Thiol Specific Fluorogenic Agents for Live Cell Subcellular Organelle Thiol Imaging

Thiols or sulfhydryl groups (-SH) play a significant role in protein structures and functions. The unique properties of a thiol group are the basis for its roles in cellular functions. Structurally, thiols are divided into protein thiols (PSH) and non-protein thiols (NPSH). NPSH include glutathione, cysteine, and homocysteine. Thiols are distributed in an uneven manner in the cell and subcellular organelles. Disturbance of thiol levels has been associated with serious diseases such as cancer, aging, cardiovascular diseases, Alzheimer disease, liver damage, and edema. The significant roles of thiols in the biological system have stimulated the development of various approaches to monitor and detect thiols. These approaches include enzymatic assays, colorimetric assays, gel electrophoresis, LC/MS, and HPLC. The major drawback of these conventional analytical approaches is that these methods require the cell to be homogenized before analysis. The breakage of cells is timeconsuming and can cause a loss of information. Thus, developing an approach to determine thiol status in intact live cells will provide a unique advantage over the conventional analytical methods. Fluorescence microscopy has been successfully used in determining analytes in intact live cells. The main challenge for using fluorescence microscopy in detecting thiols is to turn thiols into fluorescent molecules for detection. This challenge results in the necessity to develop fluorescent/fluorogenic agents that exhibit selectivity and specificity for thiols and are capable of turning thiols to fluorescent molecules to facilitate detection and quantification. Most of our current knowledge on thiols’ functions/dysfunctions at cellular or subcellular levels were derived from the data obtained from conventional analytical methods that involve breakage of cells. A fluorescence probe that can be used to detect subcellular thiols in live cells will be a valuable tool to provide better understanding of thiols’ roles in the function and dysfunction of the subcellular organelles. In this dissertation, we will present our work related to the development of thiol specific fluorogenic agents for cell surface thiol imaging and lysosomal thiol imaging in live cells. The rational design, synthesis, and determination of TBONES and TBOZEN as thiol specific fluorogenic reagents for cell surface thiol imaging in live cells will be presented. However, TBONES and TBOZEN failed to image cell surface thiols due to their inability to react with PSH. Interestingly, TBONES and TBOZEN turned out to be able to image thiols in lysosomes in live cells. Based on the work of TBONES and TBOZEN, we designed TIMBOS as a thiol specific and fluorogenic agent for lysosomal thiol imaging in live cells. TIMBOS was successfully synthesized and characterized to be thiol specific and fluorogenic. TIMBOS was demonstrated to effectively image NPSH in lysosomes in live cells and to detect NPSH change in lysosomes in a quantitative manner. In summary, we have synthesized and characterized three rationally designed thiol specific fluorogenic reagents. These three reagents were able to image NPSH and to detect NPSH change in a quantitative manner in lysosomes in live cells. These reagents will be useful tools in exploring the roles of lysosomal thiols in cellular function/dysfunction

Warranty and Maintainability Analysis for Sensor Embedded Remanufactured Products in Reverse Supply Chain Environment

Remanufactured products are very popular with consumers due to their appeal to offer the latest technology with lower prices compared to brand new products. The quality of a remanufactured product induces hesitation for many consumers, in regards to its efficacy and reliability. One stratagem that remanufacturers could employ to encourage customer security are product warranties. This paper studies and scrutinizes the impact that would be had by offering renewing warranties on remanufactured products. This study was able to determine the optimal costs of warranty for two-dimensional non-renewable warranty offered on remanufactured products using the simulation model and design of experiments

Investigation of Galectin-3 interaction with N. meningitidis and its dimerization with laminin receptor

Meningococcal meningitis from the causative organism Neisseria meningitidis is the leading cause of meningitis globally. This bacterium is among a limited number of pathogens that have the propensity to cross the blood brain barrier (BBB) vasculature causing meningitis. It has been recently demonstrated that Neisseria meningitidis targets the laminin receptor (37 LRP/67 LR) on the surface of human brain microvascular endothelial cells, and two meningococcal outer membrane proteins, PorA and PilQ, have been identified as bacterial ligands. Interestingly, this interaction is hypothesized to underlie meningococcal tropism for the central nervous system (CNS). There are two isoforms of laminin receptor; monomeric 37 kDa laminin receptor precursor (37 LRP) and mature 67 kDa laminin receptor (67 LR). The relationship between the 67 LR and its precursor 37 LRP is not completely understood, but previous observations have suggested that 37 LRP can undergo homo- and/or hetero- dimerization with Galectin-3 (Gal-3) to form mature 67 LR. Gal-3 is the only member of the chimera-type group of galectins, and has one C-terminal carbohydrate recognition domain (CRD) that is responsible for binding the ß-galactoside moieties of mono- or oligosaccharides on several host and bacterial molecules, including neisserial lipooligosaccharide (LOS). To identify the LOS-independent meningococcal ligands that bind Gal-3, binding of lactose liganded Gal-3 and CRD with meningococci was investigated using ELISA assay. Neisseria meningitidis bound lactose liganded Gal-3 significantly more than H. pylori, which is known to bind Gal-3 via LPS. This binding was not inhibited by increasing concentrations of lactose. Also the lactose liganded CRDof Gal-3 bound meningococci but to a lesser extent than full molecule. Importantly, binding of Gal-3 was conserved among 25 meningococcal clinical isolates tested in the current study. A meningococcal mutant lacking the glycosyltransferase required for chain elongation from the core lipid A-(KDO)2-Hep2 showed reduced binding to lactose-liganded Gal-3, but binding was not abolished indicating that the meningococcal-Gal-3 binding was not entirely LOS-dependant. Using a re-tagging approach, meningococcal PilQ and PilE proteins were identified as Gal-3 binding ligands. Mutation of the genes encoding either of these two molecules in strain MC58 led to a significant reduction in Gal-3 binding. PilQ is not known to be glycosylated, therefore its interaction with Gal-3 is likely to be protein-mediated. PilE is post-translationally glycosylated and deletion of the pilin glycosylation genes pglC and/or pglL dramatically reduced bacterial-Gal-3 binding. Given the binding of meningococcal PilQ to 37 LRP/67 LR and Gal-3, this study sought to investigate possible dimerization between 37 LRP and Gal-3 to form 67 LR. Double immunofluorescence staining of endogenous receptors revealed colocalization of 67 LR with its precursor and both of them with Gal-3 in HBMECs, astrocyte and COS7 cells. Moreover, co-expression of 37 LRP and Gal-3 fused to different fluorescent proteins indicated colocalization of these receptors in COS7 cells. Using bimolecular fluorescence complementation (BiFC) assays, the presence of 67 LR in homo- and hetero-dimer forms with Gal-3 has been confirmed in different cell lines. In addition, the recombinant laminin receptor bound Gal-3 and its CRD to comparable level. Further investigation for Gal-3 and 37 LRP dimerization mechanism revealed that the conserved cysteine (C173A) within the CRD of human Gal-3, which is known to abolish disulphide-mediated dimerization of murine Gal-3, is critical for Gal-3 homo- and hetero-dimerization with 37 LRP, whereas neither of the two cysteines on 37LR (cys148 and cys163) are required for dimerization. To examine the role of Gal-3 in meningococcal interaction with host cells, the adhesive and invasive capacities of meningococci were compared between Gal-3 transfected and non-transfected neuroblastoma cell line (N2a) cells. Transient expression of Gal-3 in mouse N2a cells significantly enhanced meningococcal invasion when compared with non-transfected cells. Moreover, infection of CD46-expressing transgenic mice with meningococcal strain MC58 significantly increased the expression of Gal-3 and 37 LRP in the brain. This work also attempts to study whether the 37 LRP/67 LR meningococcal ligands (rPorA, loop 4 of PorA and rPilQ) have any influence on the surface level of 67 LR and Gal-3. As indicated by flow cytometry analysis, recruitments of 67 LR and Gal-3 to the surface of HBMECs were increased in cells incubated with rPilQ, Loop 4 of PorA and more prominently rPorA. To examine these results in more detail, effect of each of these ligands on 37 LRP expression was investigated using qPCR. Loop4 of PorA and rPilQ induced 37 LRP expression significantly more than PBS. Although there was a trend for an increase in 37 LRP expression with treatment with rPorA, the difference was not statistically significant (p = 0.1507). Further investigation in future study for the effect of these bacterial adhesins on Gal-3 gene expression will be of great value. Collectively, these data revealed the capacity of Gal-3 to target meningococcal PilQ and PilE, as well as the previously known LOS and showed the importance of Gal-3 in the meningococcal-host cell interaction. This interaction may be part of host-cell defence against the organism, and/or, conversely, it may be part of a strategy adopted by the organism to modulate the host response and facilitate its invasion. Remarkably, the current findings also demonstrated the existence of 67 LR as homo- and hetero- dimer with Gal-3. This dimerization of two meningococcal host receptors may help to extend spectrum of their bacterial adhesins which may act cooperatively or synergistically at different stages of infection. Besides, the expression pattern of these receptors may suggest specific receptor repertoire in the BBB which might contribute in meningococcal tropism for the CNS

Investigation of Galectin-3 interaction with N. meningitidis and its dimerization with laminin receptor

Meningococcal meningitis from the causative organism Neisseria meningitidis is the leading cause of meningitis globally. This bacterium is among a limited number of pathogens that have the propensity to cross the blood brain barrier (BBB) vasculature causing meningitis. It has been recently demonstrated that Neisseria meningitidis targets the laminin receptor (37 LRP/67 LR) on the surface of human brain microvascular endothelial cells, and two meningococcal outer membrane proteins, PorA and PilQ, have been identified as bacterial ligands. Interestingly, this interaction is hypothesized to underlie meningococcal tropism for the central nervous system (CNS). There are two isoforms of laminin receptor; monomeric 37 kDa laminin receptor precursor (37 LRP) and mature 67 kDa laminin receptor (67 LR). The relationship between the 67 LR and its precursor 37 LRP is not completely understood, but previous observations have suggested that 37 LRP can undergo homo- and/or hetero- dimerization with Galectin-3 (Gal-3) to form mature 67 LR. Gal-3 is the only member of the chimera-type group of galectins, and has one C-terminal carbohydrate recognition domain (CRD) that is responsible for binding the ß-galactoside moieties of mono- or oligosaccharides on several host and bacterial molecules, including neisserial lipooligosaccharide (LOS). To identify the LOS-independent meningococcal ligands that bind Gal-3, binding of lactose liganded Gal-3 and CRD with meningococci was investigated using ELISA assay. Neisseria meningitidis bound lactose liganded Gal-3 significantly more than H. pylori, which is known to bind Gal-3 via LPS. This binding was not inhibited by increasing concentrations of lactose. Also the lactose liganded CRDof Gal-3 bound meningococci but to a lesser extent than full molecule. Importantly, binding of Gal-3 was conserved among 25 meningococcal clinical isolates tested in the current study. A meningococcal mutant lacking the glycosyltransferase required for chain elongation from the core lipid A-(KDO)2-Hep2 showed reduced binding to lactose-liganded Gal-3, but binding was not abolished indicating that the meningococcal-Gal-3 binding was not entirely LOS-dependant. Using a re-tagging approach, meningococcal PilQ and PilE proteins were identified as Gal-3 binding ligands. Mutation of the genes encoding either of these two molecules in strain MC58 led to a significant reduction in Gal-3 binding. PilQ is not known to be glycosylated, therefore its interaction with Gal-3 is likely to be protein-mediated. PilE is post-translationally glycosylated and deletion of the pilin glycosylation genes pglC and/or pglL dramatically reduced bacterial-Gal-3 binding. Given the binding of meningococcal PilQ to 37 LRP/67 LR and Gal-3, this study sought to investigate possible dimerization between 37 LRP and Gal-3 to form 67 LR. Double immunofluorescence staining of endogenous receptors revealed colocalization of 67 LR with its precursor and both of them with Gal-3 in HBMECs, astrocyte and COS7 cells. Moreover, co-expression of 37 LRP and Gal-3 fused to different fluorescent proteins indicated colocalization of these receptors in COS7 cells. Using bimolecular fluorescence complementation (BiFC) assays, the presence of 67 LR in homo- and hetero-dimer forms with Gal-3 has been confirmed in different cell lines. In addition, the recombinant laminin receptor bound Gal-3 and its CRD to comparable level. Further investigation for Gal-3 and 37 LRP dimerization mechanism revealed that the conserved cysteine (C173A) within the CRD of human Gal-3, which is known to abolish disulphide-mediated dimerization of murine Gal-3, is critical for Gal-3 homo- and hetero-dimerization with 37 LRP, whereas neither of the two cysteines on 37LR (cys148 and cys163) are required for dimerization. To examine the role of Gal-3 in meningococcal interaction with host cells, the adhesive and invasive capacities of meningococci were compared between Gal-3 transfected and non-transfected neuroblastoma cell line (N2a) cells. Transient expression of Gal-3 in mouse N2a cells significantly enhanced meningococcal invasion when compared with non-transfected cells. Moreover, infection of CD46-expressing transgenic mice with meningococcal strain MC58 significantly increased the expression of Gal-3 and 37 LRP in the brain. This work also attempts to study whether the 37 LRP/67 LR meningococcal ligands (rPorA, loop 4 of PorA and rPilQ) have any influence on the surface level of 67 LR and Gal-3. As indicated by flow cytometry analysis, recruitments of 67 LR and Gal-3 to the surface of HBMECs were increased in cells incubated with rPilQ, Loop 4 of PorA and more prominently rPorA. To examine these results in more detail, effect of each of these ligands on 37 LRP expression was investigated using qPCR. Loop4 of PorA and rPilQ induced 37 LRP expression significantly more than PBS. Although there was a trend for an increase in 37 LRP expression with treatment with rPorA, the difference was not statistically significant (p = 0.1507). Further investigation in future study for the effect of these bacterial adhesins on Gal-3 gene expression will be of great value. Collectively, these data revealed the capacity of Gal-3 to target meningococcal PilQ and PilE, as well as the previously known LOS and showed the importance of Gal-3 in the meningococcal-host cell interaction. This interaction may be part of host-cell defence against the organism, and/or, conversely, it may be part of a strategy adopted by the organism to modulate the host response and facilitate its invasion. Remarkably, the current findings also demonstrated the existence of 67 LR as homo- and hetero- dimer with Gal-3. This dimerization of two meningococcal host receptors may help to extend spectrum of their bacterial adhesins which may act cooperatively or synergistically at different stages of infection. Besides, the expression pattern of these receptors may suggest specific receptor repertoire in the BBB which might contribute in meningococcal tropism for the CNS

The Impacts of E-Commerce as a Service upon Fog Computing

Fog Computing is a technology that extends cloud computing and services to the edge of the network. It provides data, compute, storage and application services to the users like cloud. From kitchen equipment to aeroplane, started getting an IP address which has also been a part of internet. In the past few years, the great transmission of theoretical concept of different industries such as E-commerce into real application has been used by cloud computing. Based on adopted fog features and characteristics those are encouraging small companies that providing their E-commerce products to adopt their development into fog computing. In order to assist the E-commerce small companies with the right way to start with the basic requirements and upgrading their computing resources as their fog user base grows with time, herewith the impacts of Ecommerce as the services upon fog computing is proposed

A novel model of adoption of m-commerce in Saudi Arabia

The market of Saudi Arabia offers considerable potential for the success of mobile commerce (m-commerce) technology. In this context, this study aims to investigate the factors that influence the intentions of Saudi citizens and firms to use m-commerce technology. This study paper advances the literature by proposing a novel model to investigate the motivations behind adopting m-commerce in Saudi Arabia. The model defines factors that influence both customer and provider intentions of adopting m-commerce. The paper also outlines the methodology to be followed to evaluate the proposed model

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization

A comprehensive evaluation of safety and awareness in eyebrow microblading: a cross-sectional study

The importance of eyebrows in facial aesthetics has long been recognized, and eyebrows restoration is carried out by carefully depositing pigments into the epidermis. This cosmetic procedure is known as microblading which is very popular because of the progress in social media platforms. This study was designed to determine the awareness of eyebrow microblading, complication and laser removal in general population of Saudi Arabia. This was a cross-sectional study conducted in four different regions in Saudi Arabia (Asir, Qasim, Jawf, and Eastern province), from June 2023 to December 2023. Only females were included, and an online questionnaire was distributed. A convenient non-probability sampling method was adopted to gather data from participants. Data analysis was performed using SPSS version 21. A total of 705 participants were included in the study, with the majority 281 (39.9%) falling into the age group >35 years, and the mean age was 29.1±11.9 years. The study revealed a moderate level of awareness (65%) among participants regarding eyebrow microblading. However, alarming gaps regarding hygiene practices in microblading setups still exist. Notably, facing complications during the surgery was identified as a statistically significant factor associated with a higher level of awareness (p=0.049). Of the participants, 17.4% had undergone eyebrow microblading, with friends being the source of advice for 39.5% of them. Laser removal was chosen by more than 60% of microblading recipients, with varying degrees of pleasure and satisfaction reported. Eyebrow microblading is a successful cosmetic procedure with aesthetic advantages. However, this study also emphasizes the necessity for extensive public education regarding microblading, including its associated risks, restrictions, and appropriate practices. To ensure safe and ethical practices, stringent laws and licensing for certified professionals are also deemed essential

Perspectives, practices, and challenges of online teaching during COVID-19 pandemic: A multinational survey

The result of the movement restrictions during the COVID-19 pandemic was an impromptu and abrupt switch from in-person to online teaching. Most focus has been on the perception and experience of students during the process. The aim of this international survey is to assess staffs' perspectives and challenges of online teaching during the COVID-19 lockdown. Cross-sectional research using a validated online survey was carried out in seven countries (Brazil, Saudi Arabia, Jordan, Indonesia, India, the United Kingdom, and Egypt) between the months of December 2021 and August 2022, to explore the status of online teaching among faculty members during the COVID-19 pandemic. Variables and response are presented as percentages while logistic regression was used to assess the factors that predict levels of satisfaction and the challenges associated with online instruction. A total of 721 response were received from mainly male (53%) staffs. Most respondents are from Brazil (59%), hold a Doctorate degree (70%) and have over 10 years of working experience (62%). Although, 67% and 79% have relevant tools and received training for online teaching respectively, 44% report that online teaching required more preparation time than face-to-face. Although 41% of respondents were uncertain about the outcome of online teaching, 49% were satisfied with the process. Also, poor internet bandwidth (51%), inability to track students' engagement (18%) and Lack of technical skills (11.5%) were the three main observed limitations. Having little or no prior experience of online teaching before the COVID-19 pandemic [OR, 1.58 (95% CI, 1.35–1.85)], and not supporting the move to online teaching mode [OR, 0.56 (95% CI,0.48–0.64)] were two main factors independently linked with dissatisfaction with online teaching. While staffs who support the move to online teaching were twice likely to report no barriers [OR, 2.15 (95% CI, 1.61–2.86)]. Although, relevant tools and training were provided to support the move to online teaching during COVID-19 lockdown, barriers such as poor internet bandwidth, inability to track students’ engagement and lack of technical skills were main limitations observed internationally by teaching staffs. Addressing these barriers should be the focus of higher education institution in preparation for future disruptions to traditional teaching modes

Surgical Management of Thermal Injury: Narrative Review

Extensive burn care advanced over the past few decades to the point where burn victims can now often live. The goal of treating a severely burned patient nowadays is to help them return to their communities, families, and places of employment as fully participating members of society, rather than only preserving their life and ability to function. Burns are a common and difficult critical care issue. Specialized hospitals prioritize achieving optimal functional recovery, infection prevention, and patient stabilization. Over the past few decades, researches on burns have attracted a lot of attention. A number of significant discoveries have improved patient stability and reduced mortality, particularly in the case of younger patients and those with intermediate-degree burns. The presence of dead tissue over a burn wound hinders the healing process and serves as a breeding ground for bacteria. Consequently, clearing the eschar as soon as possible and getting a clean wound bed as soon as possible, can be regarded as the main objective to initiate the process of wound healing, either through autografting or spontaneous epithelization. This review article provides a comprehensive overview of the surgical management of thermal injuries. The article also discusses the importance of early surgical intervention, including debridement, skin grafting, and other surgical techniques. Additionally, it explores the latest advancements in surgical management and the potential future directions in this field. Overall, this review aims to provide a valuable resource for healthcare professionals involved in the care of patients with thermal injuries