Biology subjects

Ambati, J.

Publications and source records attributed to Ambati, J..

5 recordsLinked to original sources

bioRxiv2025

Sex differences in human IgG1-mediated angiogenesis inhibition depend on Y chromosome-encoded DDX3Y

Prevalent diseases of angiogenesis such as age-related macular degeneration (AMD) exhibit sex-specific prevalence, with female sex being an independent risk factor for the advanced neovascular form of AMD. The basis for these sex differences is poorly understood. Here, we quantify the impact of sex on suppression of aberrant choroidal angiogenesis by human immunoglobulin 1 (hIgG1), which possesses class-wide, antigen-independent anti-angiogenic activity. Males exhibited significantly more robust hIgG1-induced chemotaxis inhibition in primary human and mouse macrophages and anti-angiogenic activity in mouse laser-induced choroidal neovascularization (CNV). Four core genotypes and XY* Turner Syndrome mouse models revealed the Y chromosome as a sex-biasing factor contributing to hIgG1 responses. Transcriptomic and RNAi screening identified DEAD-Box Helicase 3 Y-Linked (DDX3Y) as necessary for the robust inhibitory effects of hIgG1 in males. Male mice as well as human and mouse macrophages lacking DDX3Y exhibited blunted hIgG1 responses in CNV and macrophage chemotaxis, resembling those of females. These results unveil a novel mechanism through which sex chromosome complement differences can impact key processes involved in AMD, carrying implications for various sex-related disorders and conditions involving angiogenesis.

pathology↗

bioRxiv2023

Survey of commercial antibodies targeting Y chromosome-encoded genes

Commercial antibodies are both a mainstay of biomedical research and subject to increasingly widespread scrutiny for their specificity. We evaluated the specificity of commercially available research grade antibodies by investigating those whose antigen targets are encoded by the Y chromosome. We assessed vendor-provided validation data such as immunoblotting and identified 65 commercial research grade antibodies that exhibited positive reactivity in female-derived tissues and/or cell lines. A thorough analysis of 30 commercial antibodies marketed as targeting the Y chromosome-encoded protein DDX3Y found that 16 (53%) provided no marketing data on specificity, 9 (30%) provided positive data in female or likely female materials, 4 (13%) provided only positive data in male materials, and 1 (3%) provided positive data in male materials and negative data in female materials. Together, these findings suggest that many commercial antibodies may be unsuitable to distinguish between related X and Y chromosome-encoded gene pairs and should instill caution in researchers using these tools to investigate sex chromosome-encoded proteins.

molecular biology↗

bioRxiv2021

The learning curve of murine subretinal injection among clinically trained ophthalmic surgeons

PO_SCPLOWURPOSEC_SCPLOWSubretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. MO_SCPLOWETHODSC_SCPLOWTo evaluate the LC for SRI in a murine model, we analyzed training data from 3 clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffer saline injection and the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. A Cumulative Sum (CUSUM) analysis was performed and plotted individually to monitor each surgeons simultaneous performance. RO_SCPLOWESULTSC_SCPLOWDespite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364-training case as a "cutoff" point, the predicted probability of success before and after the 364th case was 65.38% and 99.32%, respectively (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. CO_SCPLOWONCLUSIONSC_SCPLOWThis study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational RelevanceOur prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time.

neuroscience↗

bioRxiv2020

Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs endothelial corneal dystrophy (FECD), which progressively impairs vision through loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA), efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

genomics↗

bioRxiv2020

Loss of Corneal Raver2 Expression in Aniridia Associated Keratopathy

PurposeThe aim of this study was to evaluate downstream factors of Pax6 associated with the aniridic corneal phenotype. We characterize the ocular expression of Raver2, a novel heterogeneous ribonucleoprotein (hnRNP). MethodsGenome wide microarrays of corneal RNA from non-aniridic mice (C57BL/6J and MRL/MpJ) and corneas from aniridia model, Pax6+/- (SEY) mice were performed. Raver2 expression profiles were validated using qPCR and western blot. Raver2 immunohistochemistry on corneas of Pax6+/- mice and aniridic humans were compared to control specimens, and insitu-hybridization was used to analyze developmental expression of Raver2. ResultsMicroarray analysis of corneal RNA identified Raver2 as the gene with the strongest differential expression profile across non-aniridic and aniridic mouse strains. Consistent with mouse data, we found that Raver2 is expressed at high levels in healthy human corneal epithelium, and is downregulated in the corneas of human aniridia patients. ConclusionsThese findings suggest that Raver2 has an evolutionarily conserved developmental role downstream of Pax6 in corneal development and maintenance.

developmental biology↗