Development of monoclonal antibodies against Vibrio pathogens

Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria. Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios. In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals. Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates. All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios. Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS. Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.

A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood

This paper reports the development of a platform technology for measuring platelet function and aggregation based on localized strain rate micro-gradients. Recent experimental findings within our laboratories have identified a key role for strain rate micro-gradients in focally triggering initial recruitment and subsequent aggregation of discoid platelets at sites of blood vessel injury. We present the design justification, hydrodynamic characterization and experimental validation of a microfluidic device incorporating contraction–expansion geometries that generate strain rate conditions mimicking the effects of pathological changes in blood vessel geometry. Blood perfusion through this device supports our published findings of both in vivo and in vitro platelet aggregation and confirms a critical requirement for the coupling of blood flow acceleration to downstream deceleration for the initiation and stabilization of platelet aggregation, in the absence of soluble platelet agonists. The microfluidics platform presented will facilitate the detailed analysis of the effects of hemodynamic parameters on the rate and extent of platelet aggregation and will be a useful tool to elucidate the hemodynamic and platelet mechano-transduction mechanisms, underlying this shear-dependent process.

Energy expenditure in adolescents playing new generation computer games

Objective: To compare the energy expenditure of adolescents when playing sedentary and new generation active computer games.

Design: Cross sectional comparison of four computer games. Setting Research laboratories.

Participants: Six boys and five girls aged 13–15 years.

Procedure: Participants were fitted with a monitoring device validated to predict energy expenditure. They played four computer games for 15 minutes each. One of the games was sedentary (XBOX 360) and the other three were active (Wii Sports).

Main outcome measure: Predicted energy expenditure, compared using repeated measures analysis of variance.

Results: Mean (standard deviation) predicted energy expenditure when playing Wii Sports bowling (190.6 (22.2) kl/kg/min), tennis (202.5 (31.5) kl/kg/min), and boxing (198.1 (33.9) kl/kg/min) was significantly greater than when playing sedentary games (125.5 (13.7) kl/kg/min) (P<0.001). Predicted energy expenditure was at least 65.1 (95% confidence interval 47.3 to 82.9) kl/kg/min greater when playing active rather than sedentary games.

Conclusions: Playing new generation active computer games uses significantly more energy than playing sedentary computer games but not as much energy as playing the sport itself. The energy used when playing active Wii Sports games was not of high enough intensity to contribute towards the recommended daily amount of exercise in children.

Comparison of energy expenditure in adolescents when playing new generation and sedentary computer games: cross sectional study

Objective : To compare the energy expenditure of adolescents when playing sedentary and new generation active computer games.

Design : Cross sectional comparison of four computer games.

Setting : Research laboratories.

Participants : Six boys and five girls aged 13-15 years.

Procedure : Participants were fitted with a monitoring device validated to predict energy expenditure. They played four computer games for 15 minutes each. One of the games was sedentary (XBOX 360) and the other three were active (Wii Sports).

Main outcome measure : Predicted energy expenditure, compared using repeated measures analysis of variance.

Results : Mean (standard deviation) predicted energy expenditure when playing Wii Sports bowling (190.6 (22.2) kJ/kg/min), tennis (202.5 (31.5) kJ/kg/min), and boxing (198.1 (33.9) kJ/kg/min) was significantly greater than when playing sedentary games (125.5 (13.7) kJ/kg/min) (P<0.001). Predicted energy expenditure was at least 65.1 (95% confidence interval 47.3 to 82.9) kJ/kg/min greater when playing active rather than sedentary games.

Conclusions : Playing new generation active computer games uses significantly more energy than playing sedentary computer games but not as much energy as playing the sport itself. The energy used when playing active Wii Sports games was not of high enough intensity to contribute towards the recommended daily amount of exercise in children.

NMR determination of ionic structure in plasticized polyether-urethane polymer electrolytes

Solid polymer electrolytes based on amorphous polyether-urethane networks combined with lithium or sodium salts and a low molecular weight cosolvent (plasticizer) have been investigated in our laboratories for several years. Conductivity enhancements of up to two orders of magnitude can be obtained whilst still retaining solid elastomeric properties. In order to understand the effects of the plasticizers and their mechanism of conductivity enhancement, multinuclear NMR has been employed to investigate ionic structure in polymer electrolyte systems containing NaCF₃SO₃, LiCF₃SO₃ and LiClO₃ salts.

With increasing dimethyl formamide (DMF) and propylene carbonate (PC) concentration the increasing cation chemical shift with fixed salt concentration indicates a decreasing anion-cation association consistent with an increased number of charge carriers. ¹³C chemical shift data for the same systems suggests that whilst DMF also decreases cation-polymer interactions, PC does the opposite, presumably by shielding cation-anion interactions. Temperature dependent ⁷Li spin-lattice relaxation times indicate the expected increase in ionic mobility upon plasticization with a shift of the T₁ minimum to lower temperatures. The magnitude of T₁ at the minimum increases upon addition of DMF whereas there is a slight decrease when PC is added. This also supports the suggestion that the DMF preferentially solvates the cation whereas the action of PC is limited to coulomb screening, hence freeing the anion.

The advancing science by enhancing learning in the laboratory (ASELL) project : the next chapter

Most researchers agree that the laboratory experience ranks as a significant factor that influences students’ attitudes to their science courses. Consequently, good laboratory programs should play a major role in influencing student learning and performance. The laboratory program can be pivotal in defining a student's experience in the sciences, and if done poorly, can be a major contributing factor in causing disengagement from the subject area. The challenge remains to provide students with laboratory activities that are relevant, engaging and offer effective learning opportunities. The Advancing Science by Enhancing Learning in the Laboratory (ASELL) project has developed over the last 10 years with the aim of improving the quality of learning in undergraduate laboratories, providing a validated means of evaluating the laboratory experience of students and effective professional development for academic staff. After successful development in chemistry and trials using the developed principles in physics and biology, the project has now expanded to include those disciplines. This paper will discuss the activities of ASELL and provide a report about the first ASELL science workshop held at the University of Adelaide in April 2010.

Parallel developments in aprotic and protic ionic liquids : physical chemistry and applications

This Account covers research dating from the early 1960s in the field of low-melting molten salts and hydrates,which has recently become popular under the rubric of “ionic liquids”. It covers understanding gained in the principal author’s laboratories (initially in Australia, but mostly in the U.S.A.) from spectroscopic, dynamic, and thermodynamic studies and includes recent applications of this understanding in the fields of energy conversion and biopreservation. Both protic and aprotic varieties of ionic liquids are included, but recent studies have focused on the protic class because of the special applications made possible by the highly variable proton activities available in these liquids.

The epidemiology of ecstasy use and harms in New South Wales, Australia

Aims:This paper examines the epidemiology of ecstasy use and harm in Australia using multiple data sources.

Design: The data included (1) Australian Customs Service 3,4-methylenedioxymethamphetamine (MDMA) detections; (2) the National Drug Strategy Household and Australian Secondary Student Alcohol and Drug Surveys; (3) data from Australia's ecstasy and Related Drugs Reporting System; (4) the number of recorded police incidents for ecstasy possession and distribution collated by the N.S.W. Bureau of Crime Statistics and Research; (5) the number of calls to the Alcohol and Drug Information Service and Family Drug Support relating to ecstasy; (6) the Alcohol and Other Drug Treatment Services National Minimum Dataset on number of treatment episodes for ecstasy, and (7) N.S.W. Division of Analytical Laboratories toxicology data on number of deaths where MDMA was detected.

Findings: Recent ecstasy use among adults in the general population has increased, whereas among secondary students it has remained low and stable. The patterns of ecstasy consumption among regular ecstasy users have changed over time. Polydrug use and use for extended periods of time (>48 h) remain common among this group. Frequent ecstasy use is associated with a range of risk behaviours and other problems, which tend to be attributed to a number of drugs along with ecstasy. Few ecstasy users present for treatment for problems related to their ecstasy consumption.

Conclusions: Messages and interventions to reduce the risks associated with polydrug use and patterns of extended periods of use are clearly warranted. These messages should be delivered outside of traditional health care settings, as few of these users are engaged with such services.

Large-scale mechanical peeling of boron nitride nanosheets by low-energy ball milling

Mechanical cleavage by Scotch tape was the first method to produce graphene and is still widely used in laboratories. However, a critical problem of this method is the extremely low yield. We have tailored ball milling conditions to produce gentle shear forces that produce high quality boron nitride (BN) nanosheets in high yield and efficiency. The in-plane structure of the BN nanosheets has not been damaged as shown by near edge X-ray absorption fine structure measurements. The benzyl benzoate acts as the milling agent to reduce the ball impacts and milling contamination. This method is applicable to any layered materials for producing nanosheets.

The advancing science by enhancing learning in the laboratory (ASELL) project : the first Australian multidisciplinary workshop

Most science educators and researchers will agree that the laboratory experience ranks as a major factor that influences students’ attitudes to their science courses. Consequently, good laboratory programs should play a major role in influencing student learning and performance. The laboratory program can be pivotal in defining a student's experience in the sciences, and if done poorly, can be a major contributing factor in causing disengagement from the subject area. The challenge remains to provide students with laboratory activities that are relevant, engaging and offer effective learning opportunities.

The Advancing Science by Enhancing Learning in the Laboratory (ASELL) project has developed over the last 10 years with the aim of improving the quality of learning in undergraduate laboratories, providing a validated means of evaluating the laboratory experience of students and effective professional development for academic staff. After successful development in chemistry and trials using the developed principles in physics and biology, the project has now expanded to include those disciplines. This paper will discuss the activities of ASELL and provide a report about the first ASELL science workshop held at the University of Adelaide in April 2010, present some views of academic and student delegates, and make comparisons with other workshops.
Introduction

The whole-body fatty acid balance method : examples of its potential for feed efficiency and product quality optimisation in fish and poultry

Dietary lipids and fatty acids are not only fundamental in determining animal performance, but also determine the eating qualities of animal products. Several methods have been used to quantify fatty acid metabolism but most involve expensive in vitro approaches that are not suitable for most laboratories. Furthermore, there is considerable variation between methods with regard to enzyme activity, which makes comparison of results between studies difficult. The recently developed whole-body fatty acid balance method (WBFABM) is a simple and reliable in vivo method for assessing fatty acid metabolism, including rates of liponeogenesis and de novo fatty acid production, β-oxidation of fatty acids and bioconversion (elongation and desaturation) of fatty acids to long-chain polyunsaturated fatty acids. Initially developed for implementation with a fish model, the WBFABM has proven to be a simple and effective method that can be used in any laboratory equipped with a gas chromatography unit. Since its development, it has been used in several farmed finfish feeding trials and in broiler chicken feeding trials. The WBFABM is currently used at research institutions worldwide and its use is increasing in popularity among animal scientists. With this method, it is possible to track the fate of individual dietary fatty acids within the body. The WBFABM could contribute significantly to information generated by animal feeding trials.

Effects of exercise on the isoelectric patterns of erythropoietin

Objectives: Recombinant erythropoietin has a strong impact on aerobic power and is therefore one of the most potent doping agents in endurance sports. The anti-doping control of this synthetic hormone relies on the detection, in the urine, of its isoelectric pattern, which differs from that of the corresponding natural hormone, the latter being typically more acidic than the former. However, a small number of natural urinary patterns, referred to as atypical patterns, are less acidic than the dominant form. Based on anecdotal evidence, the occurrence of such patterns seems to be related to particular strenuous exercises. This study aimed to demonstrate this relation using a strenuous exercise protocol.

Design: Seven athletes took part in a training protocol including a series of supramaximal short-duration exercises. Urine and blood samples were collected throughout the protocols.

Settings: World Cycling Center, Aigle, Switzerland, and research laboratories.

Participants: Seven top-level athletes (cyclists) were involved in this study.

Main Outcome Measures: Erythropoietin (EPO) isoelectric patterns were obtained by submitting blood and urine samples to isoelectric focusing. Additional protein dosages were performed.

Results: Supramaximal short-duration exercises induced the transformation of typical urinary natural EPO patterns into atypical ones. None of the obtained atypical patterns fulfilled the 3 criteria mandatory for reporting an adverse analytical finding. Serum EPO patterns were not affected by the exercises that caused the transformation of urinary patterns.

Conclusion: An exercise-induced transient renal dysfunction is proposed as a hypothetic explanation for these observations that rely on parallel investigations of proteinuria in the same samples.

Advancing science by enhancing learning in the laboratory (ASELL)

Final report of the the Advancing Science by Enhancing Learning in the Laboratory (ASELL) project. 

Most researchers agree that the laboratory experience ranks as a significant factor that influences students’ attitudes to their science courses. Consequently, good laboratory programs should play a major role in influencing student learning and performance. The laboratory program can be pivotal in defining a student's experience in the sciences, and if done poorly, can be a major contributing factor in causing disengagement from the subject area. The challenge remains to provide students with laboratory activities that are relevant, engaging and offer effective learning opportunities.

The Advancing Science by Enhancing Learning in the Laboratory (ASELL) project has developed over the last 10 years with the aim of improving the quality of learning in undergraduate laboratories, providing a validated means of evaluating and improving the laboratory experience of students, and effective professional development for academic staff. After successful development in chemistry and trials using the developed principles in physics and biology, the project, with ALTC funding, has now expanded to include those disciplines.

The launching pad for ASELL was a multidisciplinary workshop held in Adelaide in April, 2010. This workshop involved 100 academics and students, plus 13 Deans of Science (or delegates), covering the three enabling sciences of biology, chemistry and physics. Thirty-nine undergraduate experiments were trialled over the three days of the workshop. More importantly, professional development in laboratory education was developed in the 42 academic staff that attended the workshop.

Following the workshop, delegates continued to evaluate, develop and improve both individual experiments and whole laboratory programs in their home institutions, mentored by the ASELL Team. Some highlights include:
- more than 15,000 student surveys carried out by delegates during 2010/11
- 10 whole lab programs were surveyed by delegates
- 4 new ASELL-style workshops, conducted by ASELL-trained delegates were run in 2010/11
- more than 100 ASELL-tested experiments available on the website (www.asell.org)
- ASELL workshops conducted in Philippines, Ireland in 2010, and planned in the USA and Thailand for 2011
- significant improvement in student evaluation of whole laboratory programs and individual experiments measured in universities using the ASELL approach
- high profile of ASELL activities in the Australian Council of Deans of Science (ACDS)
- research project on the misconceptions of academic staff about laboratory learning completed
- significant research on student learning in the laboratory, and staff perceptions of student learning have been carried out during 2010/11
- research results have been benchmarked against staff and students in the USA.

The biggest unresolved issue for ASELL is one of sustainability in the post-ALTC funding era. ASELL will make a series of recommendations to the ACDS, but the future of the program depends, to a large part, on how the ACDS responds.

Cane toad or computer mouse? Real and computer-simulated laboratory exercises in physiology classes

Traditional practical classes in many countries are being rationalised to reduce costs. The challenge for university educators is to provide students with the opportunity to reinforce theoretical concepts by running something other than a traditional practical program. One alternative is to replace wet labs with comparable computer simulations. These virtual experiments involve no harm to animals and require little ongoing expenditure. This study documents second-year physiology students' perceptions of and attitudes to simulations by incorporating several computer simulations into the practical program. Computer simulations met the conceptual and, to some extent, the motivational goals of university practical programs. While students enjoyed both wet labs and computer-simulated exercises, overwhelmingly the wet lab provided the more memorable and stimulating learning experience. Based on this study, students suggested that computer simulations could be effectively used to complement rather than replace practical classes where students gain laboratory skills.

Factors affecting the determination of electrochemical noise resistance

The similarity between electrochemical noise resistance (Rn) and polarization resistance (Rp) has been confirmed in many laboratories, and a new method for corrosion rate determination has been developed based upon this similarity. However, this method remains controversial. Some researchers have reported experimental results that did not support a similarity between Rp and Rn. Other researchers have found that this similarity does not hold true for every system. To address this controversy, factors affecting the measurement and calculation of Rn were investigated. Several factors - including instrument noise, direct current (DC) trend, noise sampling rates, and noise sampling duration - can affect the accuracy of Rn determination signiftcantly and, in some cases, can cause disagreement between Rn and Rp. It was recommended that a system check be carried out to avoid the influence of these factors.