Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster.

Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, ßSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av ßSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that ßSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of ßSer188 both in vivo and in vitro by characterizing the ability of Av ßSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of ßSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that ßSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.

Structures of the nitrogenase complex prepared under catalytic turnover conditions.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Reshaping Residency Recruitment: Achieving Alignment Between Applicants and Programs in Surgery.

OBJECTIVE: The residency recruitment process has become increasingly challenging for both applicants and program directors, in part, due to the inflation in the number of applications per student. As a result, it has become more daunting for programs to design processes that evaluate applicants holistically. Furthermore, the existing methods used to evaluate and select applicants do not necessarily predict success in residency and may inadvertently lend to gender, racial, and ethnic bias. This narrative review aims to identify innovative tools used in residency recruitment that will allow programs and applicants to better determine concordance of interests and achieve value alignment while supporting improved, objective evaluation of an applicant's unique attributes and experiences. DESIGN: PubMed was used to conduct a narrative review of recruitment strategies in admission processes of undergraduate and graduate medical education between 1975 and June 2021, using the designated Medical Subject Heading (MeSH0 terms. Inclusion criteria were established surrounding innovative tools to better objectively screen, evaluate, or select applicants. Strategies relying primarily on traditional metrics (United States Medical Licensing Examination (USMLE) scores, Alpha Omega Alpha status, and clerkship grades) were excluded. RESULTS: Forty-two articles met specific inclusion criteria. Using these articles, a framework was created with two specific aims: (1) to allow applicants and programs to express or assess interest and (2) to foster objective review of unique applicant attributes, skills, experiences, and competencies that align with program mission and values. The following five innovative tools for recruitment were identified: preference signaling, secondary applications, standardized letters of recommendation, situational judgment testing, and surgical simulation. CONCLUSIONS: As the number of applications continues to rise, strategies must be implemented to allow applicants and institutions to achieve better alignment or "fit," while also giving balanced consideration to all of an applicant's unique characteristics. A more holistic approach to applicant selection is a necessary tool in order to increase diversity and inclusion within the field of surgery.

Clinical Case-Conference Blogs: Integrating Clinical Librarians to Enhance Resident Education and Enforce ACGME Competencies.

Resident conferences are primary educational endeavors for trainees and faculty alike. We describe the development of collaborative clinician-librarian educational blogs within the Internal Medicine (2009), Pediatrics (2012), and General Surgery (2018) residency programs. Clinical librarians attended resident conferences and generated evidence-based blog posts based on learning topics and clinical questions encountered during the conferences. In the decade since introduction of the blogs, this partnership has resulted in over 2000 blog posts and generated over 1800 individual views per month. The development of a clinical librarian-managed blog serves as a relevant resource for promoting evidence-based practices within a case-based learning curriculum, engages interdisciplinary collaboration through existing resources, and is generalizable across various clinical practice disciplines and trainees.

Development of a Surgical Evidence Blog at Morbidity and Mortality Conferences: Integrating Clinical Librarians to Enhance Resident Education.

INTRODUCTION: Surgical morbidity and mortality (M&M) conference is a primary educational endeavor for trainees and faculty alike. Case presentations are enhanced by inclusion of clinical evidence; however, trainees are frequently limited in their ability to collect and share this type of material. METHODS: A surgical evidence web log ("blog") was created in partnership between the Clinical Librarian Service and the Department of Surgery. A clinical librarian attended weekly departmental M&M conferences and reviewed evidence in collaboration with a surgical resident. For each case presented, the clinical librarian created an evidence-based blog post based on specific learning topics encountered in the routine discussion of the M&M conference. The goal of this surgeon-librarian partnership was to enhance M&M's educational value by reinforcing learning topics, serving as a repository of case-based evidence, and involving trainees in evidence-based surgical practice. RESULTS: Blog posts included summaries of available evidence, critical reviews of seminal studies, and reviews of evidence-based guidelines framed in the context of the case. New blog posts were promoted via direct links in an existing weekly newsletter sent to all trainees and faculty in the department. Within the first year, surgical residents reported increased interaction with the literature, and 100% reported gaining knowledge that they would otherwise not receive through their standard readings, with 73% of residents using this to influence clinical practice and 87% applying knowledge in test preparation. This surgeon-librarian partnership enforces interdisciplinary collaboration through existing resources, and is highly generalizable to both surgical and medical training programs. CONCLUSIONS: In this study, the development of a surgical evidence blog represents an effective resource for promoting evidence-based practices within a case-based learning curriculum. This intervention is the first report in the literature to integrate the expertise and resources of a Clinical Librarian Service with an evidence-based resident educational curriculum in a surgical residency program.

Electron Transfer in Nitrogenase.

Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

Redox-Dependent Metastability of the Nitrogenase P-Cluster.

Molybdenum nitrogenase catalyzes the reduction of dinitrogen into ammonia, which requires the coordinated transfer of eight electrons to the active site cofactor (FeMoco) through the intermediacy of an [8Fe-7S] cluster (P-cluster), both housed in the molybdenum-iron protein (MoFeP). Previous studies on MoFeP from two different organisms, Azotobacter vinelandii ( Av) and Gluconacetobacter diazotrophicus ( Gd), have established that the P-cluster is conformationally flexible and can undergo substantial structural changes upon two-electron oxidation to the POX state, whereby a backbone amidate and an oxygenic residue (Ser or Tyr) ligate to two of the cluster's Fe centers. This redox-dependent change in coordination has been implicated in the conformationally gated electron transfer in nitrogenase. Here, we have investigated the role of the oxygenic ligand in Av MoFeP, which natively contains a Ser ligand (ßSer188) to the P-cluster. Three variants were generated in which (1) the oxygenic ligand was eliminated (ßSer188Ala), (2) the P-cluster environment was converted to the one in Gd MoFeP (ßPhe99Tyr/ßSer188Ala), and (3) two oxygenic ligands were simultaneously included (ßPhe99Tyr). Our studies have revealed that the P-cluster can become compositionally labile upon oxidation and reversibly lose one or two Fe centers in the absence of the oxygenic ligand, while still retaining wild-type-like dinitrogen reduction activity. Our findings also suggest that Av and Gd MoFePs evolved with specific preferences for Ser and Tyr ligands, respectively, and that the structural control of these ligands must extend beyond the primary and secondary coordination spheres of the P-cluster. The P-cluster adds to the increasing number of examples of inherently labile Fe-S clusters whose compositional instability may be an obligatory feature to enable redox-linked conformational changes to facilitate multielectron redox reactions.

HPV Vaccine-Related Research, Promotion and Coordination in the State of Georgia: A Systematic Review.

Human papillomavirus (HPV) vaccine was first recommended for female adolescents in the United States in 2006, and for male adolescents in 2011. In 2016, national HPV vaccine coverage was 43.4%, and slightly higher in Georgia (45.6%). Little is known about sub-state-level variation in HPV vaccination or Georgia specific research conducted to improve HPV vaccine uptake. We aimed to describe current uptake of HPV vaccination and synthesize Georgia-specific research of HPV vaccine uptake. We reviewed existing published and grey literature for different development, implementation and best practices related to vaccine uptake in adolescents. Studies measuring HPV vaccine uptake in Georgia utilizing quantitative, qualitative, or mixed-methods approaches were included alongside state-level information from National Immunization Survey-Teen, Community Health Needs Assessments, Behavioral Risk Factor Surveillance and Georgia Adolescent Immunization Surveys. Key findings across these reports were summarized. Forty-two titles and abstracts were identified through electronic database search for literature published between 2006 and 2017. Of these, five were from database sources and 37 were from additional sources. The most often mentioned barriers of HPV vaccine uptake in Georgia include cultural norms, vaccination education and a misalignment of vaccine uptake goals between the state and regional hospital community needs assessments. Increasing knowledge and uptake of HPV vaccine in Georgia requires significant research and to understand the vast socioeconomic, cultural and geographic barriers preventing uptake among adolescents. Georgia-specific efforts have been limited, however ongoing collaborations spearheaded by the Georgia Cancer Control Coalition provide opportunities for state-level support of research to address these barriers.