Radiographic comparison of eccentric stemmed vs. concentric stemless prosthetic humeral head positioning after anatomic total shoulder arthroplasty.

INTRODUCTION: Maintaining premorbid proximal humeral positioning is an essential consideration of anatomic total shoulder arthroplasty (aTSA), as malposition of the prosthetic humeral head can result in poor clinical outcomes. Stemless aTSA prosthetic heads are usually concentric, while stemmed aTSA prosthetic heads are typically eccentric in nature. Therefore, the purpose of this study was to compare the ability to restore native humeral head position between stemmed (eccentric) vs. stemless (concentric) aTSA. MATERIALS AND METHODS: Postoperative anteroposterior radiographs of 52 stemmed and 46 stemless aTSAs were analyzed. A best-fit circle was created using previously published and validated techniques to represent the premorbid humeral head position and axis of rotation. This circle was juxtaposed with another circle following the arc of the implant head. Next, the offset in center of rotation (COR), radius of curvature (RoC), and humeral head height above the greater tuberosity (HHH) were measured. Additionally, based on prior studies, an offset of >3 mm at any point between the implant head surface and premorbid best-fit circle was considered significant and further classified as overstuffed or understuffed. RESULTS: RoC deviation was significantly greater in the stemmed cohort than the stemless cohort (1.19 ± 1.37 mm vs. 0.65 ± 1.17 mm, P = .025). There was no statistically significant difference in deviation from premorbid humeral head between the stemmed and stemless cohorts for COR (3.20 ± 2.28 mm vs. 3.23 ± 2.09 mm, P = .800) or HHH (1.12 ± 3.27 mm vs. 0.92 ± 2.70 mm, P = .677). When comparing overstuffed implants to appropriately placed implants, there was a significant difference in overall COR deviation in stemmed implants (3.93 ± 2.51 mm vs. 1.92 ± 1.05 mm, P < .001). Superoinferior COR deviation (stemmed: 2.38 ± 3.01 mm vs. -0.61 ± 1.59 mm, P < .001; stemless: 2.70 ± 1.75 mm vs. -0.16 ± 1.87 mm, P < .001), mediolateral COR deviation (stemmed: 0.79 ± 2.65 mm vs. -0.62 ± 1.27 mm, P = .020; stemless: 0.40 ± 1.41 mm vs. -1.13 ± 1.96 mm, P = .020), and HHH (stemmed: 3.61 ± 2.73 mm vs. 0.50 ± 1.31 mm, P < .001; stemless: 3.98 ± 1.18 mm vs. 0.53 ± 1.41 mm, P < .001) were significantly different between overstuffed implants and appropriate implants in both the stemmed and stemless cohorts. DISCUSSION: Stemless and stemmed aTSA implants have similar rates of reproducing satisfactory postoperative humeral head COR with both producing COR deviation most commonly in the superomedial direction. Deviation in HHH contributes to overstuffing in both stemmed and stemless implants, COR deviation contributes to overstuffing in stemmed implants, while RoC (humeral head size) is not associated with overstuffing. Based on this study, it appears that neither eccentric nor concentric prosthetic heads are superior in recreating premorbid humeral head position.

Single-shot interscalene block with liposomal bupivacaine vs. non-liposomal bupivacaine in shoulder arthroplasty.

BACKGROUND: Regional anesthesia is a valuable component of multimodal pain control in total shoulder arthroplasty (TSA), and multiple interscalene block anesthetic options exist, including nonliposomal interscalene bupivacaine (NLIB) and liposomal interscalene bupivacaine (LIB). The purpose of this study was to compare pain control and opioid consumption within 48 hours postoperation in those undergoing TSA with either LIB or NLIB. METHODS: This was a retrospective cohort study at a single academic medical center including consecutive patients undergoing inpatient (>23-hour hospitalization) primary anatomic or reverse TSA from 2016 to 2020 who received either LIB or an NLIB for perioperative pain control. Perioperative patient outcomes were collected including pain levels and opioid usage, as well as 30- and 90-day emergency department (ED) visits or readmissions. The primary outcome was postoperative pain and opioid use. RESULTS: Overall, 489 patients were included in this study (316 LIB and 173 NLIB). Pain scores at 3, 6, 12, and 48 hours postoperatively were not statistically significantly different (P > .05 for all). However, the LIB group had improved pain scores at 24 and 36 hours postoperation (P < .05 all). There was no difference in the incidence of severe postoperative pain, defined as a 9 or 10 numeric rating scale-11 score, between the 2 anesthesia groups after adjusting for preoperative pain and baseline opioid use (odds ratio 1.25, 95% confidence interval 0.57-2.74; P = .57). Overall, 99 of 316 patients receiving LIB (31.3%) did not require any postoperative opioids compared with 38 of 173 receiving NLIB (22.0%); however, this difference was not statistically significant after adjusting for prior opioid use and preoperative pain (P = .33). No statistically significant differences in postoperative total morphine equivalents or mean daily morphine equivalents consumed between the groups were found during their hospital stays (P > .05 for both). Finally, no significant differences were found in 30- and 90-day ED visits or readmission rates (all P > .05). CONCLUSION: LIB and NLIB demonstrated differences in patient-reported pain scores at 24 and 36 hours postoperation, although these did not reach clinical significance. There were no statistically significant differences in opioid consumption during the hospital stay, including opioid use, total morphine equivalents, and daily mean morphine equivalents consumed during the hospital stay. Additionally, no differences were observed in 30- and 90-day ED visits or readmission rates.

Simple preoperative radiographic and computed tomography measurements predict adequate bone quality for stemless total shoulder arthroplasty.

INTRODUCTION: Although there is increased utilization of stemless humeral implants in anatomic total shoulder arthroplasty (TSA), there are inadequate objective metrics to evaluate bone quality sufficient for fixation. Our goals are to: (1) compare patient characteristics in patients who had plans for stemless TSA but received stemmed TSA due to intraoperative assessments and (2) propose threshold values of bone density, using the deltoid tuberosity index (DTI) and proximal humerus Hounsfield units (HU), on preoperative X-ray and computed tomography (CT) to allow for preoperative determination of adequate bone stock for stemless TSA. METHODS: This is an observational study conducted at an academic institution from 2019 to 2021, including consecutive primary TSAs templated to undergo stemless TSA based on 3-dimensional CT preoperative plans. Final implant selection was determined by intraoperative assessment of bone quality. Preoperative X-ray and CT images were assessed to obtain DTI and proximal humeral bone density in HU, respectively. A receiver operating characteristic curve was used to analyze the potential of preoperative X-ray and CT to classify patients as candidates for stemless TSA. RESULTS: A total of 61 planned stemless TSAs were included, with 56 (91.8%) undergoing stemless TSA and 5 (8.2%) undergoing stemmed TSA after intraoperative assessment determined that the bone quality was inadequate for stemless fixation. There were no significant differences between the 2 groups in terms of gender (P = .640), body mass index (P = .296), and race (P = .580). The stem cohort was significantly older (mean age 69 ± 12 years vs. 59 ± 10 years, P = .029), had significantly lower DTI (1.45 ± 0.13 vs. 1.68 ± 0.18, P = .007), and had significantly less proximal humeral HU (-1.4 ± 17.7 vs. 78.8 ± 52.4, P = .001). The receiver operating characteristic curve for DTI had an area under the curve (AUC) of 0.86, and bone density in HU had an AUC of 0.98 in its ability to distinguish patients who underwent stemless TSA vs. short-stem TSA. A threshold cutoff of 1.41 for DTI resulted in a sensitivity of 98% and a specificity of 60%, and a cutoff value of 14.4 HU resulted in a sensitivity of 95% and a specificity of 100%. CONCLUSIONS: Older age, lower DTI, and less proximal humeral bone density in HU were associated with the requirement to switch from stemless to short-stem humeral fixation in primary TSA. Preoperative DTI had good ability (AUC of 0.86) and preoperative HU had excellent ability (AUC of 0.98) to categorize patients as appropriate for stemless TSA. This can help surgeons adequately plan humeral fixation using standard preoperative imaging data.

Preganglionic and Postganglionic Brachial Plexus Birth Injury Effects on Shoulder Muscle Growth.

PURPOSE: Brachial plexus birth injury can differ in presentation, depending on whether the nerve ruptures distal to, or avulses proximal to, the dorsal root ganglion. More substantial contracture and bone deformity at the shoulder is typical in postganglionic injuries. However, changes to the underlying muscle structure that drive these differences in presentation are unclear. METHODS: Seventeen Sprague-Dawley rats received preganglionic or postganglionic neurectomy on a single limb on postnatal days 3 and 4. Muscles crossing the shoulder were retrieved once the rats were sacrificed at 8 weeks after birth. External rotation range of motion, muscle mass, muscle length, muscle sarcomere length, and calculated optimal muscle length were measured bilaterally. RESULTS: Average shoulder range of motion in the postganglionic group was 61.8% and 56.2% more restricted at 4 and 8 weeks, respectively, compared with that in the preganglionic group, but affected muscles after preganglionic injury were altered more severely (compared with the unaffected limb) than after postganglionic injury. Optimal muscle length in preganglionic injury was shorter in the affected limb (compared with the unaffected limb: -18.2% ± 9.2%) and to a greater extent than in postganglionic injury (-5.1% ± 6.2%). Muscle mass in preganglionic injury was lower in the affected limb (relative to the unaffected limb: -57.2% ± 24.1%) and to a greater extent than in postganglionic injury (-28.1% ± 17.7%). CONCLUSIONS: The findings suggest that the presence of contracture does not derive from restricted longitudinal muscle growth alone, but also depends on the extent of muscle mass loss occurring simultaneously after the injury. CLINICAL RELEVANCE: This study expands our understanding of differences in muscle architecture and the role of muscle structure in contracture formation for preganglionic and postganglionic brachial plexus birth injury.

Functional Grading of a Transversely Isotropic Hyperelastic Model with Applications in Modeling Tricuspid and Mitral Valve Transition Regions.

Surgical simulators and injury-prediction human models require a combination of representative tissue geometry and accurate tissue material properties to predict realistic tool-tissue interaction forces and injury mechanisms, respectively. While biological tissues have been individually characterized, the transition regions between tissues have received limited research attention, potentially resulting in inaccuracies within simulations. In this work, an approach to characterize the transition regions in transversely isotropic (TI) soft tissues using functionally graded material (FGM) modeling is presented. The effect of nonlinearities and multi-regime nature of the TI model on the functional grading process is discussed. The proposed approach has been implemented to characterize the transition regions in the leaflet (LL), chordae tendinae (CT) and the papillary muscle (PM) of porcine tricuspid valve (TV) and mitral valve (MV). The FGM model is informed using high resolution morphological measurements of the collagen fiber orientation and tissue composition in the transition regions, and deformation characteristics predicted by the FGM model are numerically validated to experimental data using X-ray diffraction imaging. The results indicate feasibility of using the FGM approach in modeling soft-tissue transitions and has implications in improving physical representation of tissue deformation throughout the body using a scalable version of the proposed approach.

Scavenging of highly reactive gamma-ketoaldehydes attenuates cognitive dysfunction associated with epileptogenesis.

Cognitive dysfunction is a major comorbidity of the epilepsies; however, treatments targeting seizure-associated cognitive dysfunction, particularly deficits in learning and memory are not available. Isoketals and neuroketals, collectively known as gamma-ketoaldehydes are formed via the non-enzymatic, free radical catalyzed oxidation of arachidonic acid and docosahexaenoic acid, respectively. They are attractive candidates for oxidative protein damage and resultant cognitive dysfunction due to their formation within the plasma membrane and their high proclivity to form cytotoxic adducts on protein lysine residues. We tested the hypothesis that gamma-ketoaldehydes mechanistically contribute to seizure-associated memory impairment using a specific gamma-ketoaldehyde scavenger, salicylamine in the kainic acid and pilocarpine rat models of temporal lobe epilepsy. We show that gamma-ketoaldehydes are increased following epileptogenic injury in hippocampus and perirhinal cortex, two brain regions imperative for learning and memory. Treatment with an orally bioavailable, brain permeable scavenger, salicylamine attenuated 1) spatial memory deficits 2) reference memory deficits and 3) neuronal loss and astrogliosis in two mechanistically distinct models of epilepsy without affecting the epileptogenic injury or the development of chronic epilepsy. We have previously demonstrated that reactive oxygen species and the lipid peroxidation biomarkers, F2-isoprostanes are produced following status epilepticus. However, which reactive species specifically mediate oxidative damage to cellular macromolecules remains at large. We provide novel data suggesting that memory impairment occurs via gamma-ketoaldehyde production in two models of epilepsy and that treatment with a gamma-ketoaldehyde scavenger can protect vulnerable neurons. This work suggests a novel target and therapy to treat seizure-induced memory deficits in epilepsy.

Tendon Transfers in Reverse Total Shoulder Arthroplasty: A Systematic Review.

PURPOSE: The purpose of this study was to evaluate clinical outcomes after tendon transfers in the setting of reverse total shoulder arthroplasty (RTSA). METHODS: PubMed and Embase were searched according to Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines to find primary clinical studies of any type of tendon transfer in the setting of RTSA. RESULTS: Overall, 17 studies (level of evidence [LOE] I: 1, LOE II: 0, LOE III: 3, LOE IV: 13) met inclusion criteria, with 300 shoulders. Most patients were female (56.7%), with an average age of 68.7 years (range 19 to 89) and a mean follow-up of 46.2 months (range 6 to 174). 11 studies reported outcomes after combined latissimus dorsi and teres major transfer (LDTM) while eight studies reported on latissimus dorsi transfer only (LD). Improvements in commonly reported subjective and functional outcome measures were as follows: external rotation +32° (LDTM) and +30° (LD), flexion +65° (LDTM) and +59° (LD), Visual Analog Score -5.4 (LDTM) and -4.5 (LD), subjective shoulder value +43.8% (LDTM) and +46.3% (LD), and overall Constant score +33.8 (LDTM) and +38.7 (LD). The overall complication rate was 11.3%, including tendon transfer ruptures (0.7%), instability (3.0%), infection (2.0%), and nerve injury (0.3%). The all-cause repeat operation rate was 7.3%, most commonly for arthroplasty revision (5.3%). Subgroup analysis revealed that lateralized implants with tendon transfer resulted in markedly greater improvements in Constant score, flexion, ER1, and ER2 while medialized implants with tendon transfer had markedly greater improvements in Visual Analog Score, subjective shoulder value, and abduction. CONCLUSION: Patients undergoing tendon transfer of either combined LDTM or latissimus dorsi alone in the setting of RTSA have markedly improved subjective and functional outcomes. A moderate incidence of complications (11.3%) was noted in this patient population.

Advancing Health Equity: A Collaborative Framework to Address HIV Disparities in Georgia and Louisiana through Data, Education, and Policy Initiatives.

The United States grapples with over 30,000 new HIV infections annually, a challenge exacerbated by delayed diagnosis and treatment. HIV stigma hinders data collection and contributes to health disparities. High-quality data and a community-driven approach are critical to reducing these disparities. The collaborative effort of the Satcher Health Leadership Institute at Morehouse School of Medicine, and the Center for Minority Health and Health Disparities Research and Education at Xavier University of Louisiana, addresses systemic barriers perpetuating the HIV epidemic in Georgia and Louisiana. We influence policy, implement an HIV education curriculum, map resources, and display data via the Health Equity Tracker. Stigma surrounding HIV/AIDS impedes disclosure, creates data gaps, and affects care quality and access. Addressing these data gaps, influencing policy, and implementing education are crucial steps to improving outcomes for Black people living with HIV/AIDS. This framework aligns with the federal Ending the HIV Epidemic program's goals, advancing health equity for vulnerable populations.

Evaluation of information from artificial intelligence on rotator cuff repair surgery.

Purpose: The purpose of this study was to analyze the quality and readability of information regarding rotator cuff repair surgery available using an online AI software. Methods: An open AI model (ChatGPT) was used to answer 24 commonly asked questions from patients on rotator cuff repair. Questions were stratified into one of three categories based on the Rothwell classification system: fact, policy, or value. The answers for each category were evaluated for reliability, quality and readability using The Journal of the American Medical Association Benchmark criteria, DISCERN score, Flesch-Kincaid Reading Ease Score and Grade Level. Results: The Journal of the American Medical Association Benchmark criteria score for all three categories was 0, which is the lowest score indicating no reliable resources cited. The DISCERN score was 51 for fact, 53 for policy, and 55 for value questions, all of which are considered good scores. Across question categories, the reliability portion of the DISCERN score was low, due to a lack of resources. The Flesch-Kincaid Reading Ease Score (and Flesch-Kincaid Grade Level) was 48.3 (10.3) for the fact class, 42.0 (10.9) for the policy class, and 38.4 (11.6) for the value class. Conclusion: The quality of information provided by the open AI chat system was generally high across all question types but had significant shortcomings in reliability due to the absence of source material citations. The DISCERN scores of the AI generated responses matched or exceeded previously published results of studies evaluating the quality of online information about rotator cuff repairs. The responses were U.S. 10th grade or higher reading level which is above the AMA and NIH recommendation of 6th grade reading level for patient materials. The AI software commonly referred the user to seek advice from orthopedic surgeons to improve their chances of a successful outcome.

Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase.

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.

Arthroscopic Distal Clavicle Bone Block Technique for Anteroinferior Instability With Critical Bone Loss.

Arthroscopic distal clavicle autograft represents a locally available source of autograft for bone block augmentation in patients with anterior shoulder instability with glenoid bone loss. Anatomic and biomechanical studies have supported distal clavicle autograft use as comparable to coracoid graft with regard to restoration of glenoid articular surface, with the theoretical advantage of minimizing complications associated with coracoid transfer procedures, such as neurologic injury and coracoid fracture. The current technique describes a modification of those previously described, including a mini-open approach for distal clavicle autograft harvest, orientation of the distal clavicle with the medial clavicle graft against the glenoid (congruent arc), an all-arthroscopic technique of graft passage, and graft placement and fixation using specialized drill guides and four suture buttons to reproducibly place and secure the graft with final capsulolabral advancement over the graft to render it extra-articular.

Chaperone Recycling in Late-Stage Flagellar Assembly.

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.

Structural basis for DNA binding by replication initiator Mcm10.

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.

Inguinal phlegmon and positive serology for syphilis after penile bite laceration: true, true and unrelated.

Among many causes of penile injury, sexual activity is infrequently reported. The present case involved delay in recognition, which led to development of a phlegmon near major vascular structures. Positive serologic evidence of syphilis was an incidental finding.

Statistical Approaches to Type Determination of the Ejector Marks on Cartridge Cases.

While type determination on bullets has been performed for over a century, type determination on cartridge cases is often overlooked. Presented here is an example of type determination of ejector marks on cartridge cases from Glock and Smith & Wesson Sigma series pistols using Naïve Bayes and Random Forest classification methods. The shapes of ejector marks were captured from images of test-fired cartridge cases and subjected to multivariate analysis. Naïve Bayes and Random Forest methods were used to assign the ejector shapes to the correct class of firearm with success rates as high as 98%. This method is easily implemented with equipment already available in crime laboratories and can serve as an investigative lead in the form of a list of firearms that could have fired the evidence. Paired with the FBI's General Rifling Characteristics (GRC) database, this could be an invaluable resource for firearm evidence at crime scenes.

Effector Mechanisms of Neutrophils within the Innate Immune System in Response to Mycobacterium tuberculosis Infection.

Neutrophils have a significant yet controversial role in the innate immune response to Mycobacterium tuberculosis (M. tb) infection, which is not yet fully understood. In addition to neutrophils' well-known effector mechanisms, they may also help control infection of M. tb through the formation of neutrophil extracellular traps (NETs), which are thought to further promote the killing of M. tb by resident alveolar macrophages. Cytokines such as IFN-γ have now been shown to serve an immunomodulatory role in neutrophil functioning in conjunction to its pro-inflammatory function. Additionally, the unique transcriptional changes of neutrophils may be used to differentiate between infection with M. tb and other bacterial and chronic rheumatological diseases such as Systemic Lupus Erythematosus. Adversely, during the innate immune response to M. tb, inappropriate phagocytosis of spent neutrophils can result in nonspecific damage to host cells due to necrotic lysis. Furthermore, some individuals have been shown to be more genetically susceptible to tuberculosis (TB) due to a "Trojan Horse" phenomenon whereby neutrophils block the ability of resident macrophages to kill M. tb. Despite these aforementioned negative consequences, through the scope of this review we will provide evidence to support the idea that neutrophils, while sometimes damaging, can also be an important component in warding off M. tb infection. This is exemplified in immunocompromised individuals, such as those with human immunodeficiency virus (HIV) infection or Type 2 diabetes mellitus. These individuals are at an increased risk of developing tuberculosis (TB) due to a diminished innate immune response associated with decreased levels of glutathione. Consequently, there has been a worldwide effort to limit and contain M. tb infection through the use of antibiotics and vaccinations. However, due to several significant limitations, the current bacille Calmette-Guerin vaccine (BCG, vaccine against TB) does not meet the criteria for universal utilization for all ages and populations across the globe. New research involving neutrophils has yielded a new vaccine called M. smegmatis-Ag85C-MPT51-HspX (mc²-CMX) that has been shown to elicit a humoral and cellular response against M. tb in mice that is superior to the BCG vaccine.

Expert consensus document: Mind the gaps­advancing research into short-term and long-term neuropsychological outcomes of youth sports-related concussions.

Sports-related concussions and repetitive subconcussive exposure are increasingly recognized as potential dangers to paediatric populations, but much remains unknown about the short-term and long-term consequences of these events, including potential cognitive impairment and risk of later-life dementia. This Expert Consensus Document is the result of a 1-day meeting convened by Safe Kids Worldwide, the Alzheimer's Drug Discovery Foundation, and the Andrews Institute for Orthopaedics and Sports Medicine. The goal is to highlight knowledge gaps and areas of critically needed research in the areas of concussion science, dementia, genetics, diagnostic and prognostic biomarkers, neuroimaging, sports injury surveillance, and information sharing. For each of these areas, we propose clear and achievable paths to improve the understanding, treatment and prevention of youth sports-related concussions.

Physical interactions between Mcm10, DNA, and DNA polymerase alpha.

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase alpha (pol alpha), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol alpha. However, the mechanism by which Mcm10 interacts with pol alpha on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID*ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286-310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol alpha within the replication fork.

Domain architecture and biochemical characterization of vertebrate Mcm10.

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.