How to become a good evidence based Bioveterinary practitioner

What is evidence-based practice

Within the field of bioveterinary science and veterinary practice and care, evidence and knowledge are forever changing. With continuing research consistently making fresh and exciting new break throughs for animal health, it is becoming increasingly difficult to keep up with what is recent and appropriate evidence. Evidence based medicine is a term that was first adopted in the 1990’s and was best defined as “a systemic approach to analyse published research as the basis of clinical decision making.” Although first adopted for use in the human medicine profession, evidence-based practice (EBP) has developed into many methodologies and tools that can be used by a variety of different professions within a diverse amount of clinical practices. EBP is a process where by a practitioner takes it upon themselves to evaluate and appraise the best evidence available to them to enhance their decision making within a clinical setting.  EBP is critical for practitioners to remain competent, relevant and clinically effective. However, acquiring evidence does not always mean scanning through pages of research journals and sifting through systematic reviews. Evidence can also consist of expert opinion, stakeholder characteristics and differing scenarios. Asking a colleague for their opinion on a situation can also be regarded as acquiring evidence. This being said, EBP has come a long way from making decisions solely based on the opinions of those who are deemed as experts or following through with a decision because “that’s the way its always been done”. The purpose of EBP is to encourage those working in the field of bioveterinary science to actively accumulate sources of evidence, evaluate and appraise that evidence, and then make appropriate decisions based on the knowledge and understanding they have gained that will be most beneficial to them and any stakeholders involved. The purpose of EBP is to reduce the risk of human bias, provide opportunities for clinicians and practitioners to become more individualised, define best practices that is supported by the most relevant forms of evidence and to overall improve the work practitioners and the services provided to involved stakeholders by allowing them to create sound and informed decisions using methodologies designed to decipher and appraise what is to be deemed as best evidence.

Acquiring evidence

So where do bioveterinary practitioners acquire evidence from? What constitutes to make something a good and valid source of evidence? The hierarchy of evidence is a tool designed for evidence-based practitioners that guides users in the appraisal of what can be deemed as best evidence for use in decision making. The hierarchy allows users to take a top-down approach to locating best evidence. In theory, this approach assumes that when practitioners are searching for evidence, they start by looking for a well-conducted and recent systematic review to answer the clinical question in hand. If a systematic review is not available, then the user will move down to the next tier of evidence and so on. The lower down the tier a piece of evidence falls, the lesser its ability to constitute towards what is deemed as best evidence. Although the hierarchy of evidence is an effective tool for evaluating physically published sources of evidence, it does not consider other constituents that make up EBP as a whole. Effective clinical decision making should always consider the best available evidence but make no assumptions that one type of evidence has overall superiority. The type of evidence needed by practitioners will vary depending on the clinical question in hand. Evidence needed will depend on the nature and activity of its purpose, so considerations must be made when determining what evidence is best for a particular clinical scenario. Clinical feasibility is the consideration of whether an activity or purpose is practical and practicable within a given context. Research into best available evidence may highlight a practice or activity as being superior to others, but if this activity cannot be performed by the practitioner then it is not the best form of evidence. The appropriateness of an activity goes hand in hand with evaluating the feasibility. Evaluating appropriateness relates to how ethical or suitable an activity relates to the context in which it is being used. Considerations into the benefits and effects is also paramount to determining best evidence. Evidence can be seen to be advantageous if those involved have gained a positive experience regarding the evidence used in a particular situation. A well-rounded evidence-based practitioner should always consider other stakeholders and factors that could have an influence in the type of evidence used in their decision making and clinical thinking.

 

nhmrc_evidence_hierarchy

 

Tools for evidence-based practice

Methodologies and EBP guidelines are a structural and efficient way to engage in evidence-based practices. One of the most commonly used EBP guidelines is the 5 steps of evidence-based practice.

  • Step 1) Practitioners are encouraged to first convert their need for information, what ever the topic or scenario may be, into a structured and answerable question. Phrasing a question correctly allows for a more organised search for evidence and reducing the chance of any important information being missed from a search.
  • Step 2) Once a clinical question has been constructed, the search for the best available evidence can then begin. The hierarchy of evidence can be used within during this step to assess the validity of evidence that has been searched for.
  • Step 3) Critical appraisal of acquired information and evidence is then necessary to evaluate considerations that may affect the use of the evidence. These include considering the feasibility and appropriateness of the evidence as well as how beneficial it will be to all stake holders involved.
  • Step 4) This step involves integrating critically appraised and appropriate best evidence with existing clinical practices and expertise. Incorporation of past experiences with current best evidence can aid in enhancing current practices. Integration of these two factors should also involve the integration of external stakeholders such as clients and patients. Considering the needs, values and opinions of external stakeholders will help to further evaluate the feasibility and appropriateness of evidence.
  • Step 5) The final step is to evaluate the effectiveness of steps 1-4. Reflecting on the process can help an evidence-based practitioner assess the effects that evidence had on influencing their final decision. Reflection may involve consideration into different application of the process, essentially refining the process for better use in the future.

 

EBP steps

 

 How to implement EBP in the work place

It is recognised that there are barriers than can affect the process of evidence-based practice within the work place. Information available to provide best evidence is usually spread over a variety of different sources and can make it very time consuming for practitioners to find information that can be used as best evidence, as well as practitioners having limited time to analyse information especially in a clinical care environment. Challenges surrounding evaluating the value of information should also be considered, even if EBP methodologies are used. To overcome these barriers, opportunities should be given to practitioners that aid in advancing their evidence-based practice skills. Such opportunities could involve educational work shops into how to correctly use EBP methodologies and how to improve skills such as; searching for appropriate literature in a timely manner, and critical appraisal of information. Providing education on how to correctly and effectively use methodologies with in turn save practitioners time in the future. Online resources such as the evidence based veterinary medicine (EBVM) toolkit, provided by RCVS knowledge, provided free information for veterinary practitioners who wish to learn more about EBP within their field of bioveterinary science. Another resource provided by RCVS knowledge is the online Knowledge Summaries. Knowledge summaries are designed to provide concise conclusions to clinical questions that be easily and quickly accessed by staff. They are summarised resources used to address knowledge needs by providing best available evidence on defined clinical questions. Knowledge summaries are particularly useful for overcoming the barrier on not being able to able EBP in a clinical care setting.

 

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Further reading

EBP is an ever-expanding topic that has many benefits for bioveterinary practitioners if it is implemented correctly. If readers wish to expand their knowledge on the topic and explore into the processes of EBP further, the following further reading resources have been provided:

Giuffrida, M. A. (2017) ‘Practical Application of Evidence-Based Practice.’ Veterinary Clinics of North America: Exotic Animal Practice. (Evidence-Based Clinical Practice in Exotic Animal Medicine), 20(3) pp. 737–748.

La Caze, A. (2016) ‘The hierarchy of evidence and quantum theory.’ Journal of Clinical Epidemiology, 72, April, pp. 4–6.

Paravattil, B., Shabana, S., Rainkie, D. and Wilby, K. J. (2019) ‘Evaluating knowledge, skills, and practice change after an accredited evidence-based medicine course for community pharmacy preceptors.’ Currents in Pharmacy Teaching and Learning, April.

Saunders, H., Vehviläinen-Julkunen, K. and Stevens, K. R. (2016) ‘Effectiveness of an education intervention to strengthen nurses’ readiness for evidence-based practice: A single-blind randomized controlled study.’ Applied Nursing Research, 31, August, pp. 175–185.

Sullivan, K. J., Wayne, C., Patey, A. M. and Nasr, A. (2017) ‘Barriers and facilitators to the implementation of evidence-based practice by pediatric surgeons.’ Journal of Pediatric Surgery, 52(10) pp. 1666–1673.

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Using in silico methods as alternatives to using protected animals within toxicology research

Introduction

Exposure to potentially harmful chemicals is always a possibility throughout varying areas of everyday life. They can be found as pesticides sprayed onto food, compounds for a new potential pharmaceutical or even as a new formula for a popular cosmetic product. Toxicology can be defined as the field of science dedicated to the investigation of the harmful effects caused by chemical substances on living organisms (Soto, 2018). Measuring the adverse effects that a specific chemical may have on a body, whether that be animal or human, is paramount for assessing the safety of a product. Measuring the dosage of chemicals is also extremely important when assessing safety. When assessing safety of chemicals, it is also imperative to evaluate how the body reacts to different dosages. The dose response curve refers to the relationship between the size of the dose given and its affect the body. In the right dosages, these affects could be very positive, but too much and the chemical could then become toxic and extremely harmful (Tsatsakis et al., 2018). Within the field of toxicology in vivo experiments involving animals have been widely used. Shukla et al., (2010) discusses how animal models have been used to investigate specific toxicological end points such as; immunotoxicity, genotoxicity and carcinogenicity. Although the outcomes of testing on animal models have provided useful information, they aren’t always consistently predictive of human biology, and they come at a large expensive. Ethical debates arise around the subject of in vivo experimentation. Where possible, the replacement of animals within research is essential. In silico is a method of testing which involves computer analysis and simulation (Raunio, 2011). Raies and Bajic, (2016) describe how in silico testing is widely used throughout toxicology to estimate the toxicity of chemicals. The aim of in silico testing is to complement existing toxicology tests by; minimising late-stage failures in drug development, prioritise chemicals and guide toxicity tests. This report will discuss how protected animals are used within research, the use of in silico technology methods as an alternative to using protected animals within toxicology research and discussions will be made into the ethics surrounding the use of research animals and why is it important to focus on the development of alternative testing methods.

The use of protected animals within research

The use of animals for scientific research has been a heavily debated subject for years. Animals have been considered good model systems for humans and human disease, therefore in vivo testing methods have been widely used within toxicology and other related research. Human obligation to consider animal ethics within research has been paramount for the development of ethical and legal guidelines that must be considered and followed by researchers (Cheluvappa et al., 2017). In 1959, William Russell and Rex Burch introduced the concept of the “3 Rs”; replacement, reduction and refinement (Ferdowsian and Beck, 2011). Miziara et al., (2012) goes on to explain how the 3 Rs were a tool designed to rationalise the use of animals in research and humanise the care of animals. Russel and Burch further argued that alternative methods such as in vitro testing and computer modelling should always be considered to replace and reduce the number of animals used where possible. Where animals must be used, techniques should be refined so as to reduce any pain or suffering caused to research animals. Such techniques included care with analgesia and postoperative antisepsis treatments. Within the UK, legislation was introduced with the purpose of protecting laboratory animals as much as possible. This legislation is known as the Animals (Scientific Procedures) Act, 1986 (ASPA). In accordance with ASPA, any living vertebrate (other than man) and any living cephalopod is deemed as “protected” once it has reached two thirds of its gestation or incubation period or is able to feed independently (‘Animals (Scientific Prodecures) Act 1986, Section 1.,’ 2019). The principles of the 3Rs and the protection of laboratory animals should also extend to the breeding, accommodation and care of any protected animals used for research (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014).

There are many different factors surrounding the husbandry of laboratory animals. If husbandry is not correct, dependent on the species, it can have detrimental effects on the animal’s health and welfare. Animals can sense the seasons even when housed in windowless rooms. Reports have indicated that seasonal variation can cause immunosuppression induced by chronic stress. Other research has also shown that mice living with a 12:12-hour photoperiod were at higher risk of death from peritonitis in the summer or autumn compared with other season, highlighting the importance of understanding seasonal variation in regards to animal health (Nevalainen, 2014). A similar study conducted by (Hawkins and Golledge, 2018) describes how it is common practice to carry out scientific procedures on rats and mice during the day time. Because rodents are nocturnal, this tends to interrupt their inactive period. Testing on rodents during their inactive periods can cause cognitive defects and induced stress. Species behaviour and species enrichment are further examples as to why species specific husbandry is so significant. Alho et al., (2016) explains how a lack of feline enrichment for indoor housed cats can lead to disorders such as; anxiety, stress, obesity and feline idiopathic cystitis. As laboratory cats must be housed in doors due to health, hygiene and other relating factors that could affect research, it is paramount for laboratory workers to ensure that there is always available enrichment to enable laboratory cats to be able to exhibit their natural behaviours.

The law states that a procedure that is undertaken on a protected animal becomes regulated if that procedure has the potential of causing pain, suffering, distress or lasting harm equivalent to, or higher than, pain induced by the insertion of a hypodermic needle according to good veterinary practice. Such procedures must only be carried out if they have a scientific or educational purpose (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014). The breeding of genetically modified animals also falls under the umbrella of regulated procedure. A home office report from 2017 indicates that approximately 0.28m (15%) genetically altered animals had experienced some form of potentially harmful effect due to the genetic alteration procedure (Great Britain and Home Office, 2018). Li et al., (2015) further explains how pig genomes are manipulated to increase the compatibility with human biology. This raises the question as to how reliable animal models can be if it is needed to manipulate their genome to make them more representative of human biology. Alternative methods may be a more feasible and ethical alternative. Further regulated procedures within toxicology include the assessment of multiple different toxicological endpoints including; acute oral toxicity and skin sensitisation. Oral toxicity studies involve the determination of the median lethal dose of a substance, otherwise known as LD50 testing (Zakari and Kubmarawa, 2016). This test determines the dose responsible for killing 50% of the participant animals within 24 hours. LD50 testing is a high debated method of endpoint testing. It requires a large number of animals in order to gain enough statistical data and is deemed as cruel and an inappropriate use of animals due to its questionable translatability to humans (Buesen et al., 2016). Skins sensitisation is another major toxicology endpoint that has to be assessed particularly in the development of dermatology products. Guinea pigs are often used as test models for skin sensitisation, but again the translatability of the results for human skin has been argued. Skin sensitisation also involve the application of an adjuvant that causes pain and distress to treated animals, highlighting the desirability of a replacement method (Basketter and Kimber, 2018).

 

The use of in silico methods as an alternative to using protected animals within research

Before a research project can begin it is required that a comprehensive project evaluation is conducted. This evaluation should consider ethical deliberations surrounding the use of animals for said project. The purpose of this ethical evaluation is to ensure that principles of the 3R’s (replacement, reduction and refinement) have been implemented within the study design (Guidance on the Operation of the Animals (Scientific Procedures) Act 1986., 2014). The 3Rs provide a useful strategy to rationally consider how animals are used in a project, without compromising the quality of the research being undertaken (Kandárová and Letašiová, 2011). Replacement is defined as a substitution of protected animals by non-sentient material or methods. Replacement does not necessarily mean that no animals are used at all. Alternative methods can be used to partially replace animals for example; replacing the use of animals for certain kind of test substances or for a particular range of toxicological hazard. Alternative methods implement the principles of reduction by decreasing the number of animals used within a project. Finally, refinement is defined as any decrease in the incidence or severity of procedures conducted for research. For example, in silico analysis may be able to predict the severity or lethality of a substance before it is tested on an animal. This allows researchers to refine their testing strategies, evidently reducing the amount of pain or suffering experienced by the animal (Mushtaq et al., 2018). In silico toxicology (IST) methods use computer resources to analyse, simulate, visualise or predict toxicity of chemicals and substances  (Myatt et al., 2018). IST methodologies aim to complement existing toxicology tests to potentially minimise the need for animal testing and improve toxicity prediction and safety assessment (Raies and Bajic, 2016). IST is a very fast expanding example of alternatives methods to animal testing used within the field of toxicology.

Toxicology testing is a necessary step in the process of all new product developments containing potentially hazardous chemicals or substances. For example; pharmaceutical drug design, food production, pesticide development and the making of cosmetic products. The developmental stage requires an analysis of different data sets to predict the toxicity of chemicals for potential new products. Data sets tend to fall into two main types of classification; single-label and multi-label classification (Yang et al., 2018). Single-label classification is used when each sample in a data set falls into only one class, or if the data set is binary and can be split into two classes. For example; carcinogenic or noncarcinogenic compounds. If a data set can be split into more than two classes, it then becomes a multi-class classification. An example of this would be classifying a compound on the degree of skin sensitisation (e.g. low, moderate, high) (Raies and Bajic, 2018). A study conducted by Zhang et al., (2015) describes the use of in silico methods used to predict chemical toxicity of pesticides within avian species. More than 663 diverse chemicals were assessed, and all chemicals were classified into three categories; highly toxic, slightly toxic and non-toxic. Although this study still used protected animals to obtain data, the prediction models significantly reduced the number of animals needed within the project. The high accuracy of the prediction models that were developed meant that pain and suffering was kept to a minimum as the highly toxic substances had already been predicted. The data that was recorded for the study can be used again for further avian toxicology research, reducing the number of animals needed in the future.

Although the development of in silico methods for toxicology testing is a seemingly popular and expanding field of research, there are some areas of limitation. There is a continuing need to ensure that new methods which are developed for use within toxicology are validated correctly. Clarity is needed about what to validate new methods against; is it already available test methods or accredited knowledge of adverse health effects of chemicals. A limitation to validating new test methods is the cost of potential time delays for the industry. For example, development of new in silico methods in parallel with traditional methods can add an extensive amount of time to a research project (Prior et al., 2019). Further limitations can include the need for whole organism interactions. Planchart et al., (2016) explains how zebra fish are still commonly used within toxicology as fish and humans share similar developmental and physiological responses to chemical exposures. Furthermore, although alternative models such as in silico are excellent for providing insight into reactions within specific areas of the body, there needs to be further development into in silico predictions regarding adverse effects to the whole organism. Although limitations are clearly apparent, in silico toxicology still has many benefits. Toxicology accounts for around 50% of failures in preclinical drug development, indicating the need for reliable prediction strategies to minimise these failures (Ford, 2016). A study conducted by Tambunan et al., (2019) explains how including in silico methods into their study design greatly reduced resource requirements in comparison to conventional methods. This is beneficial to the project as it saves time and expenses but also works in accordance with the 3Rs. Additionally, a differing study led by Passini et al., (2017) describes how in silico drug trials institute influential methodology to predict clinical risk of arrhythmias in human cardiotoxicity. The benefits shown by the methodology allow it to be integrated into existing cardiotoxicity assessment schemes, thus contributing to the reduction of animals used for experimentation.

Conclusion

Animal contributions to the field of toxicology research have provided extremely advantageous information and knowledge and have shaped the development of many life-saving drugs, safe and effective pesticides and many other advances in toxicology safety assessments. The use of in silico technology as an alternative to using animal models has been demonstrated as extremely effective and beneficial for both the research it has been involved with and to minimising the number of protected animals used within toxicology research, working in accordance with ASPA and respecting the 3Rs. Although in silico testing methods still must be used alongside animal models, the toxic predictability allows researchers to rapidly refine their studies. Predicting the toxicity of chemicals before they are tested on protected animals means that animals will experience less pain and suffering as researchers can already gauge how the animal may react to a certain chemical. Further research needs to be carried out to development more in silico models and testing methods to further increase the benefits in silico testing in the future, but needless to say in silico is an extremely positive alternative to using protected animals in research will continue to improve welfare and minimise the use of protected animals with research.

 

References              

Alho, A. M., Pontes, J. and Pomba, C. (2016) ‘Guardians’ Knowledge and Husbandry Practices of Feline Environmental Enrichment.’ Journal of Applied Animal Welfare Science, 19(2) pp. 115–125.

‘Animals (Scientific Prodecures) Act 1986, Section 1.’ (2019). Government Legislation.

Basketter, D. A. and Kimber, I. (2018) ‘Are skin sensitisation test methods relevant for proteins?’ Regulatory Toxicology and Pharmacology, 99, November, pp. 244–248.

Buesen, R., Oberholz, U., Sauer, U. G. and Landsiedel, R. (2016) ‘Acute oral toxicity testing: scientific evidence and practicability should govern Three Rs activities.’ Altern Lab Anim, 44(4) pp. 391–398.

Cheluvappa, R., Scowen, P. and Eri, R. (2017) ‘Ethics of animal research in human disease remediation, its institutional teaching; and alternatives to animal experimentation.’ Pharmacology Research & Perspectives, 5(4) p. e00332.

Ferdowsian, H. R. and Beck, N. (2011) ‘Ethical and Scientific Considerations Regarding Animal Testing and Research.’ PLOS ONE, 6(9) p. e24059.

Ford, K. A. (2016) ‘Refinement, Reduction, and Replacement of Animal Toxicity Tests by Computational Methods.’ ILAR Journal, 57(2) pp. 226–233.

Great Britain and Home Office (2018) Annual Statistics of Scientific Procedures on Living Animals Great Britain 2017.

Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. (2014).

Hawkins, P. and Golledge, H. D. R. (2018) ‘The 9 to 5 Rodent − Time for Change? Scientific and animal welfare implications of circadian and light effects on laboratory mice and rats.’ Journal of Neuroscience Methods. (Measuring Behaviour 2016), 300, April, pp. 20–25.

Kandárová, H. and Letašiová, S. (2011) ‘Alternative methods in toxicology: pre-validated and validated methods.’ Interdisciplinary Toxicology, 4(3) pp. 107–113.

Li, P., Estrada, J. L., Burlak, C., Montgomery, J., Butler, J. R., Santos, R. M., Wang, Z.-Y., Paris, L. L., Blankenship, R. L., Downey, S. M., Tector, M. and Tector, A. J. (2015) ‘Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection.’ Xenotransplantation, 22(1) pp. 20–31.

Miziara, I. D., de Matos Magalhães, A. T., Santos, M. d’Aparecida, Gomes, É. F. and de Oliveira, R. A. (2012) ‘Research ethics in animal models.’ Brazilian Journal of Otorhinolaryngology, 78(2) pp. 128–131.

Mushtaq, S., Daş, Y. K. and Aksoy, A. (2018) ‘Alternative Methods to Animal Experiments.’ Turkiye Klinikleri Journal of Laboratory Animals.

Myatt, G. J., Ahlberg, E., Akahori, Y., Allen, D., Amberg, A., Anger, L. T., Aptula, A., Auerbach, S., Beilke, L., Bellion, P., Benigni, R., Bercu, J., Booth, E. D., Bower, D., Brigo, A., Burden, N., Cammerer, Z., Cronin, M. T. D., Cross, K. P., Custer, L., Dettwiler, M., Dobo, K., Ford, K. A., Fortin, M. C., Gad-McDonald, S. E., Gellatly, N., Gervais, V., Glover, K. P., Glowienke, S., Van Gompel, J., Gutsell, S., Hardy, B., Harvey, J. S., Hillegass, J., Honma, M., Hsieh, J.-H., Hsu, C.-W., Hughes, K., Johnson, C., Jolly, R., Jones, D., Kemper, R., Kenyon, M. O., Kim, M. T., Kruhlak, N. L., Kulkarni, S. A., Kümmerer, K., Leavitt, P., Majer, B., Masten, S., Miller, S., Moser, J., Mumtaz, M., Muster, W., Neilson, L., Oprea, T. I., Patlewicz, G., Paulino, A., Lo Piparo, E., Powley, M., Quigley, D. P., Reddy, M. V., Richarz, A.-N., Ruiz, P., Schilter, B., Serafimova, R., Simpson, W., Stavitskaya, L., Stidl, R., Suarez-Rodriguez, D., Szabo, D. T., Teasdale, A., Trejo-Martin, A., Valentin, J.-P., Vuorinen, A., Wall, B. A., Watts, P., White, A. T., Wichard, J., Witt, K. L., Woolley, A., Woolley, D., Zwickl, C. and Hasselgren, C. (2018) ‘In silico toxicology protocols.’ Regulatory Toxicology and Pharmacology, 96, July, pp. 1–17.

Nevalainen, T. (2014) ‘Animal Husbandry and Experimental Design.’ ILAR Journal, 55(3) pp. 392–398.

Passini, E., Britton, O. J., Lu, H. R., Rohrbacher, J., Hermans, A. N., Gallacher, D. J., Greig, R. J. H., Bueno-Orovio, A. and Rodriguez, B. (2017) ‘Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity.’ Frontiers in Physiology, 8.

Planchart, A., Mattingly, C. J., Allen, D., Ceger, P., Casey, W., Hinton, D., Kanungo, J., Kullman, S. W., Tal, T., Bondesson, M., Burgess, S. M., Sullivan, C., Kim, C., Behl, M., Padilla, S., Reif, D. M., Tanguay, R. L. and Hamm, J. (2016) ‘Advancing Toxicology Research Using In Vivo High Throughput Toxicology with Small Fish Models.’ ALTEX, 33(4) pp. 435–452.

Prior, H., Casey, W., Kimber, I., Whelan, M. and Sewell, F. (2019) ‘Reflections on the progress towards non-animal methods for acute toxicity testing of chemicals.’ Regulatory Toxicology and Pharmacology, 102, March, pp. 30–33.

Raies, A. B. and Bajic, V. B. (2016) ‘In silico toxicology: computational methods for the prediction of chemical toxicity.’ Wiley Interdisciplinary Reviews. Computational Molecular Science, 6(2) pp. 147–172.

Raies, A. B. and Bajic, V. B. (2018) ‘In silico toxicology: comprehensive benchmarking of multi-label classification methods applied to chemical toxicity data.’ Wiley Interdisciplinary Reviews: Computational Molecular Science, 8(3) p. e1352.

Raunio, H. (2011) ‘In Silico Toxicology – Non-Testing Methods.’ Frontiers in Pharmacology, 2, June.

Shukla, S. J., Huang, R., Austin, C. P. and Xia, M. (2010) ‘The Future of Toxicity Testing: A Focus on In Vitro Methods Using a Quantitative High Throughput Screening Platform.’ Drug discovery today, 15(23–24) pp. 997–1007.

Soto, M. (2018) ‘Foreword.’ In Costa, P. M. (ed.) The Handbook of Histopathological Practices in Aquatic Environments. Academic Press, pp. vii–viii.

Tambunan, U. S. F., Parikesit, A. A., Ghifari, A. S. and Satriyanto, C. P. (2019) ‘In silico identification of 2-oxo-1,3-thiazolidine derivatives as novel inhibitor candidate of class II histone deacetylase (HDAC) in cervical cancer treatment.’ Arabian Journal of Chemistry, 12(2) pp. 272–288.

Tsatsakis, A. M., Vassilopoulou, L., Kovatsi, L., Tsitsimpikou, C., Karamanou, M., Leon, G., Liesivuori, J., Hayes, A. W. and Spandidos, D. A. (2018) ‘The dose response principle from philosophy to modern toxicology: The impact of ancient philosophy and medicine in modern toxicology science.’ Toxicology Reports, 5, January, pp. 1107–1113.

Yang, H., Sun, L., Li, W., Liu, G. and Tang, Y. (2018) ‘In Silico Prediction of Chemical Toxicity for Drug Design Using Machine Learning Methods and Structural Alerts.’ Frontiers in Chemistry, 6, February.

Zakari, A. and Kubmarawa, D. (2016) ‘Acute Toxicity (LD50) Studies Using Swiss Albino Mice and Brine Shrimp Lethality (LC50 and LC90) Determination of the Ethanol Extract of Stem Bark of Echinaceae angustifolia DC.’ Natural Products Chemistry & Research, 04(06).

Zhang, C., Cheng, F., Sun, L., Zhuang, S., Li, W., Liu, G., Lee, P. W. and Tang, Y. (2015) ‘In silico prediction of chemical toxicity on avian species using chemical category approaches.’ Chemosphere, 122, March, pp. 280–287.

                         

Reflective practice as evidence for decision making

Within this blog post, the topics reflective practice and evidence-based practice will be discussed. The importance of reflective practice and where it is typically seen being used, along with helpful models of reflection will be highlighted. Secondly, similar to reflective practice, consideration around concepts within evidence-based practice and the importance of it in a practical environment, also incorporating popular models constructing effective evidence-based practice. Finally, a comparison between the two, indication of overlaps between the practices and why it is significant for Bioveterinary scientists to use both reflective practice and evidence-based practice to enhance their professional practice.

The ability to reflect on professional experiences is seen as a key skill to advance professional practice. Husebø et al., (2015) describes reflective practice (RP) as a process of learning from experiences. The ability to compare, engage and think critically can develop an individual professionally through the assessment of experiences and evaluating how these have an impact on learning and gaining knowledge for the future (Sweet et al., 2018). RP can be seen being used within a variety of professions. Oelofsen, (2012) addresses the importance of reflective practice for nurses. It is stated that regular engagement in RP allows nurses to assess the personal and professional impact that they have on their patients, by evaluating their practice through different experiences. Sellars, (2017) also highlights the significance of RP within teaching. They state that within the active practice of teaching there is no right or wrong answer or text book to consult for every given situation. Critical reflection allows teachers to debrief said situations and make informed decisions through reflection. Reflective frameworks are more than often used to consider options and determine possible action. There are a variety of tools, in the form of models of RP, that have been established to support individuals wanting to self-reflect. A commonly used model of reflection is Gibbs Reflective Cycle Model (1988) (‘Graham Gibbs Reflective Cycle Model 1988,’ 2016) Although Gibbs is still a popular reflective model, it has been argued by Barksby et al., (2015) that it is not always easy to recall in a practice setting, and some of the stages are quite unclear. Taking this into account, the authors have developed Gibbs further and comprised a new reflective framework – The REFLECT model. Although designed for nurses and other health care practitioners, the framework could still be applied to develop professional practice for Bioveterinary scientists.

Gibbs cycle
Figure 1 – The sages of Gibbs reflective cycle. (Sheffield, 2018)
reflect.png
Figure 2 –  The seven stages of The REFLECT model. (Barksby et al., 2015)

Evidence based practice (EBP) was best described by Sackett et al., (1996) as they famously quoted “Evidence based medicine is the conscientious, explicit and judicious use of current best evidence in making decisions about the care of the individual patient.” This statement is developed further by Giuffrida, (2017) who explains that EBP is a combination of; contemporary knowledge, clinical experience and client preference. EBP has evolved from solely relying on published scientific literature for best evidence. There is no guarantee that existing knowledge will have a widespread benefit. Although a concept or treatment may have been tested and published does not mean that it will necessarily work across the board for everyone (Biglan and Ogden, 2008). Client/patient preferences has now been seen to be incorporated into EBP within professional environments. Siminoff, (2013) explains the personal impact that a patience/client can have on an intervention or treatment. Social and cultural differences, along with any previous treatments or experiences, could have a significant impact on the clinical decision. Stakeholders are extremely influential factors that should always be taken into consideration when acquiring best evidence. A beneficial and structural way to asses whether evidence is appropriate to use would be to use an EBP guide.

five_steps_of_ebp
Figure 3- 5 structural stages for assesing evidence (Turner, 2018)

Although discussed as two separate subjects, similarities can be drawn between RP and EBP. Bannigan and Moores (2016) states that by integrating the two when making decisions, a practitioner can associate their previous experiences with current, valid and relevant evidence. Looking at the stages of The REFLECT model, a comparison can be drawn between stage 5 and EBP. Stage 5 describes the process of exploring options for the future for if the individual found themselves in the same situation as what they are reflecting on. Exploring options indicates comparing different forms of available evidence to assess which is most relevant and appropriate to use to improve the outcome of the situation if it should happen again. Similarly, it could be argued that the process of reflection could be used as evidence in the future to improve clinical judgment (Mamede et al., 2008). By using the REFLECT model the user is actively indicating what they have learnt through the process of reflection. Renedo et al., (2018) explains how the relationship between experience and evidence creates a hybrid form of knowledge. Learning from experience can be used as future evidence as a practitioner can refer to the knowledge they have gained in the past and compile it with existing evidence. Past experiences could also be used to assess what is deemed as “best evidence.” For example, the available best evidence may indicate a use of a treatment that may not work on an individual for a variety of reason. The practitioner may have encountered a situation similar to this in the past and could argue why the treatment may not best to use through the knowledge they have gained through past experience. This highlights the importance of RP with the interaction between stakeholders.

The integration between RP and EBP is extremely beneficial for improving professional practice. This form of advanced learning is important for Bioveterinary scientists as it integrates a from of active learning along with the assessment of what would be the best evidence to use for different situations. Understanding the importance of learning through personal actions and experiences and then linking this with the assessment of available evidence will allow for a Bioveterinary scientist to critically determine the best course of action when being faced with challenging scenarios.

 

 

Bibliography

Bannigan, K. and Moores, A. (2016) © CAOT PUBLICATIONS ACE Key words Clinical decision making Evidence-based occupational therapy practice Reflection Curriculum development Thinking.

Barksby, J., Butcher, N. and Whyshall, A. (2015) ‘A new model of reflection for clinical practice.’ The Nursing Times.

Biglan, A. and Ogden, T. (2008) ‘The Evolution of Evidence-based Practices.’ European journal of behavior analysis, 9(1) pp. 81–95.

Giuffrida, M. A. (2017) ‘Practical Application of Evidence-Based Practice.’ Veterinary Clinics of North America: Exotic Animal Practice. (Evidence-Based Clinical Practice in Exotic Animal Medicine), 20(3) pp. 737–748.

‘Graham Gibbs Reflective Cycle Model 1988’ (2016) ELN Resources. 7th December. [Online] [Accessed on 10th November 2018] https://resources.eln.io/gibbs-reflective-cycle-model-1988/.

Husebø, S. E., O’Regan, S. and Nestel, D. (2015) ‘Reflective Practice and Its Role in Simulation.’ Clinical Simulation in Nursing. (Theory for Simulation), 11(8) pp. 368–375.

Mamede, S., Schmidt, H. G. and Penaforte, J. C. (2008) ‘Effects of reflective practice on the accuracy of medical diagnoses.’ Medical Education, 42(5) pp. 468–475.

Oelofsen, N. (2012) ‘Using reflective practice in frontline nursing.’ Nursing Times, 108(24) pp. 22–24.

Renedo, A., Komporozos-Athanasiou, A. and Marston, C. (2018) ‘Experience as Evidence: The Dialogic Construction of Health Professional Knowledge through Patient Involvement.’ Sociology, 52(4) pp. 778–795.

Sackett, D. L., Rosenberg, W. M., Gray, J. A., Haynes, R. B. and Richardson, W. S. (1996) ‘Evidence based medicine: what it is and what it isn’t.’ BMJ : British Medical Journal, 312(7023) pp. 71–72.

Sellars, M. (2017) Reflective Practice for Teachers. SAGE.

Siminoff, L. A. (2013) ‘Incorporating patient and family preferences into evidence-based medicine.’ BMC Medical Informatics and Decision Making, 13(Suppl 3) p. S6.

Sweet, L., Bass, J., Sidebotham, M., Fenwick, J. and Graham, K. (2018) ‘Developing reflective capacities in midwifery students: Enhancing learning through reflective writing.’ Women and Birth, June.

Turner, M. (n.d.) UC Library Guides: Evidence-Based Practice in Health: Introduction. [Online] [Accessed on 14th November 2018] https://canberra.libguides.com/c.php?g=599346&p=4149211.

Biochemistry experiment 4A and 4B – Time course of an enzyme catalysed reaction/ Effect of pH on the rate of an enzyme catalysed reaction

  • Introduction

Enzymes are organic molecules comprising of proteins. They are highly specific in the way in which each one catalyses a unique individual reaction. As enzymes are comprised of proteins they can denature if not kept in idyllic conditions, therefore they lose their function thus stopping any reactions (Gomes and Rocha-Santos, 2018). As enzymes are highly specific they each work differently in varying degrees of pH. (Krogdahl et al., 2015) Enzyme catalysed reaction experiments can be extremely important and beneficial, especially in some fields of medical research. Tapper et al., (2017) suggests that elevated liver enzymes within a patient can indicate detection of a wide variety of liver diseases. With the use of enzyme catalysed reaction experiments, several different tests to determine which type of disease is present can be done all at once. This is a more efficient and affordable way to obtain fast results.

 

  • Materials and methods

 

2.1 Part A: Time course of an enzyme catalysed reaction

5 sets of 3 microcentrifuge tubes were firstly labelled A1-E3. The following solutions were then added to each microcentrifuge tube; 0.2 mL distilled water, 0.25 mL sodium acetate buffer solution (pH5.0) and 0.25mL para-nitrophenol phosphate (pNPP) solution. Each tube was then left to equilibrate at room temperature for 5 minutes, then immediately transferred to a water bath of 30  where they were left for a further 5 minutes. 0.05 ml of enzyme was then added to each tube and gently agitated. In order to maintain accurate timings, the enzyme was added to each tube at 1-minute intervals. The times were recorded by a clock timer. The 5 sets of microcentrifuge tubes were incubated for 5, 10, 15, 20 and 30 minutes (i.e. tube sets A1-A3 incubated for 5 minutes, tube sets B1-B3 incubated for 10 minutes etc). At the end of each incubation period the reaction was stopped by adding 0.5 ml sodium carbonate and the tube was then placed back into the test tube rack. Before determining the absorbance of each reaction mixture, a blank solution was prepared by adding the following solutions into a microcentrifuge tube; 0.2 mL water, 0.25 mL sodium acetate buffer, 0.25 mL pNPP, 0.5 mL sodium carbonate and lastly 0.05 mL of enzyme. To determine the rate of absorption the spectrophotometer had to firstly be set to calibrated with the blank solution. The absorbance of each reaction mixture was measured at a wavelength of 410nm. Before the absorbance could be measured by the spectrophotometer, the contents of each microcentrifuge tube were transferred into a low-volume cuvette.

2.2 Part B: Effect of pH on the rate of an enzyme catalysed reaction

The same basic procedures were followed as mentioned before in section 2.1. Although, for this experiment 4 sets of 3 microcentrifuge tubes were used, with each set containing a different pH buffer. There were 4 different pH buffers provided which included; pH 3, pH 5, pH 7 and pH 9. Each of the 4 sets of microcentrifuge tubes were all placed in a water bath at 30  for 10 minutes. Following completion of incubation, the reaction was then stopped by adding 0.5 mL sodium carbonate to each tube.  The incubation period was chosen as 10 minutes sat in the centre of the linear trend line and this was deemed the most accurate choice. The blank used to calibrate the spectrophotometer in part B was prepared in the exact same way as the blank that was used in part A. After calibration of the spectrophotometer, the absorbance of each reaction mixture was again measured at a wavelength of 410nm. The raw data obtained from the spectrophotometer readings was then converted into the amount of pNP produced within 10 minutes. This data was obtained by using equation 1. Equation 2 was then used to convert the data obtained from equation 1 into the rate of pNP produced per minute. A mean average was then taken from this final set of data to be used to plot the graph that is shown in figure 3.

Equation 1 – pNP produced (μmol)= (absorbance @ 410nm-0.0035)/3.1576

 

Equation 2 – rate of reaction (μmol / min)=(amount of pNP (µmol))/(10 (min))

 

  • Results

To begin the results, the pNP calibration data provided in experiment 4A was converted into a graph as shown by figure 1. The equation provided by this first graph was then used to convert the absorbance values that were obtained in experiment 4A into concentration of pNP produced (µmol).

Figure 1

 

Figure 1 – pNP calibration data plotted to obtain equation y = 3.1576x + 0.0035.

This equation was then used to convert the recorded 3 absorption values for each set of 5 into µmol of pNP produced for each different time frame (5 minutes, 10 minutes etc.). A mean average was then taken from the 3 absorbances samples from each set. This mean average was then plotted on the graph shown in figure 2. The graph was used to calculate an accurate incubation time for the samples used in experiment 4B.

Figure 2

 

Figure 2 – Mean averages taken from the three samples recorded for the 5 different sets, plotted against the time each set was incubated for. The standard deviation from all the results recorded for each of the 5 sets was calculated to be used in the form of error bars to indicate accuracy.

It was decided that 10 minutes would be the optimum incubation time for the samples produced in experiment 4B. This decision was made as 10 minutes sits in-between the linear phase. This was deemed appropriate as 5 or 15 minutes may not have been as accurate as the sat at the very beginning and the very end of the linear phase.

Figure 3

Figure 3 –  The rate in which pNP (µmol) is being produced per minute at 4 different levels of pH, including; pH3, pH5, pH7 and pH9. The samples were incubated for an optimum time of 10 minutes in a water bath at 30.

 

  • Discussion

Acid phosphatase hydrolyses phosphate from a variety of molecules which contain a phosphate group (Wang and Liu, 2018). The substrate used within this experiment was para-nitrophenol phosphate (pNPP). When subjected to acid phosphatase, pNPP is hydrolysed to create para-nitrophenol (pNP). Within an alkaline solution pNP can be detected as it turns yellow in colouration (Jez et al., 2016). This is beneficial as the colouration can be assessed with the use of a spectrophotometer to monitor the rate of production of pNP. Production rate of any enzymes relies on specific environments.

Observing figure 3 it is clear to see that the idyllic pH for acid phosphatase to produce pNP is pH 5. As enzymes are acidic in nature it could be assumed that pH 5 would be optimum as acid phosphatase is clearly acidic in nature so would work best in an acidic environment. pH 5 is deemed acidic without it being too harsh that it becomes destructive to the enzyme. It could also be argued that pH 7 would be ineffective as the solution would still be too neutral for a productive enzyme reaction rate. This argument can be supported by Behera et al., (2017) who also carried out an enzyme assay on acid phosphatase. It is stated within this experiment that the optimum acid phosphatase activity was recorded at a temperature of 48 at a pH of 5. However, although the optimum pH for both experiments was deemed pH 5, two different temperatures were used which would have affected both results. Nevertheless, both arguments can be debated as Jennifer et al.,(2015) suggests that the optimum environment for the production of enzymes is pH 9 at a temperature of 40. The comparison of these arguments could propose that further study should be completed to investigate the effects of varying temperatures on the production of enzymes.

Optimal conditions are essential for enzyme production. A change in environment can cause an enzyme to denature, thus meaning they stop functioning, which will result in a stop of production. (Saoudi et al., 2017) Denaturation is the process in which an enzyme will undergo threatening and even destroying structural change. Although enzymes are subject to structural damage, no reaction would be strong enough to break the peptide bonds between amino acids, so the enzymes primary structure would remain unchanged (Cheng et al., 2015). As the process of denaturation causes the enzyme to change structure, this then means that the specific substrate matched with that enzyme can no longer bind as the active sight region has been impaired resulting in the substrate and enzyme no longer being a match (Witkowska et al., 2018).

 

  • Conclusion

To conclude, the production rate of pNP was measured over numerous lengths of time to investigate the most productive time span for pNP production. This information was then used to evaluate the best incubation time for the solutions used with varying degrees of pH. The experiment deemed that pH 5 was the optimum environment for pNP production. Literature suggests that this is due to the enzyme working well within an acidic environment because it itself is acidic in structure, although the pH concentration wasn’t too acidic that it would cause any structural damage to the enzyme, causing it to denature.

 

 

 

 

References

Behera, B. C., Yadav, H., Singh, S. K., Mishra, R. R., Sethi, B. K., Dutta, S. K. and Thatoi, H. N. (2017) ‘Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.’ Journal of Genetic Engineering and Biotechnology, 15(1) pp. 169–178.

Cheng, B., Wu, S., Liu, S., Rodriguez-Aliaga, P., Yu, J. and Cui, S. (2015) ‘Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases.’ Nanoscale, 7(7) pp. 2970–2977.

Gomes, A. R. and Rocha-Santos, T. A. P. (2018) ‘Enzyme Assays☆.’ In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Elsevier.

Jennifer, E. B. M., Sathishkumar, R. and Ananthan, G. (2015) ‘Screening of extracellular hydrolytic enzymes production by ascidians (Polyclinum glabrum, Microcosmus exasperates and Phallusia arabica) associated bacteria from Tuticorin, Southeast coast of India.’

Jez, J. M., Ravilious, G. E. and Herrmann, J. (2016) ‘Structural biology and regulation of the plant sulfation pathway.’ Chemico-Biological Interactions. (Special Issue on Sulfation Pathways), 259, November, pp. 31–38.

Krogdahl, Å., Sundby, A. and Holm, H. (2015) ‘Characteristics of digestive processes in Atlantic salmon (Salmo salar). Enzyme pH optima, chyme pH, and enzyme activities.’ Aquaculture. (Proceedings of the 16th International Symposium on Fish Nutrition and Feeding), 449, December, pp. 27–36.

Saoudi, O., Ghaouar, N., Ben Salah, S. and Othman, T. (2017) ‘Denaturation process of laccase in various media by refractive index measurements.’ Biochemistry and Biophysics Reports, 11, September, pp. 19–26.

Tapper, E. B., Saini, S. D. and Sengupta, N. (2017) ‘Extensive testing or focused testing of patients with elevated liver enzymes.’ Journal of Hepatology, 66(2) pp. 313–319.

Wang, L. and Liu, D. (2018) ‘Functions and regulation of phosphate starvation-induced secreted acid phosphatases in higher plants.’ Plant Science, 271, June, pp. 108–116.

Witkowska, D., Cox, H. L., Hall, T. C., Wildsmith, G. C., Machin, D. C. and Webb, M. E. (2018) ‘Analysis of substrate binding in individual active sites of bifunctional human ATIC.’ Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics, 1866(2) pp. 254–263.

 

Osmosis

Water is essential for the survival of all living beings. But, the input and output of water entering the body’s cells must be regulated. This regulation of water is known as Osmosis. Osmosis consists of a solvent and a solution, for example pure water and salt water, being separated by a semi- permeable membrane. This semi – permeable membrane allows water molecules to flow through freely from any direction, whereas the salt molecules will not be able to pass through the membrane as they are too large. Water will always move from the side with the highest water concentration to the side with the lowest water concentration, this is to equalise the concentration of constituents on both sides of the membrane. (Borg, 2017). Osmosis is known as a special type of diffusion. Osmosis is needed within animals as it keeps the water input and output within the body regulated, but osmosis itself also must be regulated. This is known as osmoregulation. Osmoregulation is responsible for monitoring the physiological processes to help maintain a concentrated balance of the body fluids inside and outside the cell. (Osmoregulation – an overview | ScienceDirect Topics, n.d.)

Water can enter the body in a variety of different ways for example; drinking, eating and living in an aquatic environment. As water can enter the body, it can also be lost from the body through; urine, faeces, vomit, lactation etc. Species have evolved over time to keep their cells alive through osmosis and osmoregulation. For example, marine fish are hypotonic to their environment. This means that their body fluids contain more water molecules and less solute molecules in comparison to their environment, which is sea water. (BioBook | Leaf: Why is osmosis so important in biology?, n.d.) If marine fish had not evolved and adapted to their environment, their cells would burst due to swelling after taking in too much water. Baldisserotto et al.,( 2007) states that as marine fish are hypotonic to their environment and lose a lot of water, they counteract this by engulfing sea water. 60-85% of this seawater is then absorbed by the intestine. The oesophagus of marine fish absorbs a lot of the salt, but it is almost impermeable to water. This makes the salt level of the water in the intestine much lower, resulting in more water and less salt being absorbed by the intestine and excess salt being excreted in any small amounts of urine that is produced. This is an adaptation that has been developed by marine fish to respond to water lose due to their environment.

On the other side of this example, there are also species that are hypertonic to their environment, such as fresh water fish. This means that the cells have a higher salt concentrations than the surrounding environment. (BioBook | Leaf: Why is osmosis so important in biology?, n.d.) Fresh water fish are opposite to marine fish so they take in water as their cells are constantly losing water. If the fish did not take in this extra water their cells would shrink up and die. (Garrey, 1916).

Terrestrial animals have numerous ways of conserving water though osmosis, such as waterproof skin, feathers fur etc. One of the more advanced adaptations for conserving water is the kidneys. The Loop of Henle resides within each nephron within the kidneys. It consists of a descending limb and an ascending limb. The descending limb is impermeable to solvents, but water can move around freely, this is where the water is absorbed back into the cells. Due to the absorption of water in the descending limb previously, it results in the filtrate in the ascending limb to become concentrated. Within the ascending limb, some solvents are reabsorbed but it is moderately impermeable to water. Any extra waste is then transported to the bladder and excreted as concentrated urine. The more dehydrated the animal is the more concentred the urine becomes. (Regulation of Water Balance, n.d.)

In summary, the evidence shown within this report reflects osmosis as a necessity for life within a whole organism. Species and cells have developed over time so that their bodies can remain hydrated in the most efficient way possible. Osmosis can be displayed differently throughout a variety of species, all with their own similar yet vastly different adaptations.

 

References

Baldisserotto, B., Mancera Romero, J. M. and Kapoor, B. G. (eds) (2007) Fish osmoregulation. Enfield, N.H: Science Publishers.

BioBook | Leaf: Why is osmosis so important in biology? (n.d.). [Online] [Accessed on 3rd December 2017] https://adapaproject.org/bbk_temp/tiki-index.php?page=Leaf%3A+Why+is+osmosis+so+important+in+biology%3F.

Borg, F. (2017) ‘What is Osmosis? Explanation and Understanding of a Physical Phenomenon.’

Garrey, W. E. (1916) ‘The Resistance of Fresh Water Fish to Changes of Osmotic and Chemical Conditions.’ American Journal of Physiology — Legacy Content, 39(3) pp. 313–329.

Osmoregulation – an overview | ScienceDirect Topics (n.d.). [Online] [Accessed on 2nd December 2017] https://www.sciencedirect.com/topics/neuroscience/osmoregulation.

Regulation of Water Balance (n.d.). [Online] [Accessed on 5th December 2017] https://courses.washington.edu/conj/bess/water/water.htm.

Concepts in molecular biology – failures in meiosis which could resulting in chromosomal disease

Meiosis is a cell division process in which haploid gamete cells are produced in a diploid organism. A diploid organism is an organism which has homologous copies of each parent chromosomes within their cells. The parents of the offspring each donate a set of chromosomes which will then equal to two sets (Xu, 2006). Humans have 46 chromosomes within their diploid cells, compared to the 23 chromosomes which can be found within haploid cells. Haploid cells can be found within the gametes of diploid organisms, otherwise known as the sperm and ovum cells or reproductive cells. Haploid cells only have one set of chromosomes because when two parent haploid cells come together, they become fertilised. This means that the offspring will gain a complete set of chromosomes and become a diploid cell or diploid organism. (Kent, 2013).

Meiosis is an incredibly significant process for maintaining life itself. MacLennan (2015) states that meiosis is essential as it allows for the reduction of chromosomes within the gamete cells to maintain the correct number of chromosomes within the offspring after fertilisation. This is to reduce chromosomal disease and maintain genetic diversity within a species. Meiosis creates genetic diversity by recombining the chromosomes genetic material. Genetic variation is then increased further when the two parent gametes come together during fertilisation, there by creating an offspring with unique DNA combinations. (Humphryes and Hochwagen, 2014).

There are two consecutive divisions that take place during meiosis and the result of the processes of meiosis is the production of four haploid daughter cells. These cells that are produced are genetically different from their parent cells and the four cells that are produced are even genetically different from each other. This is because during meiosis, a pair of homologous chromosomes can exchange genetic material before being separated. (Kent, 2013). Meiosis firstly begins with Interphase. During interphase the chromosomes and organelles within a cell begin to duplicate. This results in each chromosome consisting of two daughter chromatids. (Simon et al., 2013). Interphase is also used to store energy as ATP (Adenosine Triphosphate) which can then later be used during the meiosis process. (Kent, 2013).

The process then moves on to Prophase 1. This stage consumes around 90% of the division time as it is an extremely complex stage. During Prophase 1 the chromosomes begin to condense, the nuclear envelope disintegrates, and the spindle apparatus starts to appear. (Kent, 2013). As the chromosomes begin to condense and coil up, proteins cause the homologous chromosomes to draw together and form pairs. The results of this are structures consisting of four chromatids. Within each structure, chromatids start to swap corresponding segments. This is also known as “crossing-over”. The process of crossing over means that the genetic information present is then rearranged. This is how the cells that are produced become genetically different from their parent cells, and each other. (Simon et al., 2013).

The next step is Metaphase 1. During this stage the homologous chromosome pairs begin to line up along the equator of the cell. Spindle microtubules from one pole attach to the centromere of one chromosome, and spindle microtubules from the other pole attach to the centromere of the chromosome’s homologous pair. The centromere does not divide so the sister chromatids remain together. (Kent, 2013).

The homologous chromosomes begin to separate and migrate towards the opposite poles in which they were attached to. (Kent, 2013).  The chromosomes are separated from their homologous partners, but the sister chromatids move as a single component. (Simon et al., 2013). This process is known as Anaphase 1.

The final stage of meiosis 1 is Telophase 1 and Cytokinesis. The chromosomes finally arrive at the poles of the cell. Once they arrive each pole consists of a set of haploid chromosomes, but the chromosomes are still doubled. Cytokinesis coincides with Telophase 1 and two haploid cells are produced. (Simon et al., 2013).

The process of Meiosis 2 results in the separation of the sister chromatids pairs that were separated from their homologous pairs in Meiosis 1. Meiosis 2 begins with a haploid cell that has not undertaken duplication of chromosomes during the Interphase period. This process is considerably similar to that of Meiosis 1. (Kent, 2013). A spindle forms during Prophase 2 which pushes the chromosomes into the centre of the cell. During Metaphase 2 the chromosomes align, with spindle microtubules from opposite poles attaching to the sister chromatids of each chromosome. The centromeres of the sister chromatids separate during Anaphase 2, and the sister chromatids of each pair begin to migrate towards the opposite poles. Finally, in Telophase 2 nuclei begin to form at the poles. Cytokinesis also occurs at this time, and the final result is four haploid daughter cells, each consisting of single chromosomes. (Simon et al., 2013).

The meiotic cell division process does not always run its course perfectly. When things go wrong during meiosis, the effects can be projected through chromosomal disease. Chromosomal disease can be fatal to an embryo. Qi et al, (2018) shows that during their study 42.95% of embryo miscarriage samples, out of 149 samples, all contained at least one chromosomal abnormality. Not all cases of chromosomal abnormalities are fatal, but there are a variety of problems that can occur. Errors in meiosis are referred to as nondisjunction. This means that during the meiotic cell division phase either the homologous chromosomes or sister chromatids failed to separate properly.  Turner Syndrome is a condition that affects around 1 in every 2,500 female babies. The condition means that these female babies are born with only one X chromosome. Normally females are born with two X chromosomes. Because these females are lacking the second X chromosome, their ovaries normally degenerate before they are even born. This means that the females can not undergo puberty. The condition can be treated to an extent by providing oestrogen as a hormone treatment, these would stimulate secondary sexual characteristics such as enlarged breasts, but because the females do not have functioning ovaries they still would not be able to produce offspring. Other characteristics of Turner Syndrome can include; increased risks of cardiovascular disease, kidney defects and hearing loss. (Audesirk et al., 2017)

Another common chromosomal abnormality is Trisomy 21, or Down Syndrome. Trisomy 21 is a condition where by the offspring has accumulated an extra copy of chromosome 21. Children that are born with Trisomy 21 usually have very distinctive physical abnormalities. Some of these can include; a smaller mouth that can only open partially to try and accommodate for a much larger tongue, weak muscle tone and distinctive eye shape. These characteristics also go along side some degree of cognitive impairment (depending on the person) and learning difficulties. Over the years there has been an improvement of health and life longevity for people who suffer from Trisomy 21, but this increase in life longevity has also been linked to the rise in people with Trisomy 21 suffering from Alzheimer’s disease as they get older. Chromosomal abnormalities such as Trisomy 21, occur during meiotic cell division. Abnormalities can occur due to increased maternal age (Prenatal screening for Trisomy 21 is usually advised for mothers over the age of 35) but this isn’t always the case as babies who suffer from this condition have been born to mothers under the age of 35. (Diamandopoulos and Green, 2018).

In conclusion meiosis is vital for life to continue and succeed. Without the success of meiosis every species of living diploid organism would eventually become extinct, as organisms would not be able to further reproduce as a result of haploid gamete cells not being produced. Gamete cell production is essential for the natural continuation of a species. Although meiosis is vital for reproduction, it is also essential for genetic variability. Genetic variability allows new organisms to be produced with a completely unique set of DNA. Without genetic variability the risk of chromosomal disease can be much higher. But, chromosomal disease can occur in any new offspring, even with genetic variability present within their parents. With this in mind, chromosomal disease should continue to be researched to understand more as to why failures in meiosis can arise.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

Audesirk, T., Audesirk, G. and Byers, B. E. (2017) Biology Life on Earth. Eleventh.

Diamandopoulos, K. and Green, J. (2018) ‘Down syndrome: An integrative review.’ Journal of Neonatal Nursing, February.

Humphryes, N. and Hochwagen, A. (2014) ‘A non-sister act: Recombination template choice during meiosis.’ Experimental Cell Research. (DNA DAMAGE AND REPAIR), 329(1) pp. 53–60.

Kent, M. (2013) Advanced Biology. 2nd ed.

MacLennan, M., Crichton, J. H., Playfoot, C. J. and Adams, I. R. (2015) ‘Oocyte development, meiosis and aneuploidy.’ Seminars in Cell & Developmental Biology. (Plasma membrane repair & Development and pathology of the gonad), 45, September, pp. 68–76.

Qi, H., Xuan, Z.-L., Du, Y., Cai, L.-R., Zhang, H., Wen, X.-H., Kong, X.-D., Yang, K., Mi, Y., Fu, X.-X., Cao, S.-B., Wang, J., Chen, C.-J. and Liang, J.-B. (2018) ‘High resolution global chromosomal aberrations from spontaneous miscarriages revealed by low coverage whole genome sequencing.’ European Journal of Obstetrics & Gynecology and Reproductive Biology, 224, May, pp. 21–28.

Simon, E. J., Dickey, J. L. and Reece, J. B. (2013) Campbell Essential Biology. Fifth.

Xu, J. (2006) ‘Extracting haplotypes from diploid organisms.’ Current issues in molecular biology, 8(2) p. 113.

Professional practice, Task D – data handling reflection

During first year at university I have already gained a respectable amount of knowledge through the subjects I have been taught but I have also gained a reasonable amount of applicable skills. The ability to self-reflect has been a very accountable skill that I have gained. Self-reflection gives me the opportunity to look back to the beginning of the year and see how far I have progressed. It also allows me to identify my weaknesses, making me aware of the areas that I need to improve on if I want to be able to better myself for second year and further.

Assessing the grade I achieved from the sector studies exam indicates that there are a variety of areas that I need to improve on in order to better my studies. Comparing the mock that was completed prior to the exam, to the exam itself, I can determine that I struggled with several maths questions. In both the mock exam and the official exam I failed to complete questions referring to graphs. Pareja-Lora et al., (2016) states that mock exams are incredibly useful not just for revision purposes but to highlight strengths and weaknesses to help aid student development. Mock exams give students the opportunity to reflect on their results and gain feedback from their teachers to help develop any weaker skills. With this in mind, I should have engaged with my teacher and asked if it could have been possible for me to go over the graph question and other questions I didn’t quite understand. This would have allowed me to advance my maths skills and may have allowed me to obtain a higher grade. This argument can be developed further by Zhang and Hyland (2018) who proclaim that students who actively ask for teacher feedback gain better understanding and retention of information within that subject.

Referring to the self-reflective task that was completed at the beginning of the year, I refer to use of Self Directed Learning being a useful tool for improving skills “One way of working on a classroom flaw or struggle is to undertake Self Directed Learning (SDL)”. I still stand by this statement as SDL is extremely beneficial to students, as it involves integrity to address weaker areas of study and enables students to take extra time to work on said areas through a variety of different activities. The engagement of SDL activities leads to improved educational results for students (Kastenmeier et al., 2018). SDL can also be considered a form of continuing professional development (CPD). CPD is the act of an individual taking control of their own learning and career development. This is usually done through the act of reflection and action. If an individual has highlighted areas of improvement, they will then take action and physically do something to improve their learning i.e. courses, conferences, outside reading, work experience etc. (Megginson and Whitaker, 2017) This argument is continued further by Wareing et al.,(2017) who suggests that the act of CPD is not only beneficial to the individual, but to anyone who is involved with said individuals work. This could include, working peers, customers, patients etc depending at the career within questions.

To conclude I aim to develop myself further through the suggestions listed above. Within development I may not see a progressive change in my grades and skills set as I move into my second year. Although, these skills are not just confined to grades within university. The can be carried with me throughout my career as their will always be room for improvement and developed in whatever career path I choose.

 

 

 

 

References

Kastenmeier, A. S., Redlich, P. N., Fihn, C., Treat, R., Chou, R., Homel, A. and Lewis, B. D. (2018) ‘Individual learning plans foster self-directed learning skills and contribute to improved educational outcomes in the surgery clerkship.’ The American Journal of Surgery, January.

Megginson, D. and Whitaker, V. (2017) Continuing Professional Development. Kogan Page Publishers.

Pareja-Lora, A., Calle-Martínez, C. and Rodríguez-Arancón, P. (2016) New perspectives on teaching and working with languages in the digital era. Research-publishing.net.

Wareing, A., Buissink, C., Harper, D., Gellert Olesen, M., Soto, M., Braico, S., Van Laer, P., Gremion, I. and Rainford, L. (2017) ‘Continuing professional development (CPD) in radiography: A collaborative European meta-ethnography literature review.’ Radiography. (Radiography Education), 23, September, pp. S58–S63.

Zhang, Z. (Victor) and Hyland, K. (2018) ‘Student engagement with teacher and automated feedback on L2 writing.’ Assessing Writing. (Special Issue: The comparability of paper-based and computer-based writing: Process and performance), 36, April, pp. 90–102.

Professional development plan

 

Professional Practice Task C – Professional development plan

Whilst I am currently still only within my first year of my Bioveterinary Science degree, I have begun to think about my future career or post graduate study after completion of my first degree. I have multiple interests that have arisen since starting the course. Writing a professional development plan allows me to break these interests down into achievable goals, outlining what specific skills I will need, and how I can better myself to attain these skills. Creating a plan for my future career gives me the ability to pin point areas within my study skills that may need to be improved on to allow me to accomplish the grades I will need to either apply for particular post graduate jobs, or move onto post graduate study such as; Veterinary medicine, Masters degree, etc.

One of my academic skills that I wish to advance is my revision strategies. Developing this skill will allow me to successfully complete my degree and give me the grades I need to move onto further study, such as Veterinary Medicine. I will need to attain a 1st  class degree in Bioveterinary Science to have the chance to apply for the Veterinary Medicine accelerated course. Reflecting on exam results from earlier on in the semester, I feel as though I could have accomplished better grades. I believe I would have retained much more information if my revision skills were better. I can make this goal achievable by making small changes to the way I study. Firstly, I can create flash cards after each lecture, rather than trying to make flash cards on all the lectures after they have all been completed. This way, the information will still be fresh in my memory and it will be less time consuming. I can measure my progression by taking note of how many flash cards I get correct each week. If I am not progressing with certain cards, I will know that I will need to put aside more time to revise those certain areas of the topic. Setting aside enough time for revision will also be beneficial. Doing small sessions every week will allow me to retain more, without getting stressed about time and how much content I have left to revise. These strategies will be helpful for both my Comparative Anatomy and Physiology module and my Concepts in Molecular Biology module.

Networking is also incredibly valuable when it comes to creating a career plan. As I am still unsure of the path I want to take after completing my degree, networking and work experience is a great way to engage with scientists within different fields and learn about their own careers. There are a variety of ways that I could make this possible. The Royal Society of Biology (RSB) organise many different conferences and careers fairs that are available to student members, such as myself. As a student member it would be possible for me to volunteer at any of the conferences that take my interests. To make this possible I would have to email the RSB and explain my current situation; where and what I’m studying, why I would like to volunteer, etc. If they allow me to volunteer, it would give me to opportunity to attend one of these conferences, whilst working. It would be useful for me to bring a small notebook and pen with me to take note of any contacts of tips that may be given to me. This way, if I gain the opportunity to get talking to different scientists at a conference I could ask them about their careers and what led them down that path and what skills and grades they needed to get to where they are. Networking during conferences is also a useful tool to gain work experience. Again, by talking with scientists within different fields, it gives me the chance to ask whether it would be possible for me to gain work experience with them, or if they know of any companies/institutions that would be willing to take on students looking for work experience. Work experience is extremely helpful when creating a professional CV when looking for post graduate jobs. Having relevant work experience on my CV could put me in a different category to other candidates as it shows that I not only have the grades needed, but also relevant practical skills, experience and enthusiasm. Work experience would allow me to explore all my different interests and see what the work within that field is like. Exploring different jobs and working environments through work experience could be extremely helpful for me to put together a well thought out decision about post graduate jobs or study as it should allow me to delve into my interests and see which is the one I am most passionate about and would like to develop a career in.

Laboratory Report

 

Abstract

DNA fingerprint matching using PCR is an accurate and affective way of identifying or matching DNA to an available sample. It is used widely throughout forensic science and is spoke about very highly within further research. Throughout this report the positives of PCR are discussed, along with the use of equipment and how this method is still effective, even when not accomplished completely accurately.

 

Introduction

The purpose of this experiment was to identify which suspects DNA, out of the four samples provided, matched the DNA that was given as DNA found at a scene. The DNA matching was completed using Polymerase Chain Reaction (PCR). The DNA used within this experiment was provided in a DNA fingerprinting experiment kit, created by Edvotek (EDVOTEK, 2017). However, DNA matching using PCR is frequently used by forensic professionals to determine DNA profiling of suspects using minute amounts of DNA which has been extracted from a crime scene. (Sinelnikov and Reich, 2017). Cavanaugh and Bathrick (2018) argue that PCR amplification is one of the most effective forms of DNA matching within forensic science. PCR is found to be more effective than other methods as DNA samples can be directly added to an amplification reaction, rather than being exposed to DNA extraction, purification or quantification. All of which can damage or contaminate DNA samples. This method also allows maximum amounts of DNA to be extracted, allowing for less error when matching DNA fingerprints. This is a vital advantage when working within forensic science.

Materials

  • DNA samples (provided by EDVOTEK)
  • 5 PCR tubes
  • Primer mix
  • PCR Edvobead
  • Centrifuge
  • (50x) Buffer
  • Distilled water
  • Flask
  • Microwave
  • 7x7cm casting tray – with rubber ends and well templates
  • Electrophoresis chamber
  • Timer

Method

Firstly, we began by labelling 5 PCR tubes to ensure that the experiment was completely accurate. The tubes were labelled; Crime scene (CS), suspect 1 (S1), suspect 2 (S2) suspect 3 (S3) and suspect 4 (S4). To prepare each individual PCR reaction, each tube was filled with 20 micro litres of the primer mix provided, then 5 micro litres of the crime scene DNA (for the PCR tube labelled CS) and finally one PCR Edvobead. This was repeated for each tube, but the appropriate DNA was added to the appropriately labelled tube. Each tube was then gently shaken to mix the solution inside, and to ensure that the Edvobead was fully dissolved. Next, the samples were placed inside the centrifuge to spin and separate the DNA from the rest of the solution, making it easy to collect from the bottom of the tube. The DNA was then amplified using PCR. The PCR cycle started with initial denaturation starting at 94 degrees Celsius for 3 minutes. After, the cycle continued with conditions of 94 degrees Celsius for 30 seconds, then 55 degrees Celsius for 65 seconds and then 72 degrees Celsius for 30 seconds. This cycle was repeated 30 times. Before beginning electrophoresis, 5 micro litres of 10x gel loading solution was added to each tube.

The next step was to dilute 0.5ml of concentrated (50x) buffer with 24.5ml of distilled water into a flask. 0.25g was then added to the solution to create a total volume of 25ml. The agarose powder was then dissolved in the solution by microwaving it on high for 1 minute. The flask was taken out of the microwave and swirled to see if the agarose had dissolved. The flask continued to be heated for short 15 second bursts until the liquid was completely transparent – indicating that the agarose had been dissolved. The was set aside to cool down, whilst waiting, the rubber end caps were placed at the ends of 7x7cm the gel-casting tray that the gel would be placed in. The well template, or comb, was then clipping into the tray, ready for the solution to be poured in. Once the flask containing the solution had cooled down enough so that it could be touched without gloves, the agarose solution was poured into the gel-casting tray. The gel was left for over 20 minutes to allow enough time for setting. Once the gel was completely set the rubber end caps were removed, along with the comb. The comb had to be removed extremely carefully to ensure that none of the well moulds were damaged.

The tray containing the gel was then placed inside the electrophoresis chamber. 1x electrophoresis buffer was then poured into the chamber, until the tray was completely submerged. Lane 1 of the wells was filled in with the ladder sample. Then following that each DNA sample that was prepared earlier (CS,S1,S2,S3,S4) was placed in an individual well within the gel, in that order. The safety cover was then clipped onto the chamber, and the appropriate leads were attached to the appropriate power source (Red – Red, Black – Black). The electrophoresis chamber was then turned on for approximately 55 minutes. After the electrophoresis was complete, the gel trays were removed from the chambers, and the gel was then removed from the tray. The gels were then taken to a different lab where they were scanned by a technician to produce an image of the results.

(EDVOTEK, 2017)

Results

L S4 S3 S2 S1 CS L

(Essential Laboratory Techniques : Crime Scene Gel, n.d.)

The image above is the photo that was produced after the gel had been scanned by the technician. From order of right to left, the wells read; Ladder marker, crime scene, suspect 1, suspect 2, suspect 3, suspect 4 and the final ladder marker. As shown by this image the suspect that was the closest match to the crime scene DNA was suspect 3, as indicated by the DNA highlighted within the wells.

Discussion

From the results shown in the image provided, it can be seen quite clearly that the wells at the top of the image are more intact and more obvious to see. But towards the bottom of the image it appears the wells become less distinct. As discussed previously, the agarose gel solutions were set in gel casting trays size 7x7cm, and the electrophoresis chamber was turned on for around 55 minutes. From the results shown we can see that the wells almost taper off, towards the end of the image. As shown from the image below it is quite clear that all the wells are intact and obvious to see when the gel was scanned for an image.

(Zhang et al., 2015)

This could suggest that the agarose gel became damaged during the experiment because as the DNA was migrating during the electrophoresis (DNA migrates towards the red [positive] electrode) the gel may have become damaged if the chamber was too small and if the current was too strong. If the DNA didn’t have enough room to migrate properly the larger sections of DNA could have damaged the wells whilst travelling, creating a less clear picture. With this in mind, the electrophoresis chamber may also have been left on for too long, although the manufacturer guides were followed correctly.

Although the gel may have been damaged during the experiment, it is still clear from the results which suspect DNA matched the DNA that was found at the crime scene. This gives indication that although the experiment may not have been completed 100% correctly, it still shows how accurate PCR can be, giving and indication of why it is so popular and widely used within forensic science and other sciences. (Pounder et al., 2005)

Conclusion

In conclusion, the experiment was successful in determining which suspect DNA matched that of the crime scene DNA. DNA fingerprint recognition using PCR has been proven to be extremely successful and accurate. The method produces clear imagery and evidence, even if the process of electrophoresis isn’t always correct. This shows the benefits of PCR and how determining and matching DNA fingerprints has developed.

References

Cavanaugh, S. E. and Bathrick, A. S. (2018) ‘Direct PCR amplification of forensic touch and other challenging DNA samples: A review.’ Forensic Science International: Genetics, 32(Supplement C) pp. 40–49.

EDVOTEK (2017) ‘DNA fingerprinting using PCR.’

Essential Laboratory Techniques : Crime Scene Gel (n.d.). [Online] [Accessed on 9th January 2018] http://moodle.writtle.ac.uk/mod/resource/view.php?id=105232.

Pounder, J. I., Williams, S., Hansen, D., Healy, M., Reece, K. and Woods, G. L. (2005) ‘Repetitive-Sequence-PCR-Based DNA Fingerprinting Using the DiversiLab System for Identification of Commonly Encountered Dermatophytes.’ Journal of Clinical Microbiology, 43(5) pp. 2141–2147.

Sinelnikov, A. and Reich, K. (2017) ‘Materials and methods that allow fingerprint analysis and DNA profiling from the same latent evidence.’ Forensic Science International: Genetics Supplement Series, 6(Supplement C) pp. e40–e42.

Zhang, Y., Suehiro, Y., Shindo, Y., Sakai, K., Hazama, S., Higaki, S., Sakaida, I., Oka, M. and Yamasaki, T. (2015) ‘Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer.’ Oncology Letters, 9(1) pp. 454–458.