bluuebunny/crossref_metadata_2025 huggingface dataset:
Psychometric Testing in Interviews
Authors: Watkin Sara , Vincent Andrew
Published: November 2020

Psychometric testing is widely used outside medicine as part of an employee selection process, although its use has attracted a great deal of criticism too. In the NHS, psychometric tests are often used for selection of mangers and a growing number of Trusts are now using psychometric testing as part of the consultant interview process. This chapter looks at the implications of this and at some of the more commonly known tools. Psychometric tests include personality tests, e.g. Myers—Briggs and Insights, as well as aptitude tests designed to assess an individual’s general logical ability, and verbal, numerical and technical reasoning. Aptitude tests are thought to be more accurate in predicting job performance than personality tests but have not been widely used in medicine. The tools used within medicine tend to look at a person’s personality, how they behave within teams and how they respond to conflict. Some of the more commonly used tools are: • Myers—Briggs Type Indicator® (MBTI®) • 16 Personality Factor (16PF®) • Insights Discovery® Personality Profile (Insights) • Fundamental Interpersonal Relations Orientation (FIRO-B®) • Thomas—Kilmann Conflict Mode Instrument (TKI) • Strength Deployment Inventory® (SDI®) Two of these tools, MBTI and SDI, explicitly state that they should not be used as part of a selection process. However, they are increasingly being used, driven by the advice of unqualified or unscrupulous behavioural consultants! . . . What should I do if asked to sit a test? . . . You are who you are. Do not panic and do not try to work out what the panel want when answering questions about your personality. In general you will only get it wrong! The outcome of your assessment will not be you and the person you appear to be may be less suited for the job than the real you. Equally, this is not something you can practise—you can’t really ‘get good’ at something that is designed to simply show the real you! It’s also worth remembering that if the panel members are determined to employ someone you are not, ask yourself whether you would be happy in the role.
If You Do Not Get the Job
Authors: Watkin Sara , Vincent Andrew
Published: November 2020

If you do not get the job you are bound to feel disappointed, particularly if you have worked really hard in preparation. Remember, there can only be one appointment and it is possible you did everything right on the day and it sadly came down to something tiny. Interview panels can deliberate for hours, but the answer you get is still either ‘yes’ or ‘no’ and, if it’s the latter, it hurts. However, it is important to both learn from the experience through feedback and reflection and pick yourself up and start again, positively and proactively. This chapter looks at: • Getting effective feedback • Reflecting on that feedback • Common feedback • Picking yourself up and starting again You should always be offered feedback if you are unsuccessful at interview and it is really important that you accept this, despite how hurt you may feel in the aftermath. Indeed, if you are not offered feedback, always ask for it because it is an essential component of either improving next time or perhaps even not changing your approach at all. If someone else had a skill you just didn’t have, you can’t easily change that in short order and so if everything else was spot on, don’t break the formula! The best time to have feedback is some days after the event but close enough that you can remember what happened. Having feedback immediately after being told you were unsuccessful is likely to be less productive, as you are carrying the pain of rejection and normal defence mechanisms make it more likely that you will reject the feedback. Remember, feedback on a failure to be appointed is much like receiving bad news in medicine and, as we were taught in medical school, when given bad news most of us will not hear beyond those first few words, in our case ‘Sorry you have been unsuccessful’. By letting the dust settle and then approaching it with a mindset that says ‘This will help me learn, improve and be successful next time’, we can utilize the feedback to hone our approach to the next level.
Electricity, Magnetism and Circuits
Authors: Magee Patrick , Tooley Mark
Published: November 2020

Electricity is a broad term that includes a variety of phenomena resulting from the flow (or presence) of electric charge. It is a complex subject, but this chapter will provide the necessary background for simple electric circuits here, and the following chapters on electronics, biological signal processing, and electrical safety. Electric charge is a property of certain subatomic particles. Charge originates in the atom, in which its most familiar carriers are the proton and electron. By convention, electrons carry a negative charge while protons carry a positive charge. The protons are located in the centre of the atom and the electrons are in motion outside of the nucleus in orbits. In simplistic terms, the protons are trapped inside the nucleus making it difficult for them to ‘escape’. Under certain conditions, however, electrons can escape, and the movement of them is ‘electricity’. Charge may be transferred between bodies, either by direct contact, or by using a conducting material such as copper or wire. Static electricity refers to the imbalance of charge on a body, usually caused by friction when two dissimilar materials are rubbed together, transferring charge from one to the other. Charge is measured in coulombs (C) and one coulomb contains 6.24 × 1018 electrons. The movement of this electric charge is known as an electric current, and the magnitude of this is measured in amperes. One ampere flowing for one second passes a coulomb of charge. So the current (I) = C s−1. By historical convention, conventional current flows from the most positive part of a circuit to the negative and this concept is continued in this text and most others (but in fact the actual flow of the electrons is in the opposite direction). Current flowing through a wire causes many observable effects, for example heating and magnetism. Magnetism will be discussed later. Voltage was historically called tension or pressure, which captured the concept well, and a water analogy can further help with this understanding and is discussed later. Voltage is the electric potential energy per unit charge, measured in joules per coulomb (V, volts = J C−1).
Ultrasound and Doppler
Authors: Magee Patrick , Tooley Mark
Published: November 2020

Ultrasound has many uses in areas of medicine associated with anaesthesia. It is used for imaging, visualisation of needle and catheter positioning, therapy and, together with the Doppler effect, for measurement of flow velocity. Real-time information can be obtained with ease, and with the low energies used, diagnostic equipment exposures are not thought to be a safety issue, either for the operator or the patient. Ultrasound is a form of mechanical energy that consists of high frequency vibrations at frequencies above human hearing range (> 20 kHz) and up to frequencies in the tens of MHz range. The frequencies used are dependent on issues such as the penetration and resolution required. It is thought that low intensity ultrasound passes through living tissue without altering tissue function. Higher energy can produce heating and cavitation, both of which can alter cell function. Ultrasound is generated by electrically inducing a deformation in a piezoelectric crystal, which compresses and decompresses the medium to which it is coupled at a rate equal to the frequency of the driving voltage. The pressure changes travel through the medium in a longitudinal direction and the distance between the points of maximum pressure, or compression, is known as the wavelength. Figure 10.2 shows the relationship between the period of the wave and the wavelength. The length of this distance is dependent on the elasticity (compressibility) and the density of the medium, and the delay between the movement of adjacent particles in the medium. As shown in the figure, the wavelength (λ) and the transmission frequency (f) are related to the propagation velocity c by c = f λ. The magnitude of the wave is the difference between the maximum and minimum pressure values. The wave propagates by movement of particles: it cannot travel in a vacuum and it does not ionise the medium through which it travels. The propagation of an ultrasound wave is not constant throughout the body. Various parts allow the passage of the wave at different velocities. Also the wave is attenuated differently by the various tissue types. For example, in soft tissues the ultrasound wave has a velocity of between 1460 and 1630 m s−1 whereas in bone it is 2700–4100 m s−1.
Gas pressure, Volume and Flow Measurement
Authors: Magee Patrick , Tooley Mark
Published: November 2020

The physics of pressure, flow and the gas laws have been discussed in Chapter 7 in relation to the behaviour of gas and vapour. This section will focus on the physical principles of the measurement of gas pressure, volume and flow. Unlike a liquid, a gas is compressible and the relationship between pressure, volume and flow depends on the resistance to gas flow (or impedance if there is a frequency dependence between pressure and flow in alternating flow, see Chapter 4 for the electrical analogy of this) in conduits (bronchi, anaesthetic tubing); it also depends on the compliance of structures being filled and emptied (alveoli, reservoir bags, tubing or bellows). Normal breathing occurs by muscular expansion of the thorax, thus lowering the intrathoracic pressure, allowing air or anaesthetic gas to flow towards the alveoli down a pressure gradient from atmospheric pressure. When positive pressure ventilation occurs, gas is ‘pushed’ under pressure into the alveoli. Depending on the exact relationship between the ventilator and the lungs, different relationships exist between airway pressure (rather than alveolar pressure, which cannot easily be measured) and gas flow and volume. Gas pressure measurement devices were traditionally in the form of an aneroid barometer, a hollow metal bellows calibrated for pressure and temperature, which contracts when the external pressure on it increases, and expands when it decreases. The movement is linked to a pointer and indicator dial. It is often more convenient to make the device in the shape of part of a circular section, but the principle is the same. This is what the Bourdon gauge, which commonly measures pressure in gas cylinders, looks like. The detection of movement of the diaphragm of an aneroid barometer can take several forms. The movement can either be linked via a direct mechanical linkage to a pointer, or diaphragm movement can be linked to a capacitative or inductive element in an electrical circuit, such as a Wheatstone bridge. Airway pressure during spontaneous breathing or artificial ventilation is low. The preferred units of measurement are cm H2O and the range of values is between −20 and +20 cmH2O. The aneroid barometer to measure this will therefore be of light construction, using thin copper for the bellows material.
Blood Gas Analysis
Authors: Magee Patrick , Tooley Mark
Published: November 2020

A blood gas machine has electrodes to measure pH, pCO2 and pO2 and often measures Hb and some biochemistry as well [King et al. 2000]. Derived values from such a device include O2 saturation, O2 content, bicarbonate, base excess and total CO2. This is the Clarke electrode described in the previous section on gas analysers and is suitable for both respiratory and blood O2 analysis. A pH unit has been defined in Chapter 1 as. In words, this can be described as ‘the negative logarithm, to base ten, of the hydrogen ion concentration’. The physical principle on which the pH electrode is based depends on the fact that when a membrane separates two solutions of different [H+], a potential difference exists across the membrane. In a pH electrode, such a membrane is usually made of glass and the development of a potential difference between the two solutions is thought to be due to the migration of H+ into the glass matrix. If one solution consists of a standard [H+], the pH of the other solution can be estimated by measurement of the potential difference between them. The glass membrane used is selectively permeable to H+. No current flows in this device, which does not wear out, in contrast to the Clark electrode, in which current does flow and that does need periodic replacement. The pH measurement system is shown diagrammatically in Figure 17.1. It consists of two half cells. In one half it has an Ag/AgCl electrode and in the other a Hg/HgCl2 (calomel) electrode. Each electrode maintains a fixed electrical potential. The Ag/AgCl electrode is surrounded by a buffer solution of known pH, surrounded by the pH sensitive glass. Outside the glass membrane is the test solution, usually blood, whose pH is to be measured. It is the potential difference across the glass, between these two solutions, which is variable. The blood or other solution is separated from the calomel electrode by a porous plug and a potassium chloride salt bridge to minimise KCl diffusion. The potential difference across the system is about 60 mV per unit of pH change at 37◦C.
Cleaning and Sterilisation of Equipment
Authors: Magee Patrick , Tooley Mark
Published: November 2020

Although a proportion of anaesthetic and surgical equipment is disposable nowadays, there is still a significant amount of cleaning and sterilisation required, and with the emergence of new organisms, the methods used have come under closer scrutiny. It is worth noting, in passing, that hand cleanliness of staff coming into contact with patients has also come under close scrutiny in recent years. Cleaning of equipment involves the physical removal of as much of the infectious agent as possible, usually using water and a detergent, and is an important precursor to disinfection or sterilisation. It can be done manually where automated devices are unavailable. Ultrasonic washers are sometimes used, as are irrigation pumps for flushing out the lumina of tubes. There is a difference in definition between disinfection and sterilisation. Disinfection is merely the killing of non-spore producing micro-organisms. It kills most bacteria except mycobacteria and spores, and it kills some fungi and some viruses. A higher level of disinfection ensures the destruction of mycobacteria, and most fungi and viruses. Sterilisation is required to kill all micro-organisms, including spores, fungi and viruses. Prions are, however, resistant to most sterilisation procedures. To disinfect or sterilise the modes of heat, chemicals or radiation are used. Moist heat is much more efficacious at coagulating bacterial protein than dry heat, which requires higher temperatures for longer periods to guarantee effect. Moist heat achieves this by increasing the permeability of the organism’s cellular structure to the heat. A hot water washer or low temperature steam applied to instruments for fifteen minutes kills bacteria, but not spores. Higher temperatures are achievable by pressurising the steriliser (Boyle’s law). The modern autoclave uses steam at 134°C and 2 bar, when 3½ minutes is sufficient to kill all organisms, providing the steam can reach the instruments; however, to dry the equipment, the steam is removed and replaced with sterile air, the total cycle time being 10 minutes. Rubber and plastic materials degenerate after some time with this regime, and a combination of 121°C for fifteen minutes, or 115°C for thirty minutes can be used instead.
Ear, Nose, and Throat
Authors: Gupta Pawan
Published: November 2020

A variety of ENT disorders present to the ED on a regular basis and are seen by the ED junior doctors. The majority of these are benign, but a few may be life-threatening conditions that require immediate recognition, rapid assessment, management, and involvement of the ENT specialist, for example, severe epistaxis (especially posterior type), acute epiglottitis, angio-oedema, and Ludwig’s angina. Therefore a basic knowledge and an understanding of the diagnostic features of the common ENT conditions are vital. It would be impossible to cover the large extent of ENT conditions presenting to the ED within a small space, hence this chapter will focus on a few common ENT emergencies that a newly starting ED doctor would have to deal with on a regular basis. There is a wide variety of ENT symptoms depending on the region affected. However, it is important to keep in mind the red flag symptoms that signal urgent help of an ENT specialist is required. Some examples are: sudden unexplained sensorineural deafness, facial nerve palsy, CSF leak, difficulty swallowing with toxic appearance, and drooling saliva. The ENT physical examination is different from other systems as its components are largely inaccessible, particularly in the ED. A good headlight, an auroscope, and correct patient positioning are all important accessories. Although rod lens and flexible fibreoptic scopes for nasoendoscopy and laryngoscopy are routine investigative aids, they are outside the realm of emergency medicine. Most of the diagnosis and management, however, of ENT emergencies can be achieved by following simple rules and using basic equipment. If any patient requires more than an auroscope or standard nasal speculum for a thorough examination, they should be referred to the ENT specialist. All sorts of foreign bodies may become lodged in the ears, nose, or throat. Most of them cause discomfort but are not life-threatening. A foreign body in the throat may have the potential to compromise the airway—a fact to be borne in mind. A lot of them can be removed in the ED, particularly in adults, but children sometimes may require attention of the ENT specialist depending on their age and the capacity to cooperate.
Obstetrics and Gynaecology
Authors: Gupta Pawan
Published: November 2020

O&G is one of the broadest hospital-based specialties, encompassing medicine, surgery, and childbirth; so it is no wonder that O&G patients with a diverse range of symptoms present to the ED on a daily basis. The most common symptoms are PV bleeding, in both pregnant and non-pregnant females, PV discharge, and lower abdominal pain. The majority of these cases are benign and the patient can be discharged after reassurance and arrangement of primary care follow-up, but a few of them may have an underlying life-threatening condition. One classic example is a ruptured ectopic pregnancy. About 10% of females who present to the ED with PV bleeding or abdominal pain in the first trimester of the pregnancy have an ectopic pregnancy. Such patients may present in shock following a catastrophic haemorrhage in the abdomen or arrive at the ED well but collapse in the waiting room. Management of these emergencies requires establishment of IV fluid resuscitation, analgesia, and involvement of an O&G specialist. Such patients are often rushed to the theatre for emergency operation to control the haemorrhage. In other situations, the normal practice of taking a history and physical examination should be followed. Bear in mind that close attention to privacy and confidentiality is most crucial during the procedure. Interview the patient without the other family members if possible, as the patient may be reluctant to tell the whole story in their presence. Pelvic examination is sometimes painful, undignified, and embarrassing to many patients especially if the examining doctor is a male. This can be avoided to a large extent by explaining the process of examination, and performing it gently in the presence of a chaperone. It must not be forgotten that vaginal bleeding during pregnancy produces maternal distress, for which also the patient will need support. Patients presenting with lower abdominal pain often have a disease related to the female genital tract or the urinary system. But the lower GI tract may also contribute to the pain and should therefore be included in the differentials. With regard to the reproductive system, there are a few causes of pain during pregnancy.
Ultrasound in Lund —three world premieres
Authors: westling Håkan
Published: November 2020

In the historical section of the monograph Ultrasound in Medical Diagnosis, published in 1976, one can read that a substantial part of the early development of the ultrasound-echo method took place in the little university town of Lund in Sweden. It is not without pride that the Lundensians Inge Edler and Hellmuth Hertz comment upon this in a later review article. They also describe what happened in Lund in the 1950s which they thought contributed in a decisive way to diagnostics in cardiology, neurosurgery, and obstetrics and gynaecology. Apart from the cardiologist Edler and the physicist Hertz the pioneers were the neurosurgeon Lars Leksell and the obstetrician Bertil Sundén. Hertz and his pupils were to a high degree responsible for the technical development and the application in all three specialities. During the late 1940s and the early 1950s surgery of the heart was started in Lund. The first common lesion to be corrected in adults was mitral stenosis. The valve was opened by forced dilatation. Results were often good but there was a diagnostic problem. In some patients the symptoms were not due simply to a small valve opening. There was also a leakage of blood ‘backwards’, mitral insufficiency. This condition of course could not be improved by such a primitive operation. Instead, the procedure might make the condition worse. There was thus a great need for improved diagnostics before operation. This was when the young internist Inge Edler entered the scene. Born in 1911 he had started his career in internal medicine in Malmö but in 1950 he moved to Lund where he became responsible for the preoperative heart evaluations. He immediately focused his interest on the possibilities of making a quantitative diagnosis of mitral stenosis and to determine the existence of mitral insufficiency. Edler’s nurse was married to a physicist, Jan Cederlund. Hence it was natural for Edler to ask Cederlund if the radar technique developed during world war II could be used for examination of the heart. Cederlund forwarded the question to his friend Hellmuth Hertz, also a physicist.
Ultrasound in paediatric cardiology — state of the art
Authors: Mertens Luc L.
Published: November 2020

The development of ultrasound technology to visualize cardiac structures, based on the pioneering work by Edler and Hertz at the University of Lund in Sweden, has literally created a revolution in the field of paediatric cardiology. Before the era of cardiac catheterization and echocardiography the diagnosis of congenital heart disease was mainly based on combining physical findings, cardiac auscultation, electrocardiogram (ECG), and chest X-ray. This was largely based on the work by Helen B. Taussig at John Hopkins in the 1930s who established the field of clinical paediatric cardiology by integrating pathology knowledge with clinical findings. Diagnosis at that time was based on clinical skills and was more an art than science. The introduction of paediatric cardiac surgery in the 1950s was made possible due to the simultaneous development of cardiac catheterization and angiography which allowed an accurate description of the different cardiac lesions and the associated haemodynamics prior to surgery. For a long period catheterization was the diagnostic gold standard and all surgical patients underwent an invasive cardiac evaluation. In the 1970s, echocardiography was developed as a clinical tool and due to its non-invasive nature, was introduced quickly in paediatric cardiology. As anatomical diagnosis is challenging by M-mode echocardiography, it was really the development of two-dimensional (2-D) echocardiography in the late 1970s and early 1980s that deeply influenced the field. For the first time the congenital defects could be imaged noninvasively and the 2-D images were extensively validated by comparing them with pathological and surgical findings. Adding pulsed, continuous, and colour Doppler data to the 2-D images resulted in a complete detailed description of congenital cardiac defects and their haemodynamic consequences. Further optimization of ultrasound technology specifically for paediatric imaging, such as the development of higher-frequency probes and increasing the standard grey-scale frame rates, further improved spatial and temporal resolution and overall image quality. Based on its excellent diagnostic accuracy and its non-invasive nature, echocardiography quickly became the primary non-invasive diagnostic technique for all children with heart disease. Currently every paediatric patient with suspected heart disease will undergo an echocardiographic examination as the first (and often only) diagnostic test.
The development of echoencephalography in Sweden
Authors: Salford Leif G.
Published: November 2020

Although neurosurgery has a long history it was thanks to brave pioneering neurosurgeons such as Harvey Cushing in the United States — and in Sweden Herbert Olivecrona — that the speciality made huge progress during the first half of the 20th century. However, around 1950, the possibility to reveal pathological processes taking place inside the closed skull, was still very restricted. The only available rapid procedure was the neurological examination of the patient. X-ray of the skull is of restricted value, pneumo-encephalography was much too complicated and time-consuming for acute situations, and angiography was still in its infancy. Thus the neurosurgeon, receiving an acute patient with a suspected intracranial haematoma, had to make a qualified guess about where to start placing his trephine on the skull in order to save the life of the patient — often within minutes in the case of a bleeding between the skull bone and the dura. The mortality in those days was 40 % , often because the diagnosis was made too late. Thanks to an ingenious Swedish neurosurgeon, Lars Leksell, working at Lund University Hospital, a new approach to reveal the secrets inside the skull was introduced in clinical praxis — echoencephalography. Lars Leksell graduated from the Karolinska Institute (KI) and received his neurosurgical training in Herbert Olivecrona’s department from 1935. He volunteered as a neurosurgeon in the Finnish winter war in 1940 in Karelia. His team could operate on 24 head injuries per 24 hours and already by this point, Leksell showed his creative mind in constructing the double-action rongeur for more efficient removal of shell-splinters from the vicinity of the spinal cord. In 1941 he joined Professor Ragnar Granit (1967 Nobel laureate in Medicine) at the Institute of Neurophysiology where he presented his thesis on gamma nerve fibres in 1945. In 1946 he became the chief of the new neurosurgical unit in Lund and in 1958 he was appointed the first Professor of Neurosurgery at Lund University. In 1960 he succeeded Olivecrona as Professor and Chairman of the Neurosurgical Department at the Karolinska Institute/Hospital.
South-East Europe
Authors: O'Brien William
Published: November 2020

Copper was the first metal used by humans, a practice that began at different times in various parts of the world. The earliest evidence comes from the Near East around 10,000 years ago, when some early farming communities started to experiment with surface finds of native copper. Initially collected for their golden colour, it was soon discovered that these small pieces of pure copper could be cold-hammered into desired shapes, making them different from rock minerals. This first occurred in areas such as northern Iraq and eastern Anatolia where native copper occurs naturally. By 7000 BC there is evidence from sites such as Cayönü in Anatolia for the heating of native copper (annealing) to improve the production of beads, awls, and other small objects (Muhly 1988, 1989). In time, this led to another important discovery, namely that native copper could be melted and poured into moulds at temperatures around 1083º C. It is not certain when this first occurred, but most probably in the sixth millennium BC (see Pernicka and Anthony 2010 for overview). One of the reasons for the slow development of metallurgy in the Near East was the scarcity of native copper. The growing interest in metal eventually led to experimentation with copper minerals, such as malachite or azurite (Wertime 1973). These were initially used for non-metallurgical purposes, with malachite beads dating to the eleventh millennium BC known from a number of sites, including Shanidar Cave in northern Iraq (Solecki 1969). They were first recognized during the search for native copper, when rock outcrops were discovered bearing the distinctive green or blue staining produced by oxidation of copper minerals. The extraction of these surface minerals must have led in some instances to underground mining. It is not certain when copper ore was first smelted in the Near East. The dating of copper smelting slag at Catal Höyük in south-central Anatolia to the seventh millennium BC remains contentious. The earliest secure evidence comes from the later fifth millennium BC, at sites such as Norsuntepe in southeast Anatolia and Abu Matar in the northern Negev, Israel (Pernicka 1990; Golden et al. 2001).
Northern Europe
Authors: O'Brien William
Published: November 2020

Copper objects first circulated in Britain and Ireland around 2500 BC, thus beginning a short-lived Chalcolithic that ended with the rapid adoption of tin-bronze metallurgy after 2100 BC. Both islands have numerous sources of copper; however, these orebodies are not evenly distributed, nor were they all accessible to the prehistoric miner. This is part of the explanation why certain regions developed a strong tradition of copper mining that lasted well into the Bronze Age. Ireland has long been regarded as a significant producer of metal in the Bronze Age. This reflects the large quantities of Bronze Age metalwork found in a part of Europe with abundant sources of copper. The south-west region of Cork and Kerry was the main centre for early copper production. This began with mining at Ross Island in Killarney, where Beaker culture groups produced arsenical copper during the Chalcolithic and Early Bronze Age (c.2400–1900 BC). Farther south, there are seven copper mines now dated to the Early to Middle Bronze Age (c.1800–1400 BC) in the peninsulas of west Cork. These are known as Mount Gabriel-type mines, the name coming from the single largest concentration of such workings located on the eastern slopes of this mountain in the Mizen Peninsula (O’Brien 1994, 2003). The recent discovery of trench workings at Derrycarhoon continues the story of Bronze Age copper mining in that area to 1300–1100 BC, after which this activity seems to have ceased (O’Brien 2013). The study of these mines began during the late eighteenth/ early nineteenth centuries, when mineral prospecting led to the discovery of primitive workings at several locations in south-west Ireland. Described as ‘Dane’s Workings’ in the antiquarian literature, these mines were associated with the use of firesetting and stone hammers (see quotations from Griffith 1828 and Thomas 1850 (in O’Brien 2003) in Chapter 1). The first systematic research began in the 1930s with the discovery of the Mount Gabriel group by the geologist, Tom Duffy. These were subsequently mapped by another geologist, John Jackson, who brought these mines to wider attention when he obtained a Bronze Age date for charcoal taken from mine spoil on the mountain (Jackson 1968).
Analysis of purity
Authors: Baines Dev
Published: November 2020

In approaches to protein purification, the concepts of yield and purity are routinely used, but are often difficult to define in absolute terms. To some extent the purity of a protein sample will be defined by the final designated use of the product. In most cases, analyses will involve measurement of the mass of the protein in the sample and quantitation of specific property of the target molecule (e.g. activity) to provide values for the yield. Thus, the calculation of specific activity of given fractions through the purification process provides a valuable indication of the level of the purity attained. Although, conventionally, specific activity is used in the purification of enzymes, due to the availability of sensitive and specific assays, recent developments in fast chromatographic separations and protein mass spectrometry have led to application of these techniques to address the purity of a wider variety of biomolecules. The level of purity for any protein product requires several factors to be taken into consideration. Besides the intended use, the source of the protein will dictate the extent of analyses required, since the level of impurities present in the final product will depend not only on the purification process used but also on the starting source material. For bulk enzyme preparations (for use e.g. in biotransformations or related applications) it may only be necessary to ensure the product is free of any contaminating activities which could effect the outcome of these types of applications. For proteins required for physical studies (protein crystallography, primary sequence analysis), the purity criteria are more stringent, particularly, since the lack of purity (including sample microheterogeneity) can drastically influence the outcome of such studies. Alternatively, proteins intended for therapeutic use will have purity considerations significantly different, constrained not only by regulatory requirements but also by clinical responses that may arise from the presence of any contaminants. The nature of these contaminants, as mentioned earlier, will depend on the starting source of the target molecule (i.e. animal tissue, human serum, recombinant micro-organisms [prokaryotes, eukaryotes], and hybridomas).
Cell disintegration and extraction techniques
Authors: Cumming R. H. , Iceton G.
Published: November 2020

Disruption of cells is necessary when the desired product is intracellular. Many commercial intracellular products are proteins, such as soluble enzymes or soluble genetically engineered peptides. However, a number of genetically engineered proteins in E. coli are present in the cell as insoluble inclusion bodies; although this may be due to the chosen lysing conditions. This chapter concentrates on the extraction of soluble enzymes. To achieve a good yield of an intracellular product, it is generally a good idea to minimize the number of steps involved in the purification, as there is loss of material associated with each step. Since cell disruption is an extra procedure which itself may demand a further clarification step, it may be worth investigating if extraction of the protein can be made directly from the cell lysate (the disrupted material) without a cell debris clarification step. It may also be possible to manipulate the cell so that it excretes the protein into the surrounding growth medium, and thus, not require an extraction procedure (if the subsequent dilution of the product by the broth can be tolerated). The choice of disruption method usually follows one of the two directions: (a) Can a given disrupter be used for a particular cell type? (b) Which is the best method of extracting a product? The former often occurs in a laboratory context, whilst the latter is a question to be asked during scale-up of a process when costs are paramount. This chapter tries to accommodate both questions. Whatever type of disruption process is adopted, there are some key questions to be addressed. These are briefly discussed in the remaining sections of this overview. A recent comprehensive review of many disruption techniques is given by Middelberg. It is well known that some enzymes are more stable than others. The disruption method can impose great physical and chemical stress on the enzyme. Enzymes which are stable in the cell, perhaps by virtue of being attached to membranes, will be released into the medium during disruption.
Separation on the basis of chemistry
Authors: Hagel Lars
Published: November 2020

Most chromatographic separations are based on chemical interaction between the solute of interest or impurities to be removed and the separation medium. The exception is separations based upon physical properties such as size (e.g. size exclusion chromatography) or transport in a force field (e.g. electrochromatography). The chemical interaction may be weak (e.g. employing van der Waals forces) or very strong (e.g. involving formation of chemical bonds as in covalent chromatography). Whenever separation is based upon attractive forces between the solute and the separation medium, we talk about adsorption chromatography (also when the solute is merely retarded). The chemical interaction between the solute and the adsorbent (the chromatography medium) is governed by the surface properties of the solute and the adsorbent and is in most cases mediated by the mobile phase or additives to the mobile phase. Macromolecules such as proteins display a variety of properties and, ideally, a selected set of properties is utilized for obtaining the required selectivity (i.e. relative separation from other solutes) using a separation medium of complementary properties. This chapter briefly reviews the different types of forces of interaction between solutes and surfaces commonly employed for chromatographic purifications, important properties of solvents, and some basic surface chemical properties of proteins. This, together with a description of some common types of chromatography modes provides a basis for a rational selection of separation mechanism for the purification of proteins and the choice of mobile phase composition to regulate the relative influence of different interaction mechanisms. The separation mechanisms are focused to adsorptive modes with the exception of affinity chromatography which is discussed in Chapter 9. The different attractive forces acting between molecular and particle surfaces include (1): • dispersion forces • electrostatic dipole interactions • electron donor-acceptor forces • formation of covalent bonds All these forces are due to interactions between electric charges (permanent or induced). Dispersion, or London forces, are caused by induced dipole-induced dipole interactions and are thus classified as a non-specific interaction. This type of non-polar interaction is the dominant force promoting dissolution of non-polar solutes in organic solvents.
Crystallization in Gels and Related Methods
Authors: Robert M.-C. , Vidal O.
Published: November 2020

From the first studies showing the feasibility of macromolecular crystal growth in gels (1), an increasing attention has been paid to applications of gel techniques to the domain of biological macromolecules. Confidence in these techniques is such that kits of crystallization in gels are now commercially available (Hampton Research, Laguna Hills, CA, USA). Basically, the protein crystallization process consists of two consecutive steps: • first, the transport of growth units towards the surface of the crystals • second, the incorporation of the growth units into a crystal surface position of high bond strength. The whole growth process is dominated by the slowest of these two steps and is either transport controlled or surface controlled. Avoiding convection in the growth environment will increase the possibility of growing the crystal under slow diffusive mass transport providing that the surface interaction kinetics are faster than the characteristic diffusive flow of macromolecules (in the range of 10-6 cm2/sec for proteins). The ratio between transport to surface kinetics, which can be tuned by either enhancing or reducing transport processes in the solution, has been shown (2) to control the amplitude of growth rate fluctuations (which is thought to reduce crystal quality). These are the main reasons why gels (as well as capillaries and microgravity conducted experiments), if correctly designed, are expected to enhance the quality of crystals. This quality enhancement (3), as well as the possibility of getting crystals when conventional solution techniques failed (4), have been experimentally demonstrated. However, up to now, gel methods have been used on a rather empirical basis, as a simple transposition of solution techniques, and recent fundamental studies of nucleation and growth in gels show that the situation is not as simple as first expected (5, 6). After summarizing the main characteristics of crystal growth in gels, we will examine what are the best conditions using a gel method. Recipes for the preparation of different gel growth experiments will be given. Considering gel growth as a possible simulation of experiments under reduced gravity, recent results of space experiments will be reviewed. Mention will also be made to growth under hypergravity conditions.
Two-Dimensional Crystallization of Soluble Proteins on Planar Lipid Films
Authors: Brisson A. , Lambert O.
Published: November 2020

Electron crystallography of protein two-dimensional (2D) crystals constitutes a fast-expanding method for determining the structure of macromolecules at near-atomic resolution (1, 2). The main limitation in the application and generalization of this approach remains in obtaining highly ordered 2D crystals, as is the case of 3D crystals in X-ray crystallography. Several methods of 2D crystallization are available which can be classified into two families, depending on the type of proteins under investigation, either membrane proteins (3, 4) or soluble proteins (5, 6). In both cases, 2D crystallization is a self-organization process which spontaneously occurs between macromolecules which are restricted to diffusing by translation and rotation in a 2D space, with a fixed orientation along the normal to this plane. The scope of this chapter is restricted to the 2D crystallization of soluble proteins on planar lipid films, by the so-called ‘lipid monlayer crystallization method’ (5). Our aim is to present a step-by-step description of the experimental procedures involved in the application of this method. The method of protein 2D crystallization on planar lipid films was introduced about 15 years ago (5) and has since been successfully applied to about 30 proteins. Its principle is based on the specific interaction between soluble proteins and lipid ligands inserted in a lipid monolayer, at an air-water interface. In practice, a lipid monolayer is formed by spreading lipids dissolved in an organic solvent on a water surface. Proteins present in the aqueous subphase bind to their ligand of lipidic nature and spontaneously form 2D domains and, in favourable cases, 2D crystals. The process of 2D crystal formation relies on three successive steps: (a) Molecular recognition between a protein and its ligand. (b) Diffusion and concentration of the protein-lipid complexes in the plane of the lipid film. (c) Self-organization of the proteins into 2D crystals. As indicated in Table 1, three different types of systems can be distinguished, depending on the nature of the lipid ligand: • natural lipids • synthetic lipids made of a protein ligand coupled to a lipid molecule • charged lipids.
Synthesis of modified peptides
Authors: Mellor Sarah L , Wellings Donald A
Published: November 2020

Several important hormones such as oxytocin, secretin, and LHRH are known to be peptidyl amides. In addition to these, other peptidyl amides such as indolicidin and the protegrins have been shown to exhibit potent antimicrobial activity. The in vivo production of such compounds is via endogenous enzymatic cleavage of propeptides, making their synthesis by genetic engineering notoriously difficult. Furthermore, to facilitate the survival of synthetic peptidyl amides in vivo, an obvious defence against the action of carboxypeptidases is the N-alkylation of the carboxylic amide terminus. Such secondary amides would be expected to exhibit vastly different solubility and transport properties to primary amides, thus their chemical synthesis is of immense importance. The solid phase synthesis of peptidyl amides and peptidyl N-alkyl amides is centred around two main strategies: 1. Ammonolysis/aminolysis of resin-bound esters. 2. Use of resin-bound primary amines, which may in turn be chemically modified to generate novel secondary amine functionalized linkers for the synthesis of peptidyl N-alkyl amides. Early examples of the use of resin-bound amines for the solid synthesis of peptidyl amides involve the use of linkers such as benzhydrylamine or benzylamine. Following peptide assembly, these linkers require highly acidic (e.g. HF) mediated cleavage, and hence simultaneous removal of acid-labile sidechain protection groups occurs and may cause problems. Systematic modifications of these linker-resins by substitution with electron-donating substituents have resulted in the generation of numerous linker-resins with increased acid sensitivity. Notably, the 4-(2',4'-dimethoxyphenylaminomethyl) phenoxy derivatized (Rink) resin 1, which is cleavable by 95% v/v TFA, and the 5-(2-fluorenylmethoxycarbonylaminornethyl-3,5-dimethoxy)- phenoxyvaleric acid (PAL) linker 2 acidolysed by 75% v/v TFA. Greater acid lability has been achieved using the xanthenyl derivatized resin, 9-(fluorenylmethoxycarbonylamino)xanthen-3-yloxymethyl polystyrene (Sieber amide) resin 3, which besides being cleavable by 1% v/v TFA, holds the added advantage of readily undergoing reductive N-alkylation to afford resin-bound secondary amines for the synthesis of peptidyl N-alkyl amides. In addition to the method detailed below, a number of alternative approaches have recently been reported.
Production and analysis of ES cell aggregation chimeras
Authors: Nagy Andras , Rossant Janet
Published: November 2020

Embryonic stem (ES) cells behave like normal embryonic cells when returned to the embryonic environment after injection into a host blastocyst or after aggregation with earlier blastomere stage embryos. In such chimeras, ES cells behave like primitive ectoderm or epiblast cells (1), in that they contribute to all lineages of the resulting fetus itself, as well as to extraembryonic tissues derived from the gastrulating embryo, namely the yolk sac mesoderm, the amnion, and the allantois. However, even when aggregated with preblastocyst stage embryos, ES cells do not contribute to derivatives of the first two lineages to arise in development, namely, the extraembryonic lineages: trophoblast and primitive endoderm (2). The pluripotency of ES cells within the embryonic lineages is critical to their use in introducing new genetic alterations into mice, because truly pluripotent ES cells can contribute to the germline of chimeras, as well as all somatic lineages. However, the ability of ES cells to co-mingle with host embryonic cells, specifically in the embryonic, but not the major extraembryonic lineages, opens up a variety of possibilities for analysing gene function by genetic mosaics rather than by germline mutant analysis alone (3). There are two basic methods for generating pre-implantation chimeras in mice, whether it be embryo ↔ embryo or ES cell ↔ embryo chimeras. Blastocyst injection, in which cells are introduced into the blastocoele cavity using microinjection pipettes and micromanipulators, has been the method of choice for most ES cell chimera work (see Chapter 4). However, the original method for generating chimeras in mice, embryo aggregation, is considerably simpler and cheaper to establish in the laboratory. Aggregation chimeras are made by aggregating cleavage stage embryos together, or inner cell mass (ICM) or ES cells with cleavage stage embryos, growing them in culture to the blastocyst stage, and then transferring them to the uterus of pseudopregnant recipients to complete development. This procedure can be performed very rapidly by hand under the dissecting microscope, thus making possible high throughput production with minimal technical skill (4). In this chapter we describe some of the uses of pre-implantation chimeras, whether made by aggregation or blastocyst injection, but focus on the technical aspects of aggregation chimera generation. We also discuss the advantages and disadvantages of aggregation versus blastocyst injection for chimera production.