Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits

AbstractThe amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

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Sensitive and specific immunohistochemistry protocols for detection of SARS-CoV-2 nucleocapsid and spike proteins in formalin-fixed, paraffin-embedded COVID-19 patient tissues

10.21203/rs.3.pex-1011/v1 ◽

2020 ◽

Author(s):

Yunguang Sun ◽

Linna Ge ◽

Mary J. Rau ◽

Mollie D. Patton ◽

Alexander J. Gallan ◽

...

Keyword(s):

Lung Tissue ◽

Polyclonal Antibodies ◽

Specific Detection ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Life Threatening ◽

Negative Controls ◽

And Control ◽

Formalin Fixed ◽

Spike Proteins

Abstract Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 virus in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified two commercially available affinity-purified polyclonal antibodies raised against Nucleocapsid and Spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Protocols are presented that are mutually validated by matched detection patterns of virus-infected cells in autopsy lung tissue of COVID-19 deceased patients by the two distinctly different antibodies. Negative controls include autopsy lung tissue from patient who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. SARS-CoV-2 detection in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection as well as provide insight into clearance of virus in late COVID-19 disease stages.

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A Stepwise Data Interpretation Process For Renal Amyloidosis Subtyping by LMD-MS

10.21203/rs.3.rs-917392/v1 ◽

2021 ◽

Author(s):

Ming Ke ◽

Xin Li ◽

Lin Wang ◽

Shuling Yue ◽

Beibei Zhao

Keyword(s):

Data Interpretation ◽

Receiver Operating Curve ◽

Renal Amyloidosis ◽

Amyloid Protein ◽

Proteomic Data ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Interpretation Process ◽

Interpretation Method ◽

Formalin Fixed

Abstract Backgrounds: Systemic amyloidosis is classified according to the deposited amyloid protein, which determines its best therapeutic scheme. The laser microdissection combined with mass spectrometry (LMD-MS) technique is a promising approach for precise subtyping of amyloidosis, however, is hampered by how to interpret the MS data.Objectives: The objective of the present study is to establish a complete data interpretation procedure for LMD-MS based amyloidosis subtyping.Methods: Formalin fixed paraffin-embedded specimens from patients with renal amyloidosis were analyzed by LMD-MS for proteome quantification. Forty-two specimens were used for training the data interpretation procedure, which was validated by another 50 validation specimens. Area under receiver operating curve (AUROC) analysis of amyloid accompanying proteins (APOE, APOA4 and SAMP) for discriminating amyloidosis from non-amyloid nephropathies was performed.Results: A stepwise data interpretation procedure that include or exclude the subtypes group by group was established, in which, involvement of non-immunoglobulin amyloid protein is determined by P-score, involvement of immunoglobulin light chain is determined by variable of λ-κ, and immunoglobulin heavy chain’s participation is judged by H-score. This data interpretation method achieved a 88% accuracy in 50 validation specimens. The amyloid accompanying proteins showed significant quantitative differences between amyloidosis specimens and non-amyloid nephropathies. Each of the single accompanying protein had a AUROC value more than 0.9 for diagnosis of amyloidosis from non-amyloid control, and the averaged value of spectral count of the three accompanying proteins showed the highest AUROC (0.966), indicating it might be an alternative indicator for amyloidosis diagnosis.Conclusions: The proteomic data interpretation procedure for amyloidosis subtyping based on LMD-MS was established successfully, which has high clinical application value.

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Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues

Methods and Protocols ◽

10.3390/mps4030047 ◽

2021 ◽

Vol 4 (3) ◽

pp. 47

Author(s):

Yunguang Sun ◽

Linna Ge ◽

Sameer S. Udhane ◽

John F. Langenheim ◽

Mary J. Rau ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Polyclonal Antibodies ◽

Specific Detection ◽

Viral Reservoirs ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Life Threatening ◽

Log Linear ◽

And Control ◽

Formalin Fixed

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

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