A Nonadjuvanted Whole-Inactivated Pneumococcal Vaccine Induces Multiserotype Opsonophagocytic Responses Mediated by Noncapsule-Specific Antibodies.

Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children <5 years of age every single year, and is the leading cause of pneumonia-associated mortality globally. While the capsular polysaccharide (CPS)-based vaccine Prevnar13 prevents serious illness caused by 13 serotypes, ongoing Prevnar13 use has driven the emergence of nonincluded serotypes as major causes of infection and disease. To overcome this issue, we have developed a next-generation pneumococcal vaccine conferring serotype-independent protection. This vaccine shows equivalent or superior efficacy to Prevnar13, and performance was heightened when our vaccine was administered with no adjuvant. These findings should be considered for similar vaccines in development, as the benefit of adjuvant is often assumed and its automatic inclusion may be limiting product efficacy, resulting in potential abandonment of viable vaccine candidates, or prolonging their time to clinic.

Elucidating degradation mechanisms for a range of per- and polyfluoroalkyl substances (PFAS) via controlled irradiation studies.

Per- and polyfluoroalkyl substances (PFAS) are a challenging class of environmental pollutants due to a lack of available destructive remediation technologies. Understanding the fundamental mechanisms for degradation of PFAS is key for the development of field scalable and in-situ destructive based remediation technologies. This study aimed to elucidate and refine the current understanding of PFAS degradation mechanisms in water through a series of controlled gamma irradiation studies. Gamma irradiation of PFAS was performed using a cobalt-60 source in a batch irradiation up to 80 kGy at the Australian Nuclear Science and Technology Organisation. Perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), 6:2 fluorotelomer sulfonate (6:2 FTS), and a suite of thirteen different PFAS (including C4-C12 PFCAs, C4, C6, C8 PFSAs, and FOSA) were irradiated to investigate degradation, influence of pH, chain length, and transformation. High resolution mass spectrometry was used to identify more than 80 fluorinated transformation products throughout the degradation experiments. These included the -F/+H, -F/+OH, -F/CH2OH exchanged PFAS and n - 1 PFCA, amongst others. Given the reactive species present (hydroxyl radicals (·OH), hydrogen radicals (·H) and aqueous electrons (e-aq)), and the degradation products formed it was shown that aqueous electrons were the key reactive species responsible for initial PFAS degradation. Most importantly, based on degradation product formation, we found that the initial -F/+H does not have to occur at the α-fluoride (nearest the functional head group), rather occurring throughout the chain length leading to more complex degradation pathways than previously postulated. While our results support some of the reaction steps postulated in the literature, we have developed a unified 16 step and 3 pathway schematic of degradation supported by experimental observations.

Sterility of gamma-irradiated pathogens: a new mathematical formula to calculate sterilizing doses.

In recent years there has been increasing advocacy for highly immunogenic gamma-irradiated vaccines, several of which are currently in clinical or pre-clinical trials. Importantly, various methods of mathematical modelling and sterility testing are employed to ensure sterility. However, these methods are designed for materials with a low bioburden, such as food and pharmaceuticals. Consequently, current methods may not be reliable or applicable to estimate the irradiation dose required to sterilize microbiological preparations for vaccine purposes, where bioburden is deliberately high. In this study we investigated the applicability of current methods to calculate the sterilizing doses for different microbes. We generated inactivation curves that demonstrate single-hit and multiple-hit kinetics under different irradiation temperatures for high-titre preparations of pathogens with different genomic structures. Our data demonstrate that inactivation of viruses such as Influenza A virus, Zika virus, Semliki Forest virus and Newcastle Disease virus show single-hit kinetics following exposure to gamma-irradiation. In contrast, rotavirus inactivation shows multiple-hit kinetics and the sterilizing dose could not be calculated using current mathematical methods. Similarly, Streptococcus pneumoniae demonstrates multiple-hit kinetics. These variations in killing curves reveal an important gap in current mathematical formulae to determine sterility assurance levels. Here we propose a simple method to calculate the irradiation dose required for a single log10 reduction in bioburden (D10) value and sterilizing doses, incorporating both single- and multiple-hit kinetics, and taking into account the possible existence of a resistance shoulder for some pathogens following exposure to gamma-irradiation.

Superoxide radicals react with peptide-derived tryptophan radicals with very high rate constants to give hydroperoxides as major products.

Oxidative damage is a common process in many biological systems and proteins are major targets for damage due to their high abundance and very high rate constants for reaction with many oxidants (both radicals and two-electron species). Tryptophan (Trp) residues on peptides and proteins are a major sink for a large range of biological oxidants as these side-chains have low radical reduction potentials. The resulting Trp-derived indolyl radicals (Trp•) have long lifetimes in some circumstances due to their delocalized structures, and undergo only slow reaction with molecular oxygen, unlike most other biological radicals. In contrast, we have shown previously that Trp• undergo rapid dimerization. In the current study, we show that Trp• also undergo very fast reaction with superoxide radicals, O2•-, with k 1-2 × 109 M-1 s-1. These values do not alter dramatically with peptide structure, but the values of k correlate with overall peptide positive charge, consistent with positive electrostatic interactions. These reactions compete favourably with Trp• dimerization and O2 addition, indicating that this may be a major fate in some circumstances. The Trp• + O2•- reactions occur primarily by addition, rather than electron transfer, with this resulting in high yields of Trp-derived hydroperoxides. Subsequent degradation of these species, both stimulated and native decay, gives rise to N-formylkynurenine, kynurenine, alcohols and diols. These data indicate that reaction of O2•- with Trp• should be considered as a major pathway to Trp degradation on peptides and proteins subjected to oxidative damage.

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins.

Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.

Laparoscopic repair of a migrated adjustable gastric band connecting tube with colonic erosion.

Laparoscopic adjustable gastric bands are a popular and effective surgical option to treat morbid obesity. The overall complication rate is 10-20% and the most common complication is of 'slippage'. Although other complications such as gastric band migration and erosion have been reported, the phenomenon of a migrated gastric band connecting tube eroding into the colon (after port removal) is seldom reported in the literature. In this article we describe such a case of an incidentally found colonic erosion on colonoscopy and describe the subsequent laparoscopic repair, as well as a review of the literature.

Low Dose Gamma Irradiation Does Not Affect the Quality or Total Ascorbic Acid Concentration of "Sweetheart" Passionfruit (Passiflora edulis).

Passionfruit (Passiflora edulis, Sims, cultivar "Sweetheart") were subject to gamma irradiation at levels suitable for phytosanitary purposes (0, 150, 400 and 1000 Gy) then stored at 8 °C and assessed for fruit quality and total ascorbic acid concentration after one and fourteen days. Irradiation at any dose (≤1000 Gy) did not affect passionfruit quality (overall fruit quality, colour, firmness, fruit shrivel, stem condition, weight loss, total soluble solids level (TSS), titratable acidity (TA) level, TSS/TA ratio, juice pH and rot development), nor the total ascorbic acid concentration. The length of time in storage affected some fruit quality parameters and total ascorbic acid concentration, with longer storage periods resulting in lower quality fruit and lower total ascorbic acid concentration, irrespective of irradiation. There was no interaction between irradiation treatment and storage time, indicating that irradiation did not influence the effect of storage on passionfruit quality. The results showed that the application of 150, 400 and 1000 Gy gamma irradiation to "Sweetheart" purple passionfruit did not produce any deleterious effects on fruit quality or total ascorbic acid concentration during cold storage, thus supporting the use of low dose irradiation as a phytosanitary treatment against quarantine pests in purple passionfruit.

Predicted ionisation in mitochondria and observed acute changes in the mitochondrial transcriptome after gamma irradiation: a Monte Carlo simulation and quantitative PCR study.

It is a widely accepted that the cell nucleus is the primary site of radiation damage while extra-nuclear radiation effects are not yet systematically included into models of radiation damage. We performed Monte Carlo simulations assuming a spherical cell (diameter 11.5 µm) modelled after JURKAT cells with the inclusion of realistic elemental composition data based on published literature. The cell model consists of cytoplasm (density 1g/cm(3)), nucleus (diameter 8.5 µm; 40% of cell volume) as well as cylindrical mitochondria (diameter 1 µm; volume 0.5 µm(3)) of three different densities (1, 2 and 10 g/cm(3)) and total mitochondrial volume relative to the cell volume (10, 20, 30%). Our simulation predicts that if mitochondria take up more than 20% of a cell's volume, ionisation events will be the preferentially located in mitochondria rather than in the cell nucleus. Using quantitative polymerase chain reaction, we substantiate in JURKAT cells that human mitochondria respond to gamma radiation with early (within 30 min) differential changes in the expression levels of 18 mitochondrially encoded genes, whereby the number of regulated genes varies in a dose-dependent but non-linear pattern (10 Gy: 1 gene; 50 Gy: 5 genes; 100 Gy: 12 genes). The simulation data as well as the experimental observations suggest that current models of acute radiation effects, which largely focus on nuclear effects, might benefit from more systematic considerations of the early mitochondrial responses and how these may subsequently determine cell response to ionising radiation.

In vitro sensitivities to UVA of lymphocytes from patients with colon and melanoma cancers and precancerous states in the micronucleus and the Comet assays.

To use lymphocytes as surrogate cells to investigate their in vitro sensitivities to ultraviolet (UV) treatment in different cancers and precancerous states by comparison with lymphocytes from healthy control individuals was the main aim of this research. UV light induces precise cellular and genomic mutations. In this study, the effect of ultraviolet A (UVA) (320-400 nm) was used as a generic mutagen to evaluate in vitro different sensitivities from lymphocytes of patients with suspected melanoma (SM), malignant melanoma (MM), polyposis coli (PC) and colorectal cancer (CRC). DNA damage was evaluated by two different methods: the micronucleus (MN) assay and the Comet assay. The baseline frequency of MNs was significantly increased in lymphocytes from all patients (SM, MM, PC and CRC) when compared to healthy individuals. After UV irradiation, MN frequencies were significantly increased in lymphocytes of all groups, both patients and healthy individuals. However, the MN frequency in all patient groups was significantly higher than in the healthy individual group. Similar results for the induction of genomic DNA damage were obtained for the Comet assay. Also for the Comet assay, UVA-induced DNA damage for all four patient groups was significantly increased when compared to healthy individuals (SM, MM, PC and CRC groups: P < 0.001). Conclusively, peripheral lymphocytes from patients with cancers MM and CRC or precancerous states SM and PC are more sensitive to a generic mutagen such as UVA than lymphocytes from healthy individuals. This feature may be used as an essential biomarker to screen and diagnose precancerous states and cancers in early stages.

Radiological characterization and water equivalency of genipin gel for x-ray and electron beam dosimetry.

The genipin radiochromic gel offers enormous potential as a three-dimensional dosimeter in advanced radiotherapy techniques. We have used several methods (including Monte Carlo simulation), to investigate the water equivalency of genipin gel by characterizing its radiological properties, including mass and electron densities, photon interaction cross sections, mass energy absorption coefficient, effective atomic number, collisional, radiative and total mass stopping powers and electron mass scattering power. Depth doses were also calculated for clinical kilovoltage and megavoltage x-ray beams as well as megavoltage electron beams. The mass density, electron density and effective atomic number of genipin were found to differ from water by less than 2%. For energies below 150 keV, photoelectric absorption cross sections are more than 3% higher than water due to the strong dependence on atomic number. Compton scattering and pair production interaction cross sections for genipin gel differ from water by less than 1%. The mass energy absorption coefficient is approximately 3% higher than water for energies <60 keV due to the dominance of photoelectric absorption in this energy range. The electron mass stopping power and mass scattering power differ from water by approximately 0.3%. X-ray depth dose curves for genipin gel agree to within 1% with those for water. Our results demonstrate that genipin gel can be considered water equivalent for kilovoltage and megavoltage x-ray beam dosimetry. For megavoltage electron beam dosimetry, however, our results suggest that a correction factor may be needed to convert measured dose in genipin gel to that of water, since differences in some radiological properties of up to 3% compared to water are observed. Our results indicate that genipin gel exhibits greater water equivalency than polymer gels and PRESAGE formulations.

In-house colorectal laparoscopic preceptorship: a model for changing a unit's practice safely and efficiently.

PURPOSE: There has been steady increase in demand for laparoscopic colonic resection as benefits are manifold compared to open and include smaller incisions, less pain, quicker recovery and convalescence, reduced morbidity and reduced analgesic demands. We devised a preceptorship programme with the aim of all four coloproctologists in our unit becoming proficient colorectal laparoscopic surgeons over a period of 12 months. METHOD: The surgeon in the unit with significant experience of laparoscopic colorectal surgery acted as a preceptor to the remaining three. A prospective database was set up to allow analysis of the impact of the preceptorship on the units' elective practice and outcomes from January 2006. RESULTS: Results were analysed 106 cases to assess the success of this novel method and were more than encouraging. During this period, 57 laparoscopic resections were performed compared 49 open resections. The proportion of patients undergoing laparoscopic resection had risen from 20% to 80% (p = 0.000). This was associated with a significant drop in post-operative stay from 14 to 4 days (p = 0.000). Analysis of patient demographics, pathology and type of resection found there to be no significant difference between the open and laparoscopic groups. The conversion rate was acceptably low (10.5%) and there were no re-admissions. CONCLUSIONS: For hospitals with the facilities and an appropriately experienced preceptor, we offer this as a patient-safe, cost-neutral method of significantly increasing a units' laparoscopic practice over a relatively short period of time.

Situs inversus abdominus and malrotation in an adult with Ladd's band formation leading to intestinal ischaemia.

Situs inversus abdominus with rotational anomaly of the intestines is an extremely rare condition. Although intestinal malrotation has been recognized as a cause of obstruction in infants and children and may be complicated by intestinal ischaemia, it is very rare in adults. When it occurs in the adult patient, it may present acutely as bowel obstruction or intestinal ischaemia or chronically as vague intermittent abdominal pain. Herein, we present an acute presentation of a case of situs inversus abdominus and intestinal malrotation with Ladd's band leading to infarction of the intestine in a 32 year old woman.