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Differentiation of Microbe-microbe Interactions at Single-cell Level Through Unconventional Utilization of Expansion Microscopy

Award Number: K99DE033794
ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP
PERIOD OF PERFORMANCE START DATE: 06/10/2024
PERIOD OF PERFORMANCE END DATE: 05/31/2026

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Differentiation of Microbe-microbe Interactions at Single-cell Level Through Unconventional Utilization of Expansion Microscopy - The oral microbiome plays a crucial role not only in oral health but also in systemic health. Dysfunctional oral microbiomes have been linked to various chronic human diseases. However, understanding how the organization of the oral microbiome is connected to its function remains a major challenge. Next-generation DNA sequencing has revolutionized the study of oral microbial communities by providing comprehensive information about their composition. CLASI-FISH has contributed to our understanding by revealing the spatial organization of the microbiome. While imaging can inform us about which microbes are in proximity to each other at the micron scale, it cannot alone differentiate whether this spatial proximity is due to functional interactions or chance occurrences. To overcome this limitation, our proposed study aims to investigate microbe-microbe adhesion as a functional attribute of their interactions. We will employ a combination of expansion microscopy (ExM) and multiplex FISH imaging, which will provide a unique opportunity to globally analyze microbial interactions at the micron level. This approach will allow us to explore the functional aspects of microbial communities beyond their spatial arrangement. Furthermore, the presence of thick biofilms or aggregates within their ecological niches presents significant challenges for single-cell dissection and imaging, often leading to difficulties or ambiguities in taxonomic assignment. Our proposed study will address this concern by quantifying the relative microbial adhesion forces at the single-cell level. This will enhance our understanding of the forces governing microbial interactions within biofilms and contribute to unraveling the complexity of the oral microbiome. Overall, this study aims to investigate the functional attributes of the oral microbiome at the micron scale, bridging the gap between microbial organization and function. By employing advanced microscopy techniques and quantifying microbial adhesion forces, we hope to gain valuable insights into the intricate dynamics of the oral microbiome and its implications for human health. There are three interconnected Aims: 1) To combine multiplex FISH imaging with Expansion Microscopy. Here, the focus will be investigating the feasibility of combining multiplex peptide nucleic acid (PNA) FISH imaging with ExM on an in vitro polymicrobial biofilm. 2) To quantify relative microbe-microbe adhesion force. Here, expansion forces within the gel matrices will be calibrated through force-reference bead strategy. Following PNA FISH hybridization on the polymicrobial biofilm, the microbial adhesion forces within the biofilm will be quantified by comparison with gel expansion forces. 3) To evaluate species-level adhesion forces within natural human oral biofilms. Here, we will validate our adhesion force measurement approach using a diverse range of in vitro coaggregation pairs with established binding phenotypes. Then species-level adhesive forces within human natural oral biofilms will be obtained. This independence award (PA-20-188) will foster my independent research growth and allow me to accomplish my long-term career goal of becoming a productive, independent translational scientist in the field of oral microbiology.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $162,216 )
2025	2025	ADA FORSYTH INSTITUTE, INC.	245 1ST ST 17TH FLOOR	CAMBRIDGE	MA	02142	MIDDLESEX	USA	Oral Diseases and Disorders Research	000	2	5/27/2025	NON-COMPETING CONTINUATION	$162,216
														Subtotal = $162,216

Issue Date FY: 2024 ( Subtotal = $162,216 )
2024	2024	ADA FORSYTH INSTITUTE, INC.	245 1ST ST 17TH FLOOR	CAMBRIDGE	MA	02142	MIDDLESEX	USA	Oral Diseases and Disorders Research	000	1	6/10/2024	NEW	$162,216
														Subtotal = $162,216

Grand Total All Awards = $324,432

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Differentiation of Microbe-microbe Interactions at Single-cell Level Through Unconventional Utilization of Expansion Microscopy

Award Number: K99DE033794

ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP

PERIOD OF PERFORMANCE START DATE: 06/10/2024

PERIOD OF PERFORMANCE END DATE: 05/31/2026

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