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Departamento de Reproducción animal, Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture. © 2021 Mary Ann Liebert, Inc., publishers, Work in the Murcia laboratory was supported by MINECO-FEDER (AGL 2015-66341-R), Fundación Séneca 20040/GERM/16 and 21105/PDC/19, and a FPU fellowship (FPU16/04480) from the Spanish Ministry of Education, Culture and Sport. Work in the Oxford laboratory was supported by a PhD scholarship to Alaa Hachem funded by the Iraqi Higher Committee for Education Development, and by a PhD scholarship to Ali Hanbashi funded by the Saudi Cultural Bureau on behalf of Jazan University. The work was also supported by an International Exchanges Grant to John Parrington and Joaquin Gadea from the UK Royal Society (IE140324).

Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC–/– cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators., Highlights Two-pore channel (TPC)-mediated endolysosomal Ca2+ signalling regulates a variety of processes, including cell proliferation, differentiation, metabolism, viral infection, and cardiac function. Despite the well-established model that TPCs are Ca2+-selective channels indirectly activated by nicotinic acid adenine dinucleotide phosphate (NAADP), it has also been proposed that TPCs as Na+ channels are activated directly by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. 3D structures of mouse TPC1 and human TPC2 were recently determined, which made it possible for structure-based virtual screening methods to identify pharmacological modulators of TPC. Recent identification by high-throughput screens of pharmacological modulators that target TPCs will help reveal the molecular mechanisms underlying the role of endolysosomal Ca2+ signalling in different pathophysiological processes, and to develop new therapeutics.

In comparison with normal cells, cancer cells are equipped with a higher number of lysosomes, involved in degradative and non-degradative roles. In particular, the lysosome is a Ca2+signalling hub, and the enhancement of this interconnected machinery in cancer cells has recently prompted investigations into the role that lysosomal ion channels play in oncology. The present review reports findings about the emerging role of lysosomal Ca2+channels: Two-Pore Channels (TPCs), Transient Receptor Potential Cation Channels (TRPMLs; mucolipins), and Purinergic X Receptor 4 (P2×4R), in a variety of cancer models, highlighting their impact on crucial functions such as the regulation of autophagy and the composition of the tumour microenvironment, including the secretion-mediated interplay with immune and endothelial cells. Notably, recent evidence indicates that, by regulating tumour secretome, lysosomal Ca2+ signalling can affect the composition of the tumour-infiltrating immune cell repertoire. Intriguingly, the data so far available show that the protumoral/antitumoral role of lysosomal Ca2+ channels can differ according to the specific genetic context, types of cancer and the malignancy stage, and signals from the microenvironment.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

In comparison with normal cells, cancer cells are equipped with a higher number of lysosomes, involved in degradative and non-degradative roles. In particular, the lysosome is a Ca

Growing evidence implicates a vital role for TPC2/Ca2+ signaling in pathophysiological processes attributed to cancer, raising questions regarding the utility of TPC2 as a cancer therapeutic target. In this issue of Cell Chemical Biology, Muller et al. (2021) develop TPC2 inhibitors, SG005 and SG-094, exhibiting anti-tumor effects with potential translational relevance.

This chapter focuses on how the birth of figurative art marked a significant shift in our species’ ability to represent the world around us, and presumably, a step forward in our capacity for conscious awareness. The ability to draw a familiar object such as a cat or dog develops so naturally in children that it is easy to forget what a remarkable human capacity this is. For unlike tool use, spoken language, or perhaps even the capacity to make music, which may all have evolutionary precedents in the actions or sounds made by other species, the ability to draw appears to have no relationship to anything found in the rest of the animal kingdom. And certainly for the view presented in this book of the uniqueness of human conscious awareness, the ability of humans to both produce and appreciate visual art is something that deserves a wider discussion. In particular, abstract symbols, mathematical systems, planning diagrams, and other forms that constitute visual art, can all be viewed as further examples of the 'cultural tools' that shape our minds besides language.

This chapter explores how future technologies might impact on human consciousness. It begins by discussing how new techniques are continuing to add to the understanding of the human mind. There are many exciting technologies available now to the neuroscientist, such as genomic analysis, optogenetics, gene editing, and brain organoids. To what extent could such technologies be used to investigate the model of human consciousness outlined in this book? The chapter then considers whether artificial intelligence might come to rival that of human beings, and possible interfaces between human and machine intelligence. Our growing ability to develop functioning robots raises the question of whether an artificial human brain might be used to control such a robot, creating in effect a cyborg. However, the creation of such an entity could make a big difference in terms of an artificial brain’s sense of identity in the world, as well as its rights.

This chapter examines how ideas can change in a mass movement such as a strike, and why in some periods of history such activities can seem few and far between, while in others widespread industrial militancy is a central feature. It looks at moments in history when resistance to the status quo reached such a scale that a fundamental shift occurred in the structure of society—so-called revolutions. In so doing, the chapter relates the actions of a mass movement to what might be going on in the heads of its individual participants. It also asks what mental processes might underlie the apparently rapid shifts in social consciousness that seem to be a key feature of any genuine mass progressive movement of the people. A survey of past protest movements reveals a number of typical characteristics that may provide clues as to how human consciousness—here particularly meaning conscious awareness of oneself and one’s place in society—develops in such circumstances. The chapter also looks at how reactionary social movements can come to power, such as in Nazi Germany, and asks why some people join or acquiesce to such movements, while others actively resist them, and how this might reflect different aspects of human brain function.

This chapter explores how language helps human beings to group, distinguish, and differentiate between things in the world around them. In other words, what is the basis of conceptual thought, and how does this relate to language capacity as a whole? On the one hand, behaviourists have argued that human beings start life as a ‘blank slate’, and our language capacity is something that we learn by exposure to the language of others. On the other, Noam Chomsky proposed that humans are born with an innate capacity for language, as shown by the ease with which children rapidly learn a wide vocabulary, but also by their remarkable capacity for linking words together in complex grammatical structures. The chapter then looks at developments in the search for a biological basis for human language. Studies suggest that language in humans involves a complex interplay between a number of different genes, FOXP2 being a key one, which affect the connections between neurons in specific parts of the brain. However, it still remains unclear whether FOXP2 gene affects neurons involved in language processing per se or those that control muscles involved in speech.

This chapter studies the relationship between emotion and rational thought. Emotions have been found to be linked to chemical changes that can affect both mind and body. The brain is connected with the rest of the body through direct nerve connections to the organs, but also by brain regions like the hypothalamus, and glands linked to it such as the pituitary, that secrete hormones into the blood. As such, some scientists now argue that even human consciousness cannot be thought of as a product solely of the brain, but also of the body. A chemical that plays dual roles in the brain and the rest of the body is dopamine, which is vital for our control of movement and it also regulates many higher mental functions in humans, such as learning, concentration, planning ahead, and pleasure-seeking behaviour. The chapter then looks at the implications of Lev Vygotsky’s view of the interaction between rational thought and emotional responses for education. One important concept in this regard is what Vygotsky termed the 'zone of proximal development.

This introductory chapter begins by providing an overview of the power of the human brain, which is displayed in the wonders of modern civilization. Despite the human brain’s capacity for such intellectual and technological feats, we still know astonishingly little about how it achieves them. This deficit in understanding is a problem not only because it means we lack basic knowledge of the biological factors that underlie our human uniqueness, but also because, for all its amazing capabilities, the human mind seems particularly prone to dysfunction. Still, some would argue there is good reason to be optimistic about the prospect of developing new and better treatments for mental disorders in the not-so-distant future. Such optimism is based on the increasing potential to study how the brain works in various important new ways thanks to recent technological innovations. The chapter then considers two overly polarised views of the human mind. Ultimately, this book argues that society radically restructures the human brain within an individual person’s lifetime, and that it has also played a central role in the past history of our species, by shaping brain evolution.

This chapter evaluates the basic unit of the human brain: the nerve cell, or neuron. These cells are also the main units of the peripheral nervous system, which sends messages from the brain to the other tissues and organs that make up our bodies. Neurons are the most well-known cells in the brain but they are not the only type of cell in this organ. The other main types are the glial cells, also known as neuroglia. Recent studies of the role of glial cells in the brain are revealing potentially important differences between humans and other species in the functions of these cells. The chapter then turns to the large-scale structure of the brain. The most dramatic changes in brain size and structure occurred in the final phase of human evolutionary change. Indeed, Neanderthals had brains similar in size to those of modern humans. An important feature of the human brain is that a larger fraction of its growth occurs outside the womb. Although humans reach adult brain size in childhood, brain development continues for decades afterwards.

This chapter describes another aspect of human uniqueness—the links between consciousness, creativity, and imagination. Russian psychologist Lev Vygotsky believed that human consciousness is transformed by social interactions, particularly through language, in a similar fashion to how tools have allowed us to transform the natural world. Vygotsky saw imagination as no less influenced by this social input than other aspects of consciousness. To deal with a changing environment, he argued that the human brain ‘combines and creatively reworks elements of this past experience and uses them to generate new propositions and new behaviour [making] the human being a creature oriented toward the future, creating the future and thus altering his own present’. Vygotsky thought that imagination was particularly stimulated in the developing child through the process of play. Some aspects of adult human culture have attributes in common with play, including the link with imagination.

This chapter examines the emergence of tool use and human language in human brain evolution. Increasing use and design of tools made possible by the bipedalism of our proto-human ancestors was a key step in the development of language. Indeed, communal tool use ‘helped to bring the members of society together by increasing the cases of mutual support and joint activity’. During this process, ‘the reaction of labour and speech on the development of the brain and its attendant senses, of the increasing clarity of consciousness, power of abstraction and of conclusion, gave both labour and speech an ever renewed impulse to further development’. The chapter then considers the studies which assess the fundamental differences in terms of language capacity between humans and apes. While the behaviourist view that human language acquisition is simply an accumulation of conditioned reflexes now looks incorrect, recent studies have also challenged the view of a biological basis for a ‘universal grammar’ shared by all humans. Instead, increasing evidence points to both human biology and the process of growing up in a specific human society as being factors of equal importance in the formation of language.

This chapter assesses how much novels can reveal about the various mechanisms underlying human consciousness. Some might say, very little, and argue that only scientific study can uncover such mechanisms. However, because language plays such a key role in shaping human consciousness, the fictional explorations of the human condition that we find in novelistic literature can greatly add to our scientific understanding by concretizing that condition in its diverse forms. The chapter also explores a related question: how much do novels draw on new insights about the nature of consciousness, so increasing their ability to inform us about the human condition, and its relationship to changing forms of society? The best novels have a complexity and ambiguity of meaning that itself reflects the many contradictions in society and the individual psyche within that society. Importantly, this means there can be multiple readings of great novels, with different readers interpreting them in various ways. The chapter then examines several novels that have multiple interpretations and which also illuminate and enhance our understanding of different aspects of consciousness. These include Emily Brontë’s Wuthering Heights (1847), Henry James’s The Turn of the Screw (1898), William Golding’s Pincher Martin (1956), and Kazuo Ishiguro’s The Unconsoled (1995).

This chapter discusses how the structure of current society is relevant to discussions about human brain function. One feature of the human brain that might explain why many working people can accept and even acquiesce in their state of exploitation, and even sometimes see other workers as the primary threat, rather than as allies in campaigns for better pay and conditions at work, is the way our brains have evolved to respond to living in a potentially hostile natural environment. There is increasing evidence that many unconscious fears about a perceived threat of people of a different skin colour, sexual persuasion, or even male fears about female equality, involve the amygdala. Because of this, although inflammatory speeches by politicians or media articles may enhance such fears, thereby dividing people who have much to gain by working together for better pay and conditions, they are helped by being able to trigger deep-seated impulses in the human brain. Ultimately, the divisions in current society, by ethnicity and gender as well as class, exert a heavy toll on mental health. This chapter also looks at the phenomenon of alienation, and how this both affects our relationship to modern society and is linked to our wider state of self-conscious awareness.

This chapter begins by arguing that the different approaches to mental disorders are united in viewing such mental conditions as abnormal. Yet is it really the case that everything termed a mental ‘disorder’ is indeed so? Or could it be that in diagnosing conditions that affect the mind, we are ignoring the possibility that some ‘disorders’ may be part of the normal spectrum of human diversity? To take this argument further, could it even be the case that such diversity is an important component of human society and a source of some of its richness and achievements? The chapter looks at these possibilities, with particular reference to two quite different mental conditions—autism spectrum disorder and bipolar disorder. Autism spectrum disorders have been described as characterized by 'impairments in social interaction and both verbal and non-verbal communication, along with restricted, repetitive or stereotyped behaviour'. Meanwhile, the high and low phases of bipolar disorder are often so extreme that they interfere with everyday life; consequentially this can be a highly debilitating disorder. However, there is also an association of both types of mental disorder with great creativity, and high levels of intelligence.

This chapter investigates the relationship between the individual and society, which has been hotly disputed among philosophers and politicians through the ages. Recent studies have questioned the idea that human beings are naturally solitary individuals. Instead, they suggest that socialising with others is so central to our species that rejection is registered in the same brain regions that respond to physical pain. Other studies have undermined the idea that human beings are inherently selfish, indicating instead that altruistic acts trigger activity in the ‘reward’ region of the brain that is stimulated when a person experiences pleasure. Studies like these raise the question of how the human brain became so attuned to social cues in this way. Here there are two issues to consider. One is evidence that primates in general have evolved to be highly sensitive to social interactions with other members of their species, and this has been accompanied by enhanced brain growth in order to handle these more sophisticated interactions. Yet while social interaction may be hardwired into our brains because of evolutionary changes in our primate ancestors, some features of our strong tendency towards social interaction may be specifically human. The chapter then looks at Russian psychologist Lev Vygotsky’s novel ideas about human consciousness.

This chapter evaluates how music has been an important part of human culture for a very long time. One explanation for the centrality of music in human society is that it plays a key role in coordinating activity. Rhythmic sound ‘not only coordinates the behaviour of people in a group, it also coordinates their thinking—the mental processes of individuals in the group become synchronized’. This may explain how drums unite tribes in ceremony, why armies used to march into battle accompanied by bugle and drum, religious ceremonies are infused by song, and speech is punctuated by rhythmic emphasis on particular syllables and words. The chapter then assesses what modern neuroscience can reveal about how music affects the brain, and considers the role that music plays in the creation of meaning. It illustrates these general arguments with reference to music ranging from the classical music of Mozart and Beethoven, to mid-twentieth century jazz, through to modern pop music. The chapter also looks at how music can help us cope with some of the alienation that many people feel in modern society, and ways in which music has addressed the issue of mental illness.

This chapter returns to the various alternative views about human consciousness first mentioned at the start of this book, and assesses how this book’s account compares to, and hopefully builds on, these other viewpoints. The view of human consciousness developed in this book can explain the uniqueness of our species’ conscious awareness, but in an entirely materialistic fashion. This approach views language—the system of abstract symbols linked in a grammatical structure but also one that connects the individual to the world outside via word meaning—and other forms of human culture like music, art, and literature, as a material force that has reshaped human brain functions at every level. This has led to a qualitative shift in such functions, compared to that of every other species, including our closest animal cousins, the great apes. Unlike a purely ‘bottom up’ approach to human brain function, this view sees language, as well as other mediators of human culture, as imposing both structural and dynamic changes in our brains. Structurally, it sees the different brain regions, as well as their interconnections, as altered in humans. The chapter then reflects on what impact, if any, might this approach to understanding human consciousness have on diagnosis and treatment of mental disorders.

This book draws on the latest research on the human brain to show how it differs strikingly from those of other animals in its structure and function at molecular and cellular level. It argues that this ‘shift’, enlarging the brain, giving it greater flexibility and enabling higher functions such as imagination, was driven by tool use, but especially by the development of one remarkable tool—language. The complex social interaction brought by language opened up the possibility of shared conceptual worlds, enriched with rhythmic sounds and images that could be drawn on cave walls. This transformation enabled modern humans to generate an exceptional human consciousness, a sense of self that arises as a product of our brain biology and the social interactions we experience. Linking early work by the Russian psychologist Lev Vygotsky to the findings of modern neuroscience, the book also explores how language, culture, and society mediate brain function, and what this view of the human mind may bring to our understanding and treatment of mental illness.

This chapter highlights clinical depression and anxiety. What seemed like a major step forward both in the understanding of depression, and how to treat it, occurred in the 1950s, when evidence emerged that depression might be due to low levels of the neurochemical serotonin. In particular, neuroscientists discovered that certain chemicals that alleviated the symptoms of depression appeared to work by inhibiting the reuptake of serotonin into certain neurons in the brain. The chapter then looks at the selective serotonin reuptake inhibitors (SSRIs), which are now used to treat far more conditions than clinical depression. Recently, doubts have surfaced about the appropriateness of doctors prescribing SSRIs for such different conditions, the effectiveness of these drugs, and the mechanisms by which they affect brain function. One problem in determining the true effectiveness of SSRIs for the treatment of depression is how little is understood about the biological basis of this disorder and how drugs like SSRIs alleviate it. A complicating factor for any attempt to identify a biological basis to depression, or for that matter any mental disorder, is the important role that the social environment plays in the genesis of such disorders.

This chapter explains that the central difficulty in diagnosing and treating mental disorders is reaching a level of agreement about their underlying basis. In this respect, proponents of the view that mental disorders are primarily biological in origin were particularly excited by the Human Genome Project, as they believed this would make it possible to truly get a grip on the nature of such conditions. Genome-wide association studies (GWAS) have now been used to study a range of mental disorders. However, GWAS findings have been contradictory and confusing and this chapter looks at the reasons why, and at a number of possible biological causes of schizophrenia. The chapter then considers a very different way of explaining schizophrenia, one based on the idea that it is not genetics but the social environment that determines whether an individual succumbs to this disorder. A version of this hypothesis sees schizophrenia as primarily the result of defective family relations, and the disorder as a consequence of those distorted relations, but also as a response to wider pressures in society. One psychotherapy approach claimed to have a very good level of success in the treatment of schizophrenia is called 'Open Dialogue.

We investigated whether the endolysosomal two-pore channel TPC2 is a mediator of adipocyte differentiation. We show that Tpcn2 mRNA is expressed transiently during induction of C3H10T1/2 mesenchymal stem cells to differentiate into adipocytes, and that this expression is triggered by cAMP. This is the first demonstration of a cell signaling pathway that can regulate TPC gene expression. We also identified an important functional role for TPC2 in adipocyte differentiation. First, ectopic TPC2 expression in C3H10T1/2 cells partially rescued the block to adipocyte differentiation caused by cAMP absence. Second, inhibition of endogenous TPC2 expression in primary preadipocytes substantially reduced their ability to differentiate into adipocytes. Finally, genetic variation at the Tpcn2 locus is associated with increased upper-body fat distribution in women concomitant with reduced Tpcn2 expression in abdominal adipose tissue. Our findings implicate TPC2 as an important mediator of adipogenesis and may aid identification of new drug targets for treatment of obesity.

Simple Summary Ca2+ dyshomeostasis is implicated in several key pathophysiological processes attributed to cancer metastasis biology. Here, we decipher the role of intracellular and extracellular Ca2+ signalling pathways in processes that contribute to metastasis at the local level (involving cell proliferation, adhesion, motility, invasion, migration and the epithelial-mesenchymal transition) and also their effects on cancer metastasis globally. Ca2+ proteins are potential candidates for cancer biomarkers and druggable targets for future metastatic cancer therapy. Abstract Metastatic cancer is one of the major causes of cancer-related mortalities. Metastasis is a complex, multi-process phenomenon, and a hallmark of cancer. Calcium (Ca2+) is a ubiquitous secondary messenger, and it has become evident that Ca2+ signalling plays a vital role in cancer. Ca2+ homeostasis is dysregulated in physiological processes related to tumour metastasis and progression—including cellular adhesion, epithelial–mesenchymal transition, cell migration, motility, and invasion. In this review, we looked at the role of intracellular and extracellular Ca2+ signalling pathways in processes that contribute to metastasis at the local level and also their effects on cancer metastasis globally, as well as at underlying molecular mechanisms and clinical applications. Spatiotemporal Ca2+ homeostasis, in terms of oscillations or waves, is crucial for hindering tumour progression and metastasis. They are a limited number of clinical trials investigating treating patients with advanced stages of various cancer types. Ca2+ signalling may serve as a novel hallmark of cancer due to the versatility of Ca2+ signals in cells, which suggests that the modulation of specific upstream/downstream targets may be a therapeutic approach to treat cancer, particularly in patients with metastatic cancers.

We can now edit genomes. The technique, which involves cutting and pasting DNA code into the genome, is faster and cheaper than traditional genetic engineering and can be used on almost any animal or plant. What will this technology mean for the future? It may pave the way to banishing many diseases, and help feed the burgeoning population of the world. Woolly mammoths may again roam the tundra. But are there also risks? Might a nightmarish world of bioterrorism and rogue synthetic organisms await? John Parrington reports on the astonishing revolution underway in genetic engineering and why it matters to us all.

Despite many inequalities in the world, it is a testament to human technology that modern agriculture is able to feed the 8 billion people on the planet. However, recently extreme weather patterns linked to global warming have been having an adverse effect on crops and farmed animal production, leading to fears about whether agriculture can continue to feed all the humans on the planet. Genome editing looks set to revolutionise agriculture by making it possible to precisely edit the genomes of farm plants and animals rapidly and economically in an unprecedented way. Such editing could be used to create vegetables and meat with enhanced flavour or nutrition. It could also be used to create disease resistant plants and animals, and reduce the use of antibiotics or pesticides. Looking further into the future it might eventually be possible to use genome editing to reconfigure plants or animals to survive in increasingly extreme types of climates. Despite these positive ways of using genome editing in agriculture, concerns have been raised about the safety of food produced from genome edited animals and plants, and potential adverse effects on animal welfare. Another criticism is that genome editing may only benefit giant agribusiness companies, and not ordinary farmers and consumers. Yet against this criticism, one of the revolutionary aspects of genome editing is how easy and economical it is to use, which means that unlike previous GM technologies, there is no reason why it cannot be used in a local, sustainable, and accessible way.

We are in the middle of a scientific revolution in the biological sciences. In the 1970s scientists discovered ways to cut and paste DNA in a test tube, and then introduce gene ‘constructs’ into first bacteria and then more complex organisms such as mice. This made it possible to, for instance, produce human insulin artificially in bacteria, but also to create genetically modified mice for medical research. But such genetic engineering methods were expensive, time-consuming, and limited to only certain organisms. In contrast in recent years, new ‘genome editing’ approaches, particularly one called CRISPR/Cas, now makes it possible for the first time to precisely edit the genome of living cells from practically any species. Genome editing looks set to revolutionise medical research, clinical medicine, and agriculture, as the rest of this book will explain. But this is only one of the new technologies discussed In Redesigning Life. Others include optogenetics, which makes it possible to activate nerve cells in the brain using light, ‘organoids’, which are created from human stem cells, and have similarities to human organs like the kidney, intestines, heart, and even brain, and synthetic biology, which seeks to create artificial bacteria and yeast chromosomes from scratch and even reconfigure the genetic code. All of these technologies have great potential for improving human society, but they also raise many ethical and socio-political issues and questions about ways they might be misused, which will be explored in this book.

Modern medicine has advanced tremendously in the 20th century, yet many people still die each year from conditions like heart attacks, cancer, and infectious diseases. Genome editing looks set to transform clinical medicine over the next few decades, because it now makes it possible to alter genes either in a living person, or in an infectious agent like a virus. One particular type of disease where genome editing is likely to have a big impact is single gene disorders such as cystic fibrosis, Huntington’s disorder, and muscular dystrophy. Because these disorders are due to a mutation in a single gene, there is hope that it should be possible to use genome editing to ‘correct’ the mutation in the cells of a sufferer. But genome editing also offers much promise for the treatment of more common disorders such as cancer. In the latter case, a mutated oncogene could be corrected by genome editing, or this approach used to enhance the ability of special immune cells in the body that target cancer cells. Genome editing also has much potential for targeting viruses like as HIV. In this case, there are two possible approaches. One involves targeting the HIV genome sequence, the other involves creating a mutation in a gene coding for a human protein that HIV uses to get inside an infected person’s cells. Bacterial infections might also one day be targeted by genome editing. However, many practical obstacles remain, the main one being how to get genome editing tools into a person safely.

Given the speed of change in the development of new technologies mentioned in this book such as genome editing, optogenetics, stem cell organoids, and synthetic biology, it is hard to predict exactly how radically these technologies are likely to transform our lives in coming decades. What is clear is that as exciting as the new biotechnologies are in terms of their impact on medical research, medicine, and agriculture, they also raise a whole number of socio-political and ethical issues. These include concerns about whether monkeys engineered to have genetic similarities to humans might lead to a ‘Planet of the Apes’ scenario, and fears about ‘designer babies’ being produced in the future to have greater beauty, intelligence or sporting skill. Although one potentially positive new development is the rise of a ‘biohacker’ movement which seeks to make molecular biology more accessible to ordinary people, there are also fears that in the wrong hands genome editing might be used to create new types of biological weapons for terrorist organisations. While such fears should not be dismissed as just an overreaction, to some extent they rest on an underestimation of the complexity of the Iink between the human genome and looks, intelligence, and sporting ability, or of the difficulties involved in creating a deadly virus that is worse than naturally occurring ones. Ultimately, the best way to ensure that new technologies are used for human benefit, not harm, is to take part in an informed debate and use public lobbying to argue for them to be developed safely, ethically, and responsibly.

Bacteria are a source of many of the tools used in biotechnology. A technique called the polymerase chain reaction, or PCR, made it possible for the first time to amplify tiny starting amounts of DNA and has revolutionised medical diagnosis, testing of IVF embryos for mutations, and forensic science. PCR involves the repeated generation of DNA from a starting sequence in a cycle, one stage of which occurs at boiling point. Because of this PCR uses a DNA polymerase enzyme purified from an ‘extremophile’ bacterium that lives in hot springs. More recently scientists have constructed artificial bacterial or yeast genomes from scratch. The next step will be to create reconfigured bacteria and yeast with enhanced characteristics for use in agriculture, energy production, or generation of new materials. Some scientists are now seeking to expand the genetic code itself. The DNA code that human beings share with all other species on the planet has four ‘letters’, A, C, G, and T, which pair as A:T and C:G to join the two strands of the DNA double helix. And each particular triplet of DNA letters, for instance CGA, or TGC, codes for a specific amino acid, the 20 different amino acids joining together in a specific sequence to make up a particular protein. Scientists have now developed a new DNA letter pair, X:Y. By introducing this into an artificial bacterial genome, it is becoming possible to create many more amino acids than the current 20 naturally occurring ones, and thereby allowing many new types of proteins.

Animal ‘models’ of health and disease have been central to biomedical science since at least when William Harvey used dogs to illustrate the fact that blood is pumped by the heart through the arteries and then through the veins back to the heart. In the 1980s, a major step forward came with the discovery of embryonic stem cells and ways to manipulate these genetically and then inject into mouse embryos, resulting in the creation of knockout and knockin mice with deletions, or more subtle changes, in specific genes. Unfortunately, it has been impossible to isolate embryonic stem cells from any other species besides mice, and more recently rats and humans. Yet rodents are far from the best animals for modelling, say the body’s metabolism or heart function and disease, or brain function and mental disorders. Instead, pigs and primates are potentially far better models for these respective areas of research. CRISPR/Cas genome editing has made it possible for the first time to create precisely genome edited versions of pigs, monkeys, and any other species that may provide a better model of specific aspects of human health and disease, than rodents. So genetically modified pigs might be used to study heart disease, but also provide hearts for human transplantation, while GM monkeys might help us better understand the biological basis of mental disorders such as depression or schizophrenia. However, this area of research is raising ethical issues about the creation of monkeys with human versions of particular genes, and how this might affect their behaviour and personality.

We are currently in the middle of a revolution in the biological sciences. The genome project made it possible to read the sequence of individual genomes, but what was lacking until recently was a way to rewrite particular genes in a living cell in an accurate, rapid, and economical fashion. This is now possible thanks to the invention of molecular ‘scissors’, based on natural mechanisms in the cell, that make it possible to cut the genome at a particular point, after which the cell’s own natural repair mechanisms edit the gene as directed. The most revolutionary of the molecular scissors is one called CRISPR/Cas. This is based on a process discovered in bacteria that normally acts to destroy viruses that invade the bacterium. Reprogrammed, CRISPR/Cas now makes it possible to accurately edit the genome of any living cell of practically any species, for a fraction of the time and cost required by other genome editing approaches. Not only can this approach be used to edit the sequence of genomes but also to switch genes on or off at will. CRISPR/Cas is revolutionising medical research and looks set to do the same for clinical medicine and agriculture. But it is also the source of much controversy as recently a scientific team in China used this approach to create genome edited babies, and in general some people fear the speed at which this new technology is developing and its potential for misuse. These are all issues that are explored in subsequent chapters of this book.

Although direct engineering of animal and plant genomes only became possible from the 1970s onwards, people have been indirectly changing other species’ genetic make-up for thousands of years. This is because with the agricultural revolution that began 12,000 years ago, human beings began selecting particular variants of a species, and cultivating them as crops or breeding them as farm animals, and in the process altered a species’ genome over time. In fact this process began even earlier, with the domestication of the wolf, which eventually became our most faithful companion, the dog. And now modern genome analysis techniques are making it possible to understand how at the genetic level a dog or cat differs from their wild ancestors, or how wild boars became pigs, or wild grass could over time give rise to wheat, barley, or rice. In medical research, scientists realised that understanding how genes work could be helped by studying naturally occurring mutants. By studying how mutations affected an organism such as a fruit fly,genes involved in particular functions could be identified. A breakthrough was made when it was realised that treatment of fruit flies with X-rays dramatically increased the numbers of mutants. Today, X-rays and chemical treatments can be used to create mutant mice, and studies of such mice has identified genetic links with conditions such as congenital deafness.

Although human beings have been altering genomes by selection and breeding of particular animal or plant variants for thousands of years, and X-rays and chemicals were first used to create mutants in the early 20th century genetic engineering in the true sense only became possible much more recently. First, the discovery that genes are made of DNA, revealed the material nature of the genome. Second, scientists in the early 1970s discovered enzymes in bacteria that can be used to cut and paste DNA in the test tube. Using such molecular ‘tools‘ bacteria were engineered to produce important medical products such as human insulin for diabetics, and from this was born what would become a billion-dollar biotechnology industry. A further important development was the discovery of embryonic stem cells and manipulation of these to make ‘knockout’ mice that had a deletion of a specific gene, or ‘knockin’ mice that had subtle changes in a gene. However such an approach was still relatively expensive and time-consuming, and cold only be applied to mice. And although gene constructs could be introduced into cells in a less precise manner, the crude nature of this approach limited its application for both agriculture and gene therapy. In both areas of application there have been concerns about the safety and ethics of using such an approach. A major criticism has been the lack of precision in where a gene construct would end up the genome, leading to concerns about possible adverse effects.

Visual light, and radiation of other frequencies, are highly important for scientific research. The first light microscopes made it possible for the first time to see that organisms from plants to humans are composed of cells. Electron microscopes have allowed scientists to study the structural components of cells in great detail, and even determine the shapes of individual proteins. Many lifeforms also use light to attract a mate or prey, or deter an attacker. Following the identification of the gene coding for the fluorescent protein that makes certain jellyfish glow green it has become possible to use this to genetically label proteins in a living cell, or even a live animal. This means that now the location of proteins in a cell can be determined exactly. A major recent step forward in neuroscience came with the discovery of protein channels in algae that conduct ions in response to light. By creating transgenic mice that have these proteins in their brain neurons, it is now possible to modulate the activity of these neurons by shining light into the brain though microscopic fibre optic cables. This new science of optogenetics allows neurons to be switched on or off experimentally. The optogenetic approach has been used to uncover the neural circuits involved in memory, pain and pleasure. In the future this technique might be used to treat physical pain or depression in people. Controversially, it might be also be misused, to supress memories, or even create completely false ones in people’s heads.

Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells&rsquo, ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-&beta, II, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-&beta, II to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.