Bifunctional Molecules That Induce Both Targeted Degradation and Transcytosis of Extracellular Proteins in Brain Cells.

Targeted protein degradation (TPD) has emerged as an effective therapeutic strategy for a wide range of diseases; however, the blood-brain barrier (BBB) limits access of degraders into the central nervous system (CNS). Here, we present a new class of bifunctional small molecules, called TransMoDEs (Transcytosis-inducing molecular degraders of extracellular proteins), capable of both (1) removal of target protein via lysosomal proteolysis and (2) transcytosis of protein targets across brain endothelial cells. TransMoDEs are derived from Angiopep-2, a peptide motif previously employed as a covalent tag to facilitate receptor-mediated transcytosis across the BBB. We demonstrate that TransMoDEs containing either a biotin or chloroalkane ligand can trigger endocytosis of streptavidin or HaloTag protein, respectively. Interestingly, although low-density lipoprotein receptor-related protein 1 (LRP1) has been reported as the primary receptor for Angiopep-2, TransMoDE-mediated target uptake does not rely exclusively on this pathway. Furthermore, TransMoDE-mediated endocytosis of streptavidin in a bEnd.3 BBB model occurs in a clathrin-mediated mechanism and results in both lysosomal localization and transcytosis of the target protein. This study demonstrates that TransMoDEs can recruit, transcytose, and degrade proteins of interest in cells relevant to the CNS, supporting their further development for the removal of pathogenic neuroproteins.

Should I stay or should I go: an assessment of criteria for safe day of surgery discharge of displaced supracondylar humerus fractures.

Immobilization type and in-hospital observation following surgical management of displaced supracondylar fractures are subject to surgeon preference and training. Our goal was to determine criteria for immediate discharge and optimal type of immobilization. Medical records of 661 patients with type III, IV or flexion-type displaced supracondylar humerus fractures treated at a level 1 pediatric trauma center from January 2013 to September 2019 were reviewed. Patients were separated into 'admission appropriate' (AA = 113) and 'discharge appropriate' (DA = 548) sub-cohorts. Neurovascular deficit at presentation (P < 0.001), post-operative physical exam deterioration (P < 0.001), age (P < 0.001) and post-operative immobilization modality (P = 0.02) were significantly different between AA and DA groups. When comparing patients who presented with neurologic deficit to those neurovascularly intact, there was a significant difference in whether circumferential immobilization was used post-operatively (P < 0.001), IV medication need (P < 0.001), discharge or admission (P < 0.001), neurologic decline (P < 0.001), return to ED (P = 0.008) and vascular compromise (P = 0.05). Twenty-four of the 56 (43%) patients who were AA and had no neurovascular finding on presentation had their immobilization adjusted (bivalved or loosened) to accommodate for swelling overnight. Only 1 was initially maintained in a splint or bivalved cast; the other 23 were initially maintained post-operatively in circumferential immobilization (P = 0.01). Our findings suggest that patients with intact neurovascular exams at presentation are candidates for early discharge, and splinting or bivalved casting may be preferable, especially in patients who are discharged.

Rotation and Asymmetry of the Axial Plane Pelvis in Cerebral Palsy: A CT-Based Study.

Spinopelvic malignment is commonly seen with non-ambulatory cerebral palsy (CP). Axial plane deformation is not well described in the literature. The purpose of this study was to describe and quantify the axial plane deformity in CP using CT scans and compare it to normal controls. We retrospectively collected data using CT scans of the abdomen and pelvis of 40 patients with GMFCS IV/V CP and neuromuscular scoliosis (CPP) and normal controls (NP) matched by age and sex. Pre-operative Cobb angle was recorded for the CP patients. Pelvic anatomy was evaluated at the supra-acetabular region of bone using two angles-iliac wing angle and sacral ala angle, measured for each hemipelvis. The larger of each hemipelvis angle was considered externally rotated while the smaller angle was considered internally rotated, termed as follows-iliac wing external (IWE) and internal (IWI); sacral ala external (SAE), and internal (SAI). Differences were noted using an independent t-test while correlations with Cobb angle were performed using Pearson's correlation. Iliac wing measurements showed the externally rotated hemipelvis showed a significantly greater magnitude compared with normal controls at 47.3 ± 18.1 degrees vs. 26.4 ± 3.7 degrees in NP (p < 0.001) while no internal rotation was observed (p > 0.05). Sacral ala measurements showed greater magnitude in both external and internal rotation. SAE was 119.5 ± 9.5 degrees in CPP vs. 111.2 ± 7.7 degrees in NP (p < 0.001) while SAI was 114.1 ± 8.5 degrees in CPP vs. 107.9 ± 7.5 degrees in NP (p = 0.001). In the CP cohort, the mean Cobb angle was 61.54 degrees (n = 37/40). Cobb angle correlated with the degree of external iliac wing rotation-IWE (r = 0.457, p = 0.004) and degree of absolute difference in the rotation of the iliac wing (r = 0.506, p = 0.001). The pelvis in a patient with CP scoliosis is asymmetrically oriented exhibiting a greater external rotation of one hemipelvis relative to normal controls. The severity of neuromuscular scoliosis is related to the pelvic axial rotation in CP patients. Axial plane deformity exists in the CP pelvis and this deformity warrants consideration when considering spinopelvic instrumentation strategies and outcomes of supra-pelvic and infra-pelvic pathologies.

Clubfoot and the Ponseti Method: A Bibliometric Analysis.

Background: The aim of the present study was to perform a bibliometric analysis of research articles published on clubfoot to provide a quantitative description of the literature and to gather information on the institutions, journals, researchers, and countries publishing on this topic. Methods: This bibliometric analysis consisted of 2 Web of Science searches. The first identified all articles published prior to April 25, 2022, with "clubfoot" in the title, abstract, or keywords, and the second identified all articles with "Ponseti." Studies were exported in BibTeX format and uploaded into Biblioshiny software in RStudio. Descriptive statistics are reported for variables related to the article, author, and country in which the research was conducted. Results: A total of 2,177 articles identified using the term "clubfoot" were included. The first article was published in 1902, and there was a 3% annual growth rate. A total of 762 articles identified using the term "Ponseti" were included, with the first published in 1992 and a 13.9% annual growth rate. The Journal of Pediatric Orthopaedics accounted for almost one-quarter of all published reports. Conclusions: The literature on clubfoot has expanded in the past decades, and the percentage of studies concerning the Ponseti method has increased dramatically in the years following adoption in the U.S. and, more recently, globally. While a number of studies involved collaboration between authors in high-income and low- and middle-income countries, further collaboration will be essential to evaluate outcomes and help improve service delivery as adoption of this method increases globally. Clinical Relevance: This paper explores how the orthopaedic and scientific communities have increasingly contributed to literature on Ponseti casting and clubfoot, and discusses how contributions to the literature are becoming increasingly widespread.

When to Perform Fusion Short of the Pelvis in Patients with Cerebral Palsy?: Indications and Outcomes.

Patients with scoliosis secondary to cerebral palsy (CP) are often treated with posterior spinal fusion (PSF) with or without pelvic fixation. We sought to establish criteria to guide the decision of whether or not to perform fusion "short of the pelvis" in this population, and to assess differences in outcomes. Methods: Using 2 prospective databases, we analyzed 87 pediatric patients who underwent PSF short of the pelvis from 2008 to 2015 to treat CP-related scoliosis and who had ≥2 years of follow-up. Preoperative radiographic and clinical variables were analyzed for associations with unsatisfactory correction (defined as pelvic obliquity of ≥10°, distal implant dislodgement, and/or reoperation for increasing deformity at 2- or 5-year follow-up). Continuous variables were dichotomized using the Youden index, and a multivariable model of predictors of unsatisfactory correction was created using backward stepwise selection. Finally, radiographic, health-related quality-of-life, and clinical outcomes of patients with fusion short of the pelvis who had neither of the 2 factors associated with unsatisfactory outcomes were compared with those of 2 matched-control groups. Results: Deformity correction was unsatisfactory in 29 of 87 patients with fusion short of the pelvis. The final model included preoperative pelvic obliquity of ≥17° (odds ratio [OR], 6.8; 95% confidence interval [CI], 2.3 to 19.7; p < 0.01) and dependent sitting status (OR, 3.2; 95% CI, 1.1 to 9.9; p = 0.04) as predictors of unsatisfactory correction. The predicted probability of unsatisfactory correction increased from 10% when neither of these factors was present to a predicated probability of 27% to 44% when 1 was present and to 72% when both were present. Among matched patients with these factors who had fusion to the pelvis, there was no association with unsatisfactory correction. Patients with independent sitting status and pelvic obliquity of <17° who had fusion short of the pelvis had significantly lower blood loss and hospital length of stay, and better 2-year health-related quality-of-life scores compared with matched controls with fusion to the pelvis. Conclusions: In patients with scoliosis secondary to CP, pelvic obliquity of <17° and independent sitting status are associated with a low risk of unsatisfactory correction and better 2-year outcomes when fusion short of the pelvis is performed. These may be used as preoperative criteria to guide the decision of whether to perform fusion short of the pelvis in patients with CP. Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Pentosinane, a Post-Translational Modification of Human Proteins with Underappreciated Stability.

Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer's disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.

A dual role for CRTH2 in acute lung injury.

Acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition defined by rapid onset respiratory failure following acute lung injury (ALI). Its increased incidence due to COVID-19 and high mortality rate (∻40%) make the study of ARDS pathogenesis a crucial research priority. CRTH2 is a G protein-coupled receptor with established roles in type 2 immunity and well-characterized inhibitors. Prior studies have shown it also promotes neutrophilic inflammation, indicating that CRTH2 inhibition may be a potential therapeutic strategy for ARDS. To test this hypothesis, we first examined the expression pattern of CRTH2 on murine neutrophils. We found it is expressed on neutrophils, but only after extravasation into the lung. Next, we showed that extravasated lung neutrophils generate inflammatory responses upon stimulation with the CRTH2-specific agonist DK-PGD2, as demonstrated by reactive oxygen species (ROS) production. This response was abrogated in CRTH2 KO neutrophils. Inhibition of CRTH2 with fevipiprant suppressed baseline ROS production, indicating an autocrine PGD2-CRTH2 signaling loop. We then evaluated the role of CRTH2 in vivo using a murine model of LPS-induced ALI. Despite the pro-inflammatory effects of CRTH2 on neutrophils in vitro, we observed worsening of lung injury in CRTH2-deficient mice in terms of neutrophilic inflammation, vascular leak, and survival. Bulk RNAseq of lung tissue indicated an impairment in type 2 immune signaling; qPCR and ELISA confirmed downregulation of the key type 2 effector cytokine, IL-4. Thus, CRTH2 appears to play a dual role in ALI, directly promoting neutrophil effector responses, but indirectly suppressing lung injury and neutrophilic inflammation through type 2 immunity. These findings reveal a novel protective function for CRTH2 during lung injury and argue against the use of CRTH2 inhibitors in ARDS.

Does nutrition consultation in the year leading up to neuromuscular scoliosis surgery result in significant weight gain, or just a larger magnitude curve?

PURPOSE: Patients with neuromuscular scoliosis undergoing posterior instrumented spinal fusion can be underweight, malnourished, and have higher complication rates. A nutrition consult is common in this population and it is unclear if weight gain occurs from the consult or surgery. The purpose of the study was to determine if nutrition consultation in the year prior to spinal fusion resulted in significant differences in weight gain or percentile on the CP growth chart. The secondary aim was to determine if there would be deformity progression during that time. METHODS: Retrospective chart and radiograph review was performed for all patients with neuromuscular spinal deformity treated with posterior instrumented spinal fusion at one institution between January 1, 2009 and August 1, 2015. Inclusion criteria included < 20 years old, diagnosis of neuromuscular scoliosis, and 1-year pre-operative percentile on the CP growth chart < 50. Patient demographics, GMFCS level, weight, percentile on appropriate CP growth chart, major curve and pelvic obliquity at 1 year pre-operatively and at surgery were recorded. RESULTS: Sixty-eight patients met inclusion criteria. Thirty-seven patients had a nutrition appointment within 1 year pre-operatively, 31 patients did not. There were no significant differences between the groups when comparing increase in weight (p = 0.9), percentile on CP growth charts (p = 0.3), major deformity (p = 0.1), and pelvic obliquity (p = 0.2). Overall, there was a mean 3.2 kg weight gain, 5.2% increase on CP growth charts, 40° increase in major curve, and 5° worsening of pelvic obliquity in the year before surgery. There was an average overall increase in the pre-operative albumin value, but this was not different between groups (p = 0.6). Children who were tube fed gained on average 10.8 percentiles on the CP growth chart, whereas children without gained only 0.5 percentiles (p = 0.002). CONCLUSIONS: Nutrition consultations in the year preceding posterior instrumented spinal fusion do not lead to weight optimization prior to surgery in comparison to patients without nutrition consults. Gastrostomy tubes were found to be helpful for weight optimization and should be considered as an alternative nutrition option in pre-operative planning in underweight patients. LEVEL OF EVIDENCE: III-therapeutic study: retrospective comparative study.

Bifunctional small molecules that mediate the degradation of extracellular proteins.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

Sirtuin 2 Regulates Protein LactoylLys Modifications.

Post-translational modifications (PTMs) play roles in both physiological and pathophysiological processes through the regulation of enzyme structure and function. We recently identified a novel PTM, lactoylLys, derived through a nonenzymatic mechanism from the glycolytic by-product, lactoylglutathione. Under physiologic scenarios, glyoxalase 2 prevents the accumulation of lactoylglutathione and thus lactoylLys modifications. What dictates the site-specificity and abundance of lactoylLys PTMs, however, remains unknown. Here, we report sirtuin 2 as a lactoylLys eraser. Using chemical biology and CRISPR-Cas9, we show that SIRT2 controls the abundance of this PTM both globally and on chromatin. These results address a major gap in our understanding of how nonenzymatic PTMs are regulated and controlled.

Model studies of advanced glycation end product modification of heterograft biomaterials: The effects of in vitro glucose, glyoxal, and serum albumin on collagen structure and mechanical properties.

Glutaraldehyde cross-linked heterograft tissues, bovine pericardium (BP) or porcine aortic valves, are the leaflet materials in bioprosthetic heart valves (BHV) used in cardiac surgery for heart valve disease. BHV fail due to structural valve degeneration (SVD), often with calcification. Advanced glycation end products (AGE) are post-translational, non-enzymatic reaction products from sugars reducing proteins. AGE are present in SVD-BHV clinical explants and are not detectable in un-implanted BHV. Prior studies modeled BP-AGE formation in vitro with glyoxal, a glucose breakdown product, and serum albumin. However, glucose is the most abundant AGE precursor. Thus, the present studies investigated the hypothesis that BHV susceptibility to glucose related AGE, together with serum proteins, results in deterioration of collagen structure and mechanical properties. In vitro experiments studied AGE formation in BP and porcine collagen sponges (CS) comparing 14C-glucose and 14C-glyoxal with and without bovine serum albumin (BSA). Glucose incorporation occurred at a significantly lower level than glyoxal (p<0.02). BSA co-incubations demonstrated reduced glyoxal and glucose uptake by both BP and CS. BSA incubation caused a significant increase in BP mass, enhanced by glyoxal co-incubation. Two-photon microscopy of BP showed BSA induced disruption of collagen structure that was more severe with glucose or glyoxal co-incubation. Uniaxial testing of CS demonstrated that glucose or glyoxal together with BSA compared to controls, caused accelerated deterioration of viscoelastic relaxation, and increased stiffness over a 28-day time course. In conclusion, glucose, glyoxal and BSA uniquely contribute to AGE-mediated disruption of heterograft collagen structure and deterioration of mechanical properties.

Glycation and Serum Albumin Infiltration Contribute to the Structural Degeneration of Bioprosthetic Heart Valves.

Valvular heart diseases are associated with significant cardiovascular morbidity and mortality, and often require surgical and/or percutaneous repair or replacement. Valve replacement is limited to mechanical and biological prostheses, the latter of which circumvent the need for lifelong anticoagulation but are subject to structural valve degeneration (SVD) and failure. Although calcification is heavily studied, noncalcific SVD, which represent roughly 30% of BHV failures, is relatively underinvestigated. This original work establishes 2 novel and interacting mechanisms-glycation and serum albumin incorporation-that occur in clinical valves and are sufficient to induce hallmarks of structural degeneration as well as functional deterioration.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

The Frequency of Mediastinal Injury in Acute Posterior Sternoclavicular Dislocations: A Multicenter Study.

BACKGROUND: Acute posterior sternoclavicular dislocations (APSCD) are rare injuries that historically have prompted concern for injury to the great vessels and other mediastinal structures from initial trauma or subsequent treatment, resulting in the recommendation that a thoracic or vascular surgeon be present or available during operative treatment. The objectives of the study were to characterize the demographic, clinical, and radiographic characteristics of a large series of APSCDs in skeletally immature patients and to describe the rate and nature of any vascular or mediastinal complications that occurred during treatment. METHODS: Following Institutional Review Board approval, records of consecutive patients under 25 years of age treated for APSCD were collected from each of 6 participating centers. Only acute injuries (sustained fewer than 10 days before presentation) were included. Patient demographics, injury mechanism, associated mediastinal injuries, and need for thoracic/vascular surgery were recorded. Mediastinal structures injured or compressed by mass effect were specifically characterized by review of preoperative computed tomography imaging. RESULTS: Review identified 125 patients with a mean age of 14.7 years; 88% were male. APSCD most commonly resulted from a sporting injury (74%) followed by falls from standing height (10%) and high-energy motor vehicle trauma (10%). The most common finding on cross-sectional imaging was compression without laceration of the ipsilateral brachiocephalic vein (50%). Eleven patients had successful closed reduction, and 114 (90%) had open reduction and internal fixation, with 25 failed or unstable closed reductions preceding open treatment. There were no vascular or mediastinal injuries during reduction or fixation that required intervention. CONCLUSIONS: In this multicenter series of 125 APSCDs no injuries to the great vessels/mediastinal structures requiring intervention were identified. Although more than half of patients had evidence of extrinsic vascular compression at the time of injury, careful open reduction of acute injuries can be safely performed. Although vascular injuries following APSCD seem to be quite rare, vascular complications can be catastrophic. Treating providers should consider these data and their own institutional resources to maximize patient safety during the treatment of APSCD. LEVEL OF EVIDENCE: Level III-therapeutic case control study.

BMI change following spinal fusion for neuromuscular scoliosis surgery.

STUDY DESIGN: Retrospective descriptive, multi-center study. OBJECTIVES: We hypothesize that a post-operative weight gain will result in patients who are underweight prior to surgery. Cachexia and low body mass index is common among children with cerebral palsy (CP). Many interventions are undertaken to assist the child in nourishment and to obtain a more normal body mass. Additionally, scoliosis is common among children with CP. In our practice we have noted weight gain post operatively in severely underweight children after spinal fusion. METHODS: We underwent a retrospective review of a CP cohort from a multicenter prospective registry. Percentiles on the CP specific growth chart for which each child belonged were plotted based on the patients' age, weight, gender, GMFCS level, and tube feeding status. We then assessed percentile change in patients between pre-op visit, 1 year, 2 years and for those with available data, 5 years follow up visits. Patients with under two years of follow up, patients with GMFCS III and below, and patients without weight data were excluded. RESULTS: We identified a total of 211 potentially eligible patients from a multicenter prospective registry. 109 had complete 2 years data to analyze and 37 patients had full 5 years data to analyze. We found that patients under the 50th percentile pre-operatively increased their percentile on the CP growth chart for weight 12.1 percentiles (95% CI 6.7, 17.5 p value < 0.001) whereas patients that began at the 50th percentile or above on average lost 2.2 percentiles (95% CI -6.8, 2.3) though the change was not statistically significant (p value 0.330). These changes appeared stable at 5 years. Although regression analysis showed that Cobb correction and pelvic obliquity correction, and hyperlordosis were not independent predictors of the change, we noted that patients with residual curves after surgery of 40° or more experienced 13.3 percentile less weight gain than those with better corrections. CONCLUSIONS: Patients with CP are at risk for cachexia, malnutrition, reflux and other GI disorders. Data presented here suggests that corrective spinal surgery may improve weight percentile in patients who start out at 50th percentile and lower. Patients with 40° or greater of residual scoliosis may benefit less from spinal fusion than those with a better correction. LEVEL OF EVIDENCE: II; Prognostic retrospective cohort study.

Age-related changes in the physical properties, cross-linking, and glycation of collagen from mouse tail tendon.

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrated that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decreased with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20-week-old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

Fluorescent stem peptide mimics: In situ probes for peptidoglycan crosslinking.

Understanding the mechanisms of bacterial cell wall synthesis is essential for microbiology and medicine alike. A key step in this process is peptidoglycan crosslinking, which confers mechanical strength to the cell wall and represents a target for numerous classes of antibiotics. However, the biology of crosslinking remains poorly understood due to a lack of tools for studying the reaction in vivo. Recently, we developed a class of synthetic probes called fluorescent stem peptide mimics (FSPMs) that meet this need, allowing quantification and localization of crosslinking activity in live bacteria. We have utilized FSPMs to describe novel aspects of peptidoglycan synthesis in the human pathogen, Staphylococcus aureus. To enable wider use of our methodology, we provide detailed protocols herein for the synthesis of FSPMs, labeling of live bacteria, and evaluation of crosslinking by flow cytometry and super-resolution microscopy. We believe that FSPMs, together with complementary biosynthetic probes and traditional bacteriologic methods, will help to advance our understanding of peptidoglycan biology and accelerate the search for new antibiotics.

A low glycemic diet protects disease-prone Nrf2-deficient mice against age-related macular degeneration.

Age-related macular degeneration (AMD) is a major blinding disease, affecting over 14% of the elderly. Risk for AMD is related to age, diet, environment, and genetics. Dietary modulation of AMD risk is a promising treatment modality, but requires appropriate animal models to demonstrate advantages of diet. Mice lacking the antioxidant transcription factor Nrf2 (Nfe2l2) develop age-related retinopathy relevant to human AMD. Here we evaluated the effect of consuming high glycemic (HG) or low glycemic (LG) diets until 18-months of age on development of features relevant to AMD in Nrf2-null mice. Nrf2-null mice that consumed HG diets developed atrophic AMD, characterized by photoreceptor degeneration, retinal pigment epithelium (RPE) atrophy and pigmentary abnormalities, basal laminar deposits, and loss of the choriocapillaris. In contrast, Nrf2-null-mice that consumed LG diets did not develop retinal disease phenotypes. Consumption of HG diets was associated with accumulation of advanced glycation end-products in the RPE and systemically, whereas consumption of the LG diet was associated with increased levels of anti-glycative and anti-oxidative detoxification machinery. Together our data indicate that the Nrf2-null HG mouse is a good model for atrophic AMD studies and that the LG diet can activate protective pathways to prevent AMD, even in a genetically predisposed animal.