Post-Traumatic Periprosthetic Tibial and Fibular Fracture After Total Ankle Arthroplasty: A Case Report.

Periprosthetic fractures after total ankle arthroplasty are uncommon, with most cases occurring intraoperatively. We describe a post-traumatic periprosthetic fracture of the distal tibia and fibula after total ankle arthroplasty that was treated with minimally invasive plate osteosynthesis. It is important for orthopedic surgeons not only to recognize the risk factors for postoperative periprosthetic total ankle arthroplasty fractures, but also to be familiar with the treatment options available to maximize function and minimize complications. The design of the tibial prosthesis and surgical techniques required to prepare the ankle joint for implantation are important areas of future research to limit the risk of periprosthetic fractures.

The antimicrobial protein, CAP37, is upregulated in pyramidal neurons during Alzheimer's disease.

Inflammation is a well-defined factor in Alzheimer's disease (AD). There is a strong need to identify the molecules contributing to neuroinflammation so that therapies can be designed to prevent immune-mediated neurotoxicity. The cationic antimicrobial protein of 37 kDa (CAP37) is an inflammatory mediator constitutively expressed in neutrophils (PMNs). In addition to antibiotic activity, CAP37 exerts immunomodulatory effects on microglia. We hypothesize that CAP37 mediates the neuroinflammation associated with AD. However, PMNs are not customarily associated with the pathology of AD. This study was therefore designed to identify non-neutrophilic source(s) of CAP37 in brains of AD patients. Brain tissues from patients and age-matched controls were analyzed for CAP37 expression using immunohistochemistry (IHC). To determine factors that induce CAP37 in AD, HCN-1A primary human neurons were treated with tumor necrosis factor-alpha (TNF-α) or amyloid ß1-40 (Aß) and analyzed by IHC. Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to confirm CAP37 expression in neurons and brain tissues. IHC revealed CAP37 in cortical neurons in temporal and parietal lobes as well as CA3 and CA4 hippocampal neurons in patients with AD. CAP37 was found in more neurons in AD patients compared with age-matched controls. qRT-PCR and Western blotting showed an increase in CAP37 transcript and protein in the AD temporal lobe, a brain region that is highly impacted in AD. qRT-PCR observations confirmed CAP37 expression in neurons. TNF-α and Aß increased neuronal expression of CAP37. These findings support our hypothesis that neuronal CAP37 may modulate the neuroinflammatory response in AD.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae.

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

12q14 microdeletion associated with HMGA2 gene disruption and growth restriction.

The 12q14 microdeletion syndrome is a rare condition that has previously been characterized by pre- and postnatal growth restriction, proportionate short stature, failure to thrive, developmental delay, and osteopoikilosis. Previously reported microdeletions within this region have ranged in size from 1.83 to 10.12 Mb with a proposed 2.61 Mb smallest region of overlap containing the LEMD3, HMGA2, and GRIP1 genes. Here, we report on the identification of a 12q14 microdeletion in a female child presenting with proportionate short stature, failure to thrive, and speech delay. The genomic loss (minimum size 4.17 Mb, maximum size 4.21 Mb) contained 25 RefSeq genes including IRAK3, GRIP1, and the 3' portion of the HMGA2 gene. This is the first partial deletion of HMGA2 associated with the 12q14 microdeletion syndrome. This case further clarifies the association of LEMD3 deletions with the 12q14 microdeletion syndrome and provides additional support for the role of the HMGA2 gene in human growth.

SARS-CoV-2 infections elicit higher levels of original antigenic sin antibodies compared with SARS-CoV-2 mRNA vaccinations.

It is important to determine if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and SARS-CoV-2 mRNA vaccinations elicit different types of antibodies. Here, we characterize the magnitude and specificity of SARS-CoV-2 spike-reactive antibodies from 10 acutely infected health care workers with no prior SARS-CoV-2 exposure history and 23 participants who received SARS-CoV-2 mRNA vaccines. We found that infection and primary mRNA vaccination elicit S1- and S2-reactive antibodies, while secondary vaccination boosts mostly S1 antibodies. Using absorption assays, we found that SARS-CoV-2 infections elicit a large proportion of original antigenic sin-like antibodies that bind efficiently to the spike of common seasonal human coronaviruses but poorly to the spike of SARS-CoV-2. In converse, vaccination modestly boosts antibodies reactive to the spike of common seasonal human coronaviruses, and these antibodies cross-react more efficiently to the spike of SARS-CoV-2. Our data indicate that SARS-CoV-2 infections and mRNA vaccinations elicit fundamentally different antibody responses.

Protein kinase affinity reagents based on a 5-aminoindazole scaffold.

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.

Biochemical and pharmacological profiling of the pro-survival protein Bcl-xL.

Bcl-2 family proteins are key mediators of programmed cell death. Over-expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) has been associated with tumor progression and chemotherapeutic resistance. Pharmacological agents that neutralize the functions of anti-apoptotic Bcl-2 family proteins have emerged as a promising new class of anti-cancer agents. Biochemical analyses have demonstrated that small molecule inhibitors and some pro-apoptotic proteins exhibit distinct binding preferences for anti-apoptotic proteins. While numerous structures of anti-apoptotic proteins bound to ligands have been reported, the source of this selectivity is still unclear. Here, we present a systematic analysis of a series of Bcl-xL variants that contain mutations within the hydrophobic ligand-binding cleft. The ability of these Bcl-xL mutants to interact with both small molecule inhibitors and BH3 peptides was determined. These studies provide information on the contributions of specific residues to small molecule inhibitor binding and shed light on the ligand selectivity of these therapeutically important proteins.

Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae.

Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems.

Sero-monitoring of health care workers reveals complex relationships between common coronavirus antibodies and SARS-CoV-2 severity.

Recent common coronavirus (CCV) infections are associated with reduced COVID-19 severity upon SARS-CoV-2 infection, however the immunological mechanisms involved are unknown. We completed serological assays using samples collected from health care workers to identify antibody types associated with SARS-CoV-2 protection and COVID-19 severity. Rare SARS-CoV-2 cross-reactive antibodies elicited by past CCV infections were not associated with protection; however, the duration of symptoms following SARS-CoV-2 infections was significantly reduced in individuals with higher common betacoronavirus (ßCoV) antibody titers. Since antibody titers decline over time after CCV infections, individuals in our cohort with higher ßCoV antibody titers were more likely recently infected with common ßCoVs compared to individuals with lower antibody titers. Therefore, our data suggest that recent ßCoV infections potentially limit the severity of SARS-CoV-2 infections through mechanisms that do not involve cross-reactive antibodies. Our data are consistent with the emerging hypothesis that cellular immune responses elicited by recent common ßCoV infections transiently reduce disease severity following SARS-CoV-2 infections.

Health care worker seromonitoring reveals complex relationships between common coronavirus antibodies and COVID-19 symptom duration.

Some studies suggest that recent common coronavirus (CCV) infections are associated with reduced COVID-19 severity upon SARS-CoV-2 infection. We completed serological assays using samples collected from health care workers to identify antibody types associated with SARS-CoV-2 protection and COVID-19 symptom duration. Rare SARS-CoV-2 cross-reactive antibodies elicited by past CCV infections were not associated with protection; however, the duration of symptoms following SARS-CoV-2 infections was significantly reduced in individuals with higher common betacoronavirus (ßCoV) antibody titers. Since antibody titers decline over time after CCV infections, individuals in our cohort with higher ßCoV antibody titers were more likely recently infected with common ßCoVs compared with individuals with lower antibody titers. Therefore, our data suggest that recent ßCoV infections potentially limit the duration of symptoms following SARS-CoV-2 infections through mechanisms that do not involve cross-reactive antibodies. Our data are consistent with the emerging hypothesis that cellular immune responses elicited by recent common ßCoV infections transiently reduce symptom duration following SARS-CoV-2 infections.

NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3-SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

Cap-n-Collar Promotes Tissue Regeneration by Regulating ROS and JNK Signaling in the Drosophila melanogaster Wing Imaginal Disc.

Regeneration is a complex process that requires an organism to recognize and repair tissue damage, as well as grow and pattern new tissue. Here, we describe a genetic screen to identify novel regulators of regeneration. We ablated the Drosophila melanogaster larval wing primordium by inducing apoptosis in a spatially and temporally controlled manner and allowed the tissue to regenerate and repattern. To identify genes that regulate regeneration, we carried out a dominant-modifier screen by assessing the amount and quality of regeneration in adult wings heterozygous for isogenic deficiencies. We have identified 31 regions on the right arm of the third chromosome that modify the regenerative response. Interestingly, we observed several distinct phenotypes: mutants that regenerated poorly, mutants that regenerated faster or better than wild-type, and mutants that regenerated imperfectly and had patterning defects. We mapped one deficiency region to cap-n-collar (cnc), the Drosophila Nrf2 ortholog, which is required for regeneration. Cnc regulates reactive oxygen species levels in the regenerating epithelium, and affects c-Jun N-terminal protein kinase (JNK) signaling, growth, debris localization, and pupariation timing. Here, we present the results of our screen and propose a model wherein Cnc regulates regeneration by maintaining an optimal level of reactive oxygen species to promote JNK signaling.

Using Drosophila larvae to study epidermal wound closure and inflammation.

This methods chapter describes two methods for creating epithelial wounds in Drosophila larvae: pinch and puncture wounding. It also covers protocols for visualizing epithelial wounds, either in a dissected whole mount preparation or, using transgenic reporter larvae, in a live whole mount preparation. Finally, useful transgenic lines for live genetic screening of genes required for wound closure or inflammation are described.

Affinity reagents that target a specific inactive form of protein kinases.

A number of small-molecule inhibitors have been developed that target the catalytic domains of protein kinases that are not in an active conformation. An inactive form that has been observed in several kinases is the DFG-out conformation. This conformation is characterized by an almost 180 degrees rotation of the conserved Asp-Phe-Gly (DFG) motif in the ATP-binding cleft relative to the active form. However, the sequence and structural determinants that allow a kinase to stably adopt the DFG-out conformation are not known. Here, we characterize a series of inhibitors based on a general pharmacophore for this inactive form. We demonstrate that modified versions of these inhibitors can be used to study the thermodynamics and kinetics of ligand binding to DFG-out-adopting kinases and for enriching these kinases from complex protein mixtures.

A blood-borne PDGF/VEGF-like ligand initiates wound-induced epidermal cell migration in Drosophila larvae.

Epidermal cell migration is critical for restoration of tissue structure and function after damage. However, the mechanisms by which differentiated cells neighboring the wound sense the wound and assume a motile phenotype remain unclear. Here, we show that Pvr, a receptor tyrosine kinase (RTK) related to platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, and one of its ligands, Pvf1, are required for epidermal wound closure. Morphological comparison of wound-edge cells lacking Pvr or the Jun N-terminal kinase (JNK) signaling pathway previously implicated in larval wound closure suggests that Pvr signaling leads wound-margin epidermal cells to extend actin-based cell processes into the wound gap while JNK mediates transient dedifferentiation of cells at the wound margin. Genetic epistasis experiments reinforce the conclusion that the JNK and Pvr signaling pathways act in parallel. Tissue-specific knockdown and rescue experiments suggest that epidermally derived Pvf1 may be sequestered in the blood and that tissue damage exposes blood-borne Pvf1 to Pvr receptors on wound-edge epidermal cells and initiates the extension of cell processes into the wound gap. These results uncover a novel mechanism of sensing tissue damage and suggest that PDGF/VEGF ligands and receptors may play a conserved autocrine role in epidermal wound closure.

Active cop, passive cop: developmental stage-specific modes of wound-induced blood cell recruitment in Drosophila.

In the past few years a number of fly labs have studied wounded Drosophila embryos,(1-3) larvae(4-6) and adults7 in an effort to uncover the molecular/genetic basis of wound healing responses. The early studies in this growing field focused on the signature event of wound healing--the closure of the epidermal gap through cell migration. These studies showed that there is a conserved dichotomy between embryonic and postembryonic repair processes in flies and vertebrates: embryonic wounds heal through contraction of a supracellular actin pursestring assembled at the wound margin and postembryonic wounds heal through extension of cell processes and migration across the wound gap. Now, our group and others have begun to use these wounding assays to examine other steps of the healing process. Inflammation, the recruitment of hemocytes (blood cells) to the site of tissue damage, has been a particular focus of recent studies. This extra view article summarizes these recent findings on wound-induced inflammation, especially the curious dichotomy between modes of blood cell recruitment in embryos and larvae.

Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate.

Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.

AtGRXcp, an Arabidopsis chloroplastic glutaredoxin, is critical for protection against protein oxidative damage.

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage.