Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Sustained Arginase 1 Expression Modulates Pathological Tau Deposits in a Mouse Model of Tauopathy.

Tau accumulation remains one of the closest correlates of neuronal loss in Alzheimer's disease. In addition, tau associates with several other neurodegenerative diseases, collectively known as tauopathies, in which clinical phenotypes manifest as cognitive impairment, behavioral disturbances, and motor impairment. Polyamines act as bivalent regulators of cellular function and are involved in numerous biological processes. The regulation of the polyamines system can become dysfunctional during disease states. Arginase 1 (Arg1) and nitric oxide synthases compete for l-arginine to produce either polyamines or nitric oxide, respectively. Herein, we show that overexpression of Arg1 using adeno-associated virus (AAV) in the CNS of rTg4510 tau transgenic mice significantly reduced phospho-tau species and tangle pathology. Sustained Arg1 overexpression decreased several kinases capable of phosphorylating tau, decreased inflammation, and modulated changes in the mammalian target of rapamycin and related proteins, suggesting activation of autophagy. Arg1 overexpression also mitigated hippocampal atrophy in tau transgenic mice. Conversely, conditional deletion of Arg1 in myeloid cells resulted in increased tau accumulation relative to Arg1-sufficient mice after transduction with a recombinant AAV-tau construct. These data suggest that Arg1 and the polyamine pathway may offer novel therapeutic targets for tauopathies.

Combative Sports Injuries: An Edmonton Retrospective.

OBJECTIVE: Mixed martial arts (MMA) is an increasingly popular combative sport involving aggressive techniques that present substantial injury risk. We examined the incidence and types of injuries sustained in MMA fights and compared this with injuries sustained in boxing matches. DESIGN: Consecutive Case Series. SETTING: We used data from post-fight medical examinations on all bouts in Edmonton, Canada, between 2000 and 2013. PARTICIPANTS: The participants were 1181 MMA competitors and 550 boxers. MAIN OUTCOME MEASURES: The attending physician conducted a mandatory post-fight examination of all fighters and documented the nature of injuries sustained. RESULTS: Boxers were significantly more likely not to experience injury (49.8% vs 59.4%, P < 0.001), whereas MMA fighters were significantly more likely to experience 1 injury (typically contusion/bruising, P < 0.001). Boxers were more likely to experience loss of consciousness (7.1% vs 4.2%, P = 0.01) and serious eye injury (1.1% vs 0.3%, P = 0.02). CONCLUSIONS: The overall injury incidence in MMA competitors appears slightly higher than for boxers, but MMA fighters experience more minor contusion/bruising injuries. Boxers are more likely to experience serious injury such as concussion/head trauma involving loss of consciousness or eye injury such as retinal detachment.

TRAP display: a high-speed selection method for the generation of functional polypeptides.

Here, we describe a novel method that enables high-speed in vitro selection of functional peptides, peptidomimetics, and proteins via a simple procedure. We first developed a new cell-free translation system, the TRAP system (transcription-translation coupled with association of puromycin linker), which automatically produces a polypeptide library through a series of sequential reactions: transcription, association of puromycin-DNA linker, translation, and conjugation between the nascent polypeptide and puromycin-DNA linker. We then applied the TRAP system for the selection of macrocyclic peptides against human serum albumin. Six rounds of selection using TRAP display were performed in approximately 14 h, yielding macrocyclic peptides with nanomolar affinity to their target protein. Because TRAP display enables high-speed selection of functional polypeptides, it will facilitate the generation of various polypeptides that are useful for biological and therapeutic applications.

Diverse activation of microglia by chemokine (C-C motif) ligand 2 overexpression in brain.

BACKGROUND: The chemokine (C-C motif) ligand 2 (CCL2) is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system (CNS) inflammation. METHODS: To determine the impact of CCL2 in inflammation in vivo and to elucidate the CCL2-induced polarization of activated brain microglia, we delivered CCL2 into the brains of wild-type mice via recombinant adeno-associated virus serotype 9 (rAAV-9) driven by the chicken ß-actin promoter. We measured microglial activation using histological and chemical measurement and recruitment of monocytes using histology and flow cytometry. RESULTS: The overexpression of CCL2 in the CNS induced significant activation of brain resident microglia. CD45 and major histocompatibility complex class II immunoreactivity significantly increased at the sites of CCL2 administration. Histological characterization of the microglial phenotype revealed the elevation of "classically activated" microglial markers, such as calgranulin B and IL-1ß, as well as markers associated with "alternative activation" of microglia, including YM1 and arginase 1. The protein expression profile in the hippocampus demonstrated markedly increased levels of IL-6, GM-CSF and eotaxin (CCL-11) in response to CCL2, but no changes in the levels of other cytokines, including TNF-α and IFN-γ. Moreover, real-time PCR analysis confirmed increases in mRNA levels of gene transcripts associated with neuroinflammation following CCL2 overexpression. Finally, we investigated the chemotactic properties of CCL2 in vivo by performing adoptive transfer of bone marrow-derived cells (BMDCs) isolated from donor mice that ubiquitously expressed green fluorescent protein. Flow cytometry and histological analyses indicated that BMDCs extravasated into brain parenchyma and colabeled with microglial markers. CONCLUSION: Taken together, our results suggest that CCL2 strongly activates resident microglia in the brain. Both pro- and anti-inflammatory activation of microglia were prominent, with no bias toward the M1 or M2 phenotype in the activated cells. As expected, CCL2 overexpression actively recruited circulating monocytes into the CNS. Thus, CCL2 expression in mouse brain induces microglial activation and represents an efficient method for recruitment of peripheral macrophages.

Fabrication of maxillary overdenture supported by custom waxed and cast locator abutments: case report.

The fabrication of a maxillary overdenture supported and retained by custom waxed and cast locator attachments will be described. These angulated abutments were made necessary by a lack of maxillary bone due to advanced periodontal problems that contributed to the loss of all of the maxillary and mandibular teeth; thus, the maxillary anterior implants were placed in a labially or facially inclined position, which necessitated placement of labially inclined implant bodies. This article describes a method for correcting this angulation to create a more vertical path of placement and to allow the facially inclined implants to be used for an overdenture retentive device.

Clear Cell Ovarian Carcinoma With C1 Lateral Mass Metastasis and Pathologic Fracture: A Case Report.

Osseous metastasis (OM) in ovarian cancer (OC) are rare, with an incidence ranging from 0.8% to 2.6%, and are associated with poor prognosis. The available literature on their management and associated complications is scarce. We report a case of International Federation of Gynecology and Obstetrics (FIGO) stage IVB clear cell epithelial OC (EOC) who presented with neck pain. Imaging revealed multiple cervical spine metastases with left vertebral artery encasement and concurrent C1 lateral mass compression fracture, without neurological deficit, requiring occiput to C2 posterior instrumentation and fusion. Early OM may be associated with shorter overall survival, and survival after OM diagnosis is on the order of months. Management of OM should include a multidisciplinary team and may require surgical stabilization in addition to systemic chemotherapy, local radiotherapy, and osteoclast inhibitors.

Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface. Using the mRNA-based display technique to screen a library of in vitro-translated cyclic peptides, we identified a peptide that binds with a nanomolar affinity to TEAD. The X-ray structure of this peptide in complex with TEAD reveals that it interacts with the α-helix pocket. Under our experimental conditions, this peptide can form a ternary complex with TEAD and YAP. Furthermore, combining it with a peptide binding to the Ω-loop pocket gives an additive inhibitory effect on the YAP:TEAD interaction. Overall, our results show that it is possible to identify nanomolar inhibitors of the YAP:TEAD interaction that bind to the α-helix pocket, suggesting that developing such compounds might be a strategy to treat cancers where the Hippo pathway is deregulated.

Sequential provisional implant prosthodontics therapy.

The fabrication and long-term use of first- and second-stage provisional implant prostheses is critical to create a favorable prognosis for function and esthetics of a fixed-implant supported prosthesis. The fixed metal and acrylic resin cemented first-stage prosthesis, as reviewed in Part I, is needed for prevention of adjacent and opposing tooth movement, pressure on the implant site as well as protection to avoid micromovement of the freshly placed implant body. The second-stage prosthesis, reviewed in Part II, should be used following implant uncovering and abutment installation. The patient wears this provisional prosthesis until maturation of the bone and healing of soft tissues. The second-stage provisional prosthesis is also a fail-safe mechanism for possible early implant failures and also can be used with late failures and/or for the necessity to repair the definitive prosthesis. In addition, the screw-retained provisional prosthesis is used if and when an implant requires removal or other implants are to be placed as in a sequential approach. The creation and use of both first- and second-stage provisional prostheses involve a restorative dentist, dental technician, surgeon, and patient to work as a team. If the dentist alone cannot do diagnosis and treatment planning, surgery, and laboratory techniques, he or she needs help by employing the expertise of a surgeon and a laboratory technician. This team approach is essential for optimum results.

Clinical Characteristics, Outcomes, and Pathology Analysis in Patients With Dorsal Arachnoid Web.

BACKGROUND: Dorsal arachnoid webs (DAWs) are rare pathological abnormalities of the arachnoid layer of the spinal cord that can result in pain and myelopathy. OBJECTIVE: To present clinical, imaging, and pathological characteristics of patients diagnosed with DAW, case illustrations, and a review of the literature. METHODS: Seventeen cases of DAW between 2015 and 2019 at a tertiary medical center were retrospectively identified through a case log search. Patient characteristics, preoperative imaging, operative notes, and pathology reports were collected. Our main outcome assessed was postoperative resolution of symptoms. Odds ratios were used to determine associations between preoperative signs and symptoms with postoperative symptom resolution. RESULTS: The mean age of the cohort was 50.5 years (IQR = 16) and presented primarily with back pain (64.7%). On imaging, all patients were found to have the "scalpel sign," and nearly half had a syrinx present (41.2%). All DAWs were located in the thoracic spine, with the most common location being the midthoracic (70.6%). The mean follow-up length for all patients was 4.3 months. There were no preoperative symptoms significantly associated with postoperative symptom resolution; however, a trend was noted with the presence of a preoperative syrinx. Pathology samples consistently demonstrated fibroconnective or collagenous tissue with no evidence of inflammation or neoplasm. CONCLUSION: DAW is a rare pathology that can result in myelopathy or inappropriate interventions if misdiagnosed. Surgical intervention using laminectomy with intradural exploration should be considered in symptomatic patients with DAW because it is curative with a strong chance of preoperative symptom resolution with relatively low complication rates.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors.

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects.

Design of Thioether Cyclic Peptide Scaffolds with Passive Permeability and Oral Exposure.

Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel but independent tracks. Less precedent combines their respective attributes, thereby limiting the potential to identify permeable peptide ligands for key targets. Herein, we present novel 6-, 7-, and 8-mer cyclic peptides (MW 774-1076 g·mol-1) with passive permeability and oral exposure that feature the amino acids and thioether ring-closing common to large array formats, including DNA- and RNA-templated synthesis. Each oral peptide herein, selected from virtual libraries of partially N-methylated peptides using in silico methods, reflects the subset consistent with low energy conformations, low desolvation penalties, and passive permeability. We envision that, by retaining the backbone N-methylation pattern and consequent bias toward permeability, one can generate large peptide arrays with sufficient side chain diversity to identify permeability-biased ligands to a variety of protein targets.

A new modular radiographic classification of adult idiopathic scoliosis as an extension of the Lenke classification of adolescent idiopathic scoliosis.

PURPOSE: To propose and test the reliability of a radiographic classification system for adult idiopathic scoliosis. METHODS: A three-component radiographic classification for adult idiopathic scoliosis consisting of curve type, a lumbosacral modifier, and a global alignment modifier is presented. Twelve spine surgeons graded 30 pre-marked cases twice, approximately 1 week apart. Case order was randomized between sessions. RESULTS: The interrater reliability (Fleiss' kappa coefficient) for curve type was 0.660 and 0.798, for the lumbosacral modifier 0.944 and 0.965, and for the global alignment modifier 0.922 and 0.916, for round 1 and 2 respectively. Mean intrarater reliability was 0.807. CONCLUSIONS: This new radiographic classification of adult idiopathic scoliosis maintains the curve types from the Lenke classification and introduces the lumbosacral and global alignment modifiers. The reliability of the lumbosacral modifier and global alignment modifier shows near perfect agreement, and sets the foundation for further studies to validate the reliability, utility, and applicability of this classification system.

LPS- induced inflammation exacerbates phospho-tau pathology in rTg4510 mice.

Inflammation and microglial activation are associated with Alzheimer's disease (AD) pathology. Somewhat surprisingly, injection of a prototypical inflammatory agent, lipopolysaccharide (LPS) into brains of amyloid precursor protein (APP) transgenic mice clears some of the pre-existing amyloid deposits. It is less well understood how brain inflammation modulates tau pathology in the absence of Aß. These studies examined the role of LPS-induced inflammation on tau pathology. We used transgenic rTg4510 mice, which express the P301L mutation (4R0N TauP301L) and initiate tau pathology between 3-5 months of age. First, we found an age-dependent increase in several markers of microglial activation as these rTg4510 mice aged and tau tangles accumulated. LPS injections into the frontal cortex and hippocampus induced significant activation of CD45 and arginase 1 in rTg4510 and non-transgenic mice. In addition, activation of YM1 by LPS was exaggerated in transgenic mice relative to non-transgenic animals. Expression of Ser199/202 and phospho-tau Ser396 was increased in rTg4510 mice that received LPS compared to vehicle injections. However, the numbers of silver-positive neurons, implying presence of more pre- and mature tangles, was not significantly affected by LPS administration. These data suggest that inflammatory stimuli can facilitate tau phosphorylation. Coupled with prior results demonstrating clearance of Aß by similar LPS injections, these results suggest that brain inflammation may have opposing effects on amyloid and tau pathology, possibly explaining the failures (to date) of anti-inflammatory therapies in AD patients.

The effects of intravenous gabapentin administration on the minimum alveolar concentration of isoflurane in cats.

BACKGROUND: Gabapentin is a structural analog of gamma-aminobutyric acid, one of the inhibitory neurotransmitters of the mammalian central nervous system. It is increasingly being used preemptively to control postoperative pain. Therefore, its interaction with inhaled anesthetics is of clinical interest. In this study, we examined the effects of gabapentin on the minimum alveolar concentration (MAC) of isoflurane in cats. We hypothesized that gabapentin would decrease the MAC of isoflurane in a dose-dependent manner. METHODS: Six cats were included in the study. Gabapentin was administered IV to achieve target plasma concentrations between 0 and 16 microg/mL and the MAC of isoflurane was determined at each gabapentin concentration. Gabapentin concentrations were quantitated by liquid chromatography-mass spectrometry analysis of extracted plasma samples. MAC values at the different gabapentin plasma concentrations were analyzed by a repeated-measures analysis of variance using the Huynh-Feldt correction for violation of the sphericity assumption. RESULTS: Actual gabapentin concentrations were 0 + or - 0, 1.18 + or - 0.23, 2.25 + or - 0.23, 4.96 + or - 1.19, 10.63 + or - 1.37, and 19.69 + or - 3.97 microg/mL for the target concentrations of 0, 1, 2, 4, 8, and 16 microg/mL, respectively. The MAC of isoflurane in this study was 2.10% + or - 0.13%, 2.10% + or - 0.14%, 2.13% + or - 0.12%, 2.06% + or - 0.11%, 2.11% + or - 0.15%, and 2.09% + or - 0.25% at target plasma concentrations of 0, 1, 2, 4, 8, and 16 microg/mL, respectively. CONCLUSIONS: We conclude that gabapentin did not have a detectable effect on the MAC of isoflurane in cats.

Solutions to complications of implant therapy.

Restorative dentists face challenges when implant dentistry is performed without a surgical template. Manufactured component parts (particularly screws, gold cylinders, and the implant bodies themselves) can also cause problems during these procedures. These problems will occur regardless of the mode of retention used for the prosthesis, since the same metals are involved and nearly all implant restorations have a screw somewhere. Managing these complications is essential to avoid treatment failures.

The use of implants as retainers for removable partial dentures.

There has been little presented in the literature regarding the use of implant bodies as retainers for removable partial dentures. However, these fixtures can be a useful asset for restorative dentists, as they can be used when there is insufficient bone for a fixed prosthesis or as retainers for a provisional appliance until additional dental treatment is possible.

Fabricating a nonrotational angulated abutment for a single-tooth prosthesis.

The fabrication of a custom waxed and cast nonrotational angulated abutment will be described. Most manufacturers of implant components fabricate angulated abutments for multiunit retrievable prostheses. However, they do not manufacture any components for a single unit that is screw retained. This article covers a step-by-step technique that was developed in an attempt to resolve this problem.