Modulation of transglutaminase 2 by pituitary adenylyl cyclase activating polypeptide and nerve growth factor in neuroblastoma cells: a role in cell survival and neurite outgrowth

Several studies have reported that the multifunctional enzyme tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. Recently, TG2 enzymatic activity has been shown to be regulated by protein kinases such as PKA and PKC, modulating through G-protein coupled receptors (GPCRs). However, the regulation of TG2 following stimulation of GPCRs coupled to PKC and PKA activation are not fully understood. In neuronal cells, the neurotrophic factors; pituitary adenylate cyclase activating polypeptide (PACAP) and nerve growth factor (NGF) promote the differentiation, maturation, neurite outgrowth and survival of neurons via their GPCR's; the pituitary adenylate cyclase type 1 receptor (PAC1) and the tyrosine kinase receptor (TrKA) including multiple protein kinase signalling pathways. As some of these kinase pathways are associated with modulation of intracellular TG2 activity, it is conceivable to hypothesised that PACAP and NGF directly regulates TG2 activity. If this was found to be the case, it sought to determine whether the PACAP and/or NGF-induced neurite outgrowth and modulation of neuronal cell survival involved TG2. The main aims of this study were to investigate the molecular mechanisms underlying TG2 modulation by PACAP and NGF and its role in neurite outgrowth and neuroprotection in differentiating mouse N2a and human SHSY5Y neuroblastoma cells. In the first part, the regulation of TG2 transamidase activity by PACAP-27 and NGF in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells was assessed. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of iotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. In the second part of this study, the role of TG2 in PACAP-27 and NGF-induced cytoprotection was investigated by monitoring hypoxia-induced cell death and assessing MTT reduction, LDH release, and caspase-3 activity. The neurite outgrowth was assessed using high-throughput analysis of immunofluorescently stained cells. In differentiating N2a and SH-SY5Y cells, stimulation of PAC1 and TrKA receptor with PACAP-27 and NGF respectively, resulted in a time- and concentration- dependent activation of TG2 amine incorporation and protein crosslinking activity, which was abolished by TG2 inhibitors Z-DON and R283. Responses to PACAP-27 were attenuated mainly by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and by removal of extracellular Ca2+. Responses to NGF were abolished by MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and PKC (Ro 31-8220). Immunoprecipitation technique showed that both PACAP-27 and NGF triggered the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by inhibition of MEK1/2 and PKA for PACAP and MEK1/2, PKB, PKC and removal of extracellular Ca2+ for NGF. Fluorescence microscopy demonstrated that PACAP-27 and NGF induced in situ TG2 activity. TG2 inhibition blocked PACAP-27- and NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth. Taken together, this study highlights for the first time the importance of TG2 in the cellular functions of PACAP and NGF in neuronal cells and provides initial characterisation of the molecular mechanisms possibly involved

Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: a role in cell survival and neurite outgrowth

NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1–10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs

Assessment of the Community Pharmacists’ Knowledge and Attitudes Toward Pain and Pain Management in Saudi Arabia

Nasser M Alorfi,1 Ahmed M Ashour,1 Alanood S Algarni,1 Faris A Alsolami,2 Abdulrhman M Alansari,3 Mansour Tobaiqy4 1Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; 2Khulais General Hospital, Ministry of Health, Makkah, Saudi Arabia; 3Alsafiah Health Care Center, Ministry of Health, Madinah, Saudi Arabia; 4Department of Pharmacology, College of Medicine, University of Jeddah, Jeddah, Saudi ArabiaCorrespondence: Nasser M Alorfi, Umm Al-Qura University, Al Abdeyah, Mecca, 24381, Saudi Arabia, Email [email protected]: Pain is a global health issue that affects an individual’s quality of life. Its alleviation and management will enhance patients’ experience. Community pharmacists can help manage pain severity through their valuable roles in medical teams and by managing the consequences of pain.Objective: This study aimed to evaluate community pharmacists’ knowledge and attitudes toward pain and pain management in Saudi Arabia.Methods: A cross-sectional study was performed to evaluate community pharmacists’ knowledge and attitudes toward pain and pain management in Saudi Arabia. Pharmacists aged ≥ 21 years, with a degree in pharmacy were included in this study. Each respondent participated in an online survey covering cancer-oriented pain and assessment of pain; pharmacology; abuse of substances; and physical dependence. An independent t-test and One-way ANOVA, with least significant difference as a post-hoc test, were employed, in addition to the General Linear Regression Model using Main Effect as the model.Results: This study revealed that the pain-related knowledge and attitude among community pharmacists in Saudi Arabia were inadequate. Age (p = 0.003), work experience (p = 0.036), nature of work (p = 0.001), and work location (p = 0.003) were determined as significant factors affecting their overall knowledge and attitude toward pain.Conclusion: Overall, attempts to expand community pharmacists’ knowledge and foster an appropriate attitude toward pain management among them in Saudi Arabia are highly recommended. Additional academic courses, studies, and tailored neuroscience courses will improve their awareness and knowledge of pain and pain management.Keywords: pharmacist, pain, knowledge, attitude, practic

Shortening Door-to-Balloon Time: The Use of Ambulance versus Private Vehicle for Patients with ST-Segment Elevation Acute Myocardial Infarction

Abdulrhman Saleh Alghamdi,1,2 Abdullah Alshibani,1,2 Meshary Binhotan,1,2 Meshal Alharbi,1,2 Saleh S Algarni,2,3 Mohammed Musaed Alzahrani,1,2 Abdulmalik Nasser Asiri,1,2 Faisal Faleh Alsulami,1,2 Kamal Ayoub,2,4 Abdullah Alabdali1,2 1Department of Emergency Medical Services, College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 2King Abdullah International Medical Research Center, Riyadh, Saudi Arabia; 3Department of Respiratory Therapy, College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, 11481, Saudi Arabia; 4Ministry of the National Guard - Health Affairs, Riyadh, Saudi ArabiaCorrespondence: Abdulrhman Saleh Alghamdi, Emergency Medical Services, College of Applied Medical Sciences, Ext. 95186, Mail Code 3129, P.O. Box 3660, Riyadh, 11481, Saudi Arabia, Tel +96611 429 9999, Email [email protected]: Time is critical when dealing with acute myocardial infarction (AMI) patients in the Emergency Department (ED), as 90 min is crucial for overall health. Using non-EMS transportation for critical patients, such as patients with acute myocardial infarction, to a hospital might delay the rapid identification of the underlying medical disease and initiating definitive treatment. We aim to evaluate the association between the mode of transportation and the D2B time in patients presenting at the ED with AMI.Patients and Methods: We conducted a retrospective cohort study with patients who presented at ED with AMI and underwent percutaneous coronary intervention (PCI). The participants were patients with confirmed AMI at the ED of King Abdullah Medical City (KAMC) from January 2019 to December 2019.Results: In total, 162 AMI patients were enrolled in the study and divided based on the method of transportation. Less than half (n=65, 40.1%) were transported with an ambulance and 97 (59.9%) patients with a private car. The door-to-balloon (D2B) time for the ambulance group was 93.6± 38.31 minutes, and the private car group was 93.8± 30.88 minutes.Conclusion: There was no statistical significance when comparing the D2B time between the private car group and the ambulance group (P = 0.1870). Finally, ambulance transport significantly shortened the time to first ED physician contact. However, it was not associated with shortened D2B time when compared to private vehicle transport.Keywords: chest pain, acute myocardial infarction, ambulance, door to balloon, emergency departmen

Are Antisense Long Non-Coding RNA Related to COVID-19?

Fighting external pathogens relies on the tight regulation of the gene expression of the immune system. Ferroptosis, which is a distinct form of programmed cell death driven by iron, is involved in the enhancement of follicular helper T cell function during infection. The regulation of RNA is a key step in final gene expression. The present study aimed to identify the expression level of antisense lncRNAs (A2M-AS1, DBH-AS1, FLVCR1-DT, and NCBP2AS2-1) and FLVCR1 in COVID-19 patients and its relation to the severity of the disease. COVID-19 patients as well as age and gender-matched healthy controls were enrolled in this study. The expression level of the antisense lncRNAs was measured by RT-PCR. Results revealed the decreased expression of A2M-AS1 and FLVCR1 in COVID-19 patients. Additionally, they showed the increased expression of DBH-AS1, FLVCR1-DT, and NCBP2AS2. Both FLVCR1-DT and NCBP2AS2 showed a positive correlation with interleukin-6 (IL-6). DBH-AS1 and FLVCR1-DT had a significant association with mortality, complications, and mechanical ventilation. A significant negative correlation was found between A2M-AS1 and NCBP2AS2-1 and between FLVCR1 and FLVCR1-DT. The study confirmed that the expression level of the antisense lncRNAs was deregulated in COVID-19 patients and correlated with the severity of COVID-19, and that it may have possible roles in the pathogenesis of this disease

Exploring the Relationship Between Stress and Satisfaction During Clinical Training Among Respiratory Therapy Students: A Nationwide Cross-Sectional Survey

Rayan Siraj,1 Abdulelah M Aldhahir,2 Jaber S Alqahtani,3 Samah Bakhadlq,3 Saeed Alghamdi,4 Abdullah A Alqarni,5 Turki M Alanazi,6,7 Abdullah Alruwaili,6,7 Saleh S Algarni,8,9 Abdulrhman S Alghamd,10 Mushabbab Alahmari,11 Abdulmajeed Baogbah,12 Nawaf A Alsolami,13 Mufleh Alrougi,14 Kamal Hamed Al Khodidi,15 Fahad Alahmadi16 1Department of Respiratory Care, College of Applied Medical Sciences, King Faisal University, Al-Ahasa, 31982, Saudi Arabia; 2Respiratory Therapy Department, Faculty of Applied Medical Sciences, Jazan University, Jazan, 45142, Saudi Arabia; 3Department of Respiratory Care, Prince Sultan Military College of Health Sciences, Dammam, 34313, Saudi Arabia; 4Department of Clinical Technology, Respiratory Care Program, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia; 5Department of Respiratory Therapy, Faculty of Medical Rehabilitation Sciences, King Abdulaziz University, Jeddah, 22254, Saudi Arabia; 6College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences, Al Ahsa, 31982, Saudi Arabia; 7King Abdullah International Medical Research Center, Al Ahsa, 31982, Saudi Arabia; 8Department of Respiratory Therapy, College of Applied Medical Sciences, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 9King Abdullah International Medical Research Center, Riyadh, Saudi Arabia; 10Department of Rehabilitation Science, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia; 11Department of Respiratory Therapy, College of Applied Medical Sciences, University of Bisha, Bisha, Saudi Arabia; 12Department of Respiratory Therapy, Byrdine F. Lewis College of Nursing and Health Professions, Georgia State University, Atlanta GA, 30303, USA; 13Department of Respiratory Therapy, East Jeddah Hospital, Jeddah, Saudi Arabia; 14Department of Respiratory Care, King Abdulaziz Specialist Hospital, Taif, Saudi Arabia; 15Respiratory Therapy Unit, Children’s Hospital, Taif, Saudi Arabia; 16Respiratory Therapy Department, College of Medical Rehabilitation Sciences, Taibah University, Madinah, Saudi ArabiaCorrespondence: Rayan Siraj, Department of Respiratory Care, College of Applied Medical Sciences, King Faisal University, Al-Ahasa, 31982, Saudi Arabia, Email [email protected]: Although clinical training is an important component of healthcare education, it is nevertheless a significant source of stress for students. There is limited information on stress and satisfaction perceived by clinical-level undergraduate students studying respiratory therapy (RT) in Saudi Arabia.Methods: A descriptive, cross-sectional study design with an online questionnaire was employed to conduct this study. Data collection occurred during the academic year 2022– 2023 among RT students throughout Saudi Arabia. Patricians responded to demographic questions, the Students Stress Scale and Students Professional Satisfaction questionnaires. Descriptive, inferential, and correlational statistics were used to analyze the collected responses.Results: A total of 1001 undergraduate RT students completed the online survey. RT students and interns had an overall moderate to high stress level (mean (SD); 3.55 (0.49)), while satisfaction was perceived as mild to moderate (mean (SD): 2.56 (0.65)). In addition, 38% of the study participants have considered quitting the RT program. Female students showed higher stress levels in the following domains: inadequate knowledge and training, adverse and embarrassing experiences, clinical supervision, patients’ pain, and Education–reality conflict (p< 0.005) compared to male students. Additionally, students who considered quitting the RT program revealed higher stress levels in all domains (p< 0.005). There were negative correlations between satisfaction and stress domains: inadequate knowledge and training (r = − 0.32; p = 0.001), adverse and embarrassing experience (r = − 0.31; p = 0.025), close supervision (r = − 0.24; p = 0.001), insufficient hospital resources (r = − 0.30; p 0.002), patients’ pain and suffering (r = 0.28; p = 0.04), and education – reality conflict (r = − 0.30; p = 0.001).Conclusion: During clinical training, respiratory therapy students experience moderate to high-stress levels and low satisfaction. There need to be tailored interventions to reduce stress and intention to quit and increase students’ clinical experience satisfaction.Keywords: stress, satisfaction, clinical training, intention to qui

An extended random-sets model for fusion-based text feature selection

Selecting features that represent a specific corpus is important for the success of many machine learning and text mining applications. In information retrieval (IR), fusion-based techniques have shown remarkable performance compared to traditional models. However, in text feature selection (FS), popular models do not consider the fusion of the taxonomic features of the corpus. This research proposed an innovative and effective extended random-sets model for fusion-based FS. The model fused scores of different hierarchal features to accurately weight the representative words based on their appearance across the documents in the corpus and in several latent topics. The model was evaluated for information filtering (IF) using TREC topics and the standard RCV1 dataset. The results showed that the proposed model significantly outperformed eleven state-of-the-art baseline models in six evaluation metrics