Circulating small extracellular vesicles in Alzheimer's disease: a case-control study of neuro-inflammation and synaptic dysfunction.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aß plaques and neurofibrillary tangles. Chronic inflammation and synaptic dysfunction lead to disease progression and cognitive decline. Small extracellular vesicles (sEVs) are implicated in AD progression by facilitating the spread of pathological proteins and inflammatory cytokines. This study investigates synaptic dysfunction and neuroinflammation protein markers in plasma-derived sEVs (PsEVs), their association with Amyloid-ß and tau pathologies, and their correlation with AD progression. METHODS: A total of 90 [AD = 35, mild cognitive impairment (MCI) = 25, and healthy age-matched controls (AMC) = 30] participants were recruited. PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy. Using nanoparticle tracking analysis, the size and concentration of PsEVs were determined. Antibody-based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies. Synaptic dysfunction and neuroinflammation were evaluated with synaptophysin, TNF-α, IL-1ß, and GFAP antibodies. AD-specific markers, amyloid-ß (1-42), and p-Tau were examined within PsEVs using Western blot and ELISA. RESULTS: Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p < 0.0001). Amyloid-ß (1-42) expression within PsEVs is significantly elevated in MCI and AD compared to AMC. We could also differentiate between the amyloid-ß (1-42) expression in AD and MCI. Similarly, PsEVs-derived p-Tau exhibited elevated expression in MCI compared with AMC, which is further increased in AD. Synaptophysin exhibited downregulated expression in PsEVs from MCI to AD (p = 0.047) compared to AMC, whereas IL-1ß, TNF-α, and GFAP showed increased expression in MCI and AD compared to AMC. The correlation between the neuropsychological tests and PsEVs-derived proteins (which included markers for synaptic integrity, neuroinflammation, and disease pathology) was also performed in our study. The increased number of PsEVs correlates with disease pathological markers, synaptic dysfunction, and neuroinflammation. CONCLUSIONS: Elevated PsEVs, upregulated amyloid-ß (1-42), and p-Tau expression show high diagnostic accuracy in AD. The downregulated synaptophysin expression and upregulated neuroinflammatory markers in AD and MCI patients suggest potential synaptic degeneration and neuroinflammation. These findings support the potential of PsEV-associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression.

Fluorescence-tagged salivary small extracellular vesicles as a nanotool in early diagnosis of Parkinson's disease.

BACKGROUND: Parkinson's disease is generally asymptomatic at earlier stages. At an early stage, there is an extensive progression in the neuropathological hallmarks, although, at this stage, diagnosis is not possible with currently available diagnostic methods. Therefore, the pressing need is for susceptibility risk biomarkers that can aid in better diagnosis and therapeutics as well can objectively serve to measure the endpoint of disease progression. The role of small extracellular vesicles (sEV) in the progression of neurodegenerative diseases could be potent in playing a revolutionary role in biomarker discovery. METHODS: In our study, the salivary sEV were efficiently isolated by chemical precipitation combined with ultrafiltration from subjects (PD = 70, healthy controls = 26, and prodromal PD = 08), followed by antibody-based validation with CD63, CD9, GAPDH, Flotillin-1, and L1CAM. Morphological characterization of the isolated sEV through transmission electron microscopy. The quantification of sEV was achieved by fluorescence (lipid-binding dye-labeled) nanoparticle tracking analysis and antibody-based (CD63 Alexa fluor 488 tagged sEV) nanoparticle tracking analysis. The total alpha-synuclein (α-synTotal) in salivary sEVs cargo was quantified by ELISA. The disease severity staging confirmation for n = 18 clinically diagnosed Parkinson's disease patients was done by 99mTc-TRODAT-single-photon emission computed tomography. RESULTS: We observed a significant increase in total sEVs concentration in PD patients than in the healthy control (HC), where fluorescence lipid-binding dye-tagged sEV were observed to be higher in PD (p = 0.0001) than in the HC using NTA with a sensitivity of 94.34%. In the prodromal PD cases, the fluorescence lipid-binding dye-tagged sEV concentration was found to be higher (p = 0.008) than in HC. This result was validated through anti-CD63 tagged sEV (p = 0.0006) with similar sensitivity of 94.12%. We further validated our findings with the ELISA based on α-synTotal concentration in sEV, where it was observed to be higher in PD (p = 0.004) with a sensitivity of 88.24%. The caudate binding ratios in 99mTc-TRODAT-SPECT represent a positive correlation with sEV concentration (r = 0.8117 with p = 0.0112). CONCLUSIONS: In this study, for the first time, we have found that the fluorescence-tagged sEV has the potential to screen the progression of disease with clinically acceptable sensitivity and can be a potent early detection method for PD.

Different Biofluids, Small Extracellular Vesicles or Exosomes: Structural Analysis in Atherosclerotic Cardiovascular Disease Using Electron Microscopy Techniques.

Small extracellular vesicles (sEVs) or exosomes are secretory vesicles largely involved in cell-cell communications and found to play a role in development as well as diseases including atherosclerosis. They hold a huge potential for translational research by devising better clinical diagnostics, biomarker discovery, drug delivery, and therapeutic strategies. Variations terms of morphology and distribution are crucial to biological function integrity. Moreover, it is dependent on susceptibility to influential factors of the environment like cell stress, inflammation, and secretion by different cells in subsequent biofluids. We have observed the morphological variations in sEVs or exosomes freshly isolated from patients with atherosclerotic cardiovascular disease (AsCVD), in blood plasma, saliva, and urine biofluids compared to healthy controls. High-resolution images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) for the characterization of sEVs morphology. Western blotting and immuno-TEM gold labeling confirmed the presence of exosome markers. For the first time, we report size and shape variations, which suggest the existence of different functions of sEVs in the disease state. Morphological variations in sEVs were observed significantly in noninvasive AsCVD saliva and urine samples, important to understand the cell behavior and physiological state. These variations will be useful to investigate their possible role in the disease process.

A comprehensive proteomic profiling of urinary exosomes and the identification of early non-invasive biomarker in patients with coronary artery disease.

Urinary small extracellular vesicles or exosomes (uEVs) source could be an emerging trove of biomarkers in coronary artery disease (CAD). It is a chronic inflammatory disease having a long asymptomatic phase of fatty-fibrous development in arteries leading to angina, myocardial infarction, and death. Our study was aimed at identifying differential protein expression profiling of uEVs in CAD. We collected urine samples of CAD patients (n = 41) age 18-65 years and gender matched healthy controls (n = 41). We isolated uEVs using differential ultracentrifugation. Further, uEV samples were characterized by western blotting exosome markers (Flotillin, TSG, CD63, and CD9), nano tracking analysis, and transmission and scanning electron microscopy. A total of 508 proteins were identified by iTRAQ-based mass spectrometry. We observed protein expression levels of AZGP1, SEMG1/2, ORM1, IGL, SERPINA5, HSPG2, prosaposin, gelsolin, and CD59 were upregulated, and UMOD, KNG1, AMBP, prothrombin, and TF were downregulated. Protein-protein interactions, gene ontology and pathway analysis were performed to functionally annotate identified uEVs proteins. A novel uEVs differential protein signature is shown. On validating UMOD protein by ELISA in two clinically different CAD, stable-CAD patients had lower levels than healthy controls whereas recent myocardial infarction patients had lowest. Our findings suggest UMOD importance as early diagnostic biomarker. SIGNIFICANCE: Coronary artery disease is a chronic inflammatory disease caused by gradual deposition of cholesterol and fat along with other proteins to develop plaque inside arteries. This further leads to blockage of artery, heart attack and death. There are no identifiable early biomarkers to diagnose this. For the first time, we have identified the differentially expressed proteins isolated from non-invasive uEV of CAD patients compared to healthy controls by using MS Orbitrap and iTRAQ labelling of peptides. We have identified decreased levels of UMOD protein in CAD. These findings have been confirmed by ELISA. Furthermore, the levels of UMOD were observed as more highly decreased in recent myocardial infarction CAD patients, indicating the importance of this protein as an early diagnostic biomarker. Conclusively, our study represents a non-invasive urinary EVs trove of differentially expressed proteins in CAD. This will form a groundwork for understanding the pathophysiology of CAD and will help in future translational research utilizing uEVs.

Identification of a novel inhibitor of SARS-CoV-2 main protease: an in silico, biochemical, and cell-based approach.

The recurrent nature of coronavirus outbreaks, severity of the COVID-19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA-approved and natural product libraries using in silico approach for potential hits against the SARS-CoV-2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell-based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS-CoV-2-based cell-free assays, followed by testing in the HCoV-229E-based culture system. Of the tested compounds, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1-isopropyl-1H-benzofuro[3,2-b]pyrazolo[4,3-e]pyridin-3(2H)-one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS-CoV-2-culture system, where STL522228 demonstrated superior antiviral activity (EC50 = 0.44 µm) compared to Remdesivir (EC50 = 0.62 µm). Based on these findings, we report the strong anti-coronavirus activity of STL522228, and propose that it as a promising pan-coronavirus Mpro inhibitor for further experimental and preclinical validation.

Circulating plasma miR-23b-3p as a biomarker target for idiopathic Parkinson's disease: comparison with small extracellular vesicle miRNA.

Background: Parkinson's disease (PD) is an increasingly common neurodegenerative condition, which causes movement dysfunction and a broad range of non-motor symptoms. There is no molecular or biochemical diagnosis test for PD. The miRNAs are a class of small non-coding RNAs and are extensively studied owing to their altered expression in pathological states and facile harvesting and analysis techniques. Methods: A total of 48 samples (16 each of PD, aged-matched, and young controls) were recruited. The small extracellular vesicles (sEVs) were isolated and validated using Western blot, transmission electron microscope, and nanoparticle tracking analysis. Small RNA isolation, library preparation, and small RNA sequencing followed by differential expression and targeted prediction of miRNA were performed. The real-time PCR was performed with the targeted miRNA on PD, age-matched, and young healthy control of plasma and plasma-derived sEVs to demonstrate their potential as a diagnostic biomarker. Results: In RNA sequencing, we identified 14.89% upregulated (fold change 1.11 to 11.04, p < 0.05) and 16.54% downregulated (fold change -1.04 to -7.28, p < 0.05) miRNAs in PD and controls. Four differentially expressed miRNAs (miR-23b-3p, miR-29a-3p, miR-19b-3p, and miR-150-3p) were selected. The expression of miR-23b-3p was "upregulated" (p = 0.002) in plasma, whereas "downregulated" (p = 0.0284) in plasma-derived sEVs in PD than age-matched controls. The ROC analysis of miR-23b-3p revealed better AUC values in plasma (AUC = 0.8086, p = 0.0029) and plasma-derived sEVs (AUC = 0.7278, p = 0.0483) of PD and age-matched controls. Conclusion: We observed an opposite expression profile of miR-23b-3p in PD and age-matched healthy control in plasma and plasma-derived sEV fractions, where the expression of miR-23b-3p is increased in PD plasma while decreased in plasma-derived sEV fractions. We further observed the different miR-23b-3p expression profiles in young and age-matched healthy control.

Deciphering the mechanism of p73 recognition of p53 response elements using the crystal structure of p73-DNA complexes and computational studies.

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3' and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.

Cell surface proteins in archaeal and bacterial genomes comprising "LVIVD", "RIVW" and "LGxL" tandem sequence repeats are predicted to fold as beta-propeller.

Proteins that share even low sequence homologies are known to adopt similar folds. The beta-propeller structural motif is one such example. Identifying sequences that adopt a beta-propeller fold is useful to annotate protein structure and function. Often, tandem sequence repeats provide the necessary signal for identifying beta-propellers in proteins. In our recent analysis to identify cell surface proteins in archaeal and bacterial genomes, we identified some proteins that contain novel tandem repeats "LVIVD", "RIVW" and "LGxL". In this work, based on protein fold predictions and three-dimensional comparative modeling methods, we predicted that these repeat types fold as beta-propeller. Further, the evolutionary trace analysis of all proteins constituting amino acid sequence repeats in beta-propellers suggest that the novel repeats have diverged from a common ancestor.

Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases.

Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

Functional and structural analysis of mice TRPC6 with human analogue through homology modelling.

Homology models are increasingly used to determine structural and functional relationships of genes and proteins in biomedical research. In the current study, for the first time, we compared the TRPC6 gene in mouse and human. The protein encoded by this gene forms a receptor activated calcium channel in cell membrane. Defects in this gene have been implicated in a wide range of diseases including glioblastomas. To determine the structural similarities in mouse and human TRPC6, we used standard bioinformatics tools such as fold prediction to identify the protein 3D structure, sequence-structure comparison, and prediction of template and protein structure. We also used glioblastoma cell line U373MG and human glioblastoma tumour tissues to study the expression of TRPC6 in disease conditions to implicate this gene in pathological ailment. Based on the results we conclude that human TRPC6 contains 90% identity and 93% similarity with mouse TRPC6, suggesting that this protein is well conserved in these two species. These isoforms likely demonstrate similar mechanisms in regulating gene expression; thus TRPC6 studies in mice may be extrapolated to humans.

Computational analysis of human and mouse CREB3L4 Protein.

CREB3L4 is a member of the CREB/ATF transcription factor family, characterized by their regulation of gene expression through the cAMP-responsive element. Previous studies identified this protein in mice and humans. Whereas CREB3L4 in mice (referred to as Tisp40) is found in the testes and functions in spermatogenesis, human CREB3L4 is primarily detected in the prostate and has been implicated in cancer. We conducted computational analyses to compare the structural homology between murine Tisp40α human CREB3L4. Our results reveal that the primary and secondary structures of the two proteins contain high similarity. Additionally, predicted helical transmembrane structure reveals that the proteins likely have similar structure and function. This study offers preliminary findings that support the translation of mouse Tisp40α findings into human models, based on structural homology.

Highly conserved Asp-204 and Gly-776 are important for activity of the quinoprotein glucose dehydrogenase of Escherichia coli and for mineral phosphate solubilization.

Gram-negative bacteria membrane-bound glucose dehydrogenase (m-GDH) has pyrroloquinoline quinone [PQQ (2,7,9,-tricarboxyl-1H-pyrrolo[2,3-f]quinoline-4,5-dione)] as its prosthetic group, transferring electrons to ubiquinone (UQ) in the membrane. Based on the sequence homology of the C-terminal catalytic domain (151-796 amino acid residues) we have modeled the 3D structure of Escherichia coli GDH. The geometrical parameters of the homology model structure, validated using the Ramachandran plot, revealed 95.8% of residues in the allowed regions and 2.2% of the residues in disallowed regions. From the model, we have identified five different amino acids that are specifically involved in maintaining the PQQ in the correct configuration along with a Ca(2+) ion in the active site, and two amino acids on the surface of the protein that might be involved in UQ binding or transfer of electrons to the UQ. Site-directed mutants R201A, D204A, E217L, E217A, R266Q, R266E, E591L, E591Q, E591K, L712W, L712R, G776K, G776D and G776L lost their GDH activity, while E217Q and G776A retained their function similar to that of wild-type GDH, both in terms of specific activity and mineral phosphate solubilization. Our conclusions are consistent with those previously based on model GDH produced by a different method and using a different template X-ray structure.

Docking of phosphonate and trehalose analog inhibitors into M. tuberculosis mycolyltransferase Ag85C: Comparison of the two scoring fitness functions GoldScore and ChemScore, in the GOLD software.

The Ag85 family enzymes are responsible for the synthesis of cell wall components in mycobacterial species. Inhibitors to these enzymes are potential antimycobacterial agents. We have carried out the docking of phoshonate and trehalose analog inhibitors into the three dimensional structure of mycolyltransferase enzyme, Ag85C of M. tuberculosis using the GOLD software. The inhibitor binding positions and affinity were evaluated using both the scoring fitness functions- GoldScore and ChemScore. We observed that the inhibitor binding position identified using the GoldScore was marginally better than the ChemScore. A qualitative agreement between the reported experimental biological activities (IC50) and the GoldScore was observed. We identified that amino acid residues Arg541, Trp762 are important for inhibitor recognition via hydrogen bonding interactions. This information can be exploited to design Ag85C specific inhibitors.