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Objective: To survey ophthalmic surgeons' opinions comparing a novel three-dimensional (3D) heads-up display system with a conventional surgical microscopy for minimally invasive glaucoma surgery (MIGS) on an artificial eye model., Materials and Methods: Twenty-one ophthalmologists at the 2021 Canadian Ophthalmological Society Annual Meeting in Halifax, Nova Scotia, underwent a 90-minute skills-transfer course on MIGS. Using an artificial eye model (SimulEYE iTrack Model; InsEYE LLC, Westlake Village, Calif.), participants engaged in hands-on practice of MIGS via both a 3D heads-up display system (3D HUDS) (Zeiss Artevo 800; Carl Zeiss Meditec, Jena, Germany) and a conventional surgical microscope. Following completion, participants and instructors answered a 16-question survey comparing the 2 systems (3D HUDS vs conventional surgical microscope). Survey responses were recorded on a 9-point double-headed Likert scale ranging from strongly favour 3D HUDS (1) to strongly favour conventional surgical microscopy (9). Mann-Whitney U nonparametric analysis was used to compare instructor versus participants and experts versus nonexperts., Results: Survey ratings favoured the 3D HUDS over the conventional surgical microscopy, with respondent ratings for all survey questions ranging from a response of 1 (strongly favour 3D HUDS) to 5 (equal). Mann-Whitney U statistical analysis revealed no significant difference between instructor versus participant as well as between expert versus nonexpert. Most ratings for the 3D HUDS were received for ergonomic setup of the surgical modality, depth of field (or) field of view, and usefulness in training residents for MIGS. Equal ratings for the 3D HUDS and conventional surgical microscope were received for system malfunctions and lag during surgery., Conclusions: The 3D HUDS was favoured over conventional microscopy for the performance of simulated MIGS by ophthalmologists with varying levels of experience. The survey results suggest that the 3D HUDS in an artificial eye model is useful for teaching minimally invasive glaucoma surgery, particularly with the advent of competency-based ophthalmology education programs., Competing Interests: Footnotes and Disclosure Jordan J. Huang, Derek Waldner, Jaxon J. Huang, Joshua M. Huang, and Paul Huang have no financial disclosures. Patrick Gooi is a consultant for Alcon, Abbvie, Bausch and Lomb, Glaukos, Santen, and Labtician. Patrick Gooi has a patent on a glaucoma device that is not commercialized. Joshua C. Teichman is a consultant or advisor for Aequus, Alcon, Allergan, Bausch and Lomb, Labtician-Théa, Novartis, Santen, Shire, and Sun Pharma. Mahshad Darvish-Zargar is a consultant or advisor for Alcon, Allergan, Avir Pharma, Johnson & Johnson, Labtician, and Sun Pharma., (Copyright © 2023 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Desmoplastic small round cell tumour (DSRCT) is an ultra-rare soft tissue sarcoma that is characterised by aggressive disease and dismal patient outcomes. Despite multi-modal therapy, prognosis remains poor and there are currently no effective targeted therapies available for patients with this disease. Advances in comprehensive molecular profiling approaches including next generation sequencing and proteomics hold the promise of identifying new therapeutic targets and biomarkers. In this review, we provide an overview of the current status of molecular profiling studies in DSRCT patient specimens and cell lines, highlighting the key genomic, epigenetic and proteomic findings that have contributed to our biological knowledge base of this recalcitrant disease. In-depth analysis of these molecular profiles has led to the identification of promising novel and repurposed candidate therapies that are suitable for translation into clinical trials. We further provide a perspective on how future integrated studies including proteogenomics could further enrich our understanding of this ultra-rare entity and deliver progress that will ultimately impact the outcomes of patients with DSRCT., Competing Interests: Declarations of interest none., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nitric oxide (NO) is a well-known gaseous mediator that maintains vascular homeostasis. Extensive evidence supports that a hallmark of endothelial dysfunction, which leads to cardiovascular diseases, is endothelial NO deficiency. Thus, restoring endothelial NO represents a promising approach to treating cardiovascular complications. Despite many therapeutic agents having been shown to augment NO bioavailability under various pathological conditions, success in resulting clinical trials has remained elusive. There is solid evidence of diverse beneficial effects of the treatment with low-power near-infrared (NIR) light, defined as photobiomodulation (PBM). Although the precise mechanisms of action of PBM are still elusive, recent studies consistently report that PBM improves endothelial dysfunction via increasing bioavailable NO in a dose-dependent manner and open a feasible path to the use of PBM for treating cardiovascular diseases via augmenting NO bioavailability. In particular, the use of NIR light in the NIR-II window (1000-1700 nm) for PBM, which has reduced scattering and minimal tissue absorption with the largest penetration depth, is emerging as a promising therapy. In this review, we update recent findings on PBM and NO., Competing Interests: Declaration of competing interest All authors: No reported conflicts., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To investigate the quality of information related to glaucoma procedures found online using 2 different assessment tools., Design: Cross-sectional survey of 100 web sites found via Google search engine., Methods: The terms "peripheral iridotomy" and "trabeculectomy" along with synonymous keywords were inputted into Google's search engine. The first 50 functional websites for each term were assessed by 2 independent raters using the DISCERN instrument as well as a quality assessment tool by the Journal of the American Medical Association (JAMA). Statistical analysis included an evaluation of intra-rater reproducibility and interclass correlation between the 2 scales., Main Outcome Measures: (i) Quality of web site content based on DISCERN and JAMA scores, (ii) quality of web site based on categorization of web site (iii), intra-rater reproducibility of each scale, and (iv) interclass correlation between the 2 rating scales., Results: Only 22% of the web sites for peripheral iridotomy and 34% of the web sites for trabeculectomy met all the criteria for JAMA's quality assessment. The mean DISCERN scores for peripheral iridotomy and trabeculectomy were 44 and 43.7, respectively, indicating poor quality. For the DISCERN scale, level of agreement between raters for each question ranged from κ = 0.550 (95% confidence interval [CI] 0.700-1.026) to κ = 0.884 (95% CI 0.751-1.017). For the JAMA 4 scale, level of agreement for each question ranged from κ = 0.874 (95% CI 0.734-1.01) to κ = 1.00., Conclusion: Our study indicates that information found online for two common ophthalmic procedures is of variable and poor quality. Thus, patients may be receiving misinformation online and better measures need to be implemented to avoid the dissemination of low-quality health information., (Copyright © 2021 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma is a rare, primary tumour of bone. Curative treatment consists of multi-agent chemotherapy and complete surgical resection. Despite the use of multi-agent chemotherapy, the risk of recurrence is high. Survival outcomes for patients with osteosarcoma have not changed since the 1980's. Based on biologic rationale, there has been interest in adding immunotherapies to upfront curative intent chemotherapy, including mifamurtide (a macrophage activator) and interferon. However, results to date have been disappointing. In the metastatic setting, checkpoint inhibitors alone have not proven effective. Ongoing translational work is needed to further understand which patients may benefit from immune-oncology approaches with standard cytotoxic chemotherapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: Primary leiomyosarcoma (LMS) of the gastrointestinal (GI) tract is rare. Limited literature exists regarding the clinical characteristics and outcome for patients with localised and metastatic disease., Methods: A retrospective chart review was performed for patients greater than 18 years of age diagnosed with GI LMS at The Royal Marsden Hospital between 1 January 2000-1 May 2020. Descriptive statistics were performed. Patients were censored at data cut-off date of 27 June 2020., Results: Forty-six patients with a median age at diagnosis of 54 years (range 25-85) were identified. Fifteen percent (n = 7) of patients previously received abdominal radiation for an unrelated cancer. All patients with localised disease (n = 36) had resection with oncological margins. For patients who underwent potentially curative surgery, median recurrence-free survival (mRFS) was 13 months (0.4-183 months), and half of these patients (n = 18) developed recurrent disease post resection (distant n = 16, local n = 2). Median overall survival (mOS) was 27 months for patients with distant recurrence. Twenty-one percent (n = 10) of patients presented with synchronous metastatic disease and their mOS was 19 months. Median progression-free survival (mPFS) for patients treated with conventional chemotherapy ranged from 2.0 to 8.0 months., Conclusion: The risk of recurrence is significant, and recurrence-free survival was short even with complete oncologic resection. The relationship of prior abdominal radiotherapy to the development of GI LMS warrants further investigation. Outcomes with systemic therapy for metastatic disease were poor and there is a need for the development of more effective systemic therapies., Competing Interests: Declaration of competing interest RLJ is the recipient of grants/research support from MSD, GSK. AJH and MJS are the recipient of research support from Amgen. RLJ is the recipient of consultation fees from Adaptimmune, Athenex, Blueprint, Clinigen, Eisai, Epizyme, Daichii, Deciphera, Immunedesign, Lilly, Merck, Pharmamar, UptoDate. MJFS has been an advisory board member for Amgen. All other authors have no conflicts of interest to declare., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Soft tissue sarcomas (STS) are a group of rare and heterogeneous cancers. While large-scale genomic and epigenomic profiling of STS have been undertaken, proteomic analysis has thus far been limited. Here we utilise sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for proteomic profiling of formalin fixed paraffin embedded (FFPE) specimens from a cohort of STS patients (n = 36) across four histological subtypes (leiomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma). We quantified 2951 proteins across all cases and show that there is a significant enrichment of gene sets associated with smooth muscle contraction in leiomyosarcoma, RNA splicing regulation in synovial sarcoma and leukocyte activation in undifferentiated pleomorphic sarcoma. We further identified a subgroup of STS cases that have a distinct expression profile in a panel of proteins, with worse survival outcomes when compared to the rest of the cohort. Our study highlights the value of comprehensive proteomic characterisation as a means to identify histotype-specific STS profiles that describe key biological pathways of clinical and therapeutic relevance; as well as for discovering new prognostic biomarkers in this group of rare and difficult-to-treat diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Neurovascular training models include animal models, synthetics, or computer simulation. In vivo models are expensive and require significant resources. Synthetic/computer models do not reflect the elasticity of fresh vessels. We describe an endovascular and microsurgical training model using a chicken thigh/leg., Methods: A total of 20 chicken thigh/leg models were obtained. Angiography was used to understand the anatomy. Proximal cannulation with a 5-French catheter was achieved and connected to a hemostatic valve with a pump to simulate pulsatile flow. Aneurysms were created at the thigh-leg junction. For clipping training, 3 types of aneurysms were created to reproduce anatomy seen in middle cerebral, anterior communicating, and posterior communicating aneurysms., Results: The average cost per specimen was $1.70 ± $0.30. The diameter of the proximal femoral artery was 2.4 mm ± 0.2 mm. The length from the proximal femoral artery to the aneurysm was 9.5 cm ± 0.7 cm. Distal catheterization was successful in all cases (n = 6). Successful deployment of coils and a stent was achieved under fluoroscopic guidance. Gross oversizing of coils and other mistakes led to aneurysm rupture. Each examiner performed an exploration of the pulsatile aneurysm, application and reapplication of a variety of clips, and then the final inspection of branching vessels to confirm patency., Conclusions: The chicken thigh/leg model provides training opportunities in microsurgical suturing, endovascular techniques for aneurysm obliteration, and microsurgical reconstruction of aneurysms. It combines affordability, time efficiency, and reproducibility. Further studies measuring improvement in technical aneurysm management and comparison with other training models are warranted., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15-20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SIGNIFICANCE: The matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Discoidin Domain Receptor 2 (DDR2) is a collagen-binding receptor tyrosine kinase that initiates delayed and sustained tyrosine phosphorylation signalling. To understand the molecular basis of this unique phosphorylation profile, here we utilise fluorescence microscopy to map the spatiotemporal localisation of DDR2 and tyrosine phosphorylated proteins upon stimulation with collagen. We show that cellular phosphorylated proteins are localised to the interface where DDR2 is in contact with collagen and not in the early endosomes or lysosomes. We find that DDR2 localisation is independent of integrin activation and the key DDR2 signalling effector SHC1. Structure-function analysis reveals that DDR2 mutants defective for collagen binding or kinase activity are unable to localise to the cell surface, demonstrating for the first time that both collagen binding and kinase functions are required for spatial localisation of DDR2. This study provides new insights into the underlying structural features that control DDR2 activation in space and time., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Marfan syndrome (MFS) is a genetic disorder that frequently leads to aortic root dissection and aneurysm. Despite promising preclinical and pilot clinical data, a recent large-scale study using antihypertensive angiotensin II (AngII) receptor type 1 (ATR1) blocker losartan has failed to meet expectations at preventing MFS-associated aortic root dilation, casting doubts about optimal therapy. To study the deleterious role of normal ATR1 signaling in aortic root widening, we generated MFS mice lacking ATR1a expression in an attempt to preserve protective ATR2 signaling. Despite being hypotensive and resistant to AngII vasopressor effects, MFS/ATR1a-null mice showed unabated aortic root enlargement and remained fully responsive to losartan, confirming that blood pressure lowering is of minor therapeutic value in MFS and that losartan's antiremodeling properties may be ATR1 independent. Having shown that MFS causes endothelial dysfunction and that losartan can activate endothelial function in mice and patients, we found that nitric oxide synthase (NOS) inhibition renders losartan therapeutically inactive, whereas multiple transgenic and pharmacologic models of endothelial NOS activation block aortic root dilation by correcting extracellular signal-regulated kinase signaling. In vitro, losartan can increase endothelial NO release in the absence of AngII and correct MFS NO levels in vivo. Our data suggest that increased protective endothelial function, rather than ATR1 inhibition or blood pressure lowering, might be of therapeutic significance in preventing aortic root disease in MFS., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Acquired drug resistance impacts the majority of patients being treated with tyrosine kinase inhibitors (TKIs) and remains a key challenge in modern anti-cancer therapy. The lack of clinically effective therapies to overcome resistance represents an unmet need. Understanding the signalling that drives drug resistance will facilitate the development of new salvage therapies to treat patients with secondary TKI resistance. In this study, we utilise mass spectrometry to characterise the global phosphoproteomic alterations that accompany the acquisition of resistance to two FDA-approved TKIs, pazopanib and dasatinib, in the A204 rhabdoid tumour cell line. Our analysis finds that only 6% and 9.7% of the quantified phosphoproteome is altered upon the acquisition of pazopanib and dasatinib resistance, respectively. Pazopanib resistant cells display elevated phosphorylation in cytoskeletal regulatory pathways while dasatinib resistant cells show an upregulation of the insulin receptor/IGF-1R signalling pathway. Drug response profiling rediscovers several previously reported vulnerabilities associated with pazopanib and dasatinib resistance and identifies a new dependency to the second generation HSP90 inhibitor NVP-AUY-922. This study provides a useful resource detailing the candidate signalling determinants of acquired TKI resistance; and reveals a therapeutic approach of inhibiting HSP90 function as a means of salvage therapy to overcome pazopanib and dasatinib resistance., Significance: Pazopanib and dasatinib are tyrosine kinase inhibitors (TKIs) approved for the treatment of multiple cancer types. Patients who are treated with these drugs are prone to the development of drug resistance and consequently tumour relapse. Here we use quantitative phosphoproteomics to characterise the signalling pathways which are enriched in cells that have acquired resistance to these two drugs. Furthermore, targeted drug screens were used to identify salvage therapies capable of overcoming pazopanib and dasatinib resistance. This data advances our understanding of the mechanisms of TKI resistance and highlights candidate targets for cancer therapy., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Delays in patient transfers are associated with worse outcomes for some neurosurgical conditions. One of the primary causes of transfer delay is lack of neurosurgery intensive care unit bed availability. In the present study, we characterize the results of implementing an interhospital transfer protocol to reduce unnecessary transfers and improve bed availability., Methods: A transfer protocol was implemented in July 2012 at the Bellevue Hospital Department of Neurosurgery that screened for and prevented transfer of low-risk patients who were unlikely to require specialized inpatient neurosurgical care. The impact of this protocol was assessed with prospectively recorded data on all potential interhospital transfers from May 2011 through June 2016., Results: Of the 1978 calls (regarding 1886 individual patients), 402 occurred before the implementation of the transfer protocol and 1576 occurred after. Before the protocol, 84.1% of transfer requests were accepted, but 15.2% were subsequently denied for bed unavailability. After the protocol, a smaller share of transfer requests were accepted after protocol screening (71.8%, P < 0.001), but only 1.9% (P < 0.001) were subsequently denied because of bed unavailability. The diagnosis demographics changed significantly (P < 0.001), with a larger share of arriving transfers suffering from aneurysms or tumors after the protocol and a smaller share suffering from stenosis/disc disease without neurological symptoms., Conclusions: The transfer protocol implemented in the present study allowed transfer determination based on the need for specialized neurosurgical care rather than chance unavailability of beds. Developing interhospital transfer protocols may be an effective strategy to efficiently allocate limited hospital resources and improve transfer systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Despite the recent approval of third-generation therapies, overcoming resistance to epidermal growth factor receptor (EGFR) inhibitors remains a major challenge in non-small cell lung cancer. Conceptually, synthetic lethality holds the promise of identifying non-intuitive targets for tackling both acquired and intrinsic resistance in this setting. However, translating these laboratory findings into effective clinical strategies continues to be elusive. Here, we provide an overview of the synthetic lethal approaches that have been employed to study EGFR inhibitor resistance and review the oncogene and non-oncogene signalling mechanisms that have thus far been unveiled by synthetic lethality screens. We highlight the potential challenges associated with progressing these discoveries into the clinic including context dependency, signalling plasticity, and tumour heterogeneity, and we offer a perspective on emerging network biology and computational solutions to exploit these phenomena for cancer therapy and biomarker discovery. We conclude by presenting a number of tangible steps to bolster our understanding of fundamental synthetic lethality mechanisms and advance these findings beyond the confines of the laboratory., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: ARUBA (A Randomized Trial of Unruptured Brain Arteriovenous Malformations) found better short-term outcomes after conservative management compared with intervention for unruptured arteriovenous malformations (AVMs). However, because Spetzler-Martin (SM) grade I-II AVMs have the lowest treatment morbidity, sufficient follow-up of these lesions may show a long-term benefit from intervention. The aim of this multicenter, retrospective cohort study is to assess the outcomes after stereotactic radiosurgery (SRS) for ARUBA-eligible SM grade I-II AVMs., Methods: We pooled SRS data for patients with AVM from 7 institutions and selected ARUBA-eligible SM grade I-II AVMs with ≥12 months follow-up for analysis. Favorable outcome was defined as AVM obliteration, no post-SRS hemorrhage, and no permanently symptomatic radiation-induced changes., Results: The ARUBA-eligible SM grade I-II AVM cohort comprised 232 patients (mean age, 42 years). The mean nidus volume, SRS margin dose, and follow-up duration were 2.1 cm 3 , 22.5 Gy, and 90.5 months, respectively. The actuarial obliteration rates at 5 and 10 years were 72% and 87%, respectively; annual post-SRS hemorrhage rate was 1.0%; symptomatic and permanent radiation-induced changes occurred in 8% and 1%, respectively; and favorable outcome was achieved in 76%. Favorable outcome was significantly more likely in patients treated with a margin dose >20 Gy (83%) versus ≤20 Gy (62%; P < 0.001). Stroke or death occurred in 10% after SRS., Conclusions: For ARUBA-eligible SM grade I-II AVMs, long-term SRS outcomes compare favorably with the natural history. SRS should be considered for adult patients harboring unruptured, previously untreated low-grade AVMs with a minimum life expectancy of a decade., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The phosphatase SHP-2 plays an essential role in growth factor signaling and mutations in its locus is the cause of congenital and acquired pathologies. Mutations of SHP-2 are known to affect the activation of the RAS pathway. Gain-of-function mutations cause the Noonan syndrome, the most common non-chromosomal congenital disorder. In order to obtain a holistic picture of the intricate regulatory mechanisms underlying SHP-2 physiology and pathology, we set out to characterize perturbations of the cell phosphorylation profile caused by an altered localization of SHP-2. To describe the proteins whose activity may be directly or indirectly modulated by SHP-2 activity, we identified tyrosine peptides that are differentially phosphorylated in wild type SHP-2 cells and isogenic cells expressing a non-functional SHP-2 variant that cannot dephosphorylate the physiological substrates due to a defect in cellular localization upon growth factor stimulation. By an iTRAQ based strategy coupled to mass spectrometry, we have identified 63 phosphorylated tyrosine residues in 53 different proteins whose phosphorylation is affected by SHP-2 activity. Some of these confirm already established regulatory mechanisms while many others suggest new possible signaling routes that may contribute to the modulation of the ERK and p38 pathways by SHP-2. Interestingly many new proteins that we found to be regulated by SHP-2 activity are implicated in the formation and regulation of focal adhesions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Management of nonemergent, nonacute subdural hematomas (SDHs) ranges from observation to burr-hole evacuation or craniotomy, but recurrence rates are high. We evaluated the safety and efficacy of tranexamic acid (TXA) for the treatment of residual SDHs after bedside twist-drill evacuation., Methods: We performed a retrospective analysis of a prospectively maintained database from November 2013 to November 2014 for all patients who underwent placement of a bedside subdural evacuating port system (SEPS) followed by treatment with oral TXA (650 mg daily). All demographics, evidence of venous thromboembolism, and volumes of pertinent computed tomography were obtained., Results: Twenty subdural hematomas in 14 patients met the inclusion criteria for this study. Most SDHs were mixed density. Mean SDH volume on presentation was 145.96 ± 40.22 cm(3) with a mean midline shift of 9.44 ± 4.84 mm. Mean volumes decreased to 80.00 ± 31.96 cm(3) and midline shift improved to 4.44 ± 3.29 mm after SEPS placement (P < 0.0001 and P = 0.0046). All patients were placed on TXA after their procedure. Mean follow-up with computed tomography was 92.1 ± 27.5 days, and mean SDH volume at last follow-up was 7.41 ± 15.54 cm(3) with a mean midline shift of 0.19 ± 0.69 mm (P < 0.0001 and P = 0.0002). Percent volume reduction was significantly higher after TXA than after SEPS (91.31% vs. 40.74%; P < 0.0001). No increase or delayed recurrence of the SDH was noted during TXA treatment. All but 1 clinical presenting symptom improved at follow-up. No venous thromboembolisms were noted among the patients., Conclusions: In our pilot study, chronic SDH volumes were reduced by 40.74% after SEPS drainage. The residual volume was reduced by an additional 91.31% during oral TXA treatment. No patients developed delayed recurrence or expansion of their SDHs. Further prospective studies are needed to evaluate the role of TXA for adjunctive treatment of chronic SDHs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Discoidin domain receptor 2 (DDR2) is an atypical receptor tyrosine kinase that binds to and is activated by collagen in the extracellular matrix. Recent exon sequencing studies have identified DDR2 to be mutated with a 3% to 4% incidence in squamous cell cancers of the lung. This article summarizes the current state of knowledge of DDR2 biology and signaling in lung squamous cell cancer. It also explores the context-dependent role of this receptor as both an oncogene and a tumor suppressor in cancer cells. Promising therapeutic opportunities based on existing and novel targeted small molecule inhibitors against DDR2 may provide new strategies for treating lung squamous cell cancer patients.

Phosphorylation of endothelial nitric oxide synthase (eNOS) is an important regulator of its enzymatic activity. We generated knockin mice expressing phosphomimetic (SD) and unphosphorylatable (SA) eNOS mutations at S1176 to study the role of eNOS phosphorylation. The single amino acid SA mutation is associated with hypertension and decreased vascular reactivity, while the SD mutation results in increased basal and stimulated endothelial NO production. In addition to these vascular effects, modulation of the S1176 phosphorylation site resulted in unanticipated effects on metabolism. The eNOS SA mutation results in insulin resistance, hyperinsulinemia, adiposity, and increased weight gain on high fat. In contrast, the eNOS SD mutation is associated with decreased insulin levels and resistance to high fat-induced weight gain. These results demonstrate the importance of eNOS in regulation of insulin sensitivity, energy metabolism, and bodyweight regulation, and suggest eNOS phosphorylation as a novel target for the treatment of obesity and insulin resistance., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objective: We previously reported that deletion of brain type neuronal nitric oxide synthase-alpha (nNOS-alpha) accelerates atherosclerosis in apolipoproteinE (apoE) knockout (ko) mice. The regulation of nNOS expression is complex, involving the generation of mRNA splice variants. The current study investigates occurrence and distribution of nNOS variants in atherosclerotic lesions of apoE ko and apoE/nNOS-alpha double ko (dko) animals., Methods: Mice were fed a high fat diet for 20 weeks. Immunohistochemistry and western blot analysis were performed using antibodies detecting the carboxy terminal-, or amino terminal-residue of the nNOS protein. Confocal microscopy and in situ hybridization were used to identify the compartment of cellular expression., Results: In situ hybridization revealed the presence of nNOS-alpha and -gamma mRNA variants in apoE ko plaques, while only nNOS-gamma was detectable in apoE/nNOS dko plaques. Consistent with mRNA expression nNOS-alpha protein can be detected in the neointima of apoE ko, but not apoE/nNOS dko animals. In contrast, the carboxy terminal antibody stained the neointima and media in apoE ko vessels and showed residual nNOS immunoreactivity in apoE/nNOS dko lesions. Confocal microscopy showed predominant nNOS expression in vascular smooth muscle cells, while colocalization with macrophages was less pronounced., Conclusions: Our study shows that nNOS-alpha and -gamma splice variants are expressed in atherosclerotic plaques of apoE ko mice. nNOS variants colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells. Since nNOS-alpha is atheroprotective, other nNOS splice variants which differ in enzyme kinetic and subcellular localization may also influence plaque formation.

Background: Noninvasive cardiac imaging plays an important role in the diagnosis and management of coronary artery disease (CAD). Prior studies have focused on the diagnostic performance of noninvasive modalities using angiographically significant stenoses as the reference standard. Recent trends in evidence-based medicine and increasing imaging utilization call for validation of diagnostic algorithms with well-designed, controlled trials with clinical outcome endpoints., Objective: To compare the performance of stress radionuclide myocardial perfusion imaging (MPI) and coronary computed tomography angiography (CTA) in terms of outcomes., Methods: We designed a single-center, randomized controlled trial that compares MPI and CTA as the initial modality for the evaluation of patients hospitalized for chest pain without known CAD or acute myocardial infarction. Patients with intermediate-risk characteristics and a clinical need for noninvasive imaging are included. The primary outcome measured is the incidence of conventional angiography not leading to subsequent coronary revascularization within 1 year. The study is powered to detect a reduction from 11% to 3% in catheterization not leading to an intervention with a sample size of 400. Secondary outcomes include procedural complications and posttest renal dysfunction (safety outcomes), major adverse cardiovascular events, length of hospital stay, subsequent hospitalizations and imaging, changes in medical management, and tolerability of the noninvasive test., Conclusions: The results of this trial will further our understanding of the relative appropriateness of CTA and MPI in evaluating intermediate-risk patients hospitalized with chest pain. It will also have implications for the design and probability of success of multicentered trials that are currently being planned.

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.

We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article.

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.

Nitric oxide (NO) is an important mediator of cerebral blood flow and metabolism. As a vasodilator, NO regulates cerebral blood flow, and couples regional brain perfusion with metabolic activity. Following cerebral ischemia, NO levels rise significantly due to activation of neuronal nitric oxide synthase by NMDA receptor mediated calcium entry. Depending on its tissue and enzymatic source, NO may be protective or toxic. This article reviews the effects of NO following cerebral ischemia, the signaling pathways through which NO acts, and its potential roles in cerebral ischemic preconditioning.

We investigated the antiviral activity of olive leaf extract (OLE) preparations standardized by liquid chromatography-coupled mass spectrometry (LC-MS) against HIV-1 infection and replication. We find that OLE inhibits acute infection and cell-to-cell transmission of HIV-1 as assayed by syncytia formation using uninfected MT2 cells co-cultured with HIV-1-infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose dependent, with EC(50)s of around 0.2 microg/ml. In the effective dose range, no cytotoxicity on uninfected target cells was detected. The therapeutic index of OLE is above 5000. To identify viral and host targets for OLE, we characterized gene expression profiles associated with HIV-1 infection and OLE treatment using cDNA microarrays. HIV-1 infection modulates the expression patterns of cellular genes involved in apoptosis, stress, cytokine, protein kinase C, and hedgehog signaling. HIV-1 infection up-regulates the expression of the heat-shock proteins hsp27 and hsp90, the DNA damage inducible transcript 1 gadd45, the p53-binding protein mdm2, and the hedgehog signal protein patched 1, while it down-regulates the expression of the anti-apoptotic BCL2-associated X protein Bax. Treatment with OLE reverses many of these HIV-1 infection-associated changes. Treatment of HIV-1-infected cells with OLE also up-regulates the expression of the apoptosis inhibitor proteins IAP1 and 2, as well as the calcium and protein kinase C pathway signaling molecules IL-2, IL-2Ralpha, and ornithine decarboxylase ODC1.

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.

ZYMV-AGII (zucchini yellow mosaic virus-AGII) is a recombinant nonpathogenic potyvirus-based vector system for the expression of foreign genes in cucurbit plants and their edible fruits, including squash, cucumber, melon, watermelon, and pumpkin. MAP30 (Momordica anti-HIV protein, 30 kDa) and GAP31 (Gelonium anti-HIV protein 31 kDa) are multifunctional plant proteins with activity against HIV-1 virus. These proteins are also effective against other viruses, tumor cells, and microbes. We report here the production and characterization of biologically active MAP30 and GAP31 in squash plant by expression of their genes using the ZYMV-AGII vector. Recombinant expressed MAP30 and GAP31 exhibit comparable antiviral, antitumor, and antimicrobial activities as their counterparts from their original plant sources, with EC(50)s in the ranges of 0.2-0.3 nM for HIV-1. These results demonstrate for the first time the amplification and production of therapeutic proteins, MAP30 and GAP31, in common vegetables. This provides valuable alternative food sources of these antiviral, antitumor, and antimicrobial agents for therapeutic applications., ((c)2002 Elsevier Science (USA).)