DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

A blood-based multi-pathway biomarker assay for early detection and staging of Alzheimer's disease across ethnic groups.

INTRODUCTION: Existing blood-based biomarkers for Alzheimer's disease (AD) mainly focus on its pathological features. However, studies on blood-based biomarkers associated with other biological processes for a comprehensive evaluation of AD status are limited. METHODS: We developed a blood-based, multiplex biomarker assay for AD that measures the levels of 21 proteins involved in multiple biological pathways. We evaluated the assay's performance for classifying AD and indicating AD-related endophenotypes in three independent cohorts from Chinese or European-descent populations. RESULTS: The 21-protein assay accurately classified AD (area under the receiver operating characteristic curve [AUC] = 0.9407 to 0.9867) and mild cognitive impairment (MCI; AUC = 0.8434 to 0.8945) while also indicating brain amyloid pathology. Moreover, the assay simultaneously evaluated the changes of five biological processes in individuals and revealed the ethnic-specific dysregulations of biological processes upon AD progression. DISCUSSION: This study demonstrated the utility of a blood-based, multi-pathway biomarker assay for early screening and staging of AD, providing insights for patient stratification and precision medicine. HIGHLIGHTS: The authors developed a blood-based biomarker assay for Alzheimer's disease. The 21-protein assay classifies AD/MCI and indicates brain amyloid pathology. The 21-protein assay can simultaneously assess activities of five biological processes. Ethnic-specific dysregulations of biological processes in AD were revealed.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). METHODS: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. RESULTS: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. CONCLUSIONS: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract.

Who Knew? Dopamine Transporter Activity Is Critical in Innate and Adaptive Immune Responses.

The dopamine transporter (DAT) regulates the dimension and duration of dopamine transmission. DAT expression, its trafficking, protein-protein interactions, and its activity are conventionally studied in the CNS and within the context of neurological diseases such as Parkinson's Diseases and neuropsychiatric diseases such as drug addiction, attention deficit hyperactivity and autism. However, DAT is also expressed at the plasma membrane of peripheral immune cells such as monocytes, macrophages, T-cells, and B-cells. DAT activity via an autocrine/paracrine signaling loop regulates macrophage responses to immune stimulation. In a recent study, we identified an immunosuppressive function for DAT, where blockade of DAT activity enhanced LPS-mediated production of IL-6, TNF-α, and mitochondrial superoxide levels, demonstrating that DAT activity regulates macrophage immune responses. In the current study, we tested the hypothesis that in the DAT knockout mice, innate and adaptive immunity are perturbed. We found that genetic deletion of DAT (DAT-/-) results in an exaggerated baseline inflammatory phenotype in peripheral circulating myeloid cells. In peritoneal macrophages obtained from DAT-/- mice, we identified increased MHC-II expression and exaggerated phagocytic response to LPS-induced immune stimulation, suppressed T-cell populations at baseline and following systemic endotoxemia and exaggerated memory B cell expansion. In DAT-/- mice, norepinephrine and dopamine levels are increased in spleen and thymus, but not in circulating serum. These findings in conjunction with spleen hypoplasia, increased splenic myeloid cells, and elevated MHC-II expression, in DAT-/- mice further support a critical role for DAT activity in peripheral immunity. While the current study is only focused on identifying the role of DAT in peripheral immunity, our data point to a much broader implication of DAT activity than previously thought. This study is dedicated to the memory of Dr. Marc Caron who has left an indelible mark in the dopamine transporter field.

Epithelial-Mesenchymal Transition Suppresses AMPK and Sensitizes Cancer Cells to Pyroptosis under Energy Stress.

Epithelial-mesenchymal transition (EMT) is implicated in tumor metastasis and therapeutic resistance. It remains a challenge to target cancer cells that have undergone EMT. The Snail family of key EMT-inducing transcription factors directly binds to and transcriptionally represses not only epithelial genes but also a myriad of additional genomic targets that may carry out significant biological functions. Therefore, we reasoned that EMT inherently causes various concomitant phenotypes, some of which may create targetable vulnerabilities for cancer treatment. In the present study, we found that Snail transcription factors bind to the promoters of multiple genes encoding subunits of the AMP-activated protein kinase (AMPK) complex, and expression of AMPK genes was markedly downregulated by EMT. Accordingly, high AMPK expression in tumors correlated with epithelial cell markers and low AMPK expression in tumors was strongly associated with adverse prognosis. AMPK is the principal sensor of cellular energy status. In response to energy stress, AMPK is activated and critically reprograms cellular metabolism to restore energy homeostasis and maintain cell survival. We showed that activation of AMPK by energy stress was severely impaired by EMT. Consequently, EMT cancer cells became hypersensitive to a variety of energy stress conditions and primarily underwent pyroptosis, a regulated form of necrotic cell death. Collectively, the study suggests that EMT impedes the activation of AMPK signaling induced by energy stress and sensitizes cancer cells to pyroptotic cell death under energy stress conditions. Therefore, while EMT promotes malignant progression, it concurrently induces collateral vulnerabilities that may be therapeutically exploited.

A Retrospective Study Characterizing the Radiographic Progression of Hallux Valgus.

BACKGROUND: Hallux valgus deformity is the lateral deviation of the metatarsophalangeal (MTP) joint and is the most common pathology of the great toe, affecting 2%-4% of the population. It is commonly believed that the condition progresses over time both in the magnitude of deformity and development of arthritic changes. To our knowledge, there are rare studies describing the rate of deformity progression and the development of arthritic changes. Our aim is to quantify the progression of hallux valgus and associated arthritic changes in an adult population using radiographs. METHODS: Patients who are 18 years of age and older (mean age: 61.7 years and range: 18.6-94.2) who presented to our institutions between January 1, 2004, and December 1, 2019, were included. Patients were included in the study if they had hallux valgus on weight-bearing radiographs and two such radiographs at least six months apart. Hallux valgus angle (HVA), intermetatarsal angle (IMA), metatarsophalangeal (MTP) arthritis, tarsometatarsal (TMT) osteoarthritis, Hardy-Clapham sesamoid position, and round sign were collected per patient in the initial and final radiographs. Included cases were first studied together in a whole group. Then, cases were separated into three groups based on the time between initial and final radiographs. RESULTS: A total of 52 radiographic records for 43 patients were included. HVA and IMA progress with time; however, progression does not follow a direct relationship with the time elapsed between initial and final radiographs (p = 0.92 and p = 0.35, respectively). The progression of TMT osteoarthritis, sesamoid position, and round sign do not show a dependence on the time elapsed (p = 0.20, p = 0.11, and p = 0.42, respectively). An increase of one sesamoid unit position over baseline is associated with a 0.85-degree increase in IMA. A one-unit progression of sesamoid position at baseline raises the odds of MTP osteoarthritis progression at the follow-up visit by 2.14 (OR = 2.14, p = 0.0007, CI = [1.35, 3.83]). A HVA increase of one degree increases the odds of TMT osteoarthritis progression at follow-up by a factor of 1.17 (OR = 1.17, p = 0.0005, CI = [1.07, 1.34]). Patients with MTP arthritis at the initial visit have 3.77 times higher odds of round sign progression on their follow-up visit (OR = 3.77, p = 0.027, CI = [1.16, 13.13]). DISCUSSION: Hallux valgus progression can be quantified. Upon their first visit, a patient's hallux valgus parameters can be utilized to demonstrate expected progression. Progression of the deformity and arthritic changes is slow. Nonetheless, the results should be considered by surgeons and patients when developing a treatment plan with patients.

Sensitization of FOLFOX-resistant colorectal cancer cells via the modulation of a novel pathway involving protein phosphatase 2A.

The treatment of colorectal cancer (CRC) with FOLFOX shows some efficacy, but these tumors quickly develop resistance to this treatment. We have observed increased phosphorylation of AKT1/mTOR/4EBP1 and levels of p21 in FOLFOX-resistant CRC cells. We have identified a small molecule, NSC49L, that stimulates protein phosphatase 2A (PP2A) activity, downregulates the AKT1/mTOR/4EBP1-axis, and inhibits p21 translation. We have provided evidence that NSC49L- and TRAIL-mediated sensitization is synergistically induced in p21-knockdown CRC cells, which is reversed in p21-overexpressing cells. p21 binds with procaspase 3 and prevents the activation of caspase 3. We have shown that TRAIL induces apoptosis through the activation of caspase 3 by NSC49L-mediated downregulation of p21 translation, and thereby cleavage of procaspase 3 into caspase 3. NSC49L does not affect global protein synthesis. These studies provide a mechanistic understanding of NSC49L as a PP2A agonist, and how its combination with TRAIL sensitizes FOLFOX-resistant CRC cells.

Anticancer Agents Derived from Cyclic Thiosulfonates: Structure-Reactivity and Structure-Activity Relationships.

Reported are structure-property-function relationships associated with a class of cyclic thiosulfonate molecules-disulfide-bond disrupting agents (DDAs)-with the ability to downregulate the Epidermal Growth Factor Receptor (HER) family in parallel and selectively induce apoptosis of EGFR+ or HER2+ breast cancer cells. Recent findings have revealed that the DDA mechanism of action involves covalent binding to the thiol(ate) from the active site cysteine residue of members of the protein disulfide isomerase (PDI) family. Reported is how structural modifications to the pharmacophore can alter the anticancer activity of cyclic thiosulfonates by tuning the dynamics of thiol-thiosulfonate exchange reactions, and the studies reveal a correlation between the biological potency and thiol-reactivity. Specificity of the cyclic thiosulfonate ring-opening reaction by a nucleophilic attack can be modulated by substituent addition to a parent scaffold. Lead compound optimization efforts are also reported, and have resulted in a considerable decrease of the IC50 /IC90 values toward HER-family overexpressing breast cancer cells.

Biological effect of dysregulated LBX1 on adolescent idiopathic scoliosis through modulating muscle carbohydrate metabolism.

BACKGROUND CONTEXT: Abnormal energy metabolism such as lower body weight and body mass index (BMI) and less fat mass is widely reported in patients with adolescent idiopathic scoliosis (AIS) and has been implicated in deformity development. However, the underlying mechanism is largely unclear. LBX1 is one of the promising AIS predisposing genes validated by multicenter studies. PURPOSE: This study aimed to identify differentially expressed proteins (DEPs) relating to energy metabolism in AIS by using proteomic and metabolic analysis and to explore if the expression of these DEPs is associated with clinical parameters and modulated by LBX1. STUDY DESIGN: This is a cross-sectional study using clinical data and biological samples followed by basic study using a cellular model. PATIENT SAMPLE: Plasma samples were collected from Chinese girls with nonprogressive and progressive AIS (N=7 and 8, respectively) and age-matched healthy girls (N=50). Paraspinal muscle tissues were collected intraoperatively from concave and convex side of the apex of the major spinal curve in AIS (N=24) and either side from nonscoliosis patients (N=14). OUTCOME MEASURES: Radiological Cobb angle and basic anthropometric data of recruited subjects were measured. The DEPs and metabolites were compared in plasma using proteomics and metabolomics technique. The relative expression of selected genes was measured in muscles. METHODS: Plasma samples from AIS were collected at first clinical visit and were further divided into nonprogressive or progressive groups according to Cobb angle changes in 6-year follow-up. Age-matched healthy girls were recruited as control. High-performance liquid chromatography-mass spectrometry based proteomic analysis was carried out in three groups to identify DEPs and their annotated metabolic pathways. An independent cohort was used for validation by gas chromatography-mass spectrometry based metabolomic analysis. Paraspinal muscles were subjected to quantitative polymerase chain reaction (qPCR) followed by correlation analysis. Human skeletal muscle myoblast (HSMM) was used as the cellular model. RESULTS: The likelihood of aberrant galactose metabolism and glycolysis was found to be associated with AIS curve progression as evidenced by the thirteen DEPs and seven related metabolites according to proteomic and metabolomic analysis. Some of the DEPs showed significantly altered expression in AIS concave and convex sides paraspinal muscles compared with those in nonscoliosis control. Four DEPs were found significantly and negatively correlated with LBX1 in AIS convex side paraspinal muscles. Overexpressing LBX1 in HSMM cells led to increased expression of three DEPs and decreased expression of three DEPs, respectively. CONCLUSIONS: This is the first integrated proteomic and metabolomic analysis on AIS. Our findings show dysregulated galactose metabolism and glycolysis pathways in progressive group of AIS, suggesting the presence of abnormal energy metabolism at early stage of this disease, and their association with higher risk of progressing into more severe curvature. Evidence from ex vivo study with human muscle biopsies and in vitro study with human myoblast cells propose the possible effect of LBX1 on these two pathways in skeletal muscles. The present study provides new evidence of LBX1 function in AIS via modulating effect on the expression of energy metabolism related genes. This study might provide new insights into etiopathogenesis and development of novel treatment strategy targeting on abnormal body weight and BMI in patients with AIS. Additionally, the plasma proteomic and metabolomic studies suggested new candidates as biomarkers for establishing predictive model for AIS onset/progression.

Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Understanding Patient Preference of Providers to Treat Foot and Ankle Disorders.

Objective: The purpose of this study is to evaluate patients' knowledge of the professional training between doctors of podiatric medicine (DPM) and orthopedic surgery doctors (MD or DO), patients' confidence in understanding these differences, and factors that are important to patients when selecting a provider.Design: A 28-question survey was administered to new patients who were referred to the foot and ankle service in an orthopedic department. Survey questions included data on patient demographics, patient opinion, and knowledge of differences between podiatrists and orthopedic surgeons.Setting: Patient surveys were administered at Froedtert Memorial Lutheran Hospital in Wauwatosa, Wisconsin USA and Mayo Clinic in Jacksonville, Florida USA.Participants: 186 patients completed the survey.Methods: Study population characteristics and survey results were analyzed with variance (ANOVA), Fisher's Exact test, binomial tests, and chi-square tests.Results: Of the 186 patients who completed the entire survey, 108 chose "orthopedic surgeon" as their provider of preference for any foot or ankle injury. Patients preferred an orthopedic surgeon over a DPM for ankle (65.7% vs. 9.6%, P<0.01) and knee injuries (86.0% vs. 5.0%, P<0.01), while they preferred a DPM for toe pain (29.4% vs. 42.2%, P<0.03). 80.8% of patients thought orthopaedic surgeons and podiatrists undergo the same professional training.Conclusions: Patients have poor understanding of the different provider training between a DPM and orthopedic surgeon. Patients showed a preference based on anatomic location with podiatrist favored for conditions affecting the toes. Those patients that demonstrate a higher level of knowledge of the specialties were more likely to prefer an orthopaedic surgeon. Other factors, such as physician interpersonal skills and provider availability, may be more important for patients than training backgrounds.

Multi-segment foot kinematics during gait following ankle arthroplasty.

Ankle arthritis is a debilitating disease marked by pain and limited function. Total ankle arthroplasty improves pain while preserving motion and offers an alternative to the traditional treatment of ankle fusion. Gait analysis and functional outcomes tools can provide an objective balanced analysis of ankle replacement for the treatment of ankle arthritis. Twenty-nine patients with end-stage ankle arthritis were evaluated before and after ankle arthroplasty. Multi-segment foot and ankle kinematics were assessed annually following surgery (average 3.5 years, range 1-6 years) using the Milwaukee Foot Model and a Vicon video motion analysis system. Functional outcomes (American Orthopedic Foot and Ankle Society [AOFAS] ankle/hindfoot scale, short form 36 [SF-36] questionnaire) and temporal-spatial parameters were also assessed. Kinematic results were compared to findings from a previously collected group of healthy ambulators. AOFAS and SF-36 mean scores improved postoperatively. Walking speed and stride length increased after surgery. There were significant improvements in tibial sagittal range of motion in terminal stance and hindfoot sagittal range of motion in preswing. Decreased external rotation of the tibia and increased external rotation of the hindfoot were noted throughout the gait cycle. Pain and function improved after ankle replacement as supported by better outcomes scores, increased temporal-spatial parameters, and significant improvement in tibial sagittal range of motion during terminal stance and hindfoot sagittal range of motion during preswing. While multi-segment foot kinematics were improved, they were not restored to control values. Statement of clinical significance: Total ankle arthroplasty does not fully normalize mutli-segment gait kinematics despite improved patient-reported outcomes and gait mechanics.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

PURPOSE: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. EXPERIMENTAL DESIGN: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. RESULTS: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. CONCLUSIONS: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

Inhibition of cotranslational translocation by apratoxin S4: Effects on oncogenic receptor tyrosine kinases and the fate of transmembrane proteins produced in the cytoplasm.

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases.

Gene editing enables rapid engineering of complex antibiotic assembly lines.

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Open Treatment of Osteochondral Lesions of the Talus With Bone Grafting and Particulated Juvenile Cartilage Allografting.

BACKGROUND: Large cystic osteochondral lesions of the talus (OLT) are challenging pathological conditions to treat, but particulated juvenile cartilage allografts (PJCAs) supplemented with bone grafts are a promising therapeutic option. The purpose of this project was to further elucidate the role of PJCA with concomitant bone autografts for treating large cystic OLTs with extensive subchondral bone involvement (greater than 150 mm2 in area and/or deeper than 5 mm). METHODS: We identified 6 patients with a mean OLT area of 307.2 ± 252.4 mm2 and a mean lesion depth of 10.85 ± 6.10 mm who underwent DeNovo PJCA with bone autografting between 2013 and 2017. Postoperative outcomes were assessed with radiographs, Foot and Ankle Outcome Scores (FAOS), and visual pain scale scores. RESULTS: At final follow-up (27.0 ± 12.59 weeks), all patients had symptomatic improvement and incorporation of the graft on radiographs. At an average of 62 ± 20.88 months postoperatively, no patients required a revision surgery. All patients contacted by phone in 2018 and 2020 reported they would do the procedure again in retrospect and reported an improvement in their symptoms relative to their preoperative state, especially with pain and in the FAOS activities of daily living subsection (91.93 ± 9.04 in 2018, 74.63 ± 26.86 in 2020). CONCLUSION: PJCA with concomitant bone autograft is a viable treatment option for patients with large cystic OLTs. LEVELS OF EVIDENCE: Level IV.

Repurposing Tranexamic Acid as an Anticancer Agent.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

First Tarsometatarsal Joint Loading After Sequential Correction of Hallux Valgus Using a Proximal Opening Wedge Metatarsal Osteotomy and Distal Soft Tissue Procedure.

BACKGROUND: The proximal opening wedge osteotomy (POWO) of the first metatarsal (TMT-1) is commonly performed in the operative treatment of hallux valgus. Limited work has been dedicated to study POWO's effect on the TMT-1 joint, however. The purpose of this study is to evaluate the changes in TMT-1 joint contact stress following POWO of the first metatarsal. METHODS: Five fresh-frozen cadaveric below-knee specimens (mean age: 73 years) with hallux valgus deformities (mean hallux valgus angle [HVA]: 37.4 ± 8.5 degrees) were studied. The specimens were loaded to 400 N on an MTS servohydraulic load frame. Joint contact characteristics at TMT-1 joint were measured with a Tekscan pressure sensor (Model 6900, 1100 psi; Tekscan Inc, Boston, MA) with various opening wedge sizes of 3, 5, and 7 mm both without and with a distal soft tissue release (DSTR). The contact force, area, and peak contact stress were compared among groups using analysis of variance and post hoc multiple comparisons over the untreated (Dunnett test, P < .05). RESULTS: The mean contact force was 47.7 ± 33.5 N for untreated specimens. This increased sequentially with opening wedge size and reached statistical significance for 7-mm opening wedge (129.7 ± 62.3 N, P = .01) and 7-mm wedge + DSTR (134.8 ± 60.5 N, P = .008). The mean peak contact stress was 2.8 ± 1.3 MPa for the untreated specimens and increased incrementally with wedge size to 5.7 ± 3.0 MPa for 7-mm wedge only (P = .03) and 5.6 ± 2.5 MPa for 7-mm wedge + DSTR (P = .05). The contact area increased with corrections, but none reached significance. CONCLUSION: With increasing opening wedge size, loading of the TMT-1 joint increases. Joint stresses higher than 4.7 MPa have been shown to be chondrotoxic, potentially predisposing patients to arthritic joint changes following POWO. LEVEL OF EVIDENCE: XXXXXX.

Factors used in Applicant Ranking of Orthopedic Foot and Ankle Fellowships and the Availability of Online Information.

BACKGROUND: The Internet is often the first resource used by applicants to evaluate fellowship programs. However, information on these websites can be often incomplete, inaccessible, and/or inaccurate. The primary objective of this study was to examine key factors that orthopedic foot and ankle fellowship applicants use to rank programs. The secondary objective was to assess both the accessibility and availability of the information on orthopedic foot and ankle fellowship program websites. METHODS: A Qualtrics survey was distributed via e-mail to those who matched into an orthopedic foot and ankle fellowship position from years 2008-2020. A comprehensive list of orthopedic foot and ankle fellowship programs was created. Program websites were evaluated for accessibility as well as the quality of recruitment and educational content. RESULTS: There were a total of 114 survey responses out of 644 invites (17.7%). The most important factors for establishing a rank list were operative experience, current faculty, and program reputation. Eighty-five percent (41/48) of orthopedic foot and ankle fellowship websites were directly accessible using Google. On average, accessible orthopedic foot and ankle fellowship websites contained only 57% (11.5/20) of the content deemed desirable. CONCLUSION: Orthopedic foot and ankle websites are widely accessible and have higher recruitment and educational quality content scores compared with previously published data. The most important factors for establishing a rank list are consistent with previous literature. Those who ranked operative experience as one of the most important factors when establishing a rank list did not complete more operative cases than those who did not. LEVEL OF EVIDENCE: Level IV.