Probing protein aggregation through spectroscopic insights and multimodal approaches: A comprehensive review for counteracting neurodegenerative disorders.

Aberrant accumulation of protein misfolding can cause aggregation and fibrillation and is one of the primary characteristic features of neurodegenerative diseases. Because they are disordered, misfolded, and aggregated proteins pose a significant setback in drug designing. The structural study of intermediate steps in these kinds of aggregated proteins will allow us to determine the conformational changes as well as the probable pathways encompassing various neurodegenerative disorders. The analysis of protein aggregates involved in neurodegenerative diseases relies on a diverse toolkit of biophysical techniques, encompassing both morphological and non-morphological methods. Additionally, Thioflavin T (ThT) assays and Circular Dichroism (CD) spectroscopy facilitate investigations into aggregation kinetics and secondary structure alterations. The collective application of these biophysical techniques empowers researchers to comprehensively unravel the intricate nature of protein aggregates associated with neurodegeneration. Furthermore, the topics covered in this review have summed up a handful of well-established techniques used for the structural analysis of protein aggregation. This multifaceted approach advances our fundamental understanding of the underlying mechanisms driving neurodegenerative diseases and informs potential therapeutic strategies.

Amyloid-induced neurodegeneration: A comprehensive review through aggregomics perception of proteins in health and pathology.

Amyloidosis of protein caused by fibrillation and aggregation are some of the most exciting new edges not only in protein sciences but also in molecular medicines. The present review discusses recent advancements in the field of neurodegenerative diseases and therapeutic applications with ongoing clinical trials, featuring new areas of protein misfolding resulting in aggregation. The endogenous accretion of protein fibrils having fibrillar morphology symbolizes the beginning of neuro-disorders. Prognostic amyloidosis is prominent in numerous degenerative infections such as Alzheimer's and Parkinson's disease, Amyotrophic lateral sclerosis (ALS), etc. However, the molecular basis determining the intracellular or extracellular evidence of aggregates, playing a significant role as a causative factor in neurodegeneration is still unclear. Structural conversions and protein self-assembly resulting in the formation of amyloid oligomers and fibrils are important events in the pathophysiology of the disease. This comprehensive review sheds light on the evolving landscape of potential treatment modalities, highlighting the ongoing clinical trials and the potential socio-economic impact of novel therapeutic interventions in the realm of neurodegenerative diseases. Furthermore, many drugs are undergoing different levels of clinical trials that would certainly help in treating these disorders and will surely improve the socio-impact of human life.

Biochemical Characterization of Laccase from Spirulina CPCC-695 and Their Role in Estrone Degradation.

The addition of exogenous endocrine disrupting compounds (EDCs) like estrone, in the food chain through the aquatic system, disrupts steroid biosynthesis and metabolism by altering either the genomic or non-genomic pathway that eventually results in various diseases. Thus, bioremediation of these compounds is urgently required to prevent their addition and persistence in the environment. Enzymatic degradation has proven to be a knight in shining armour as it is safe and generates no toxic products. The multicopper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase), laccase with the potential to degrade both phenolic and non-phenolic substrates has recently gained attention. In this study, the laccase was purified, characterized, and used to study estrone degradation. The culture filtrate (crude laccase) was concentrated and precipitated using cold-acetone and dialyzed against tris buffer (50 mM) giving a four-fold partially purified form, with 45.56% yield and 204.14 U/mg as specific activity and a single peak at 250-300 nm. The partially purified laccase was approximately 80 kDa as estimated by SDS-PAGE preferred ABTS as substrate. Both crude and partially purified laccase showed maximum activity at pH 3.0, 40 °C, and 4 mM ABTS. Kinetic constants (Km, Vmax) of crude and partially purified laccase were found to be 0.83 mM; 494.31 mM/min, and 0.58 mM; 480.54 mM/min respectively. Iron sulphate and sodium azide inhibited laccase maximally. Crude and partially purified laccase degradation efficiency was 87.55 and 91.35% respectively. Spirulina CPCC-695 laccase with efficient estrone degradation ability renders them promising candidates for EDCs bioremediation.

Optimized culture conditions facilitate the estrone biodegradation ability and laccase activity of Spirulina CPCC-695.

Endocrine disrupting compounds (EDCs) are emerging contaminants that persist and contaminate the environment. They mimic hormones, block hormones, or modulate their synthesis, metabolism, transport, and action, affecting living organisms and their progeny. Steroid hormones from exogenous sources like water bodies are important EDCs. Their biodegradation is an urgent global need. The present study is a preliminary work to maximize the estrone degradation potential of Spirulina CPCC-695 and study the effect of optimized conditions on its laccase activity. It was observed that the exponential phase culture at pH 10.0, 30 ℃, and 200 rpm of agitation speed resulted in the maximum growth, estrone degradation efficiency (93.12%), and highest laccase activity (74%) of Spirulina CPCC-695.

Exogenous salicylic acid mediated herbicide (Paraquat) resistance in cyanobacterial biofertilizer Microchaete sp. NCCU-342.

Paddy field farming remains the dominant form of growing rice in modern times as the rice is the staple food for over half the world's population and is closely associated with food security and political stability of many countries. Record increase in rice production have been observed since the start of the Green Revolution. India is one of the largest paddy producer and exporter in the world. However, constant use of chemical herbicide like paraquat had shown adverse impact on the rice yield. Non-target organisms of the habitat including cyanobacterial paddy biofertilizer face the herbicide toxicity and are unable to perform efficiently their role as biofertilizer. Therefore, in the present study, an attempt has been made to enhance the paraquat resistance in rice biofertilizer (Microchaete sp. NCCU-342) by exogenous addition of salicylic acid. Paraquat showed toxicity in Microchaete sp. NCCU-342 in a dose-dependent manner. Concentration of paraquat >1.0 µM exhibited lethal effect since the beginning. Through successive narrow range experiment, LD50 value of paraquat was obtained as 0.6 µM. Biomass exposed to paraquat (LD50 value) and salicylic acid (0.3 mM) showed mitigation in free radical production (2.20 % MDA and 1.69 % H2O2) and enhancement in the activity of the antioxidant enzymes activity, i.e. SOD, CAT, APX (137.76 %, 87.45 %, 118 %, respectively) and osmolytes (3.8 % proline and 21.51% sucrose). Thus, for sustainable agricultural practice, especially for paddy field cyanobacterial biofertilizer, application of salicylic acid or organism with higher salicylic acid production ability may be an alternative to overcome the paraquat toxicity.

Biosynthesis, characterization and biomedical potential of Arthrospira indica SOSA-4 mediated SeNPs.

The use of aqueous cyanobacterial extracts for selenium nanoparticle (SeNP) synthesis is considered green, cost-effective, and eco-friendly technology that is more advanced than physical and chemical methods. In the current study, an aqueous extract of Arthrospira indica SOSA-4 was used as a reducing and stabilizing agent for the green synthesis of SeNPs. The UV-Visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-Ray diffraction, Raman spectroscopy, Atomic force microscopy (AFM), Scanning electron microscopy-Energy Dispersive X-Ray spectroscopy(SEM-EDX), and Transmission electron microscopy (TEM) were performed to characterize the biosynthesized SeNPs. Gas chromatography-Mass spectrometry (GC-MS) was also performed to know the composition of the cyanobacterial extract. SEM, TEM, and AFM showed the average size of SeNPs to be 8.5 nm, 9 nm, and 8.7 nm respectively. FT-IR analysis demonstrated the presence of functional groups on the SeNPs that acted as stabilizing agents. XRD pattern and Raman spectroscopy showed the amorphous nature of SeNPs. Synthesized SeNPs showed significant antioxidant activity in DPPH, FRAP, SOR, and ABTS assay. SeNPs showed good anti-microbial activity against Staphylococcus aureus, Escherichia coli, Candida albicans, Candida glabrata, and Candida tropicalis and good anti-cancer activity in MTT assay, Trypan assay, and Flow cytometry analysis against MCF-7, SiHa, and SW480 cell lines. Non-toxicity of SeNPs against normal cell line (HEK-293) was an additional property that affirmed its potential as a bio-compatible nanomaterial.

Biochemical Characterization of Novel Phenylalanine Ammonia-Lyase from Spirulina CPCC-695.

Phenylalanine ammonia lyase (PAL) catalyzes the deamination of phenylalanine to cinnamic acid and ammonia. It plays a crucial role in the formation of secondary metabolites through the phenylpropanoid pathway. Recently there has been growing interest in exploring the biochemical properties of PAL for its clinical and commercial applications. PAL as a key component has been used in metabolic engineering and synthetic biology. Due to its high substrate specificity and catalytic efficacy, PAL has opened a new area of interest in the biomedical field. PAL has been frequently used in the enzyme replacement therapy of phenylketonuria, cancer treatment and microbial production of l-phe the precursor of noncalorific sweetener aspartame (Methyl L-α-aspartyl-l-phenylalaninate), antimicrobial and health supplements. PAL occurs in few plants, fungi, bacteria, and cyanobacteria. The present investigation is a preliminary study in which an attempt has been made for the isolation, partial purification, and biochemical characterization of PAL (crude and partially purified) from Spirulina CPCC-695. Partially purified PAL exhibited higher enzymatic activity and protein content than the crude enzyme. Molecular weight of the crude and partially purified PAL was ~ 66 kDa. The optimum temperature and pH for PAL activity was observed as 30 â„ƒ and 8.0 respectively. l-Phe was the most preferred substrate (100 mM) whereas gallic acid showed maximum inhibition of PAL activity. Enzyme kinetics suggested good catalytic efficacy of the PAL enzyme and affinity towards substrate. Both the enzyme (crude and partially purified) showed less than 5% haemolysis suggesting the biocompatible nature of PAL.

Mitigative effect of biosynthesized SeNPs on cyanobacteria under paraquat toxicity.

Increasing population has resulted in increased food demand. Pesticides like paraquat (PQ) have been used indiscriminately to increase the growth and yield of crops. However, this has adversely affected a wide spectrum of non-target organisms like cyanobacteria that are used as a bio-fertilizer in the rice field. In the present study, biogenic- Gloeocaspa gelatinosa NCCU -430 mediated selenium nanoparticles (SeNPs) were synthesized and characterized using different techniques including UV-Visible spectroscopy, XRD, FTIR, TEM and SEM-EDX for their use as PQ toxicity mitigator in cyanobacterial biofertilizer (Anabaena variabilis NCCU-442). Therefore, a comparative study was performed among control, PQ, SeNPs and SeNPs+PQ to check the efficacy of SeNPs in mitigation of PQ induced toxicity. Supplementation of SeNPs in PQ treated culture enhanced antioxidant enzymes activity i.e., SOD (7.55%), CAT (57.94%), APX (17.45%) and GR (14.72%) as compared to only PQ treated culture. The outcomes of the present study suggested that SeNPs can ameliorate the PQ induced stress that may be used in sustainable rice cultivation needed for filing the gap between requirement and supply.

Biomedical potential of Anabaena variabilis NCCU-441 based Selenium nanoparticles and their comparison with commercial nanoparticles.

Selenium nanoparticles (SeNPs) are gaining importance in the field of medicines due to their high surface area and unique properties than their other forms of selenium. In this study, biogenic selenium nanoparticles (B-SeNPs) were synthesized using cyanobacteria and their bioactivities (antioxidant, antimicrobial, anticancer and biocompatibility) were determined for comparison with commercially available chemically synthesized selenium nanoparticles (C-SeNPs). Color change of reaction mixture from sky blue to orange-red indicated the synthesis of biogenic SeNPs (B-SeNPs). UV-Vis spectra of the reaction mixture exhibited peak at 266 nm. During optimization, 30 °C of temperature, 24 h of time and 1:2 concentration ratio of sodium selenite and cell extract represented the best condition for SeNPs synthesis. Various functional groups and biochemical compounds present in the aqueous extract of Anabaena variabilis NCCU-441, which may have possibly influenced the reduction process of SeNPs were identified by FT-IR spectrum and GC-MS. The synthesized cyanobacterial SeNPs were orange red in color, spherical in shape, 10.8 nm in size and amorphous in nature. The B-SeNPs showed better anti-oxidant (DPPH, FRAP, SOR and ABTS assays), anti-microbial (antibacterial and antifungal) and anti-cancer activitities along with its biocompatibility in comparison to C-SeNPs suggesting higher probability of their biomedical application.

Biomedical applications of environmental friendly poly-hydroxyalkanoates.

Biological polyesters of hydroxyacids are known as polyhydroxyalkanoates (PHA). They have proved to be an alternative, environmentally friendly and attractive candidate for the replacement of petroleum-based plastics in many applications. Many bacteria synthesize these compounds as an intracellular carbon and energy compound usually under unbalanced growth conditions. Biodegradability and biocompatibility of different PHA has been studied in cell culture systems or in an animal host during the last few decades. Such investigations have proposed that PHA can be used as biomaterials for applications in conventional medical devices such as sutures, patches, meshes, implants, and tissue engineering scaffolds as well. Moreover, findings related to encapsulation capability and degradation kinetics of some PHA polymers has paved their way for development of controlled drug delivery systems. The present review discusses about bio-plastics, their characteristics, examines the key findings and recent advances highlighting the usage of bio-plastics in different medical devices. The patents concerning to PHA application in biomedical field have been also enlisted that will provide a brief overview of the status of research in bio-plastic. This would help medical researchers and practitioners to replace the synthetic plastics aids that are currently being used. Simultaneously, it could also prove to be a strong step in reducing the plastic pollution that surged abruptly due to the COVID-19 medical waste.

Exploring algae and cyanobacteria as a promising natural source of antiviral drug against SARS-CoV-2.

The present outburst of coronavirus-associated (SARS-CoV-2) acute respiratory disease coronavirus disease 19 (COVID-19) in December 2019 in Wuhan, China is the third recognised spill over due to the zoonotic transmission. SARS-CoVs are about 29.7 kb positive, single stranded (ss) RNA viruses that are considered as zoonotic pathogens, bat being their natural reservoirs and also shows transmission within humans. The rapidly increasing COVID-19 cases and need of best and efficient drug/vaccine/strategy to counteract the virus entry and its pathogenesis has made it a Herculean challenge for scientists. Synthetic drugs associated complications has attracted scientific attention for natural product-based drugs. Chemo-diversity of algae and cyanobacteria offers a novel approach and can be recognized as a relevant source for developing a future natural "antiviral drug". The aim of this review is to highlight important features of SARS-CoV-2/COVID-19 and the antiviral compounds recognized in algae and cyanobacteria, with their mechanisms of actions. Algae possess both immunity improving capacity and suppresses many viruses. Thus, they can be recommended as a preventive and curative remedy against SARS-CoV-2.

Estrone degrading enzymes of Spirulina CPCC-695 and synthesis of bioplastic precursor as a by-product.

Estrone, a steroidal estrogen that is persistently contaminating the surface water has been classified as an endocrine disruptor and as Group-1 carcinogen by the World Health Organization. Long-term exposure to estrone-contaminated water disrupt physiology, behaviour and sexual development of living organisms that lead to many disorders. So, it has to be eliminated from our surrounding. Its biological degradation is a cost effective and eco-friendly approach. The present study targets to predict the degradation pathway and understand the role of cyanobacterial enzymes: oxidoreductases (laccase, peroxidase) and esterase in estrone degradation. Poly-ß-hydroxy butyrate (PHB) was also quantified as a by-product of estrone biodegradation. The estrone degradation pathway was predicted using EAWAG-BBD/PPS database. Spirulina CPCC-695 was grown in different concentration of estrone (20 mg/l, 50 mg/l, 100 mg/l and 200 mg/l). The culture without estrone was considered as control. The culture supernatant was used for testing laccase and esterase activity whereas the biomass was used to test peroxidase activity and quantify by-product (PHB). The enzymes showed concentration-dependent activities. Maximum enzyme activities were seen at 20 mg/l estrone. Spirulina CPCC-695 utilizes estrone as a carbon source and degrades it to produce pyruvate which forms acetyl CoA that undergo condensation, reduction and polymerization to form PHB. Maximum PHB (169 µg) was also produced at 20 mg/l as a by-product during degradation.

Protein aggregation, misfolding and consequential human neurodegenerative diseases.

Proteins are major components of the biological functions in a cell. Biology demands that a protein must fold into its stable three-dimensional structure to become functional. In an unfavorable cellular environment, protein may get misfolded resulting in its aggregation. These conformational disorders are directly related to the tissue damage resulting in cellular dysfunction giving rise to different diseases. This way, several neurodegenerative diseases such as Alzheimer, Parkinson Huntington diseases and amyotrophic lateral sclerosis are caused. Misfolding of the protein is prevented by innate molecular chaperones of different classes. It is envisaged that work on this line is likely to translate the knowledge into the development of possible strategies for early diagnosis and efficient management of such related human diseases. The present review deals with the human neurodegenerative diseases caused due to the protein misfolding highlighting pathomechanisms and therapeutic intervention.

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies, enzyme, and cell assays.

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 106, M-1 (±.21). However, UBA-domain of MARK4-F3 doesn't show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC50 values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.

Exploring Missense Mutations in Tyrosine Kinases Implicated with Neurodegeneration.

Protein kinases are one of the largest families of evolutionarily related proteins and the third most common protein class of human genome. All the protein kinases share the same structural organization. They are made up of an extracellular domain, transmembrane domain and an intra cellular kinase domain. Missense mutations in these kinases have been studied extensively and correlated with various neurological disorders. Individual mutations in the kinase domain affect the functions of protein. The enhanced or reduced expression of protein leads to hyperactivation or inactivation of the signalling pathways, resulting in neurodegeneration. Here, we present extensive analyses of missense mutations in the tyrosine kinase focussing on the neurodegenerative diseases encompassing structure function relationship. This is envisaged to enhance our understanding about the neurodegeneration and possible therapeutic measures.

Protein aggregation and neurodegenerative diseases: From theory to therapy.

The study of protein misfolding and aggregation saw resurgence in the last decade. Aggregation is the main cause of several human neurodegenerative diseases which makes this field as the leading edge in the science today. Protein aggregation is a highly complex process resulting in formation of a variety of aggregates with different structures and morphologies. Many of them are highly cytotoxic. In-depth knowledge about structure, mechanism of formation, and physiological effects of aggregates will shed new light on the aggregation-mediated cell toxicity, and helps in deciphering new target for drug design and development. This review summarizes the existing information on the molecular mechanism of protein misfolding and aggregation involved in neurodegeneration stressing on the possible therapeutic intervention in neurodegenerative diseases. As our knowledge about the relation between the protein misfolding and disease pathogenesis will be enhanced, more specific and promising treatment opportunities will come into existence.

Ubiquitin-associated domain of MARK4 provides stability at physiological pH.

Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1-310) and only kinase domain (residues 59-310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions.