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

Tectono-geomorphological evolution of the Northern Red Sea margins

The geodynamic evolution of continental rifts and rifted margins influences petroleum prospectivity. Rift studies have tended to focus on offshore domains, whilst a more holistic approach would also consider sub-aerial data, providing information on rift flank uplift, drainage evolution and sediment routing. The Neogene northern Red Sea rift allows examination of the interaction between geodynamics, tectonics and geomorphology. Here, its tectono-geomorphic evolution is assessed by integrating drainage inverse modelling, drainage analysis, low-temperature thermochronology and structural mapping. On the margin scale, inverse modelling shows an early uplift (~22-15 Ma) in the southern part of the northeastern Red Sea and northern Gulf of Suez margins, and a later uplift (~14-0 Ma) along the northeastern Red Sea/Gulf of Aqaba, Sinai and northern Egyptian Red Sea margins. A smaller scale (20-30 km) study using low-temperature thermochronology and structural mapping reveals that pre-existing structures of suitable orientation do not all show resolvable reactivation during Red Sea rifting. The present-day drainage records the interplay of basement heterogeneities, rift-related uplift and later uplift. North-directed pre-rift drainage was modified forming transfer, hangingwall and footwall catchments. Later uplift reorganised drainage by reversal and capture, changing catchment sizes and relocating catchment outlets. The early uplift is interpreted to have been driven by rifting with possible mantle support and the later uplift was driven largely by transform tectonics and dynamic support by mantle flow. The catchment distribution indicates that early northern Red Sea rifting was accommodated by SW-dipping faults, with polarity changing further north into, and within, the Gulf of Suez. This study benefits from the integration of several datasets, and highlights rift geodynamic complexity and the necessity to integrate surface and subsurface data to constrain sediment pathways for petroleum exploration

The Effects of Instrument Lubricants on the Physical and Mechanical Properties of Resin-Based Composites

Ph. D. ThesisUncured resin-based composites (RBCs) tend to stick to the placement instrument instead of cavity walls, potentially increasing void formation and margin discrepancy. Instrument lubricants (ILs) are used to overcome this problem, but they may affect the properties of RBC restorations. One hundred registered UK dentists were surveyed using a bespoke questionnaire to investigate their perspective on IL use and understand why and how they used them. Additionally, different laboratory investigations were conducted, based on the survey data, to test the effects of ILs on the physical and mechanical properties of RBCs. Two RBCs were treated with three classes of ILs —solvents, bonding agents and wetting resins —to investigate the effects different ILs have on the physical and mechanical properties of RBCs. Several areas were tested: degree of conversion, water uptake, Martens hardness, diametral tensile strength, microtensile bonding strength (μTBS), and the appearance of changes at the increment interface. The survey revealed that about 50% of the dentists used lubricants (32% response rate), of which bonding agents (67%) and wetting resins (33%) were most common. These were applied with microbrushes (47%) and by wiping the placement instruments (40%). The solvents, bonding agents and wetting resins created significant reductions in diametral tensile strength and Martens hardness, and increased water uptake compared to control groups fabricated without lubricants. The μTBS significantly reduced following treatment with solvents and bonding agents, but there was no reduction from the wetting resins. The respondents used lubricants to aid manipulation during placement. However, these materials have an impact upon physical and mechanical properties with solvents and bonding agents having the greater effect. Therefore, the use of ILs to manipulate the RBC should be limited or avoided.King Khalid Universit

Evaluation of multiple and single emission peak light emitting diode light curing units effect on the degree of conversion and microhardness of resin-based pit and fissure sealant

Indiana University-Purdue University Indianapolis (IUPUI)Objective: The objective was to assess a multiple emission peak light-emitting-diode (LED) light-curing unit (LCU) by measuring the polymerization efficiency through the degree of conversion (DC) and Knoop microhardness (KHN) of a resin-based pit and fissure sealant at various light curing times and two distances compared to a single emission peak LED LCU. Method: Sixty disks of resin-based pit and fissure sealant (Delton, DENTSPLY, York, PA) samples (6x1mm) were fabricated (n=5/LCU/group). Prepared samples were polymerized using 10, 20 and 40 second curing time at 2 or 4 mm curing distances. The irradiance and radiant exposure received on the top/bottom surfaces of the samples were measured using the Managing Accurate Resin Curing-Resin Calibrator (MARC-RC) system. The samples were stored at 37°C for one hour. Then, the DC (n=3/surface) and KHN (n=5/surface) measurements were collected on the top and bottom surfaces using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) and a microhardness tester (Instron) utilizing 25-gm at 10 seconds dwell time, respectively. Multiple-way ANOVA was performed followed by Tukey test (α=0.05). Result: The irradiance from the multiple emission peak LED LCU was significantly higher than the single emission peak LED LCU (1312.6 and 768.3 mW/cm2) respectively. Moreover, the multiple emission peak LED LCU displayed significantly higher DC (82.5%) and microhardness (26.2 KHN) compared to the single emission peak LED LCU (75.5% DC and 21.2 KHN) when curing samples at 2 and 4 mm curing distances assessed using 10, 20 and 40-second curing times. The 10 second cure at 4 mm showed significantly lower DC and KHN values compared to the other groups. Conclusion: The multiple emission peak LED LCU demonstrated significantly higher irradiance, DC and KHN compared to the single emission peak LED LCU on a resin-based pit and fissure sealant at 2 and 4 mm curing distances and 10, 20 and 40 second curing times. Therefore, the multiple emission peak LED LCU performed higher than the single emission peak LED LCU

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 co-existence of NAFLD and CHB is associated with suboptimal viral and biochemical response to CHB antiviral therapy: a systematic review and meta-analysis

Background and aimsChronic hepatitis B (CHB) and non-alcoholic fatty liver disease (NAFLD) are leading causes of liver-related morbidity and mortality. The interaction between these two disease processes is poorly defined and the impact of NAFLD on HBV-related cirrhosis and HCC remains unclear. The aim of this study was to evaluate the impact of NAFLD on response to antiviral CHB therapy to inform the debate on changing CHB treatment thresholds for these comorbid patients.MethodsStudies with a minimum of 50 adult CHB patients on nucleoside analogue therapy with or without concurrent NAFLD were identified from PubMed/Medline and EMBASE to February 21, 2023. Data extraction from each study included HBeAg and treatment status, diagnostic method of NAFLD, frequency of monitoring intervals, patient age, gender, grade of hepatic steatosis, BMI and metabolic comorbidities. The outcomes of interest, complete virological response (CVR), biochemical response (BR) and HBeAg loss/seroconversion, were recorded at each available monitoring interval. Comparing CHB-NAFLD and CHB-only groups, pooled odds ratios (OR) and 95% confidence intervals (CI) were calculated using random- or fixed-effects models depending on heterogeneity.ResultsFrom a search of 470 citations, we identified 32 potentially relevant papers. Overall, 11 studies, comprising 2580 unique patients, met the inclusion criteria of the meta-analysis. CHB-NAFLD patients exhibited significantly lower rates of CVR compared to CHB-only patients. This was demonstrated by an OR of 0.59 (0.38-0.93, p=0.001, I2 = 72%) at 12 months, which tapered off to an OR of 0.67 (0.48-0.95, p=0.02) at 60 months. CHB-NAFLD patients also exhibited significantly lower rates of BR compared to CHB-only patients, as demonstrated by ORs of 0.39 (0.24-0.62, p<0.0001, I2 = 53%) at 12 months and 0.33 (0.17-0.63, p=0.0008) at 24 months.ConclusionPatients with concurrent CHB and NAFLD experience delayed CVR to antiviral therapy and more persistent biochemical abnormalities in comparison to patients with CHB only. This supports the argument for earlier antiviral therapy in order to avert CHB complications in these multi-morbid patients, as the global disease burden of NAFLD continues to increase

STRESS REACTIONS AND COPING STRATEGY AMONG HEALTHCARE PROFESSIONALS DURING COVID-19 OUTBREAK

Background: Psychological impacts among healthcare professionals have increased significantly due to the increasing number of COVID-19 cases. This study aimed to identify stress and coping strategies among healthcare professionals in Saudi Arabia during the COVID-19 outbreak. Subjects and methods: A cross-sectional study online survey was conducted for health care professionals during a peak of COVID-19 from March to June 2020 at different healthcare institutions at KSA (n=342). Results: Sixty-five percent of responders often and always feel fears about infection and subsequent effects on themselves, the patient, and the family. 57% of them stated that they felt sometimes depressed mode and 47% anxiety during the outbreak. Eightyfour percent of the respondent always focusing on prevention as the first biosecurity measures such as hand-washing habits and using hand sanitizer, and 38.3% of them make sometimes relax and rest. While half of the responses (50%) sometimes had physical exercise. Also, thirty-eight percent joined sometimes community and/or group online chat groups, and 56.1% always keep contact with family and friends through social messaging or phone calls. Conclusion: Understanding this topic is important for healthcare organizations, effective strategies, and programs is needed to provide holistic staff care and wellbeing during outbreaks that focus on the value of mental and emotional support

STRESS REACTIONS AND COPING STRATEGY AMONG HEALTHCARE PROFESSIONALS DURING COVID-19 OUTBREAK

Background: Psychological impacts among healthcare professionals have increased significantly due to the increasing number of COVID-19 cases. This study aimed to identify stress and coping strategies among healthcare professionals in Saudi Arabia during the COVID-19 outbreak. Subjects and methods: A cross-sectional study online survey was conducted for health care professionals during a peak of COVID-19 from March to June 2020 at different healthcare institutions at KSA (n=342). Results: Sixty-five percent of responders often and always feel fears about infection and subsequent effects on themselves, the patient, and the family. 57% of them stated that they felt sometimes depressed mode and 47% anxiety during the outbreak. Eightyfour percent of the respondent always focusing on prevention as the first biosecurity measures such as hand-washing habits and using hand sanitizer, and 38.3% of them make sometimes relax and rest. While half of the responses (50%) sometimes had physical exercise. Also, thirty-eight percent joined sometimes community and/or group online chat groups, and 56.1% always keep contact with family and friends through social messaging or phone calls. Conclusion: Understanding this topic is important for healthcare organizations, effective strategies, and programs is needed to provide holistic staff care and wellbeing during outbreaks that focus on the value of mental and emotional support