Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity

Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.Dr. El-Agnaf’s laboratory was funded by Qatar Biomedical Research Institute under the Start-up Fund SF 2017– 007. The Newcastle Brain Tissue Resource is funded in part by a grant from the UK Medical Research Council, by NIHR Newcastle Biomedical Research Centre awarded to the Newcastle upon Tyne NHS Foundation Trust and Newcastle University, and by a grant from the Alzheimer’s Society and Alzheimer’s Research UK as part of the Brains for Dementia Research Project

Signal transducer and activator of transcription 3 (Stat3) suppresses stat1/interferon signaling pathway and inflammation in senescent preadipocytes

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin‐resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3‐L1 preadipocytes. By employing transcriptomics analyses, we discovered up‐regulation of key pro‐inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate‐Adenosine Monophosphate (GMP‐AMP) synthase‐stimulator of interferon genes (cGAS‐STING) to drive the expression of interferon β (IFNβ), C‐X‐C motif chemokine ligand 10 (CXCL10), and interferon signaling‐related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS‐STING signaling—it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway

Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity.

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders

Prognostic tools and candidate drugs based on plasma proteomics of patients with severe COVID-19 complications

COVID-19 complications still present a huge burden on healthcare systems and warrant predictive risk models to triage patients and inform early intervention. Here, we profile 893 plasma proteins from 50 severe and 50 mild-moderate COVID-19 patients, and 50 healthy controls, and show that 375 proteins are differentially expressed in the plasma of severe COVID-19 patients. These differentially expressed plasma proteins are implicated in the pathogenesis of COVID-19 and present targets for candidate drugs to prevent or treat severe complications. Based on the plasma proteomics and clinical lab tests, we also report a 12-plasma protein signature and a model of seven routine clinical tests that validate in an independent cohort as early risk predictors of COVID-19 severity and patient survival. The risk predictors and candidate drugs described in our study can be used and developed for personalized management of SARS-CoV-2 infected patients. 2022, The Author(s).The authors would like to thank all the patients, volunteers, and the healthcare co-workers from Allergy and Immunology Section-HMC, and Dr. Mohamed G.H. Mohamedali, Mr. Hassen Maatoug, and Mr. Ahmed Soliman from Hezm Mebairek General Hospital-HMC for developing disposable racks for samples transportation, tubes labeling, blood collection, and handling. We thank the support provided by Qatar University Biomedical Research Centre, Biosafety Level 3, and Associate Professor Hadi M. Yassine (M.Sc., Ph.D.). We also acknowledge the help of the Anti-Doping Lab-Qatar (ADLQ) and Qatar Red Crescent (QRC) for recruiting control samples. This work was supported by a grant fund from Hamad Medical Corporation (fund number MRC-05-003) and core funding from Qatar Biomedical Research Institute (QBRI).Scopu

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population

Inflammatory protein signatures in individuals with obesity and metabolic syndrome

There is variability in the metabolic health status among individuals presenting with obesity; some may be metabolically healthy, while others may have developed the metabolic syndrome, a cluster including insulin resistance, hypertension, dyslipidemia, and increased risk of cardiovascular disease and type 2 diabetes. The mechanisms contributing to this metabolic heterogeneity are not fully understood. To address this question, plasma samples from 48 individuals with BMI ≥ 35 kg/m2 were examined (27 with and 21 without metabolic syndrome). Fasting plasma samples were subjected to Olink proteomics analysis for 184 cardiometabolic and inflammation-enriched proteins. Data analysis showed a clear differentiation between the two groups with distinct plasma protein expression profiles. Twenty-four proteins were differentially expressed (DEPs) between the two groups. Pathways related to immune cell migration, leukocyte chemotaxis, chemokine signaling, mucosal inflammatory response, tissue repair and remodeling were enriched in the group with metabolic syndrome. Functional analysis of DEPs revealed upregulation of 15 immunological pathways. The study identifies some of the pathways that are altered and reflect metabolic health in individuals with obesity. This provides valuable insights into some of the underlying mechanisms and can lead to identification of therapeutic targets to improve metabolic health in individuals with obesity.Research conducted on Qatar cohort was funded by Qatar Metabolic Institute (QMI), Hamad Medical Corporation, Doha Qatar. Qatar National Library funded the publication of this article

Blood Proteomics Analysis Reveals Potential Biomarkers and Convergent Dysregulated Pathways in Autism Spectrum Disorder: A Pilot Study

Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician’s assessment and family’s awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted.This project is funded by QBRI’s internal fund and GSRA-QNRF (GSRA6-1-0616-19097)

Blood Proteomics Analysis Reveals Potential Biomarkers and Convergent Dysregulated Pathways in Autism Spectrum Disorder: A Pilot Study

Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician’s assessment and family’s awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted

Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity

Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.Other Information Published in: Scientific Reports License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41598-020-65035-8</p

Prognostic tools and candidate drugs based on plasma proteomics of patients with severe COVID-19 complications

COVID-19 complications still present a huge burden on healthcare systems and warrant predictive risk models to triage patients and inform early intervention. Here, we profile 893 plasma proteins from 50 severe and 50 mild-moderate COVID-19 patients, and 50 healthy controls, and show that 375 proteins are differentially expressed in the plasma of severe COVID-19 patients. These differentially expressed plasma proteins are implicated in the pathogenesis of COVID-19 and present targets for candidate drugs to prevent or treat severe complications. Based on the plasma proteomics and clinical lab tests, we also report a 12-plasma protein signature and a model of seven routine clinical tests that validate in an independent cohort as early risk predictors of COVID-19 severity and patient survival. The risk predictors and candidate drugs described in our study can be used and developed for personalized management of SARS-CoV-2 infected patients.</div