Biotechnological applications of a surfactant protein, ranaspumin-2

Surfactant activity is generally associated with small molecules rather than biological macromolecules like proteins. Only a few proteins have good intrinsic surfactant activity, an example being the natural surfactant ranaspumin 2 (Rsn2) from the foam nests of the túngara frog. In solution, Rsn2 has a hydrophobic core and hydrophilic exterior, but when Rsn2 comes in contact with an air-water interface, it changes conformation to expose its hydrophobic core to interact with the air and present a hydrophilic face to the water. The unique combination of biocompatibility along with surface activity offers the possibility of developing biomedical applications based on Rsn2. Some of the possible applications, including cell patterning, functionalising scaffolds and stabilising droplets, have been explored in the work described in this thesis. The ability of Rsn2 to coat hydrophobic surfaces persistently, rendering them wettable and the nature of coating and interaction with the surfaces were investigated. This formed the basis for the development of a method to coat a range of hydrophobic polymers, which are commonly used for biomedical applications. These Rsn2 coated surfaces were tested for their capability to control cell adhesion on surfaces which usually do not support cell adhesion. Rsn2 coating was demonstrated to promote, and thus allowed the spatial control over, cell adhesion on otherwise non-cell compatible surfaces. The potential of Rsn2 to be used as a protein fusion partner for the production of further functionalised cell engineering substrates was explored by constructing five different integrin binding sequence (IBS)-Rsn2 conjugates. Specific IBS-Rsn2 proteins proved successful in increasing the adhesion and biomineralising potential of osteoblasts isolated from neonatal rats. In addition, Rsn2's ability to stabilise microscopic oil droplets were investigated. Rsn2 stabilised oil droplets were stable for more than six months. Thus, the surfactant properties of Rsn2 can be used for many potential biomedical applications

Distinct and Shared Roles of β-Arrestin-1 and β-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

Background: The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of β-arrestin-1/β-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires β-arrestins. The role of β-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. Methodology/Principal Findings: We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of β-arrestin-1 and β-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing β-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing β-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of β-arrestin-1, β-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10-30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in β-arrestin1/2 knockdown cells. Conclusion/Significance: This study demonstrates distinct roles for β-arrestins-1 and β-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both β-arrestin-1 and β-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by β-arrestins in human mast cells. © 2011 Vibhuti et al

Procedural and physical interventions for vaccine injections systematic review of randomized controlled trials and quasi-randomized controlled trials

Background: This systematic review evaluated the effectiveness of physical and procedural interventions for reducing pain and related outcomes during vaccination. Design/Methods: Databases were searched using a broad search strategy to identify relevant randomized and quasi-randomized controlled trials. Data were extracted according to procedure phase (preprocedure, acute, recovery, and combinations of these) and pooled using established methods. Results: A total of 31 studies were included. Acute infant distress was diminished during intramuscular injection without aspiration (n=313): standardized mean difference (SMD) -0.82 (95% confidence interval [CI]: -1.18, -0.46). Injecting the most painful vaccine last during vaccinations reduced acute infant distress (n=196): SMD -0.69 (95%CI: -0.98, -0.4). Simultaneous injections reduced acute infant distress compared with sequential injections (n=172): SMD -0.56 (95%CI: -0.87, -0.25). There was no benefit of simultaneous injections in children. Less infant distress during the acute and recovery phases combined occurred with vastus lateralis (vs. deltoid) injections (n=185): SMD -0.70 (95%CI: -1.00, -0.41). Skin-to-skin contact in neonates (n=736) reduced acute distress: SMD -0.65 (95% CI: -1.05, -0.25). Holding infants reduced acute distress after removal of the data from 1 methodologically diverse study (n=107): SMD -1.25 (95% CI: -2.05, -0.46). Holding after vaccination (n=417) reduced infant distress during the acute and recovery phases combined: SMD -0.65 (95% CI: -1.08, -0.22). Self-reported fear was reduced for children positioned upright (n=107): SMD -0.39 (95% CI: -0.77, -0.01). Non-nutritive sucking (n=186) reduced acute distress in infants: SMD -1.88 (95% CI: -2.57, -1.18). Manual tactile stimulation did not reduce pain across the lifespan. An external vibrating device and cold reduced pain in children (n=145): SMD -1.23 (95% CI: -1.58, -0.87). There was no benefit of warming the vaccine in adults. Muscle tension was beneficial in selected indices of fainting in adolescents and adults. Conclusions: Interventions with evidence of benefit in select populations include: no aspiration, injecting most painful vaccine last, simultaneous injections, vastus lateralis injection, positioning interventions, non-nutritive sucking, external vibrating device with cold, and muscle tension

Distinct and Shared Roles of β-Arrestin-1 and β-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

BACKGROUND: The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of β-arrestin-1/β-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires β-arrestins. The role of β-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of β-arrestin-1 and β-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing β-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing β-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of β-arrestin-1, β-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10-30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in β-arrestin1/2 knockdown cells. CONCLUSION/SIGNIFICANCE: This study demonstrates distinct roles for β-arrestins-1 and β-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both β-arrestin-1 and β-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by β-arrestins in human mast cells

An asymptomatic presentation of gastric outlet obstruction secondary to congenital antral web in an extremely preterm infant

A case of gastric outlet obstruction secondary to antral web in a preterm infant born at 25 weeks gestation is reported. The diagnosis was suspected on plain abdominal radiograph performed postnatally to confirm position of the umbilical catheters. On the initial radiograph (at age 1 h), a dilated stomach with a gasless abdomen was noted. A repeat chest and abdominal radiograph performed 24 h later due to increased ventilatory requirements showed persistence of this finding and upper gastrointestinal obstruction was suspected. An upper gastrointestinal contrast study confirmed the diagnosis of gastric outlet obstruction. The infant underwent a curative pyloroplasty on day 11 of life. The postoperative course was uneventful

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed. Copyright (c) 2006 John Wiley & Sons, Ltd

The role of context in shaping HIV testing and prevention engagement among urban refugee and displaced adolescents and youth in Kampala, Uganda: findings from a qualitative study

OBJECTIVE: To explore experiences, preferences and engagement with HIV testing and prevention among urban refugee and displaced adolescents and youth in Kampala, Uganda, with a focus on the role of contextual factors in shaping access and uptake. METHODS: This qualitative community‐based study with urban refugee and displaced youth aged 16–24 living in Kampala’s informal settlements involved five focus groups (FG), including two with young women, two with young men, and one with sex workers from March to May 2019. We also conducted five in‐depth key informant interviews. We conducted thematic analysis informed by Campbell and Cornish’s conceptualisation of material and symbolic contexts. RESULTS: Refugee/displaced youth participants (n = 44; mean age: 20.25, SD: 2.19; men: n = 17; women: n = 27) were from the Democratic Republic of Congo (n = 29), Rwanda (n = 11), Burundi (n = 3) and Sudan (n = 1). Participant narratives reflected material and symbolic contexts that shaped HIV testing awareness, preferences and uptake. Material contextual factors that presented barriers to HIV testing and prevention engagement included transportation costs to clinics, overcrowded living conditions that limited access to private spaces, low literacy and language barriers. Symbolic contexts that constrained HIV testing engagement included medical mistrust of HIV testing and inequitable gender norms. Religion emerged as an opportunity to connect with refugee communities and to address conservative religious positions on HIV and sexual health. CONCLUSION: Efforts to increase access and uptake along the HIV testing and prevention cascade can meaningfully engage urban refugee and displaced youth to develop culturally and contextually relevant services to optimise HIV and sexual health outcomes