How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication.

During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.

A conserved mechanism for regulating replisome disassembly in eukaryotes.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase1-3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4-7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8-10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

Structure of a human replisome shows the organisation and interactions of a DNA replication machine.

The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45-MCM-GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading-strand polymerase Pol ε, together with TIMELESS-TIPIN, CLASPIN and AND-1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Šcryo-EM structure of a human replisome comprising CMG, Pol ε, TIMELESS-TIPIN, CLASPIN and AND-1 bound to replication fork DNA. The structure permits a detailed understanding of how AND-1, TIMELESS-TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS-TIPIN with replication fork DNA suggests a mechanism for strand separation.

Enhanced Mitochondrial Targeting and Inhibition of Pyroptosis with Multifunctional Metallopolyphenol Nanoparticles in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a significant contributor to low back pain, characterized by excessive reactive oxygen species generation and inflammation-induced pyroptosis. Unfortunately, there are currently no specific molecules or materials available to effectively delay IVDD. This study develops a multifunctional full name of PG@Cu nanoparticle network (PG@Cu). A designed pentapeptide, bonded on PG@Cu nanoparticles via a Schiff base bond, imparts multifunctionality to the metal polyphenol particles (PG@Cu-FP). PG@Cu-FP exhibits enhanced escape from lysosomal capture, enabling efficient targeting of mitochondria to scavenge excess reactive oxygen species. The scavenging activity against reactive oxygen species originates from the polyphenol-based structures within the nanoparticles. Furthermore, Pyroptosis is effectively blocked by inhibiting Gasdermin mediated pore formation and membrane rupture. PG@Cu-FP successfully reduces the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome by inhibiting Gasdermin protein family (Gasdermin D, GSDMD) oligomerization, leading to reduced expression of Nod-like receptors. This multifaceted approach demonstrates higher efficiency in inhibiting Pyroptosis. Experimental results confirm that PG@Cu-FP preserves disc height, retains water content, and preserves tissue structure. These findings highlight the potential of PG@Cu-FP in improving IVDD and provide novel insights for future research in IVDD treatments.

New approach to testing treatments for osteoarthritis: FastOA.

Animal models of post traumatic osteoarthritis have shown many promising treatments for disease, but human trials have mostly failed to identify effective treatments. This viewpoint suggests that the frequent failure of drug and treatment development in osteoarthritis is due, in part, to the advanced stage of disease of patients in trials and suggests that mirroring the animal model approach might be more successful. It suggests a path forward by enriching trial enrollees with those likely to develop post traumatic OA quickly.

Patients with advanced cancer and their spouses parenting minor children: The role of the relationship context in parenting concerns.

OBJECTIVE: Patients with advanced cancer who parent minor children report parenting concerns and increased psychological distress. This cross-sectional study seeks to understand parenting-related issues in patients and spousal caregivers from a relationship perspective. METHODS: Patients with a metastatic solid malignancy and their spouses independently completed cross-sectional assessments of psychological distress (Hospital Anxiety and Depression Scale), parenting concerns (Parenting Concern Questionnaire) and efficacy (Cancer-Related Parenting Self-Efficacy Scale), and relationship measures (DAS-7, Couples' Illness Communication Scale, and Family Relationship Index). RESULTS: Of the 51 patients (57% female, 49% NHW, mean age 42 years) and spouses (43% female, 43% NHW, mean age of 42 years), approximately 50% couples endorsed psychological distress and were at risk for family dysfunction. Spouses reported significantly higher levels of parenting-related concerns (t = -2.0, p < 0.05) and anxiety (t = -2.8, p < 0.001) than patients. Parenting concerns were significantly associated with illness communication (r = -0.56, p < 0.001) and family function (r = -0.38, p < 0.001). Although the expected interactions between parenting concerns and relationship variables (i.e., illness communication, dyadic adjustment, and family function) were significant for depressive symptoms at p < 0.05, the associations were not in the expected direction. Relationship function buffered against depressive symptoms for those with low rather than high parenting concerns. CONCLUSIONS: Not only patients but also spouses report cancer-related parenting concerns. The associations between parenting concerns and distress were stronger for spouses than patients. Dual caregiving appears to be a particularly stressful role. Because relationship function was associated with parenting concerns, we suggest that parent support programs that are couple-based and include both parenting-specific and relationship-specific content may be most effective in reducing distress for this vulnerable population.

Does intervertebral disc degeneration in adolescent idiopathic scoliosis correlate with patient-reported pain scores? A review of 968 cases.

PURPOSE: Report the rate and severity of degenerative disc disease (DDD) in non-surgical adolescent idiopathic scoliosis (AIS) patients and correlate these findings with patient-reported symptomatology scores. Additionally, to quantify the rate of concurrent pathological radiological findings in this group. METHODS: This was a retrospective chart review study at a single tertiary centre. AIS patients aged 10-16 who had received a whole spine MRI between September 2007 and January 2019 and who had not received surgical intervention to their spine were included. MRI scan reports were screened to extract those who had evidence of DDD. These were then reviewed by a blinded second reviewer who graded every disc using the Pfirrmann grading system. SRS-22 scores were extracted for patients when available. RESULTS: In total, 968 participants were included in the study. Of these, 93 (9.6%) had evidence of DDD, which was Pfirrmann grade ≥ 3 in 28 (2.9%). The most commonly affected level was L5/S1 (59.1% of DDD cases). A total of 55 patients (5.7%) had evidence of syringomyelia, 41 (3.4%) had evidence of spondylolisthesis (all L5/S1), 14 (1.4%) had bilateral L5 pars defects, and 5 (0.5%) had facet joint degeneration. Spondylolisthesis and bilateral pars defects were more common in patients with DDD identified on MRI scan (p < 0.001 and p = 0.04, respectively). Function (p = 0.048) and pain (p = 0.046) scores were worse in patients with DDD. CONCLUSION: We present a baseline for the rate and severity of DDD in the non-operative AIS cohort. This should assist in decision-making and counselling of patients prior to surgery. LEVEL OF EVIDENCE: III.

Emergent bilingual children during the silent period: A scoping review of their communication strategies and classroom environments.

This scoping review aimed to investigate the communication strategies utilized by children who acquire a minority language (L1) and subsequently learn a community language (L2) during what is commonly referred to as the "silent period." Electronic database searches were conducted using keywords such as "silent period" and "bilingual children," resulting in the inclusion of 40 studies in the review. The findings revealed that emergent bilingual children utilize various communication strategies, including nonverbal communication, private speech, and their L1, to communicate within classroom environments. The findings shed light on the adaptability of emergent bilingual children during early stage of L2 acquisition. Furthermore, our review provides information about the classroom contexts such as teacher support and peer interactions where children develop their L2 skills. From a clinical perspective, recognizing these strategies and classroom contexts could significantly enhance the screening process for emergent bilingual children.

Patient-specific 3D printing to replace components of a rib-to pelvis "Eiffel Tower" vertebral expanding prosthetic titanium rib system in an infant: a case report.

PURPOSE: To address the anatomical challenges facing complex and revision spinal surgery, patient-specific 3D-printed models (3D-PMs) have received growing attention worldwide, primarily in adults. We report the use of a 3D-PM in the treatment of a case of wound breakdown over a component of a VEPTR (Vertical Expandable Prosthetic Titanium Rib; DePuy-Synthes) system, requiring replacement of Dunn-McCarthy hook and sleeve with components contoured to a patient-specific 3D-PM of the spine. METHOD: A two-year-old born with myelomeningocele (MMC), repaired at birth, developed progressive MMC-associated kyphoscoliosis. Elective insertion of a rib-to-pelvis 'Eiffel Tower' bilateral VEPTR growing rods construct was performed without initial complication. Prominence of the right VEPTR sleeve and Dunn-McCarthy hook side-to-side connector resulted in breakdown of overlying poor-quality soft tissues, necessitating washout, partial implant removal, intravenous antibiotic therapy and delayed primary wound closure. A patient-specific 3D-PM, utilising pre-operative CT spine and pelvis 3D-reconstructions, allowed pre-operative formation of a contoured implant, which was inserted without need for further revision. RESULTS: The patient underwent further VEPTR lengthening without recurrent infection, wound breakdown or implant failure at 24-month follow-up. Satisfactory control of the deformity has been achieved with continued improvement in sitting height and radiographic indices. CONCLUSION: This case illustrates the possibility, in certain cases, of using 3D-PM to develop complex components of spinal implant systems pre-operatively, removing the time and difficulty of intra-operative contouring. Consequently, custom-contoured implants may be produced, sterilised and implanted. This technique may be an option, in infants, including MMC-associated kyphoscoliosis, where midline fixation is not possible.

Thermogenic carbon release from the Central Atlantic magmatic province caused major end-Triassic carbon cycle perturbations.

The Central Atlantic magmatic province (CAMP), the end-Triassic mass extinction (ETE), and associated major carbon cycle perturbations occurred synchronously around the Triassic-Jurassic (T-J) boundary (201 Ma). Negative carbon isotope excursions (CIEs) recorded in marine and terrestrial sediments attest to the input of isotopically light carbon, although the carbon sources remain debated. Here, we explore the effects of mantle-derived and thermogenic carbon released from the emplacement of CAMP using the long-term ocean-atmosphere-sediment carbon cycle reservoir (LOSCAR) model. We have tested a detailed emission scenario grounded by numerous complementary boundary conditions, aiming to model the full extent of the carbon cycle perturbations around the T-J boundary. These include three negative CIEs (i.e., Marshi/Precursor, Spelae/Initial, Tilmanni/Main) with sharp positive CIEs in between. We show that a total of ∼24,000 Gt C (including ∼12,000 Gt thermogenic C) replicates the proxy data. These results indicate that thermogenic carbon generated from the contact aureoles around CAMP sills represents a credible source for the negative CIEs. An extremely isotopically depleted carbon source, such as marine methane clathrates, is therefore not required. Furthermore, we also find that significant organic carbon burial, in addition to silicate weathering, is necessary to account for the positive δ13C intervals following the negative CIEs.

ER-associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.

Hematopoietic stem cells (HSC) self-renew to sustain stem cell pools and differentiate to generate all types of blood cells. HSCs remain in quiescence to sustain their long-term self-renewal potential. It remains unclear whether protein quality control is required for stem cells in quiescence when RNA content, protein synthesis, and metabolic activities are profoundly reduced. Here, we report that protein quality control via endoplasmic reticulum-associated degradation (ERAD) governs the function of quiescent HSCs. The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyperproliferation, which leads to complete loss of HSC self-renewal and HSC depletion. Mechanistically, ERAD deficiency via Sel1L knockout leads to activation of mammalian target of rapamycin (mTOR) signaling. Furthermore, we identify Ras homolog enriched in brain (Rheb), an activator of mTOR, as a novel protein substrate of Sel1L/Hrd1 ERAD, which accumulates upon Sel1L deletion and HSC activation. Importantly, inhibition of mTOR, or Rheb, rescues HSC defects in Sel1L knockout mice. Protein quality control via ERAD is, therefore, a critical checkpoint that governs HSC quiescence and self-renewal by Rheb-mediated restriction of mTOR activity.

Selection of parenchymal preserving or total pancreatectomy with/without islet cell autotransplantation surgery for patients with chronic pancreatitis.

BACKGROUND: The selection of surgery between parenchymal preserving (PPS) and total pancreatectomy (TP) with/without islet cell autotransplantation (IAT) for chronic pancreatitis (CP) patients varies based on multiple factors with a scarcity in literature addressing both at the same time. The aim of this manuscript is to present an algorithm for the surgery selection based on dominant area of disease, ductal dilatation, and glycemic control and compare outcomes. METHODS: From 2017 to 2021, CP patients offered surgery at a single institution were retrospectively evaluated. RESULTS: 51 patients underwent surgery (20 [39.2%] TPIAT, 4 [7.8%] TP, and 27 [52.9%] PPS - 9 Whipple procedures, 15 distal pancreatectomies, and 3 duct drainage procedures). No significant difference was observed in baseline characteristics or perioperative outcomes except median length of stay (8 days [IQR 6-10] vs. 13 days [IQR 9-15.5], p < 0.001), attributed to insulin requirement and education for TPIAT group. No differences in postoperative complications, such as clinically significant leak and intrabdominal fluid collection (3 [11.1%] vs 2 [10%], p = 1.0), hemorrhage (0 vs. 2 [10.0%], p = 0.2), delayed feeding (1 [3.7%] vs. 5 [25.0%], p = 0.07), or wound infection (4 [14.8%] vs. 0, p = 0.1) between PPS and TPIAT groups, respectively, were observed nor requirement of long-acting insulin at discharge (2 [15.4%] vs. 7 [43.8%], p = 0.1) for pre-operatively non-diabetic patients. No significant difference in weaning off narcotics and no mortality observed. CONCLUSION: The most appropriate selection of surgery based on the algorithm yields good and comparable outcomes.

Imaging of Patients Suspected of SLAP Tear: A Cost-Effectiveness Study.

BACKGROUND. Superior labrum anterior and posterior (SLAP) tears are a common shoulder pathology. Although MRI is the imaging reference standard for diagnosis of this pathology, the cost-effectiveness of common MRI strategies is unclear. OBJECTIVE. The primary objective of our study was to determine the cost-effectiveness of the common MRI-based strategies used for the diagnosis of SLAP tears. METHODS. We created decision analytic models from the perspective of the U.S. health care system over a 2-year time horizon for a hypothetical population of 25-year-old patients with a previous diagnosis of SLAP tear. We used the decision models to compare the differences in incremental cost-effectiveness of the common MRI strategies, which included combinations of 1.5-T and 3-T MR arthrography (MRA) and unenhanced MRI protocols, and the resulting treatment applied for these patients. Input data on cost, probability, and utility estimates were obtained through a comprehensive literature search. The primary effectiveness outcome was quality-adjusted life years. Costs were estimated in 2017 U.S. dollars. RESULTS. When all imaging strategies were considered, the unenhanced 3-T MRI-based imaging strategy was the preferred and dominant option over 3-T MRA and 1.5-T imaging (MRI and MRA). When the model was run without 3-T imaging as an option, 1.5-T MRA was the favored option. Probabilistic sensitivity analyses confirmed the same preferred imaging strategy results. CONCLUSION. An unenhanced 3-T MRI-based strategy is the most cost-effective imaging option for patients with suspected SLAP tear. When 3-T imaging is not available, 1.5-T MRA is more cost-effective than 1.5-T MRI. The main driver of these results is the fact that 3-T MRI and 1.5-T MRA are the most specific tests in these respective scenarios, which results in fewer false-positives, prevents unnecessary surgeries, and leads to decreased costs. CLINICAL IMPACT. Our cost-effectiveness model findings complement prior diagnostic accuracy work, helping produce a more comprehensive approach to define imaging utility for radiologists, clinicians, and patients with SLAP tears who have access to various types of MRI options.

Use of Continuous Glucose Monitoring for evaluation of hypoglycemia pre- and post-operatively for patients undergoing Total Pancreatectomy with Autologous Islet Cell Transplant.

INTRODUCTION: Total pancreatectomy with autologous islet transplant (TPAIT) is indicated for patients with chronic pancreatitis to improve quality of life while reducing complications from hypoglycemia. Continuous glucose monitoring (CGM) was used to assess overall islet function and the incidence of hypoglycemia pre- and post-operatively. METHODS: Nineteen patients who underwent TPAIT at a single center from 2018 to 2020 were included. Pre-operatively, patients were defined by diabetic status. HbA1c, stimulated C-peptide, and CGM were used to characterize glycemic function. RESULTS: Pre-operatively, three patients had diabetes, and 16 patients did not have diabetes. Eight out of 16 non-diabetic patients were insulin independent (50%). Of six non-diabetic patients with > 10% hypoglycemia on pre-operative CGM, 33% were insulin-independent post-operatively (P = .3). Of non-diabetic patients with ≥ 80% time in the euglycemic range, 62% were insulin-independent post-operatively (P = .2). For patients without diabetes, the median percent time in hypoglycemic range was reduced from 8% to 1% (P = .001). Delta C-peptide had a positive correlation with islet yield (P = .03). DISCUSSION: Conventional evaluation of TPAIT patients assesses primarily beta cell function. As pancreatogenic diabetes is concerning principally for the risk of hypoglycemia, assessment of alpha cell function can improve the quality of care. CGM better captures islet function and increases the identification of hypoglycemia.

Index radiographic measurements in the prediction of progression in infantile idiopathic scoliosis: a comparative analysis and description of a novel predictive model.

PURPOSE: To assess the comparative accuracy of commonly utilised index radiological measurements in the prognosis of infantile idiopathic scoliosis (IIS) and build a parsimonious prognostic model utilising these measurements. METHODS: This was a retrospective analysis of a UK population of patients with IIS. Index radiological parameters were analysed, and outcome of their condition was determined over long-term follow-up. Comparative accuracy of each radiological measurement category was determined by logistic regression analyses and the corresponding receiver operating characteristic (ROC) curve. A predictive model of IIS progression using these measurements was then created. RESULTS: All three radiological measurement categories were predictive of IIS progression. However, on pairwise comparison of ROC curves and multivariate analysis, the index Cobb angle proved the most significant predictor of curve progression. Using the index Cobb angle only, a predictive model of curve progression achieved an accuracy of 81.18% with a cut-off Cobb angle of 34.5° found to be the optimal threshold to discriminate a progressive from resolving curve. CONCLUSION: Of the three analysed index radiological parameters commonly used by surgeons in the prognosis of IIS, we found that the Cobb angle is the most accurate predictive measure. Further, neither addition of the RVAD nor convex RVA provided significant further prognostic value in a multivariate model of progression. However, not even the Cobb angle model was accurate in all cases; emphasising caution should be applied when relying on index radiological measurements to predict IIS outcomes.

Comparison of Clinical and Semiquantitative Cartilage Grading Systems in Predicting Outcomes After Arthroscopic Partial Meniscectomy.

OBJECTIVE. Cartilage loss on preoperative knee MRI is a predictor of poor outcomes after arthroscopic partial meniscectomy. The purpose of this study was to compare the ability to predict outcomes after arthroscopic partial meniscectomy with a clinically used modified Outerbridge system versus a semiquantitative MRI Osteoarthritis Knee Score system for grading cartilage loss. MATERIALS AND METHODS. Patients who underwent preoperative knee MRI within 6 months of arthroscopic partial meniscectomy and who had outcomes available from the time of surgery and 1 year later were eligible for inclusion. Cases were evaluated by two radiologists and one radiology fellow with the use of both grading systems. The accuracy of each system in discriminating between surgical success and failure was estimated using the ROC curve (AUC) with 95% CIs. A Wald test was used to assess noninferiority of the clinical grading system. Interreader agreement regarding the accuracy of the grading systems in predicting outcomes was also compared. RESULTS. A total of 78 patients (38 women and 40 men; mean age, 56.6 years) were included in the study. A prediction model using clinical grading (AUC = 0.695; 95% CI, 0.566-0.824) was noninferior (p = 0.047) to a model using MRI Osteoarthritis Knee Score grading (AUC = 0.683; 95% CI, 0.539-0.827). Both MRI prediction models performed better than a model using demographic characteristics only (AUC = 0.667; 95% CI, 0.522-0.812). Inter-reader agreement with clinical grading (80.8%) was higher than that with MRI Osteoarthritis Knee Score grading (65.0%; p = 0.012). CONCLUSION. A clinically used system to grade cartilage loss on MRI is as effective as a semiquantitative system for predicting outcomes after arthroscopic partial meniscectomy, while also offering improved interreader agreement.

Functional retinal outcomes in patients with prediabetes and type 2 diabetes.

PURPOSE: Type 2 diabetes (T2DM) is a leading cause of visual impairment. Its precursor, prediabetes (preDM), is growing in numbers every year. While it is well known that T2DM causes changes in retinal function early in the disease process, it is likely that some of these changes emerge during the preDM stage. This study evaluates retinal function measures in patients with preDM to determine if there are differences in colour vision, contrast sensitivity (CS), and multifocal electroretinogram (mfERG) measures present before T2DM is diagnosed. METHODS: The L'Anthony desaturated D-15 test, Mars Chart CS test, and mfERG were administered on the right eye of 43 participants; 15 controls (HbA1c ≤ 5.6%), 17 with preDM (HbA1c 5.7%-6.4%), and 11 with T2DM (either physician diagnosed or with untreated HbA1c ≥ 6.5%). HbA1c values were measured at the time of the other tests. Colour vision confusion scores (CVCS) were calculated from the D-15 using the method developed by Torok. Multivariate regression (which controlled for age differences) was used to evaluate the relationship of HbA1c and functional measures. Kruskal-Wallis tests were also used to evaluate differences between groups with post-hoc analysis. RESULTS: CVCSs were significantly different between the three groups (p = 0.009). There was an association between higher CVCS and higher HbA1c values across all groups as well as specifically within the preDM group when controlling for age (R2  = 0.29, p = 0.01 and R2  = 0.39, p = 0.02 respectively). Multivariate regression of all of the functional tests together and HbA1c found only colour vision remained significant, indicating that the functional examination metrics may provide redundant data, with similar changes in prediabetes where colour vision may be the strongest indicator early in the process. CONCLUSIONS: Patients with prediabetes have functional changes that can be measured in the retina before the diagnosis of diabetes, with the L'Anthony D-15 colour vision test providing the strongest association with glucose dysregulation in this population. This has important implications for follow up and screening for diabetes within optometric practices. Further studies are needed to follow these patients over time to see how and when these metrics change.

Radical S-adenosylmethionine maquette chemistry: Cx3Cx2C peptide coordinated redox active [4Fe-4S] clusters.

The synthesis and characterization of short peptide-based maquettes of metalloprotein active sites facilitate an inquiry into their structure/function relationships and evolution. The [4Fe-4S]-maquettes of bacterial ferredoxin metalloproteins (Fd) have been used in the past to engineer redox active centers into artificial metalloenzymes. The novelty of our study is the application of maquettes to the superfamily of [4Fe-4S] cluster and S-adenosylmethionine-dependent radical metalloenzymes (radical SAM). The radical SAM superfamily enzymes contain site-differentiated, redox active [4Fe-4S] clusters coordinated to Cx3Cx2C or related motifs, which is in contrast to the Cx2Cx2C motif found in bacterial ferredoxins (Fd). Under an optimized set of experimental conditions, a high degree of reconstitution (80-100%) was achieved for both radical SAM- and Fd-maquettes. Negligible chemical speciation was observed for all sequences, with predominantly [4Fe-4S]2+ for the 'as-reconstituted' state. However, the reduction of [4Fe-4S]2+-maquettes provides low conversion (7-17%) to the paramagnetic [4Fe-4S]+ state, independent of either the spacing of the cysteine residues (Cx3Cx2C vs. Cx2Cx2C), the nature of intervening amino acids, or the length of the cluster binding motif. In the absence of the stabilizing protein environment, the reduction process is proposed to proceed via [4Fe-4S]2+ cluster disassembly and reassembly in a more reduced state. UV-Vis and EPR spectroscopic techniques are employed as analytical tools to quantitate the as-reconstituted (or oxidized) and one-electron reduced states of the [4Fe-4S] clusters, respectively. We demonstrate that short Fd and radical SAM derived 7- to 9-mer peptides containing appropriate cysteine motifs function equally well in coordinating redox active [4Fe-4S] clusters.

Validity and efficiency of a smartphone-based electronic data collection tool for operative data in rotator cuff repair.

BACKGROUND: This study tested validity and efficiency of Orthopaedic Minimal Data Set (OrthoMiDaS) Episode of Care (OME). METHODS: We analyzed 100 isolated rotator cuff repair cases in the OME database. Surgeons completed a traditional operative note and OME report. A blinded reviewer extracted data from operative notes and implant logs in electronic medical records by manual chart review. OME and electronic medical record data were compared with data counts and agreement between 40 variables of rotator cuff disease and repair procedures. Data counts were assessed using raw percentages and McNemar test (with continuity correction). Agreement of categorical variables was analyzed using Cohen κ (unweighted) and of numerical variables using the concordance correlation coefficient (CCC). Efficiency was assessed by median time to complete. RESULTS: OME database had significantly higher data counts for 25% (10/40) of variables. A high level of proportional and statistical agreement was demonstrated between the data. Among 35 categorical variables, proportional agreement was perfect for 17%, almost perfect (0.81 ≤ κ ≤ 1.00) for 37%, substantial (0.61 ≤ κ ≤ 0.80) for 20%, moderate (0.41 ≤ κ ≤ 0.60) for 14%, fair (0.21 ≤ κ ≤ 0.40) for 6%, and slight (0.0 ≤ κ ≤ 0.20) for 6%. Of 5 numerical variables, agreement was almost perfect (CCC > 0.99) for 20% and poor (CCC < 0.90) for 80%. Median OME completion time was 161.5 seconds (interquartile range, 116-224.5). CONCLUSION: OME is an efficient, valid tool for collecting comprehensive, standardized data on rotator cuff repair.