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Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases with no approved therapeutics. TSE pathology is characterized by abnormal accumulation of amyloidogenic and infectious prion protein conformers (PrPSc) in the central nervous system. Herein, we examined the role of gallate group in green tea catechins in modulating the aggregation of human prion protein (HuPrP) using two green tea constituents i.e., epicatechin 3-gallate (EC3G; with intact gallate ring) and epigallocatechin (EGC; without gallate ring). Molecular docking indicated distinct differences in hydrogen bonding and hydrophobic interactions of EC3G and EGC at the β2-α2 loop of HuPrP. These differences were substantiated by 44-fold higher KD for EC3G as compared to EGC with the former significantly reducing Thioflavin T (ThT) binding aggregates of HuPrP. Conformational alterations in HuPrP aggregates were validated by particle sizing, AFM analysis and A11 and OC conformational antibodies. As compared to EGC, EC3G showed relatively higher reduction in toxicity and cellular internalization of HuPrP oligomers in Neuro-2a cells. Additionally, EC3G also displayed higher fibril disaggregating properties as observed by ThT kinetics and electron microscopy. Our observations were supported by molecular dynamics (MD) simulations that showed markedly reduced α2-α3 and β2-α2 loop mobilities in presence of EC3G that may lead to constriction of HuPrP conformational space with lowered β-sheet conversion. In totality, gallate moiety of catechins play key role in modulating HuPrP aggregation, and toxicity and could be a new structural motif for designing therapeutics against prion diseases and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti- Clostridium chauvoei- specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). The study indicated the production of conformational recombinant flagellin—rFliA(C)—antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei- specific antibodies in BQ-recovered and/or vaccinated animals. [Display omitted] • Construct design based on structural features of flagellin of Clostridium chauvoei. • Native purification of highly soluble recombinant FliA(C) by a single-step process. • Elicitation of rFliA(C) specific conformational antibodies in rabbit & guinea pig. • In ELISA, rFliA(C) detected antibodies against Clostridium chauvoei in bovine sera. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) of proteins play a significant role in normal protein function but can also be instrumental in disease pathogenesis. One critical yet under-studied PTM in disease is ubiquitination. Ubiquitin chain addition and substrate specificity are determined by a large spectrum of ubiquitin-ligating and -modifying enzymes, E3 ligases, whose expression levels and activities are tightly regulated in a cell-specific manner. While most ubiquitin chains can target proteins for proteasomal degradation, ubiquitination can contribute to other functions within the cell, including protein localization, protein activity, endocytosis, transcription, and autophagy. One E3 ligase, UBE3A, has garnered much attention because of its involvement in learning and memory, as well as its association with neurodevelopmental autism spectrum disorders (ASDs). However, more recent findings have suggested a potential involvement of UBE3A in neurodegenerative proteinopathies, where reduced UBE3A levels can lead to an enhanced rate of aggregate formation and cell death. Here, we review the literature on UBE3A in neurodevelopment, function, and neurodegenerative diseases and demonstrate that UBE3A could play a critical role in disease progression and cognitive function. [ABSTRACT FROM AUTHOR]

Simple Summary: A pseudogene, by definition, is a segment of DNA that structurally resembles an established gene but has undergone mutation drift or genomic translocation where it no longer codes for an active protein. An example of this type of relationship is seen with the oncofetal protein Cripto-1 (CR1) and its pseudogene homolog Cripto-3 (CR3). The CR1 gene is expressed on chromosome 3 and CR3 on the X chromosome. They both code for a 188AA protein but differ by six AAs. Given the closeness of their protein structure, commercially available antibodies cannot differentiate CR1 from CR3. Hence, CR3 remains a pseudogene in human biology and its relationship with tumor malignancies underdetermined. Our newly generated discriminatory anti-CR1/anti-CR3 monoclonal antibodies have now proven that CR3 is a translated protein found in diverse human cancers, tracking with disease severity, involved with cell signaling and expressed as a cell-tethered or soluble entity, thus negating its pseudogene status. Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) and more than twenty other dementias, termed tauopathies, are pathologically defined by insoluble aggregates of the microtubule-associated protein tau (MAPT). Although tau aggregation correlates with AD symptomology, the specific tau species, i.e., monomers, soluble oligomers, and insoluble aggregates that induce neurotoxicity are incompletely understood. We developed a light-responsive tau protein (optoTAU) and used viscosity-sensitive AggFluor probes to investigate the consequence(s) of tau aggregation in human neurons and identify modifiers of tau aggregation in AD and other tauopathies. We determined that optoTAU reproduces biological and structural properties of tau aggregation observed in human brains and the pathophysiological transition in tau solubility in live cells. We also provide proof-of-concept for the utilization of optoTAU as a pharmacological platform to identify modifiers of tau aggregation. These findings have broad implications for the characterization of aggregation-prone proteins and investigation of the complex relationship between protein solubility, cellular function, and disease progression. A study to develop an optogenetic model of tau aggregation provides proof-of-concept for utilization of optoTAU to investigate the consequences of tau aggregation in human neurons and identify modifiers of tau aggregation in tauopathies. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection. [ABSTRACT FROM AUTHOR]

Pathological tau aggregates cause cognitive decline in neurodegenerative tauopathies, including Alzheimer's disease (AD). These aggregates are prevalent within intracellular compartments. Current tau immunotherapies have shown limited efficacy in clearing intracellular tau aggregates and improving cognition in clinical trials. In this study, we developed toxic tau conformation–specific monoclonal antibody-2 (TTCM2), which selectively recognized pathological tau aggregates in brain tissues from patients with AD, dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). TTCM2 potently inhibited tau-seeding activity, an essential mechanism underlying tauopathy progression. To effectively target intracellular tau aggregates and ensure rapid delivery to the brain, TTCM2 was loaded in micelles (TTCM2-ms) and administered through the intranasal route. We found that intranasally administered TTCM2-ms efficiently entered the brain in hTau-tauopathy mice, targeting pathological tau in intracellular compartments. Moreover, a single intranasal dose of TTCM2-ms effectively cleared pathological tau, elevated synaptic proteins, and improved cognitive functions in aged tauopathy mice. Mechanistic studies revealed that TTCM2-ms cleared intracellular, synaptic, and seed-competent tau aggregates through tripartite motif-containing 21 (TRIM21), an intracellular antibody receptor and E3 ubiquitin ligase known to facilitate proteasomal degradation of cytosolic antibody-bound proteins. TRIM21 was found to be essential for TTCM2-ms–mediated clearance of tau pathology. Our study collectively provides evidence of the effectiveness of nasal tau immunotherapy in targeting and clearing intracellular tau pathology through TRIM21 and enhancing cognition in aged tauopathy mice. This study could be valuable in designing effective tau immunotherapies for AD and other tauopathies. Editor's summary: Tau pathology is a common denominator for neurodegenerative diseases such as Alzheimer's disease or frontotemporal dementia, but selectively targeting toxic tau remains a challenge. Here, Gaikwad et al. developed a conformation-specific antibody for tau and demonstrated selective targeting of toxic tau aggregates in patient-derived tissue and tau oligomers in vitro. Packed into lipophile micelles, the antibody could readily reach the brains of tauopathy mice upon intranasal administration, where it ameliorated tau pathology and improved cognitive function. These results suggest a new immunotherapeutic strategy for neurodegenerative diseases with tau pathology. —Daniela Neuhofer [ABSTRACT FROM AUTHOR]

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies. [ABSTRACT FROM AUTHOR]

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies. The microbiota of the intestinal tract is considered a large biofilm formed by myriads bacteria that have a considerable impact in health and disease. Here, the authors show that biofilm-associated proteins from intestinal microbiota form amyloid-like structures that exacerbate alpha-synuclein pathologies. [ABSTRACT FROM AUTHOR]

Tau protein misfolding and aggregation are pathological hallmarks of Alzheimer's disease and over twenty neurodegenerative disorders. However, the molecular mechanisms of tau aggregation in vivo remain incompletely understood. There are two types of tau aggregates in the brain: soluble aggregates (oligomers and protofibrils) and insoluble filaments (fibrils). Compared to filamentous aggregates, soluble aggregates are more toxic and exhibit prion-like transmission, providing seeds for templated misfolding. Curiously, in its native state, tau is a highly soluble, heat-stable protein that does not form fibrils by itself, not even when hyperphosphorylated. In vitro studies have found that negatively charged molecules such as heparin, RNA, or arachidonic acid are generally required to induce tau aggregation. Two recent breakthroughs have provided new insights into tau aggregation mechanisms. First, as an intrinsically disordered protein, tau is found to undergo liquid-liquid phase separation (LLPS) both in vitro and inside cells. Second, cryo-electron microscopy has revealed diverse fibrillar tau conformations associated with different neurodegenerative disorders. Nonetheless, only the fibrillar core is structurally resolved, and the remainder of the protein appears as a "fuzzy coat". From this review, it appears that further studies are required (1) to clarify the role of LLPS in tau aggregation; (2) to unveil the structural features of soluble tau aggregates; (3) to understand the involvement of fuzzy coat regions in oligomer and fibril formation. [ABSTRACT FROM AUTHOR]

The glycosylation of viral envelope proteins plays an important role in virus biology and the immune response of the host to infection. Hepatitis C virus (HCV) envelope proteins E1 and E2, key players in virus entry and spread, are highly N-glycosylated and possess 4 (5 in certain genotypes) to 11 conserved glycosylation sites, respectively. Many published results based on recombinant proteins indicate that the glycan shield can mask the epitopes targeted by neutralizing antibodies. Glycan shifting within the conserved linear E2 region (412–423) could be one of the escape strategies used by HCV. In the present report, we isolated E2 genes from samples (collected before the IFN-RBV therapy) originating from pediatric patients infected with HCV gt 1a. We analyzed the biochemical properties of cloned E2 glycoprotein variants and investigated their glycosylation status. The sequencing of E2 genes isolated from patients who did not respond to therapy revealed mutations at N-glycosylation sites, thus leading to a lower molecular weight and a low affinity to both linear and conformational neutralizing antibodies. The loss of the glycosylation site within the conserved epitope (amino acid 417) impaired the binding with AP33, an antibody that potently neutralizes all genotypes of HCV. Our findings, based on clinical samples, confirm the influence of N-glycosylation aberrations on the antigenic and conformational properties of HCV E1/E2, which may possibly correlate with the outcome of therapy in patients. [ABSTRACT FROM AUTHOR]

Introduction: Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display. Methods: To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, wewere able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner. Results: Microspheres coatedwithHIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation. Discussion: In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required. [ABSTRACT FROM AUTHOR]

Decades of research have demonstrated an incontrovertible role of amyloid-ß (Aß) in the etiology of Alzheimer's disease (AD). However, the overemphasis on the pathological impacts of Aß may obscure the role of its metabolic precursor, amyloid precursor protein (APP), as a significant hub in the occurrence and progression of AD. The complicated enzymatic processing, ubiquitous receptor-like properties, and abundant expression of APP in the brain, as well as its close links with systemic metabolism, mitochondrial function and neuroinflammation, imply that APP plays multifaceted roles in AD. In this review, we briefly describe the evolutionarily conserved biological characteristics of APP, including its structure, functions and enzymatic processing. We also discuss the possible involvement of APP and its enzymatic metabolites in AD, both detrimental and beneficial. Finally, we describe pharmacological agents or genetic approaches with the capability to reduce APP expression or inhibit its cellular internalization, which can ameliorate multiple aspects of AD pathologies and halt disease progression. These approaches provide a basis for further drug development to combat this terrible disease. [ABSTRACT FROM AUTHOR]

Chemokines are cytokines whose primary role is cellular activation and stimulation of leukocyte migration. They perform their various functions by interacting with G protein-coupled cell surface receptors (GPCRs) and are involved in the regulation of many biological processes such as apoptosis, proliferation, angiogenesis, hematopoiesis or organogenesis. They contribute to the maintenance of the homeostasis of lymphocytes and coordinate the function of the immune system. However, chemokines and their receptors are sometimes hijacked by some pathogens to infect the host organism. For a given chemokine receptor, there is a wide structural, organizational and conformational diversity. In this review, we describe the evidence for structural variety reported for the chemokine receptor CCR5, how this variability can be exploited by HIV-1 to infect its target cells and what therapeutic solutions are currently being developed to overcome this problem. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) in the brain and progressive loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Although the role of neuroinflammation and cellular immunity in PD has been extensively studied, the involvement of humoral immunity mediated by antibodies and B cells has received less attention. This article provides a comprehensive review of the current understanding of humoral immunity in PD. Here, we discuss alterations in B cells in PD, including changes in their number and phenotype. Evidence mostly indicates a decrease in the quantity of B cells in PD, accompanied by a shift in the population from naïve to memory cells. Furthermore, the existence of autoantibodies that target several antigens in PD has been investigated (i.e., anti-α-syn autoantibodies, anti-glial-derived antigen antibodies, anti-Tau antibodies, antineuromelanin antibodies, and antibodies against the renin-angiotensin system). Several autoantibodies are generated in PD, which may either provide protection or have harmful effects on disease progression. Furthermore, we have reviewed studies focusing on the utilization of antibodies as a potential treatment for PD, both in animal and clinical trials. This review sheds light on the intricate interplay between antibodies and the pathological processes in PD, including complement system activation., (© 2024 John Wiley & Sons Ltd.)

Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents. [ABSTRACT FROM AUTHOR]

Abnormal assembly of tau, α-synuclein, TDP-43 and amyloid-β proteins into amyloid filaments defines most human neurodegenerative diseases. Genetics provides a direct link between filament formation and the causes of disease. Developments in cryo-electron microscopy (cryo-EM) have made it possible to determine the atomic structures of amyloids from postmortem human brains. Here we review the structures of brain-derived amyloid filaments that have been determined so far and discuss their impact on research into neurodegeneration. Whereas a given protein can adopt many different filament structures, specific amyloid folds define distinct diseases. Amyloid structures thus provide a description of neuropathology at the atomic level and a basis for studying disease. Future research should focus on model systems that replicate the structures observed in disease to better understand the molecular mechanisms of disease and develop improved diagnostics and therapies.Structural studies of amyloid filaments purified from brains of people with neurodegenerative diseases link specific amyloid folds with distinct diseases and provide a basis for the development of models of neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the Dabie bandavirus, [or SFTS virus (SFTSV)] that has become increasingly widespread since it was first reported in 2009. The SFTSV comprises three essential single-stranded RNA gene segments, with the S segment encoding the nucleocapsid (N) protein. Since the N protein is the most abundant and stable viral protein, it is a useful diagnostic marker of infection. Various SFTSV N-protein-based detection methods have been developed. However, given the limited research on antibodies of an SFTSV N-protein, here we report the characterization of the antibodies against SFTSV N protein especially their mapping results which is essential for more efficient and optimized detection of SFTSV. Methods: To generate SFTSV-N-protein-specific monoclonal antibodies, recombinant full-length SFTSV N protein was expressed in E. coli, and the purified N protein was immunized to mice. The binding epitope positions of the antibodies generated were identified through binding-domain mapping. An antibody pair test using a lateral flow immunoassay (LFIA) was performed to identify effective diagnostic combinations of paired antibodies. Results: Nine monoclonal antibodies specific for the SFTSV N protein were generated. Antibodies #3(B4E2) and #5(B4D9) were specific for sequential epitopes, while the remainder were specific for conformational epitopes. Antibody #4(C2G1) showed the highest affinity for the SFTSV N protein. The binding domain mapping results indicated the binding regions of the antibodies were divided into three groups. The antibody pair test demonstrated that #3(B4E2)/#4(C2G1) and #4(C2G1)/#5(B4D9) were effective antibody pairs for SFTSV diagnosis. Conclusions: Effective virus detection requires at least two strong antibodies recognizing separate epitope binding sites of the virus antigen. Here, we generated SFTSV-N-protein-specific monoclonal antibodies and subsequently performed epitope mapping and an antibody pair test to enhance the diagnostic efficiency and accuracy of SFTSV. Confirmation of epitope mappings and their combination immune response to the N protein provide valuable information for effective detection of SFTSV as well as can respond actively to detect a variant SFTSV. [ABSTRACT FROM AUTHOR]

Alpha-synucleinopathies (α-synucleinopathies) such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are all characterized by aggregates of alpha-synuclein (α-syn), but display heterogeneous clinical and pathological phenotypes. The mechanism underlying this heterogeneity is thought to be due to diversity in the α-syn strains present across the diseases. α-syn obtained from the post-mortem brain of patients who lived with these conditions is heterogenous, and displays a different protease sensitivity, ultrastructure, cytotoxicity, and seeding potential. The primary aim of this review is to summarize previous studies investigating these concepts, which not only reflect the idea of different syn strains being present, but demonstrate that each property explains a small part of a much larger puzzle. Strains of α-syn appear at the center of the correlation between α-syn properties and the disease phenotype, likely influenced by external factors. There are considerable similarities in the properties of disease-specific α-syn strains, but MSA seems to consistently display more aggressive traits. Elucidating the molecular underpinnings of heterogeneity amongst α-synucleinopathies holds promise for future clinical translation, allowing for the development of personalized medicine approaches tackling the root cause of each α-synucleinopathy. [ABSTRACT FROM AUTHOR]

Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer's disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens. [ABSTRACT FROM AUTHOR]

Like many neurodegenerative diseases, Parkinson's disease (PD) is characterized by the formation of proteinaceous aggregates in brain cells. In PD, those proteinaceous aggregates are formed by the α-synuclein (αSyn) and are considered the trademark of this neurodegenerative disease. In addition to PD, αSyn pathological aggregation is also detected in atypical Parkinsonism, including Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), as well as neurodegeneration with brain iron accumulation, some cases of traumatic brain injuries, and variants of Alzheimer's disease. Collectively, these (and other) disorders are referred to as synucleinopathies, highlighting the relation between disease type and protein misfolding/aggregation. Despite these pathological relationships, however, synucleinopathies cover a wide range of pathologies, present with a multiplicity of symptoms, and arise from dysfunctions in different neuroanatomical regions and cell populations. Strikingly, αSyn deposition occurs in different types of cells, with oligodendrocytes being mainly affected in MSA, while aggregates are found in neurons in PD. If multiple factors contribute to the development of a pathology, especially in the cases of slow-developing neurodegenerative disorders, the common presence of αSyn aggregation, as both a marker and potential driver of disease, is puzzling. In this review, we will focus on comparing PD, DLB, and MSA, from symptomatology to molecular description, highlighting the role and contribution of αSyn aggregates in each disorder. We will particularly present recent evidence for the involvement of conformational strains of αSyn aggregates and discuss the reciprocal relationship between αSyn strains and the cellular milieu. Moreover, we will highlight the need for effective methodologies for the strainotyping of aggregates to ameliorate diagnosing capabilities and therapeutic treatments. [ABSTRACT FROM AUTHOR]

α-synuclein (α-syn) is an intrinsically disordered protein abundant in the central nervous system. Physiologically, the protein regulates vesicle trafficking and neurotransmitter release in the presynaptic terminals. Pathologies related to misfolding and aggregation of α-syn are referred to as α-synucleinopathies, and they constitute a frequent cause of neurodegeneration. The most common α-synucleinopathy, Parkinson's disease (PD), is caused by abnormal accumulation of α-syn in the dopaminergic neurons of the midbrain. This results in protein overload, activation of endoplasmic reticulum (ER) stress, and, ultimately, neural cell apoptosis and neurodegeneration. To date, the available treatment options for PD are only symptomatic and rely on dopamine replacement therapy or palliative surgery. As the prevalence of PD has skyrocketed in recent years, there is a pending issue for development of new disease-modifying strategies. These include anti-aggregative agents that target α-syn directly (gene therapy, small molecules and immunization), indirectly (modulators of ER stress, oxidative stress and clearance pathways) or combine both actions (natural compounds). Herein, we provide an overview on the characteristic features of the structure and pathogenic mechanisms of α-syn that could be targeted with novel molecular-based therapies. [ABSTRACT FROM AUTHOR]

Drug development in recent years is increasingly focused on developing personalized treatments based on blocking molecules selective for therapeutic targets specifically present in individual patients. In this perspective, the specificity of therapeutic targets and blocking agents plays a crucial role. Monoclonal antibodies (mAbs) and their surrogates are increasingly used in this context thanks to their ability to bind therapeutic targets and to inhibit their activity or to transport bioactive molecules into the compartments in which the targets are expressed. Small antibody-like molecules, such as Fabs, are often used in certain clinical settings where small size and better tissue penetration are required. In the wake of this research trend, we developed a murine mAb (3D1) neutralizing the activity of Nodal, an oncofetal protein that is attracting an ever-increasing interest as a selective therapeutic target for several cancer types. Here, we report the preparation of a recombinant Fab of 3D1 that has been humanized through a computational approach starting from the sequence of the murine antibody. The Fab has been expressed in bacterial cells (1 mg/L bacterial culture), biochemically characterized in terms of stability and binding properties by circular dichroism and bio-layer interferometry techniques and tested in vitro on Nodal-positive cancer cells. [ABSTRACT FROM AUTHOR]

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery. [ABSTRACT FROM AUTHOR]

Neuroinformatics is a research field that focusses on software tools capable of identifying, analysing, modelling, organising and sharing multiscale neuroscience data. Neuroinformatics has exploded in the last two decades with the emergence of the Big Data phenomenon, characterised by the so‐called 3Vs (volume, velocity and variety), which provided neuroscientists with an improved ability to acquire and process data faster and more cheaply thanks to technical improvements in clinical, genomic and radiological technologies. This situation has led to a 'data deluge', as neuroscientists can routinely collect more study data in a few days than they could in a year just a decade ago. To address this phenomenon, several neuroimaging‐focussed neuroinformatics platforms have emerged, funded by national or transnational agencies, with the following goals: (i) development of tools for archiving and organising analytical data (XNAT, REDCap and LabKey); (ii) development of data‐driven models evolving from reductionist approaches to multidimensional models (RIN, IVN, HBD, EuroPOND, E‐DADS and GAAIN BRAIN); and (iii) development of e‐infrastructures to provide sufficient computational power and storage resources (neuGRID, HBP‐EBRAINS, LONI and CONP). Although the scenario is still fragmented, there are technological and economical attempts at both national and international levels to introduce high standards for open and Findable, Accessible, Interoperable and Reusable (FAIR) neuroscience worldwide. [ABSTRACT FROM AUTHOR]

In the elderly, a debilitating condition known as dementia, which is a major health concern, is caused by Alzheimer's disease (AD). Despite promising advances by researchers, there is currently no way to completely cure this devastating disease. It is illustrated by the deposition of amyloid β-peptide (Aβ) plaques that are followed by neural dysfunction and cognitive decline. Responses against AD activate an immune system that contributes to and accelerates AD pathogenesis. Potential efforts in the field of pathogenesis have prompted researchers to explore novel therapies such as active and passive vaccines against Aβ proteins (Aβ immunotherapy), intravenous immunoglobulin, and tau immunotherapy, as well as targets that include microglia and several cytokines for the treatment of AD. Aims are now underway by experts to begin immunotherapies before the clinical manifestation, which is made possible by improving the sensitivity of biomarkers used for the diagnosis of AD to have better outcome measures. This review provides an overview of approved immunotherapeutic strategies for AD and those currently being investigated in clinical trials. We examine their mechanisms of action and discuss the potential perspectives and challenges associated with immunotherapies for AD. [ABSTRACT FROM AUTHOR]

Human papillomavirus belongs to papovaviridae family papillomavirus A, a spherical deoxyribonucleic acid (DNA) virus, which can cause the proliferation of squamous epithelial cells of human skin or mucous membranes. With the rapid increase in the incidence of condyloma acuminatum among STDs and the increase in diseases caused by HPV infection, HPV infection has seriously endangered human health. In this paper, the in vitro detection of HPV E1 protein was realized using AgNCs-dsDNA. And through the test of this detection method, we calculated that the detection limit of this method is 0.886 nM. Compared with other methods for detecting E1 protein in vitro, this method has high sensitivity and simple operation. In addition, the detection method also has good anti-interference and selectivity, and can realize the detection of E1 in serum samples. The transfection efficiency of BLV-miR-B4-3p mimics at different time points was determined by quantitative real-time PCR (qPCR); the transcriptome sequencing of lymphocytes transfected with different concentrations of BLV-miR-B4-3p mimics was performed, and differential gene clustering was performed on the sequencing results. And the BLV-miR-B4-3p target gene prediction and transcriptome analysis results were verified by qPCR. The effects of BLV-miR-B4-3p on the transcriptional levels of immune-related cytokines in human lymphocytes were analyzed. Transcriptome sequencing analysis showed that after BLV-miR-B4-3p entered lymphocytes, a total of 556 differentially expressed genes were obtained. GO enrichment and KEGG analysis results showed that BLV-miR-B4-3p could independently activate influenza. The signaling pathway ultimately affects the body's immune system process, stress response, defense response, immune response, and other biological processes. After BLV-miR-B4-3p enters lymphocytes, it will lead to abnormal lymphocyte immune function, including the mRNA expression of TNF-α in Th1 cytokines which was significantly increased (P < 0.05), and the expression of IL-10 in Th2 cytokines was significantly increased (P < 0.05). The mRNA expression was significantly decreased (P < 0.05), and the mRNA expression of IL-27 was significantly increased (P < 0.001), which did not affect the mRNA expression of lymphocyte proliferation and activation-related regulators. The tumor suppressor breast cancer 1 (BRCA1) and antimicrobial peptide CAMP were significantly increased, and decreased (P < 0.001), and the expression of pro-apoptotic factor Caspase9 showed a significant downward trend (P < 0.05). [ABSTRACT FROM AUTHOR]

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV. [ABSTRACT FROM AUTHOR]

During the initial stages of gastrulation during embryonic differentiation and wound healing, Cripto-1 is a critical protein for human growth. The epithelial adhesion molecules' downregulation, the mesenchymal overexpression, and mobile proteins are important mechanisms by which Cripto-1 initiates epithelial to mesenchymal transition (EMT). As a result, the function of Cripto-1 for inducing EMT to increase cell migration is advantageous during embryogenesis; however, it is deleterious during the formation, growth, and malignant tumor metastasis. The majority of malignancies are reported to have elevated levels of Cripto-1. Cripto-1 can modify cancerous cells through its function in EMT, which enables these cells to migrate via the extracellular matrix, bloodstream, and lymphatic vessels, on their way for metastasizing to other organs. The goal of this review is to explain what role Cripto-1 plays in common cancers and to summarize how therapeutic strategies are used to interfere with this molecule to target cancers. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by intracerebral deposition of abnormal proteinaceous assemblies made of amyloid-β (Aß) peptides or tau proteins. These peptides and proteins induce synaptic dysfunctions that are strongly correlated with cognitive decline. Intracerebral infusion of well-defined Aβ seeds from non-mutated Aβ1-40 or Aβ1-42 peptides can increase Aβ depositions several months after the infusion. Familial forms of AD are associated with mutations in the amyloid precursor protein (APP) that induce the production of Aβ peptides with different structures. The Aβ Osaka (Aβosa mutation (E693Δ)) is located within the Aβ sequence and thus the Aβosa peptides have different structures and properties as compared to non-mutated Aβ1-42 peptides (Aβwt). Here, we wondered if a single exposure to this mutated Aβ can worsen AD pathology as well as downstream events including cognition, cerebral connectivity and synaptic health several months after the inoculation. To answer this question we inoculated Aβ1-42-bearing Osaka mutation (Aβosa) in the dentate gyrus of APPswe/PS1dE9 mice at the age of two months. Their cognition and cerebral connectivity were analyzed at 4 months post-inoculation by behavioral evaluation and functional MRI. Aβ pathology as well as synaptic density were evaluated by histology. The impact of Aβosa peptides on synaptic health was also measured on primary cortical neurons. Remarkably, the intracerebral administration of Aβosa induced cognitive and synaptic impairments as well as a reduction of functional connectivity between different brain regions, 4 months post-inoculation. It increased Aβ plaque depositions and increased Aβ oligomers. This is the first study showing that a single, sporadic event as Aβosa inoculation can worsen the fate of the pathology and clinical outcome several months after the event. It suggests that a single inoculation of Aβ regulates a large cascade of events for a long time. [ABSTRACT FROM AUTHOR]

Background: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. Methods: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. Results: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. Conclusions: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts. [ABSTRACT FROM AUTHOR]

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity. [ABSTRACT FROM AUTHOR]

Potency is a critical quality attribute for controlling quality consistency and relevant biological properties of vaccines. Owing to the high demand for animals, lengthy operations and high variability of in vivo methods, in vitro alternatives for human vaccine potency assays are extensively developed. Herein, in vivo and in vitro methods for potency assays of previously licensed human vaccines were sorted, followed by a brief description of the background for substituting in vivo methods with in vitro alternatives. Based on the analysis of current research on the substitution of vaccine potency assays, barriers and suggestions for substituting were proposed. Owing to the variability of in vivo methods, the correlation between in vivo and in vitro methods may be low. One or more in vitro method(s) that determine the vaccine antigen content and functions, should be established. Since the substitution involves with the change of critical quality attributes and specifications, the specifications of in vitro methods should be appropriately set to maintain the efficacy of vaccines. For novel vaccines in research and development, in vitro methods for monitoring the consistency and relevant biological properties, should be established based on reflecting the immunogenicity of vaccines. [ABSTRACT FROM AUTHOR]

Membrane proteins (MPs) play key roles in cellular signaling pathways and are responsible for intercellular and intracellular interactions. Dysfunctional MPs are directly related to the pathogenesis of various diseases, and they have been exploited as one of the most sought-after targets in the pharmaceutical industry. However, working with MPs is difficult given that their amphiphilic nature requires protection from biological membrane or membrane mimetics. Polymersomes are bilayered nanovesicles made of self-assembled block copolymers that have been widely used as cell membrane mimetics for MP reconstitution and in engineering of artificial cells. This review highlights the prevailing trend in the application of polymersomes in MP study and drug discovery. We begin with a review on the techniques for synthesis and characterization of polymersomes as well as methods of MP insertion to form proteopolymersomes. Next, we review the structural and functional analysis of the different types of MPs reconstituted in polymersomes, including membrane transport proteins, MP complexes, and membrane receptors. We then summarize the factors affecting reconstitution efficiency and the quality of reconstituted MPs for structural and functional studies. Additionally, we discuss the potential in using proteopolymersomes as platforms for high-throughput screening (HTS) in drug discovery to identify modulators of MPs. We conclude by providing future perspectives and recommendations on advancing the study of MPs and drug development using proteopolymersomes. [ABSTRACT FROM AUTHOR]

Alzheimer's disease neuropathologic change (ADNC) is clinically heterogenous and can present with a classic multidomain amnestic syndrome or focal non-amnestic syndromes. Here, we investigated the distribution and burden of phosphorylated and C-terminally cleaved tau pathologies across hippocampal subfields and cortical regions among phenotypic variants of Alzheimer's disease (AD). In this study, autopsy-confirmed patients with ADNC, were classified into amnestic (aAD, N = 40) and non-amnestic (naAD, N = 39) groups based on clinical criteria. We performed digital assessment of tissue sections immunostained for phosphorylated-tau (AT8 detects pretangles and mature tangles), D421-truncated tau (TauC3, a marker for mature tangles and ghost tangles), and E391-truncated tau (MN423, a marker that primarily detects ghost tangles), in hippocampal subfields and three cortical regions. Linear mixed-effect models were used to test regional and group differences while adjusting for demographics. Both groups showed AT8-reactivity across hippocampal subfields that mirrored traditional Braak staging with higher burden of phosphorylated-tau in subregions implicated as affected early in Braak staging. The burden of phosphorylated-tau and TauC3-immunoreactive tau in the hippocampus was largely similar between the aAD and naAD groups. In contrast, the naAD group had lower relative distribution of MN423-reactive tangles in CA1 (β = − 0.2, SE = 0.09, p = 0.001) and CA2 (β = − 0.25, SE = 0.09, p = 0.005) compared to the aAD. While the two groups had similar levels of phosphorylated-tau pathology in cortical regions, there was higher burden of TauC3 reactivity in sup/mid temporal cortex (β = 0.16, SE = 0.07, p = 0.02) and MN423 reactivity in all cortical regions (β = 0.4–0.43, SE = 0.09, p < 0.001) in the naAD compared to aAD. In conclusion, AD clinical variants may have a signature distribution of overall phosphorylated-tau pathology within the hippocampus reflecting traditional Braak staging; however, non-amnestic AD has greater relative mature tangle pathology in the neocortex compared to patients with clinical amnestic AD, where the hippocampus had greatest relative burden of C-terminally cleaved tau reactivity. Thus, varying neuronal susceptibility to tau-mediated neurodegeneration may influence the clinical expression of ADNC. [ABSTRACT FROM AUTHOR]

The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson's disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain β-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA. [ABSTRACT FROM AUTHOR]

Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or b-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 1 and 2 infections cause high unmet disease burdens worldwide. Mainly HSV-2 causes persistent sexually transmitted disease, fatal neonatal disease and increased transmission of HIV/AIDS. Thus, there is an urgent requirement to develop effective vaccines. We developed nucleic acid vaccines encoding a novel virus entry complex stabilising cell membrane fusion, 'virus-like membranes', VLM. Two dose intramuscular immunisations using DNA expression plasmids in a guinea pig model gave 100% protection against acute disease and significantly reduced virus replication after virus intravaginal challenge. There was also reduced establishment of latency within the dorsal root ganglia and spinal cord, but recurrent disease and recurrent virus shedding remained. To increase cellular immunity and protect against recurrent disease, cDNA encoding an inhibitor of chemokine receptors on T regulatory cells was added and compared to chemokine CCL5 effects. Immunisation including this novel human chemokine gene, newly defined splice variant from an endogenous virus genome, 'virokine immune therapeutic', VIT, protected most guinea pigs from recurrent disease and reduced recurrent virus shedding distinct from a gD protein vaccine similar to that previously evaluated in clinical trials. All DNA vaccines induced significant neutralising antibodies and warrant evaluation for new therapeutic treatments. [ABSTRACT FROM AUTHOR]