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Article ; Online: The winding journey of biotech capital raising: deciphering the jargon.

Frame, Nicholas / Casasanta, Michael / Ben-Joseph, Oded

2023 Volume 41, Issue 10, Page(s) 1213–1215

Abstract: Ensuring biotech companies are sufficiently capitalized to propel innovation and development remains a central focus for management. In this article, we draw on our broad perspective interacting with venture capitalists to offer thoughts on investor ... ...

Abstract	Ensuring biotech companies are sufficiently capitalized to propel innovation and development remains a central focus for management. In this article, we draw on our broad perspective interacting with venture capitalists to offer thoughts on investor feedback. Understanding venture capitalists' mindsets and investment theses will increase the probability of securing needed capital.
MeSH term(s)	Capital Financing ; Investments ; Biotechnology
Language	English
Publishing date	2023-07-12
Publishing country	England
Document type	Journal Article
ZDB-ID	47474-5
ISSN	1879-3096 ; 0167-7799
ISSN (online)	1879-3096
ISSN	0167-7799
DOI	10.1016/j.tibtech.2023.06.011
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Article ; Online: A Vector Suite for the Overexpression and Purification of Tagged Outer Membrane, Periplasmic, and Secreted Proteins in E. coli.

Casasanta, Michael A / Slade, Daniel J

Methods in molecular biology (Clifton, N.J.)

2019 Volume 1960, Page(s) 123–138

Abstract: Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to ... ...

Abstract	Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.
MeSH term(s)	Bacterial Outer Membrane Proteins/genetics ; Bacterial Outer Membrane Proteins/isolation & purification ; Bacterial Outer Membrane Proteins/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/isolation & purification ; Escherichia coli Proteins/metabolism ; Periplasm/metabolism ; Protein Sorting Signals/genetics ; Protein Sorting Signals/physiology
Chemical Substances	Bacterial Outer Membrane Proteins ; Escherichia coli Proteins ; Protein Sorting Signals
Language	English
Publishing date	2019-02-23
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-9167-9_11
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Article ; Online: Expression of concern: Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Casasanta, Michael A / Jonaid, G M / Kaylor, Liam / Luqiu, William Y / Solares, Maria J / Schroen, Mariah L / Dearnaley, William J / Wilson, Jarad / Dukes, Madeline J / Kelly, Deborah F

Nanoscale

2024 Volume 16, Issue 9, Page(s) 4919

Abstract: ... using cryo-electron microscopy' by Michael A. Casasanta ...

Abstract	Expression of concern for 'Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy' by Michael A. Casasanta
Language	English
Publishing date	2024-02-29
Publishing country	England
Document type	Expression of Concern
ZDB-ID	2515664-0
ISSN	2040-3372 ; 2040-3364
ISSN (online)	2040-3372
ISSN	2040-3364
DOI	10.1039/d4nr90037e
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Article ; Online: Type IV pili facilitated natural competence in Fusobacterium nucleatum.

Sanders, Blake E / Umaña, Ariana / Nguyen, Tam T D / Williams, Kevin J / Yoo, Christopher C / Casasanta, Michael A / Wozniak, Bryce / Slade, Daniel J

Anaerobe

2023 Volume 82, Page(s) 102760

Abstract: Objectives: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first ... ...

Abstract	Objectives: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. Methods: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. Results: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. Conclusions: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.
MeSH term(s)	Infant, Newborn ; Humans ; Female ; Fusobacterium nucleatum/metabolism ; Base Composition ; Sequence Analysis, DNA ; Phylogeny ; Premature Birth ; RNA, Ribosomal, 16S ; Fusobacterium ; DNA, Bacterial/genetics ; Fusobacterium Infections/microbiology
Chemical Substances	RNA, Ribosomal, 16S ; DNA, Bacterial
Language	English
Publishing date	2023-07-13
Publishing country	England
Document type	Journal Article
ZDB-ID	1237621-8
ISSN	1095-8274 ; 1075-9964
ISSN (online)	1095-8274
ISSN	1075-9964
DOI	10.1016/j.anaerobe.2023.102760
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Article ; Online: The winding journey of biotech capital raising: deciphering the jargon

Frame, Nicholas / Casasanta, Michael / Ben-Joseph, Oded

Trends in Biotechnology. 2023 July 12,

2023

Abstract	Ensuring biotech companies are sufficiently capitalized to propel innovation and development remains a central focus for management. In this article, we draw on our broad perspective interacting with venture capitalists to offer thoughts on investor feedback. Understanding venture capitalists’ mindsets and investment theses will increase the probability of securing needed capital.
Keywords	biotechnology ; capital ; probability
Language	English
Dates of publication	2023-0712
Publishing place	Elsevier Ltd
Document type	Article ; Online
Note	Pre-press version
ZDB-ID	47474-5
ISSN	1879-3096 ; 0167-7799
ISSN (online)	1879-3096
ISSN	0167-7799
DOI	10.1016/j.tibtech.2023.06.011
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Article: Delineating Conformational Variability in Small Protein Structures Using Combinatorial Refinement Strategies.

Kelly, Deborah F / Jonaid, G M / Kaylor, Liam / Solares, Maria J / Berry, Samantha / DiCecco, Liza-Anastasia / Dearnaley, William / Casasanta, Michael

Micromachines

2023 Volume 14, Issue 10

Abstract: As small protein assemblies and even small proteins are becoming more amenable to cryo-Electron Microscopy (EM) structural studies, it is important to consider the complementary dynamic information present in the data. Current computational strategies ... ...

Abstract	As small protein assemblies and even small proteins are becoming more amenable to cryo-Electron Microscopy (EM) structural studies, it is important to consider the complementary dynamic information present in the data. Current computational strategies are limited in their ability to resolve minute differences among low molecular weight entities. Here, we demonstrate a new combinatorial approach to delineate flexible conformations among small proteins using real-space refinement applications. We performed a meta-analysis of structural data for the SARS CoV-2 Nucleocapsid (N) protein using a combination of rigid-body refinement and simulated annealing methods. For the N protein monomer, we determined three new flexible conformers with good stereochemistry and quantitative comparisons provided new evidence of their dynamic properties. A similar analysis performed for the N protein dimer showed only minor structural differences among the flexible models. These results suggested a more stable view of the N protein dimer than the monomer structure. Taken together, the new computational strategies can delineate conformational changes in low molecular weight proteins that may go unnoticed by conventional assessments. The results also suggest that small proteins may be further stabilized for structural studies through the use of solution components that limit the movement of external flexible regions.
Language	English
Publishing date	2023-09-29
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2620864-7
ISSN	2072-666X
ISSN	2072-666X
DOI	10.3390/mi14101869
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Article ; Online: Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests.

Casasanta, Michael A / Jonaid, G M / Kaylor, Liam / Luqiu, William Y / DiCecco, Liza-Anastasia / Solares, Maria J / Berry, Samantha / Dearnaley, William J / Kelly, Deborah F

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada

2023 Volume 29, Issue 2, Page(s) 649–657

Abstract: The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling ... ...

Abstract	The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.
MeSH term(s)	Humans ; SARS-CoV-2 ; Cryoelectron Microscopy ; COVID-19/diagnosis ; Models, Structural
Language	English
Publishing date	2023-09-25
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1385710-1
ISSN	1435-8115 ; 1431-9276
ISSN (online)	1435-8115
ISSN	1431-9276
DOI	10.1093/micmic/ozac036
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Article ; Online: Type IV pili facilitated natural competence in Fusobacterium nucleatum

Sanders, Blake E. / Umaña, Ariana / Nguyen, Tam T.D. / Williams, Kevin J. / Yoo, Christopher C. / Casasanta, Michael A. / Wozniak, Bryce / Slade, Daniel J.

Anaerobe. 2023 Aug., v. 82 p.102760-

2023

Abstract: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV ...

Abstract	Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.
Keywords	DNA ; DNA methylation ; Fusobacterium nucleatum ; bacteria ; bioinformatics ; chromosomes ; fimbriae ; gene transfer ; genes ; genomics ; homologous recombination ; pathogens ; periodontitis ; premature birth ; virulence ; Fusobacterium ; DNA methyltransferase ; Natural competence ; Type IV pili ; Transformation ; Bacterial genetics
Language	English
Dates of publication	2023-08
Publishing place	Elsevier Ltd
Document type	Article ; Online
ZDB-ID	1237621-8
ISSN	1075-9964
ISSN	1075-9964
DOI	10.1016/j.anaerobe.2023.102760
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Article ; Online: Automated Tools to Advance High-Resolution Imaging in Liquid.

Jonaid, G M / Casasanta, Michael A / Dearnaley, William J / Berry, Samantha / Kaylor, Liam / Dressel-Dukes, Madeline J / Spilman, Michael S / Gray, Jennifer L / Kelly, Deborah F

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada

2022 , Page(s) 1–10

Abstract: Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel ...

Abstract	Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5–7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.
Language	English
Publishing date	2022-01-20
Publishing country	England
Document type	Journal Article
ZDB-ID	1385710-1
ISSN	1435-8115 ; 1431-9276
ISSN (online)	1435-8115
ISSN	1431-9276
DOI	10.1017/S1431927621013921
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Article ; Online: Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle.

Trunk, Thomas / Casasanta, Michael A / Yoo, Christopher C / Slade, Daniel J / Leo, Jack C

The Biochemical journal

2019 Volume 476, Issue 18, Page(s) 2657–2676

Abstract: Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly ... ...

Abstract	Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of
MeSH term(s)	Bacteria/genetics ; Bacteria/metabolism ; Bacteria/pathogenicity ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Bacterial Secretion Systems/genetics ; Bacterial Secretion Systems/metabolism ; Humans ; Lipase/genetics ; Lipase/metabolism ; Protein Transport/physiology ; Signal Transduction/physiology ; Virulence Factors/genetics ; Virulence Factors/metabolism
Chemical Substances	Bacterial Proteins ; Bacterial Secretion Systems ; Virulence Factors ; Lipase (EC 3.1.1.3)
Language	English
Publishing date	2019-09-24
Publishing country	England
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2969-5
ISSN	1470-8728 ; 0006-2936 ; 0306-3275 ; 0264-6021
ISSN (online)	1470-8728
ISSN	0006-2936 ; 0306-3275 ; 0264-6021
DOI	10.1042/BCJ20190136
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