rational design of peptide inhibitors against amyloid formation based on the amyloidogenic-prone region of the protein. In the current study, we have designed and identified an Asp-containing rationally designed hexapeptide (SqP4) as an excellent inhibitor of hen egg-white lysozyme (HEWL) amyloid progression in vitro. First, SqP4 showed strong affinity toward the native monomeric HEWL leading to the stabilization of the native form and restriction in the unfolding process of monomeric HEWL. Second, SqP4 was found to arrest the amyloidogenic misfolded structure of HEWL in a nonfibrillar monomer-like stage. We also observed the differential effect of the protonation state of the charged amino acid (Asp) within the peptide inhibitor on the amyloid formation of HEWL and explored the reason behind the observations. The findings of this study can be implemented in future strategies for the development of potent therapeutics against other amyloid-related diseases.

Carboxylic Group Functionalized Carbon Quantum Dots inhibit Hen Egg White Lysozyme Amyloidogenesis, leading to the Formation of Spherical Aggregates with Reduced Toxicity and ROS Generation.

INTRODUCTION: Proteinopathies are a group of diseases where the protein structure has been altered. These alterations are linked to the production of amyloids, which are persistent, organized clumps of protein molecules through inter-molecular interactions. Several disorders, including Alzheimer's and Parkinson's, have been related to the presence of amyloids. Highly ordered beta sheets or beta folds are characteristic of amyloids; these structures can further self- -assemble into stable fibrils. METHOD: Protein aggregation is caused by a wide variety of environmental and experimental factors, including mutations, high pH, high temperature, and chemical modification. Despite several efforts, a cure for amyloidosis has yet to be found. Due to its advantageous semi-conducting characteristics, unique optical features, high surface area-to-volume ratio, biocompatibility, etc., carbon quantum dots (CQDs) have lately emerged as key instruments for a wide range of biomedical applications. To this end, we have investigated the effect of CQDs with a carboxyl group on their surface (CQD-CA) on the in vitro amyloidogenesis of hen egg white lysozyme (HEWL). RESULT: By generating a stable compound that is resistant to fibrillation, our findings show that CQD-CA can suppress amyloid and disaggregate HEWL. In addition, CQD-CA caused the creation of non-toxic spherical aggregates, which generated much less reactive oxygen species (ROS). CONCLUSION: Overall, our results show that more research into amyloidosis treatments, including surface functionalized CQDs, is warranted.

Role of Sam68 as an adaptor protein in inflammatory signaling.

Sam68 is a ubiquitously expressed KH-domain containing RNA-binding protein highly studied for its involvement in regulating multiple steps of RNA metabolism. Sam68 also contains multiple protein-protein interaction regions such as proline-rich regions, tyrosine phosphorylation sites, and arginine methylation sites, all of which facilitate its participation as an adaptor protein in multiple signaling pathways, likely independent of its RNA-binding role. This review focuses on providing a comprehensive report on the adaptor roles of Sam68 in inflammatory signaling and inflammatory diseases. The insights presented here have the potential to open new avenues in inflammation research and justify targeting Sam68 to control aberrant inflammatory responses.

Exploring the potential of amyloids in biomedical applications: A review.

Amyloid is defined as a fibrous quaternary structure formed by assembling protein or peptide monomers into intermolecularly hydrogen linked ß-sheets. There is a prevalent issue with protein aggregation and the buildup of amyloid molecules, which results in human neurological illnesses including Alzheimer's and Parkinson's. But it is now evident that many organisms, like bacteria, fungi as well as humans, use the same fibrillar structure to carry out a variety of biological functions, such as structure and protection supporting interface transitions and cell-cell recognition, protein control and storage, epigenetic inheritance, and memory. Recent discoveries of self-assembling amyloidogenic peptides and proteins, based on the amyloid core structure, give rise to interesting biomaterials with potential uses in numerous industries. These functions dramatically diverge from the initial conception of amyloid fibrils as intrinsically diseased entities. Apart from the natural ability of amyloids to spontaneously arrange themselves and their exceptional material characteristics, this aspect has prompted extensive research into engineering artificial amyloids for generating various nanostructures, molecular substances, and combined materials. Here, we discuss significant developments in the artificial design of useful amyloids as well as how amyloid materials serve as examples of how function emerges from protein self-assembly at various length scales.

Formation of hen egg white lysozyme derived amyloid-based hydrogels using different gelation agents: A potential tool for drug delivery.

Amyloids are highly stable protein fibrillar aggregates that get deposited in various parts of our body and cause detrimental diseases. But in nature, the presence of functional amyloids is also noted in bacteria that help them by forming hyphae, biofilm, protein reservoirs, signalling messengers, etc. Keeping this perspective in mind, the idea behind this research was to develop functional amyloids in the form of hydrogel and analyse its potential in the biomedical sector as a drug-delivery tool. The synthesis and characterisation of three types of amyloid-based hydrogels have been reported in this work. Hen Egg-White Lysozyme (HEWL) protein was chosen as the principal ingredient as it is extensively used as a standard protein for studying amyloidogenesis and has inherent antibacterial properties. Comparative studies of different hydrogel properties exhibited variations in the hydrogels based on compositional differences in them. Finally, a drug release assay was done on the synthesized hydrogels to explore their potential as drug delivery tools.

Amine Group Surface-Functionalized Carbon Quantum Dots Exhibit Anti-amyloidogenic Effects Towards Hen Egg White Lysozyme by Inducing Formation of Nontoxic Spherical Aggregates.

The tendency of polypeptide chains to deviate from their conventional protein folding pathway and instead get trapped as off-pathway intermediates, has been a matter of great concern. These off-pathway intermediates eventually lead to the formation of insoluble, ordered fibrillar aggregates called amyloids, which are responsible for a host of neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Type II diabetes. In spite of extensive research, development of an effective therapeutic strategy against amyloidosis still remains elusive. In recent times, carbon quantum dots (CQD) have grabbed the attention of researchers against amyloidogenesis due to their ease of preparation, aqueous soluble nature, unique optical properties, high surface to volume ratio, physio-chemical properties, semi-conducting nature and mainly biocompatible. In the current study, we have reported an easy-to-prepare procedure for synthesis of amine group surface functionalized CQDs from commonly available kitchen spices with anti-oxidant properties. The as-synthesized CQDs were evaluated for their anti-amyloidogenic properties towards Hen Egg White Lysozyme (HEWL). Our results clearly show that the surfaced functionalized CQDs were able to interact with HEWL, thereby forming a stable complex, which was resistant towards amyloid formation and instead lead to the formation of non-toxic globular aggregates.

Hydrogen-Bonding Trends in a Bithiophene with 3- and/or 4-Pyridyl Substituents.

To improve the charge-carrier transport capabilities of thin-film organic materials, the intermolecular electronic couplings in the material should be maximized. Decreasing intermolecular distance while maintaining proper orbital overlap in highly conjugated aromatic molecules has so far been a successful way to increase electronic coupling. We attempted to decrease the intermolecular distance in this study by synthesizing cocrystals of simple benzoic acid coformers and dipyridyl-2,2'-bithiophene molecules to understand how the coformer identity and pyridine N atom placement affected solid-state properties. We found that with the 5-(3-pyridyl)-5'-(4-pyridyl)-isomer, the 4-pyridyl ring interacted with electrophiles and protons more strongly. Synthesized cocrystal powders were found to have reduced average crystallite size in reference to the parent compounds. The opposite was found for the intermolecular electronic couplings, as determined via density functional theory (DFT) calculations, which were relatively large in some of the cocrystals.

A Comprehensive Review on Inorganic Nanoparticles as Effective Modulators of Amyloidogenesis.

Many degenerative disorders have started to develop as a result of the deposition of insoluble protein fibrillar clumps known as amyloid. This deposition mostly limits normal cellular function and signaling. This build-up of amyloid in vivo results in a variety of illnesses in the body, including type 2 diabetes, several neurodegenerative diseases (such as Alzheimer's disease and spongiform encephalopathy), and Alzheimer's disease. Growing interest has been demonstrated in nanoparticles as a potential treatments for amyloidosis throughout the past few decades. Inorganic nanoparticles are one of them and have also been in substantial research as a potential anti-amyloid drug. Inorganic nanoparticles have emerged as a good study candidates because of their nano size, distinctive physical characteristics, and capacity to traverse the blood-brain barrier. In the current review, we have focused on the effects of different types of inorganic nanoparticles on amyloidogenesis and attempted to understand their underlying mechanism of action.

Inhibition of SARS-CoV-2 infection in cellular systems using engineered trimeric receptor-binding domain of spike protein.

Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest. For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1.

Identification of Novel Peptides as Potential Modulators of Aß42 Amyloidogenesis: An in silico Approach.

AIMS: Alzheimer's disease is a neurodegenerative disease for which no cure is available. The presence of amyloid plaques in the extracellular space of neural cells is the key feature of this fatal disease. BACKGROUND: The proteolysis of Amyloid Precursor Protein by presenilin leads to the formation of Amyloid-beta peptides (Aß 42/40). Deposition of 42 residual Aß peptides forms fibril's structure, disrupting neuron synaptic transmission, inducing neural cell toxicity, and ultimately leading to neuron death. OBJECTIVE: Various novel peptides have been investigated via molecular docking and molecular dynamic simulation studies to investigate their effects on Aß amyloidogenesis. METHODS: The sequence-based peptides were rationally designed and investigated for their interaction with Aß42 monomer and fibril, and their influence on the structural stability of target proteins was studied. RESULTS: Analyzed docking results suggest that the peptide YRIGY (P6) has the highest binding affinity with Aß42 fibril amongst all the synthetic peptides, and the peptide DKAPFF (P12) similarly shows a better binding with the Aß42 monomer. Moreover, simulation results also suggest that the higher the binding affinity, the better the inhibitory action. CONCLUSION: These findings indicate that both the rationally designed peptides can modulate amyloidogenesis, but peptide (P6) has better potential for the disaggregation of the fibrils. In contrast, peptide P12 stabilizes the native structure of the Aß42 monomer more effectively and hence can serve as a potential amyloid inhibitor. Thus, these peptides can be explored as therapeutic agents against Alzheimer's disease. Experimental testing of these peptides for immunogenicity, stability in cellular conditions, toxic effects and membrane permeability can be the future research scope of this study.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH.

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross ß sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

Elucidating the inhibitory effects of rationally designed novel hexapeptide against hen egg white lysozyme fibrillation at acidic and physiological pH.

Inhibition of highly ordered cross-ß-sheet-rich aggregates of misfolded amyloid proteins using rationally designed sequence-based short peptides is a promising therapeutic strategy for the treatment of neurodegenerative diseases. Here, we have explored the anti-amyloidogenic potency of a rationally designed hexapeptide (Tyr-Pro-Gln-Ile-Pro-Asn) on in vitro hen egg white lysozyme (HEWL) amyloid fibril formation at acidic pH and physiological pH using computational docking as well as various biophysical techniques such as fluorescence spectroscopy, UV-vis spectroscopy, FTIR spectroscopy, confocal microscopy and TEM. The peptide was designed based on the aggregation-prone region (APR) of HEWL and thus referred to as SqP1 (Sequence-based Peptide 1). SqP1 showed over 70% inhibition of HEWL amyloid formation at pH 2.2 and approximately 50% inhibition at pH 7.5. We propose that SqP1 binds to the APR of HEWL and interacts strongly with the Trp62/Trp63, ultimately stabilizing monomeric HEWL at both the pH conditions and preventing conformation changes in the structure of HEWL, leading to the formation of amyloidogenic fibrillar structures. A sequence-based peptide inhibitor of HEWL amyloid formation was not reported previously, making this a critical study that will further emphasize the importance of short synthetic peptides as amyloid inhibitors.

A human centred approach to digital technologies in health care delivery among mothers, children and adolescents.

BACKGROUND: Healthcare outcomes in child, adolescent and maternal in Tanzania are poor, and mostly characterised by fragmentary service provision. In order to address this weakness, digital technologies are sought to be integrated in the milieu of health as they present vast opportunities especially in the ability to improve health information management and coordination. Prior to the design and implementation of the Afya-Tek digital intervention, formative research was carried out to ensure that the solution meets the needs of the users. The formative research aimed to examine: the burden of disease and related health seeking behaviour; workflow procedures and challenges experiencing healthcare actors; adolescent health and health seeking behaviour; and lastly examine technological literacy and perceptions on the use of digital technologies in healthcare delivery. This paper therefore, presents findings from the formative research. METHODS: The study employed exploratory design grounded in a qualitative approach. In-depth interview, focus group discussion, participant observation and documentary review methods were used for collecting data at different levels. The analysis was done thematically, whereby meaning was deduced behind the words which the participants used. RESULTS: Findings suggest that the perceived burden of diseases and health seeking behaviour differ across age and social group. Multiple work-related challenges, such as lack of proper mechanism to track referrals and patient's information were noted across healthcare actors. There was a keen interest in the use of technologies shown by all study participants to improve care coordination and health outcomes among health system actors. Participants shared their views on how they envision the digital system working. CONCLUSION: The formative research provided insightful background information with regard to the study objectives. The findings are used for informing the subsequent phases of the co-development and implementation of the Afya-Tek digital health intervention; with a view to making it relevant to the needs of those who will use it in the future. As such, the findings have to a large extent met the purpose of the current study by envisaging the best ways to design digital intervention tailored to meet the needs of those who will be using it.

Myxovirus resistance (Mx) Gene Diversity in Avian Influenza Virus Infections.

Avian influenza viruses (AIVs) pose threats to animal and human health. Outbreaks from the highly pathogenic avian influenza virus (HPAIV) in indigenous chickens in Bangladesh are infrequent. This could be attributed to the Myxovirus resistance (Mx) gene. To determine the impact of Mx gene diversity on AIV infections in chicken, we assessed the Mx genes, AIVs, and anti-AIV antibodies. DNA from blood cells, serum, and cloacal swab samples was isolated from non-vaccinated indigenous chickens and vaccinated commercial chickens. Possible relationships were assessed using the general linear model (GLM) procedure. Three genotypes of the Mx gene were detected (the resistant AA type, the sensitive GG type, and the heterozygous AG type). The AA genotype (0.48) was more prevalent than the GG (0.19) and the AG (0.33) genotypes. The AA genotype was more prevalent in indigenous than in commercial chickens. A total of 17 hemagglutinating viruses were isolated from the 512 swab samples. AIVs were detected in two samples (2/512; 0.39%) and subtyped as H1N1, whereas Newcastle disease virus (NDV) was detected in the remaining samples. The viral infections did not lead to apparent symptoms. Anti-AIV antibodies were detected in 44.92% of the samples with levels ranging from 27.37% to 67.65% in indigenous chickens and from 26% to 87.5% in commercial chickens. The anti-AIV antibody was detected in 40.16%, 65.98%, and 39.77% of chickens with resistant, sensitive, and heterozygous genotypes, respectively. The genotypes showed significant association (p < 0.001) with the anti-AIV antibodies. The low AIV isolation rates and high antibody prevalence rates could indicate seroconversion resulting from exposure to the virus as it circulates. Results indicate that the resistant genotype of the Mx gene might not offer anti-AIV protection for chickens.

A comprehensive mini-review on amyloidogenesis of different SARS-CoV-2 proteins and its effect on amyloid formation in various host proteins.

Amyloidogenesis is the inherent ability of proteins to change their conformation from native state to cross ß-sheet rich fibrillar structures called amyloids which result in a wide range of diseases like Parkinson's disease, Alzheimer's disease, Finnish familial amyloidosis, ATTR amyloidosis, British and Danish dementia, etc. COVID-19, on the other hand is seen to have many similarities in symptoms with other amyloidogenic diseases and the overlap of these morbidities and symptoms led to the proposition whether SARS-CoV-2 proteins are undergoing amyloidogenesis and whether it is resulting in or aggravating amyloidogenesis of any human host protein. Thus the SARS-CoV-2 proteins in infected cells, i.e., Spike (S) protein, Nucleocapsid (N) protein, and Envelope (E) protein were tested via different machinery and amyloidogenesis in them were proven. In this review, we will analyze the pathway of amyloid formation in S-protein, N-protein, E-protein along with the effect that SARS-CoV-2 is creating on various host proteins leading to the unexpected onset of many morbidities like COVID-induced Acute Respiratory Distress Syndrome (ARDS), Parkinsonism in young COVID patients, formation of fibrin microthrombi in heart, etc., and their future implications.

Realist evaluation in times of decolonising global health.

Realist evaluation (RE) is a theory-driven evaluation approach inspired by scientific realism. It has become increasingly popular in the field of global health where it is often applied in low- and middle-income countries. This makes it timely to discuss RE's relationship to the emerging decolonisation of global health movement. In this short perspective, we argue that the principles and practices that underpin RE have great potential to contribute to the decolonisation endeavour. Both the focus on the inclusion of local stakeholders and the openness to the rival theories these stakeholders bring to the fore, are promising. However, in practice, we see that a lack of acknowledgement of power imbalances and different ontologies and an overreliance on Western-based theories thwart this potential. We therefore suggest that realist evaluations performed by external researchers, especially in the field of global health, should actively engage with issues of (power) inequities. This is not only the just thing to do, but will also contribute to a better understanding of the intervention and may facilitate the emancipation of the disenfranchised. One way of doing this is through the adoption of participatory (action) research methods, currently underused in realist evaluations. We finally give a short example of an evaluation that combines emancipatory and participatory practice development with a realist approach. The Afya-Tek project in Tanzania has an innovative bottom-up approach throughout the full evaluation cycle and shows the possible strength of the proposed combination to create better interventions, more empowered stakeholders, and more illuminating programme theories.

Integration of perinatal mental health care into district health services in Uganda: Why is it not happening? The Four Domain Integrated Health (4DIH) explanatory framework.

The Sustainable Development Goals recognise mental health and well-being as a target area, however, mobilizing funding and prioritisation of the same remains a challenge. Perinatal mental health care has the potential for incorporation and integration across the overall maternal health agenda, and can be especially relevant for low- and middle-income countries in their overall health systems strengthening strategies. This study aimed at qualitatively situating the extent to which integration of perinatal mental health care into maternal health care was considered desirable, possible and opportune within the existing policy and service-delivery environment in Uganda. A total of 81 in-depth interviews and nine focus group discussions (N = 64) were conducted with a variety of national, district, health system and community-level stakeholders. Data were analysed thematically using theory- and data-driven codebooks in NVivo 11. Analysis of the desirability, possibility and opportunity for integrating perinatal mental health care within the Ugandan district health system, highlights that concerned stakeholders perceive this as a worthwhile endeavour that would benefit the communities as well as the health system as a whole. Based on these current realities and ideal scenarios, a tentative explanatory framework that brings together various perspectives - that is, the perceived nature of the health problem, local and national health system issues, alternative systems of care and support, and international global perspectives - was constructed. The framework needs further validation but already hints at the need for global, national and local forces to concurrently rally behind the inclusion and integration of perinatal mental health care, especially at the primary care level in low- and middle-income contexts. If the global health community is poised to achieve high quality, women-centered care and people-centered health systems across the lifespan, then the sustainable integration of mental health care into general health care, is a commitment that can no longer be delayed.

The role of intra and inter-molecular disulfide bonds in modulating amyloidogenesis: A review.

All proteins have the inherent ability to undergo transformation from their native structure to a ß sheet rich fibrillar structure, called amyloid when subjected to specific conditions. Proteins with a high propensity to form amyloid fibrils have been implicated in a variety of disorders like Alzheimer's disease, Parkinson's disease, Type II diabetes, Amyotrophic Lateral Sclerosis (ALS) and prion diseases. Among the various critical factors that modulate the process of amyloid formation, disulfide bonds have been identified as one of the key determinants of amyloid propensity in proteins. Studies have shown that intra-molecular disulfide bonds impart stability to the native structure of a protein and decrease the tendency for amyloid aggregation, whereas intermolecular disulfide bonds aid in the process of aggregation. In this review, we will analyze the varying effects of both intra as well as inter-molecular disulfide bonds on the amyloid aggregation propensities of a few proteins associated with amyloid disorders.

Inhibitory effects of carbon quantum dots towards hen egg white lysozyme amyloidogenesis through formation of a stable protein complex.

Proteins, under certain circumstances such as defective quality control mechanism, mutations and altered environmental conditions, undergo misfolding and assemble into highly ordered beta-sheet structured fibrillar aggregates called amyloid fibrils. Formation of amyloid is seen in most of the protein linked degenerative diseases like Alzheimer's disease, Parkinson's disease, Huntington's disease, Type II diabetes mellitus and many more. Amyloid fibril forms via intermediate state(s), and is known to follow a nucleated condensation polymerization mechanism. Though extensive research is being carried out towards finding a therapeutic solution to the amyloidosis, an effective treatment to these diseases still remains elusive and also the mechanism of amyloidogenesis largely remains unclear. In recent times, carbon quantum dots (CQDs) are gaining the attention of researchers due to their semi-conductive nature, excellent physio-chemical properties, high surface to volume ratio, optical properties and mainly bio-compatibility. In the current study, we have synthesized CQDs from commonly available kitchen spice mix and explored their role in amyloidogenesis using hen egg white lysozyme (HEWL) as a model protein. The results clearly demonstrate the amyloid inhibitory as well as disaggregation potential of CQD by forming a stable complex with HEWL and thereby increasing the energy barrier for the aggregation process.

Human Serum Albumin Aggregation and its Modulation Using Nanoparticles: A Review.

Amyloid fibrils are highly stable protein fibrillar aggregates believed to be involved in various neurodegenerative diseases, which include Alzheimer's disease, Parkinson's disease, and prion diseases. Inhibiting the aggregation process is a potential strategy to prevent diseases caused by amyloid formation. In this regard, nanoparticles have emerged as promising candidates owing to their unique physical/chemical properties of small size, large surface area, biocompatibility, biodegradability, non-toxicity, and ease of functionalization. Human Serum Albumin (HSA) is a soluble multidomain monomeric protein that interacts with various ligands and hormones, aiding in their transport, distribution, metabolism in the circulatory system, and also plays a vital role in extracellular fluid volume stabilization. Under certain in vitro conditions, HSA has been reported to undergo conformational changes leading to fibril formation and hence acts as a suitable model for studying amyloidogenesis. In this review, we have explored the effects of various nanoparticles on HSA aggregation and their mechanism of action. The study will throw light on the mechanistic details of nanoparticle-mediated amyloid modulation, which will help in the development of effective therapeutics against amyloidosis.