Current status of eye-lens dosimetry in Canada.

For occupational exposures in planned exposure situations International Commission on Radiological Protection (ICRP) publication 118 recommends an equivalent dose limit for the lens of the eye of 20 mSv yr-1averaged over five years with no single year exceeding 50 mSv. This constitutes a reduction from the previous limit of 150 mSv yr-1. The Canadian nuclear regulator, the Canadian Nuclear Safety Commission, responded to the ICRP recommendation by initiating amendments to theRadiation Protection Regulationsthrough a discussion paper which was published for comment by interested stakeholders in 2013. The revised equivalent dose limit of 50 mSv in a one-year dosimetry period for nuclear energy workers came into effect in January 2021. This paper presents the outcome of discussions with Canadian stakeholders in diverse fields of radiological work which focused on the implementation of the reduced occupational equivalent dose limit for the lens of the eye in their respective workplaces. These exchanges highlighted the existing practices for monitoring doses to the lens of the eye and identified current technological gaps. The exchanges also identified that, in many cases, the lens of the eye dose is anticipated to be well within the new dose limit despite some of the gaps in technology. The paper also presents the monitoring and eye-lens dose assessment solutions that are available based on different methods for eye-lens monitoring; presented together with criteria for their use.

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.

Oral mucositis-case series of a rare adverse effect associated with immunotherapy.

Immune checkpoint inhibitors, such as pembrolizumab and nivolumab, act by inhibiting programmed death-1 and activating the T cells against cancer. An imbalance in this immune response, however, could lead to immune-related adverse events (irAEs) involving multiple organs like rash, fatigue, hypo and hyperthyroidism, pneumonitis, hepatitis, and colitis, among others. Oral irAEs are not uncommon among immune checkpoint inhibitors which include xerostomia, dysgeusia, and lichenoid reactions; however, oral mucositis is rarely seen or reported in patients receiving PD-1 inhibitors. We present 3 cases of this rare complication in varying grades of severity. The patients were managed with steroids, either topical or systemic, depending on the severity of the lesions with either postponement or withholding therapy due to toxicity. Through this article, we hope to bring to light this overlooked and underdiagnosed oral adverse event associated with the use of immunotherapy and various treatment options for its management.

Oxygen gradients can determine epigenetic asymmetry and cellular differentiation via differential regulation of Tet activity in embryonic stem cells.

Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally 'poised' promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.

Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling.

The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production.

Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription.

Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1-3week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2'-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth.

An approach of selecting appropriate markers from the primary tumor to enable detection of circulating tumor cells in patients with non-small cell lung cancer.

PURPOSE: Circulating tumor cells (CTCs) are rare and difficult to isolate, and require selecting minimal but appropriate markers. The aim of this study was to identify markers in the primary non small cell lung cancer (NSCLC) tissue to guide isolation of CTCs from the peripheral blood of patients with lung cancer. METHODS: The expression of CK-19, EGFR and MUC-1 was evaluated by RT-PCR in the NSCLC tumor and paired adjacent normal tissues from 27 patients. The normal cytology, and the neoplastic and fibrotic pathology of the tissue were analyzed by histochemistry. The expression of the markers was analyzed in relation to the stage and grade of disease. RESULTS: Expression analysis showed that 42% of the tumors were positive for CK-19, whereas 85% for both EGFR and MUC-1. Ninety two percent of the tumors expressed any one marker. All (100%) adjacent normal tissues were CK-19 negative, 52% EGFR negative and 44% MUC-1 negative. CK-19 expression was specific to the tumor tissue but it was expressed by only 42% of them, manifesting a need for at least three markers to guide the detection of CTCs isolated from the peripheral blood of NSCLC patients. Histopathology demonstrated that 58% were adenocarcinomas, 35% squamous cell carcinomas and 7% had mixed pathology. CONCLUSIONS: This data serves as a prelude and emphasizes the importance of selecting markers expressed in the primary tumor tissue to facilitate and enable enumeration of CTCs.

Protein-Specific Signal Peptides for Mammalian Vector Engineering.

Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese hamster ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive endoplasmic reticulum (ER) translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination of in silico and targeted design rule-based in vitro testing can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers ≥1.8×, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlined in silico/in vitro testing processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products.

IL-10 production in non-small cell lung carcinoma patients is regulated by ERK, P38 and COX-2.

Immune dysfunction is hallmark of patients with non-small cell lung carcinoma (NSCLC). The molecular mechanism involved in COX-2- and PGE2-mediated production of immunosuppressive cytokine IL-10 is not well-understood. Our study addresses the involvement of T cell downstream signalling intermediates, cytokines (IL-10 and IFN-γ) and their transcription factors (T-bet and GATA-3) in COX-2-mediated regulation of lymphocyte functions in NSCLC patients. In comparison to healthy individual, a marked decrease in lymphocyte proliferation to anti-CD3 MAb was observed in NSCLC patients by thymidine incorporation assay. Using flow cytometry, decrease in intracellular calcium release with increase in reactive oxygen species was observed in lymphocytes of NSCLC patients. These patients showed increased IL-10 and PGE2 with reduced IFN-γ production by ELISA. Results demonstrated defect in regulation of transcription factors T-bet and GATA-3 as analysed by Western blotting (WB), immunoprecipitation and EMSA. Overexpression of p-p38, p-ERK and COX-2 were observed with diminished p-JNK by WB. IL-10/IFN-γ levels were found to be differentially regulated via p38 and ERK mitogen-activated protein kinase (MAPK) pathways in cooperation with COX-2. Inhibition of these pathways using selective inhibitors lead to increased lymphocyte proliferative response to anti-CD3 MAb and IFN-γ production with decrease in IL-10 production. Studies showed involvement of ERK, p38 and COX-2 pathways in high IL-10 production, driven by lung tumour derived PGE2. The selective COX-2 inhibitor rofecoxib showed ability to alter the cytokine balance by affecting regulation of T-bet and GATA-3 transcription factors.

Mutational Analysis of EGFR Mutations in Non-Small Cell Lung Carcinoma-An Indian Perspective of 212 Patients.

Lung cancer is the world's leading cause of cancer-related deaths. Epidermal growth factor receptor (EGFR) is one of the critical oncogenes and plays a significant role in tumor proliferation and metastasis. Patients with sensitizing mutations in the EGFR gene have better clinical outcomes when treated with tyrosine kinase inhibitors (TKI). This study expands our knowledge of the spectrum of EGFR mutations among lung cancer patients in the Indian scenario. This is a retrospective descriptive study of all newly diagnosed patients with lung cancer in tertiary care hospital in India. All the samples were subjected to real-time PCR (q-PCR) analysis and confirmation of rare novel mutations was done using Sanger sequencing. Clinicopathological characteristics, mutational EGFR status, and location on the exon and metastatic sites were evaluated. An analysis of total 212 samples showed mutations in 38.67% of cases. Among these, five (5.9%) samples had mutations in exon 18, 41 (48.8%) samples had mutations in exon 19, 12 (14.28%) samples had mutations in exon 20, and 26 (30.95%) samples had mutations in exon 21. Eleven (13.41%) were found to be uncommon EGFR mutations. Additionally, six (21.4%) samples that had EGFR mutations were also positive for brain metastasis. Future testing on bigger panels will help to characterize the incidence of genetic mutations and to determine the appropriate targeted treatment choices for NSCLC patients.

Hyperkalemia during surgery: is it an early warning of propofol infusion syndrome?

We present a case of severe hyperkalemia in a 48-year-old man after short-term infusion of an average dose of propofol. We suspected that the hyperkalemia in this patient was a sign of propofol infusion syndrome. The patient was undergoing a video-assisted esophagectomy, for which one-lung ventilation, with air/oxygen, isoflurane, and continuous epidural analgesia was supplemented with propofol infusion. In the intraoperative period, the patient developed severe hyperkalemia with mild acidosis but no cardiovascular failure. There were no other evident causes of hyperkalemia as documented by laboratory data. The procedure was abandoned and the patient was taken to postoperative recovery, where his potassium levels returned to normal at the end of 10 h.

Novel cost-effective method of laparoscopic feeding-jejunostomy.

A feeding jejunostomy tube placement is required for entral feeding in a variety of clinical scenarios. It offers an advantage over gastrostomies by eliminating the risk of aspiration. Standard described laparoscopic methods require special instrumentation and expensive custom-made tubes. We describe a simple cost-effective method of feeding jejunostomy using regular laparoscopic instruments and an inexpensive readily available tube. The average operating time was 35 min. We had no intra-operative complications and only one post-operative complication in the form of extra-peritoneal leakage of feeds due to a damaged tube. No complications were encountered while pulling out the tubes after an average period of 5-6 weeks.

Redox-Dependent Regulation of Sulfur Metabolism in Biomolecules: Implications for Cardiovascular Health.

SIGNIFICANCE: Sulfur-containing amino acids are integral to the molecular mechanisms that underlie many aspects of cellular function and homeostasis, facilitated by reversible changes in the oxidation states of sulfur atoms. Sulfur-containing amino acids are metabolically linked by interacting pathways that impact the one-carbon metabolic cycle and generation of methyl groups, the folate cycle, and maintenance of the major cellular redox buffer; glutathione. Dysregulation of these pathways is associated with diverse pathologies, notably of the cardiovascular (CV) system, which are typically characterized by inappropriate plasma levels of sulfur-containing amino acids. Recent Advances: Perhaps not surprisingly, the cellular redox state has emerged as a major regulator of many enzymatic processes within these metabolic cycles. The metabolism of cysteine can also result in the production of hydrogen sulfide (H2S), a signaling molecule whose activity is potentially linked to intracellular levels of both reactive oxygen species (ROS) and molecular oxygen. CRITICAL ISSUES: In most cases, the endogenous physiological sources of ROS that might mediate the interlinked metabolic pathways of sulfur-containing biomolecules remain unknown. However, the family of NADPH oxidases, and Nox4 in particular, is emerging as a likely candidate. FUTURE DIRECTIONS: This review focuses on the current knowledge of key aspects of sulfur metabolism, which are regulated by redox-based chemical reactions, and the likely intracellular oxidant sources that might mediate this regulation. This knowledge will be important to guide future targeted therapeutic interventions in diverse CV disorders.

Stereotactic body radiotherapy for lung tumors: Dosimetric analysis and clinical outcome.

INTRODUCTION: Stereotactic body radiotherapy (SBRT) has emerged as an important modality in malignant lung tumor treatment both in early localized primary and oligometastatic setting. This study aims to present the results of lung SBRT both in terms of dosimetry and clinical outcome. MATERIALS AND METHODS: Twenty-seven patients were assessed from 2012 to 2016. Both the primary and oligometastatic lung tumors were evaluated. Respiratory motion management was done employing ANZAI (Siemens, Germany) based four-dimensional computed tomography (CT). Commonly used fractionations were 60 Gy/5 fractions for peripheral tumors and 48 Gy/6 fractions for central tumors. Radiation Therapy Oncology Group toxicity criteria were used for toxicity and whole-body positron emission tomography-CT scan was done at follow-up for response evaluation. RESULTS: Twenty-seven patients were evaluated, 18 (66.7%) patients had a primary, and 9 (33.3%) patients had metastatic lung tumors. The male-to-female ratio for the entire cohort was 2:1. The median age at diagnosis was 65.8 years. Mean planning target volume (PTV) D2cc was 54.9 ± 9.04 Gy and mean internal target volume diameter was 3.0 ± 1.07 cm. Mean V20 Gy, V10 Gy, and V5 Gy of (lungs total-PTV) and (Lung ipsilateral - PTV) were 5.4 ± 4% and 10.9 ± 7.9%, 11.7 ± 5.8% and 24.2 ± 14.0%, and 22.05 ± 12.4% and 33.2 ± 15.3%, respectively. In total 21 (84%) patients and 4 patients (16%) showed a complete and partial response, respectively. One (3%) patient developed Gr 3 radiation pneumonitis. One year local control was in 18 (81%) patients whereas 4 (14%) patients progressed and three patients did not report. A higher prescribed dose significantly correlated with 1 year tumor control (P = 0.036). CONCLUSION: This study infers the feasibility and a favorable outcome for lung cancer amenable to SBRT in addition to being one of the largest clinical experiences for lung stereotactic treatment in our country.

Leiomyoma of esophagus.

Leiomyomas are rare benign esophageal neoplasms with an indolent clinical course. Symptoms mimic that of esophageal cancer. Esophagoscopy and endoscopic ultrasonography are the main diagnostic methods. Symptomatic and large leiomyomas should be treated surgically while small, asymptomatic lesions may be managed by regular follow up and repeated endoscopies.

Redox regulation of gasotransmission in the vascular system: A focus on angiogenesis.

Reactive oxygen species have emerged as key participants in a broad range of physiological and pathophysiological processes, not least within the vascular system. Diverse cellular functions which have been attributed to some of these pro-oxidants within the vasculature include the regulation of blood pressure, neovascularisation and vascular inflammation. We here highlight the emerging roles of the enzymatically-generated reaction oxygen species, O2- and H2O2, in the regulation of the functions of the gaseous signalling molecules: nitric oxide (NO), carbon monoxide (CO), and hydrogen sulphide (H2S). These gasotransmitters are produced on demand from distinct enzymatic sources and in recent years it has become apparent that they are capable of mediating a number of homeostatic processes within the cardiovascular system including enhanced vasodilation, angiogenesis, wound healing and improved cardiac function following myocardial infarction. In common with O2- and/or H2O2 they signal by altering the functions of target proteins, either by the covalent modification of thiol groups or by direct binding to metal centres within metalloproteins, most notably haem proteins. The regulation of the enzymes which generate NO, CO and H2S have been shown to be influenced at both the transcriptional and post-translational levels by redox-dependent mechanisms, while the activity and bioavailability of the gasotransmitters themselves are also subject to oxidative modification. Within vascular cells, the family of nicotinamide adenine dinucleotide phosphate oxidases (NAPDH oxidases/Noxs) have emerged as functionally significant sources of regulated O2- and H2O2 production and accordingly, direct associations between Nox-generated oxidants and the functions of specific gasotransmitters are beginning to be identified. This review focuses on the current knowledge of the redox-dependent mechanisms which regulate the generation and activity of these gases, with particular reference to their roles in angiogenesis.

Giant mediastinal liposarcoma: a case report.

Liposarcomas are extremely rare in the mediastinum. They may achieve considerable size before causing any symptoms. Mediastinal liposarcomas may invade surrounding structures like the pericardium or the superior vena cava. Complete surgical excision is the optimal treatment in resectable cases. Excision of adjacent structures like the pericardium may be needed if the tumor infiltrates them. We report on a case of a giant liposarcoma of the mediastinum involving both hemithoraces and extending into the neck, which was successfully managed by complete surgical excision.

Superior vena cava syndrome: A radiation oncologist's perspective.

Superior vena cava syndrome is referred to as a constellation of symptoms and signs caused by obstruction of superior vena cava. It can occur due to both benign and malignant causes with the latter being the predominant. There is a paradigm shift in the approach to manage this condition. It is no longer considered a medical emergency and histological diagnosis is necessary before treatment. This article reviews the causes, symptoms, pathophysiology, and overall management policy which have changed over decades.

Heat shock protein induced TCR gammadelta gene rearrangements in patients with oral cancer.

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.

Juvenile nasopharyngeal angiofibroma: a single institution study.

BACKGROUND: Juvenile nasopharyngeal angiofibroma (JNA) is a rare tumor of adolescent males and there is a paucity of Indian studies on this subject. AIMS: To present the experience of management of JNA at a single institution. SETTING AND DESIGN: This is a retrospective observational study of patients with JNA who presented at the Tata Memorial Hospital between May 1988 and August 2001. MATERIALS AND METHODS: Thirty-two patients with JNA were treated in the study period. Since the time period was prolonged and diagnostic and therapeutic protocols had undergone many changes, the patients were divided into two groups, namely 1988-1996 and 1997-2001. The age distribution, disease patterns, management approaches and treatment outcomes of patients in the two groups were recorded. Statistical analyses were done using students 't' test and test for proportion. RESULTS: The mean age at presentation was 16 years and more than 90% of the patients had Stage III or IV disease. Preoperative embolization was carried out in 19 patients. The surgical approaches used were median maxillectomy, infratemporal fossa, transpalatal, maxillary swing and craniofacial approach. The recurrence rate, complete resection rate and cure rates were 12.5%, 41% and 63% respectively. CONCLUSION: Surgery is the mainstay of treatment of JNA. Preoperative embolization and newer surgical approaches result in less blood loss and complete resection. Aggressive re-resection should be done for resectable recurrences reserving radiotherapy for unresectable, recurrent/ residual disease.