10 research outputs found
Production of Recombinant Injectosome and Outer Membrane Proteins from Yersinia Pestis KIM5
Molecular biology techniques and low cost reagents have lowered the barriers to entry for development of biological arsenals by non-state and state groups. Additionally, genetic engineering of epitope targets from such pathogens as Y. pestis would annul current detection methods and therapeutic treatments. Vaccines often have short shelf lives and are of minimal utility if not used prior to exposure to BW agents. Camelidae, including camels and llamas produce unique antibodies termed Nanobodies® (Nbs) or antigen specific fragments (VHH) which are much smaller than traditional antibodies (15 vs. ~150 kDal) yet seem to attach with the same selectivity and affinity as full antibodies. VHH are more stable than the fragile, more bulky antibodies and maintain their structure and function even at high temperature and humidity. It is thought that, due to these unique characteristics, VHH could be reconstituted from a lyophilized pellet and used as a real time injectable immunotherapeutic to be used when warfighters have been exposed to BW. Two logical candidates for VHH production are the low-calcium-response V protein (LcrV) and the needle-like Yop (Yersinia Outer-membrane Protein) Secretion Protein F (YscF). This effort successfully produced quantities greater than 1-mg purified native recombinant LcrV and YscF proteins as antigens for VHH production
A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF
[FeFe]-hydrogenases are nature’s
most prolific hydrogen
catalysts, excelling at facilely interconverting H<sub>2</sub> and
protons. The catalytic core common to all [FeFe]-hydrogenases is a
complex metallocofactor, referred to as the H-cluster, which is composed
of a standard [4Fe-4S] cluster linked through a bridging thiolate
to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide,
and cyanide ligands. This 2Fe subcluster is synthesized and inserted
into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF,
and HydG. HydE and HydG are radical <i>S</i>-adenosylmethionine
enzymes and synthesize the nonprotein ligands of the H-cluster. HydF
is a GTPase that functions as a scaffold or carrier for 2Fe subcluster
production. Herein, we utilize UV–visible, circular dichroism,
and electron paramagnetic resonance spectroscopic studies to establish
the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound
to HydF. We have used spectroelectrochemical titrations to assign
iron–sulfur cluster midpoint potentials, have shown that HydF
purifies with a reduced [2Fe-2S] cluster in the absence of exogenous
reducing agents, and have tracked iron–sulfur cluster spectroscopic
changes with quaternary structural perturbations. Our results provide
an important foundation for understanding the maturation process by
defining the iron–sulfur cluster content of HydF prior to its
interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster
of HydF either acts as a placeholder for HydG-derived Fe(CO)<sub>2</sub>CN species or serves as a scaffold for 2Fe subcluster assembly
Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation
Nature utilizes [FeFe]-hydrogenase
enzymes to catalyze the interconversion
between H<sub>2</sub> and protons and electrons. Catalysis occurs
at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated
2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis
of this unique metallocofactor is accomplished by three maturase enzymes
denoted HydE, HydF, and HydG. HydE and HydG belong to the radical <i>S</i>-adenosylmethionine superfamily of enzymes and synthesize
the nonprotein ligands of the H-cluster. These enzymes interact with
HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe
subcluster assembly. Prior characterization of HydF demonstrated the
protein exists in both dimeric and tetrameric states and coordinates
both [4Fe-4S]<sup>2+/+</sup> and [2Fe-2S]<sup>2+/+</sup> clusters
[Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick,
J. B. (2016) <i>Biochemistry 55</i>, 3514–3527].
Herein, electron paramagnetic resonance (EPR) is utilized to characterize
the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters bound to
HydF. Examination of spin relaxation times using pulsed EPR in HydF
samples exhibiting both [4Fe-4S]<sup>+</sup> and [2Fe-2S]<sup>+</sup> cluster EPR signals supports a model in which the two cluster types
either are bound to widely separated sites on HydF or are not simultaneously
bound to a single HydF species. Gel filtration chromatographic analyses
of HydF spectroscopic samples strongly suggest the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters are coordinated to the dimeric
form of the protein. Lastly, we examined the 2Fe subcluster-loaded
form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase
activation. Together, the results indicate a specific role for the
HydF dimer in the H-cluster biosynthesis pathway
[FeFe]-Hydrogenase Maturation: Insights into the Role HydE Plays in Dithiomethylamine Biosynthesis
HydE and HydG are radical <i>S</i>-adenosyl-l-methionine enzymes required for the
maturation of [FeFe]-hydrogenase
(HydA) and produce the nonprotein organic ligands characteristic of
its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster)
is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains
two carbon monoxides, three cyanides, and a bridging dithiomethylamine
as ligands. While recent studies have shed light on the nature of
diatomic ligand biosynthesis by HydG, little information exists on
the function of HydE. Herein, we present biochemical, spectroscopic,
bioinformatic, and molecular modeling data that together map the active
site and provide significant insight into the role of HydE in H-cluster
biosynthesis. Electron paramagnetic resonance and UV–visible
spectroscopic studies demonstrate that reconstituted HydE binds two
[4Fe-4S] clusters and copurifies with <i>S</i>-adenosyl-l-methionine. Incorporation of deuterium from D<sub>2</sub>O
into 5′-deoxyadenosine, the cleavage product of <i>S</i>-adenosyl-l-methionine, coupled with molecular docking experiments
suggests that the HydE substrate contains a thiol functional group.
This information, along with HydE sequence similarity and genome context
networks, has allowed us to redefine the presumed mechanism for HydE
away from BioB-like sulfur insertion chemistry; these data collectively
suggest that the source of the sulfur atoms in the dithiomethylamine
bridge of the H-cluster is likely derived from HydE’s thiol
containing substrate
International consensus statement on allergy and rhinology: Sinonasal tumors
BACKGROUND: Sinonasal neoplasms, whether benign and malignant, pose a significant challenge to clinicians and represents a model area for multidisciplinary collaboration in order to optimize patient care. The International Consensus Statement on Allergy and Rhinology: Sinonasal Tumors (ICSNT) aims to summarize the best available evidence and presents 48 thematic and histopathology-based topics spanning the field. METHODS: In accordance with prior ICAR documents, ICSNT assigned each topic as an Evidence-Based Review with Recommendations, Evidence-Based Review, and Literature Review based on level of evidence. An international group of multidisciplinary author teams were assembled for the topic reviews using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses format, and completed sections underwent a thorough and iterative consensus-building process. The final document underwent rigorous synthesis and review prior to publication. RESULTS: The ICNST document consists of 4 major sections: general principles, benign neoplasms and lesions, malignant neoplasms, and quality of life and surveillance. It covers 48 conceptual and/or histopathology-based topics relevant to sinonasal neoplasms and masses. Topics with a high level of evidence provided specific recommendations, while other areas summarized the current state of evidence. A final section highlights research opportunities and future directions, contributing to advancing knowledge and community intervention. CONCLUSION: As an embodiment of the multidisciplinary and collaborative model of care in sinonasal neoplasms and masses, ICSNT was designed as a comprehensive, international, and multidisciplinary collaborative endeavor. Its primary objective is to summarize the existing evidence in the field of sinonasal neoplasms and masses
Recommended from our members
International Consensus Statement on Allergy and Rhinology: Sinonasal Tumors
BACKGROUND: Sinonasal neoplasms, whether benign and malignant, pose a significant challenge to clinicians and represents a model area for multidisciplinary collaboration in order to optimize patient care. The International Consensus Statement on Allergy and Rhinology: Sinonasal Tumors (ICSNT) aims to summarize the best available evidence and presents 48 thematic and histopathology-based topics spanning the field. METHODS: In accordance with prior ICAR documents, ICSNT assigned each topic as an Evidence-Based Review with Recommendations, Evidence-Based Review, and Literature Review based on level of evidence. An international group of multidisciplinary author teams were assembled for the topic reviews using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses format, and completed sections underwent a thorough and iterative consensus-building process. The final document underwent rigorous synthesis and review prior to publication. RESULTS: The ICNST document consists of 4 major sections: general principles, benign neoplasms and lesions, malignant neoplasms, and quality of life and surveillance. It covers 48 conceptual and/or histopathology-based topics relevant to sinonasal neoplasms and masses. Topics with a high level of evidence provided specific recommendations, while other areas summarized the current state of evidence. A final section highlights research opportunities and future directions, contributing to advancing knowledge and community intervention. CONCLUSION: As an embodiment of the multidisciplinary and collaborative model of care in sinonasal neoplasms and masses, ICSNT was designed as a comprehensive, international, and multidisciplinary collaborative endeavor. Its primary objective is to summarize the existing evidence in the field of sinonasal neoplasms and masses
International consensus statement on allergy and rhinology: Sinonasal tumors.
BACKGROUND: Sinonasal neoplasms, whether benign and malignant, pose a significant challenge to clinicians and represents a model area for multidisciplinary collaboration in order to optimize patient care. The International Consensus Statement on Allergy and Rhinology: Sinonasal Tumors (ICSNT) aims to summarize the best available evidence and presents 48 thematic and histopathology-based topics spanning the field.
METHODS: In accordance with prior ICAR documents, ICSNT assigned each topic as an Evidence-Based Review with Recommendations, Evidence-Based Review, and Literature Review based on level of evidence. An international group of multidisciplinary author teams were assembled for the topic reviews using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses format, and completed sections underwent a thorough and iterative consensus-building process. The final document underwent rigorous synthesis and review prior to publication.
RESULTS: The ICNST document consists of 4 major sections: general principles, benign neoplasms and lesions, malignant neoplasms, and quality of life and surveillance. It covers 48 conceptual and/or histopathology-based topics relevant to sinonasal neoplasms and masses. Topics with a high level of evidence provided specific recommendations, while other areas summarized the current state of evidence. A final section highlights research opportunities and future directions, contributing to advancing knowledge and community intervention.
CONCLUSION: As an embodiment of the multidisciplinary and collaborative model of care in sinonasal neoplasms and masses, ICSNT was designed as a comprehensive, international, and multidisciplinary collaborative endeavor. Its primary objective is to summarize the existing evidence in the field of sinonasal neoplasms and masses. This article is protected by copyright. All rights reserved
International consensus statement on allergy and rhinology: Sinonasal tumors
Background: Sinonasal neoplasms, whether benign and malignant, pose a significant challenge to clinicians and represents a model area for multidisciplinary collaboration in order to optimize patient care. The International Consensus Statement on Allergy and Rhinology: Sinonasal Tumors (ICSNT) aims to summarize the best available evidence and presents 48 thematic and histopathology-based topics spanning the field. Methods: In accordance with prior ICAR documents, ICSNT assigned each topic as an Evidence-Based Review with Recommendations, Evidence-Based Review, and Literature Review based on level of evidence. An international group of multidisciplinary author teams were assembled for the topic reviews using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses format, and completed sections underwent a thorough and iterative consensus-building process. The final document underwent rigorous synthesis and review prior to publication. Results: The ICNST document consists of 4 major sections: general principles, benign neoplasms and lesions, malignant neoplasms, and quality of life and surveillance. It covers 48 conceptual and/or histopathology-based topics relevant to sinonasal neoplasms and masses. Topics with a high level of evidence provided specific recommendations, while other areas summarized the current state of evidence. A final section highlights research opportunities and future directions, contributing to advancing knowledge and community intervention. Conclusion: As an embodiment of the multidisciplinary and collaborative model of care in sinonasal neoplasms and masses, ICSNT was designed as a comprehensive, international, and multidisciplinary collaborative endeavor. Its primary objective is to summarize the existing evidence in the field of sinonasal neoplasms and masses. This article is protected by copyright. All rights reserved