A Guide for Gentlemen Studying Medicine at the University of Edinburgh (1792). According to Gregory, this was ‘a knavish pamphlet, published under a false name . . . intended to promote the pecuniary interest of one man, and to injure the fame and fortune of another, both of them my colleagues in this University’.In 1792, James Gregory (1753–1821), Professor of the Practice of Physic at the University of Edinburgh, became involved in a highly public dispute with a family of man midwives. The row centred on a text attributed to ‘J. Johnson Esq’ entitled As this episode suggests, James Gregory was not averse to engaging in public quarrels with his medical colleagues. Even in the highly fractious world of medicine in late Georgian Edinburgh, he was notorious for his belligerence. Henry Cockburn 1779–1854), the celebrated Whig jurist, claimed that, whatever his virtues, ‘a disposition towards personal attack was his besetting sin’., the celTo date, most scholarly accounts of the life and works of John Bell have tended to view him primarily as the erstwhile mentor of his more famous younger brother, Charles Bell 1774–1842).74–1842.7For the most part, then, historians have understood this episode in terms of intra‐professional and institutional politics. Quite right too; as we shall see, the Gregory–Bell dispute spans three distinct yet interrelated historiographical fields: firstly, the political culture of medicine in Edinburgh during this period; secondly, the relations between physicians and surgeons at a time when the latter were growing in number and increasingly asserting their status; and thirdly, as I have explored elsewhere, the contested politics of charitable medical institutions in the early nineteenth century.Additional Memorial to the Managers of the Royal Infirmary (1803), for example, exhibits a forensic attention to the minutiae of Bell's writings which more than borders on the tedious. By contrast, though Bell was praised by Gregory for his ‘extraordinary talents’ as an ‘Advocate’ for his fellow surgeons, his publications possess a superior literary quality, so that even Gregory admitted that reading them was ‘an agreeable amusement’.Letters on Professional Character and Manners: On the Education of a Surgeon, and the Duties and Qualifications of a Physician: Addressed to James Gregory (1810), a work which not only set out his vision of surgical identity, but did so in explicit contrast to that of the physician. Furthermore, it was against the backdrop of this conflict that Bell produced his magnum opus, The Principles of Surgery (1801–6), a work that would continue to be read as a standard text for decades to come.However, one aspect of the dispute which has yet to be considered is its longer‐term implications for surgery and surgical identity. Gregory's published contributions to the row have a legalistic quality. His 513‐page This dispute, and John Bell's contribution to it, is thus highly worthy of scholarly analysis, not only because he was regarded as ‘the only true surgeon in Edinburgh’feeling and emotion. As Thomas Dixon has argued, the term ‘emotion’ has a complex history. In the eighteenth century it functioned ‘either as an undefined and general term for any kind of mental feeling or agitation, or sometimes as a stylistic variant for central theoretical terms such as “passion” and “affection”’.This article contends that what made Bell's work so appealing to large numbers of surgeons in the late Georgian period was its articulation of a culturally resonant professional identity shaped, to a profound degree, by The history of emotion is now a well‐established field of study but it is only in relatively recent years that historians of medicine and science have begun to take emotions seriously in their consideration of professional practice, identity and representation.In the context of the Gregory–Bell dispute, this critique of affectation centred on the meanings assigned to embodied knowledge and practice. In his work on the history of emotion as a concept, Thomas Dixon mistakenly refers to Charles Bell as a physician, rather than a surgeon. This is intriguing, because those embodied, physiological qualities, such as the motion of the heart and lungs, which Bell brought to the ‘modern’ concept of the emotions, were a direct product of his surgical world‐view.The medical world of late eighteenth‐ and early nineteenth‐century Edinburgh has been so well documented by historians that little more needs to be said here, save as context.some of the most skilful surgeons’.One of the singular features of Edinburgh medical education was clinical instruction, a model of teaching imported from Leiden. To that end, a small six‐bedroom infirmary was opened in 1729 followed in 1741 by a much larger purpose‐built structure, now known as the Royal Infirmary, in Thomson's Yards.It is important to note the wording employed by the Incorporation in their appeal to the managers; they claimed that the system of universal attendance was necessary ‘in order to preserve an equality among the surgeons of Edinburgh’.science.Although formal membership of the Incorporation/Royal College fluctuated across the century, by the later 1700s the number of surgeons in Edinburgh was increasing. More importantly, perhaps, surgical knowledge was also on the rise. During the long years of war with France (1793–1815), there was increased demand from the army and navy for medical practitioners. What each regiment or ship‐of‐the‐line needed was not a physician but a surgeon, or rather surgeon‐apothecary, capable both of performing operations and of attending to the quotidian medical needs of the men.Remarks on Mr John Bell's Anatomy of the Heart and Arteries.System of Surgery (1783–8) of another (unrelated) surgical rival, Benjamin Bell (1749–1806).The difference betwixt your description and that of a bold operator, is just that which distinguishes an assassin from a brave man! You write bloodily, though not boldly: you speak not like a regular surgeon . . . but like a desperate man, careless of everything, and afraid only of being affronted, or, in other words, ‘embarrassed’ in the midst of a public exhibition! You write like one who had been often caught and entangled in difficulties from which he had no other way of disengaging himself than by a slap‐dash stroke of the knife . . . You are enfuriated [sic] by opposition! the words adhesion, stricture, gut, and sac, excite proportioned fury! and you exclaim, tear, cut, clip, destroy – Tear the adhesions, cut every thing; – surgery consists in cutting! and the best surgery is to cut every thing!!!Despite his eminence, Bell was not without his critics. Nor were the surgeons of Edinburgh a united body. Indeed, the period immediately preceding the dispute between Bell and Gregory was characterized by the latter as one of internecine ‘chirurgical warfare’.Quite how Bell came to be involved in this dispute is not entirely clear. The evidence suggests that he was a ‘pugnacious’ and quarrelsome man; ‘No formidable insect delighted in his sting so much as he did’, wrote Cockburn.If being the son of an impoverished and marginalized clergyman made John Bell something of an outsider, then James Gregory was the quintessential insider. He was born in 1753 to John Gregory, physician and sometime lecturer in philosophy at King's College, Aberdeen. His father was appointed Professor of the Practice of Physic at Edinburgh in 1766, becoming celebrated for both his medical and moral writings.It was perhaps the entitlement and security which came from such privileged connections that gave Gregory the confidence to engage in as many feuds as he did. In 1798 he was elected President of the Royal College of Physicians, a post which gave him an automatic seat on the governing council of the Royal Infirmary. By this time the issue of surgical attendance at the Infirmary had still not been resolved. In addition to four salaried posts, created in 1769, the surgeons attended by rotation for a period not normally exceeding two months. However, their subordinate status was evident in the fact they were not allowed to undertake any operation ‘of importance’ without the permission of the physicians‐in‐ordinary and, as a result, many of the more senior surgeons declined to attend.Memorial to the Managers of the Royal Infirmary (1800) in which Gregory proposed the permanent appointment of ‘a number of ordinary attending Surgeons’ as well as ‘a regular supply of younger Surgeons’ and a ‘sufficient number of extraordinary or consulting surgeons . . . to give their advice and assistance in all extraordinary and difficult cases’.some of the most skilful Chirurgeons’ was significant because:more and others less skilful. This, I presume, many people would very readily believe without the evidence, either of a ghost or of a Royal Charter . . . it is generally known to be the case with the individuals of many different professions; very remarkably among lawyers and wig‐makers, shoemakers and tailors, milliners, cooks, fiddlers, dancing‐masters, postilions, and physicians.It implies, that, of the Surgeons in Edinburgh, some may be This intervention was a tract entitled all members of the Royal College of Physicians, not ‘some of the most skilful’. That proviso was reserved for surgeons.Several tropes that would become recurrent themes of Gregory's argument are worth noting here: firstly, the reference to ‘a ghost’, an allusion to the fatal consequences of surgical incompetence; secondly, the rhetorically expedient lumping together of high‐status ‘professions’, such as law, with lower status trades such as cookery; and thirdly, the disingenuousness of including physicians in this list. After all, the Charter granted the right to attend the Infirmary to entirely in the hands of the youngest and most inexperienced surgeons’.This invidious distinction between individual competence and collective authority is essential for understanding both Gregory's argument and the reaction to it. While he repeatedly claimed that the ‘merits or demerits of any individual’ were ‘unnecessary for the object which I have in view’, his refusal to single out individuals meant that his criticisms necessarily fell upon the surgeons as a body. In particular, they fell upon the mostly junior members of the College of Surgeons who, unlike many of their senior brethren, ‘rigorously avail[ed] themselves’ of their right of attendance at the Infirmary. The practice of surgery in the Infirmary was, Gregory claimed, ‘Despite his refusal to name names, Gregory peppered his text with sensational examples of surgical incompetence, particularly in regard to manual dexterity, a quality which, he claimed, could only be gained by experience.our families, and our children, when we act as Managers of the Hospital: They have a right to our paternal care; they have a right to obtain from us the best assistance, and every relief and comfort which we can procure for them.The poorest patients in the Hospital have the understanding, the feelings, and the rights of men. They know and feel, though they may not have the words to express it, that misery is sacred. They are In his criticism of the system of surgical rotation, Gregory resorted not only to sensationalism but also to moral outrage. Writing in the languages of Christian charity and paternalism, given particular inflection by the cultures of sensibility and humanitarianism, he stated:expense of the poor patients, I mean by any unnecessary suffering, or danger, or harm to them, is injustice and cruelty instead of charity, benevolence, and brotherly love to the sufferers; it is a breach of trust on those who do it, or permit it; it is an outrage on human nature.Whatever is learned; or whatever real or supposed improvement is acquired at the Therefore:fault of a youth of four‐and‐twenty, just going to begin the practice of his profession, that he has not the skill, and useful experience, and manual dexterity of a surgeon fifty years of age’, he clearly thought it was the fault of surgeons as a corporate body. Indeed, he repeatedly claimed that the agreement made by Kennedy and the Incorporation of Surgeons with the managers of the Infirmary in 1738 was a wilful act of self‐interested cruelty: ‘I doubt’, he wrote, ‘whether there was ever in this world, or ever can be, a more direct, avowed, and disgraceful opposition between the interests, real or supposed, of a corporation, and the interests and the rights of humanity.’While Gregory claimed that it was ‘not the two hundred, or one hundred, or even forty of them established in such a town . . . Physicians and Surgeons are by no means on the same footing, in point of political economy, with corn and cattle; a superfluity of which, in the course of nature, is soon converted into an additional number of men and women, to the most essential benefit of the state. They cannot even be applied to various uses, like a superfluous quantity of wool, and flax, and iron, and other raw materials used in manufactures; nor can they be exported and bartered by way of trade for other valuable articles, like a super‐abundance of highly‐manufactured goods, beyond what their own country can consume. The superfluous physicians and surgeons are absolutely useless and helpless . . .But it would be no advantage to the public, any more than to the Faculty, to have Gregory could have closed his argument there, but he did not. Rather than limiting himself to the issue of surgical rotation at the Infirmary, he proceeded to critique surgery as a whole and to question its place within the social landscape and political economy of the city. The Incorporation's stated desire to ‘preserve an equality among the surgeons of Edinburgh’ was anathema to Gregory. The reason why they were so keen to preserve ‘equality’, he claimed, was because there was simply too many of them. A certain level of competition was necessary to ensure a tolerable standard of competence, perhaps twenty physicians and twenty surgeons in a city where only ten of each were really needed:ancien régime, an aristocrat in outlook, if not in law. Just as his conception of charity was rooted in a paternalistic benevolence which was being challenged by the twin forces of political economics and utilitarianism, his understanding of society was similarly shaped by the face‐to‐face relations of the pre‐modern world, rather than the abstractions of social and cultural modernity.demand than a subjective assessment of social worth. Deprecating the notion of surgical ‘equality’, he claimed that he preferred ‘inequality’ and looked with barely concealed disdain on the growing ranks of surgeon‐apothecaries emerging from the private anatomy schools:Their number would soon increase rapidly, by the addition of all who had the requisite qualifications, and so little activity, or spirit, or honourable ambition, as to be contented for life with the miserable pittance and degrading situation which such a system of equality would insure to them. Surgery in Edinburgh would soon cease to be a learned or a liberal profession. Those who practised it would not be ranked in public estimation with physicians, or merchants, or lawyers; nor would any of them be known and esteemed as authors of learned, scientific, and useful works on anatomy, chemistry, physic, or surgery . . . In one short sentence, by such a system of equality, the Surgeons of Edinburgh would soon become Barbers again.Gregory's awkward fusion of the political economics of Adam Smith  with the mercantilism it would displace is indicative of his innate conservatism in the face of profound social, economic and intellectual change. Gregory was a man of the per se. He counted a number among his close friends. But while he admired a talented few, the rest he dismissed as ‘arrant Drones’.No person can be so stupid as . . . to wish that a perfect equality were established among physicians and surgeons respectively, so that in case of need, he might call a physician or a surgeon, without knowing his character, or even his name; just as he might call a porter or hackney coach, without enquiring the name of the porter or the character of the coachman.Gregory did not mind surgeons For Gregory, the true signifier of character and, hence, social worth was wealth. In this sense, the surgeons of Edinburgh were in a highly precarious position. A minority, he suggested, ‘are perpetually rolling about the streets on four wheels, while three or four times as many of them walk about the streets on their two hinder legs in true primitive simplicity’. And while ‘it is of little or no moment to [the patient] whether the operator come to his house on foot or in a gilded chariot . . . I should rather think it some comfort . . . in those anxious and fearful moments, to know that his surgeon has such an extensive and successful practice that he can afford to keep such a chariot’. Indeed, if the surgeons were inclined to pursue equality, Gregory argued, ‘In a few years, not one of them would be able to live in a genteel, or what at present they think a decent and comfortable manner, nor would any of them be admitted into the company of gentlemen.’Many of our greatest heroes, both in red coats and in blue, men who would boldly march up against a battery of cannon, or joyfully obey an Admiral's signal for close action and breaking the line of battle of an enemy's fleet, will grow pale at the sight of only two or three Surgeons, when these come to consult about them. And many a poor patient, when he is set down in midst of five and twenty Surgeons in this hospital, I have no doubt, feels all the horror, without the faith of the Prophet Daniel, when first he took his seat in the den of lions.As is clear, Gregory repeatedly goaded surgeons about their origins in trade and suggested the possibility, even likelihood, of social regression. But, just as importantly, he played upon another well‐established stigma associated with surgery: cruelty. More than simply drawing attention to what he perceived to be the cruelty of allowing inexperienced surgeons to attend the Infirmary, he went so far as to imply that surgery as a whole was inherently cruel. Drawing upon an appropriate metaphor for the period, he claimed that:I never am present at any operation in private practice, unless at the patient's particular desire; of course I see such operations very seldom. Though I have been intimately connected with the Infirmary as a clinical professor for more than three and twenty years, I have not during all that time been present at a public operation in the Theatre, where the junior Surgeons chiefly operate, but one day.Such observations about the emotional demands of surgery were, as we shall see, not uncommonly made by surgeons themselves, but coming from a physician, especially one so openly disdainful of surgeons and surgical science, they were doubtless galling. Gregory even wrote that:In a later text, Gregory stated that he ‘neither like[d] the sight nor the smell of a putrid mangled human body’,Memorial would be dissected by the denizens of Surgeons’ Hall, but he can have had little idea that this task would fall to perhaps the most gifted anatomist in Edinburgh. Bell had formerly expressed himself loath to comment on the issue of surgical attendance at the Infirmary, but he was sufficiently offended by the tone of Gregory's text to agree to a request, made by the junior members of the College of Surgeons, that he publish a response.Answer for the Junior Members of the Royal College of Surgeons of Edinburgh to the Memorial of Dr James Gregory (1800), Bell claimed that the surgeons were a ‘public body’ who ‘demand respect’. Instead, their profession had been ‘cruelly traduced’. ‘He mocks at all dignity’, Bell claimed, ‘at all semblance of science, at all professional skill, faith, honesty, or honour; and we and our cruelties are his constant theme.’Gregory was correct to assume that his To become skilled [in surgery], a man must live among the sick: he must have lively feelings, and a sympathizing nature; his mind and senses must be deeply impressed with the character of every kind of suffering; he must have that inward sympathy with the distresses of his fellow‐creature[s], which fills the mind with sincere and affectionate interest. What can more aggravate sickness, than to tell the long tale of misery to one who merely listens, who betrays no touch of compassion, whose cold and formal inquiries imply no interest, and end with a prescription in form[?] Such a man never learnt his profession, will never learn it: he has no feelings towards his individual patients, and can have no enthusiasm towards his general duty. In our profession, young men should have instilled into their minds that sympathy with the sufferings of their patient, and that keen spirit of investigation should be roused in them, which refines every sense, and quickens the intellect.This emphasis on cruelty is revealing. Bell was particularly sensitive to the charge that surgery was, in his words, ‘a cruel trade’.To be initiated into our profession, is not merely to be taught the principles of Chemistry, and the Anatomy of the human body; but it is . . . to feel an interest in the fate of each patient; to form apprehensions for his safety which perhaps he himself does not feel . . . to be alarmed by changes of voice, pulse, and countenance, which make no impression even on the patient's friends. This is the true initiation in to our profession; and he, who is once full of these sympathies, takes an interest in every case, and studies with unremitting diligence.Thus, in contrast to Gregory's accusations of cruelty, accusations which tapped into well‐worn negative stereotypes, Bell suggested that the identity of the surgeon was positively predicated on sensibility, on the capacity for compassion and sympathy. These were not simply manners, mere ornamental social graces. Rather, according to Bell, a highly attuned sensitivity to the feelings of one's patients was a vital clinical skill, and any surgeon who did not possess ‘lively feelings, and a sympathizing nature’ had not ‘leant his profession’. Indeed, not only was the surgeon to feel compassion for his patients, he was also to become what I have called elsewhere a kind of ‘emotional savant’, able to read the most subtle expressions of feeling in others and act with calming reassurance.Memorial drew heavily upon the same rhetoric of Christian charity and sensibility that had been deployed by his father. However, Bell questioned the sincerity of such expressions, especially when compared to the embodied experiences of the surgeon:Has his mind been thus keenly touched, almost disordered, at the miseries of his fellow creatures? No, no! his strong sensibilities we hold but lightly: He never passed a sleepless night, reflecting what was to be done on the morrow; never witnessed the severities of the surgeon; never strained hard his breath, nor involuntarily clenched his hands at the sight of another's agony; nor blanched with fear, nor felt the palpitations of anxiety, in the midst of an eventful operation? Let a man feel the things he can feel, and his sensibilities will be applauded. This sensibility is not of the right stamp: he coolly collects his jests, when he would be witty; and as coolly strains out a lamentation when he would be thought humane.In presenting sensibility as an integral part of surgical practice and identity, Bell sought not merely to deflect accusations of cruelty, but also to question Gregory's own public identity. As we have seen, Gregory's Bell's use of the phrase ‘right stamp’ is telling. In this period a stamp might suggest the mark made upon goods on which duty had been paid, something which, whatever its formal purpose, was often construed, by manufacturer and public alike, as a signifier of authenticity.The Man of Feeling. Perhaps the quintessential sentimental novel, its protagonist, Harley, embodies an ‘ideal sensibility’, and ‘sheds tears over the misfortunes of the people he meets’.Bell's critique of Gregory's emotional affectations was rooted in wider debates about the merits of sensibility. As Markman Ellis has argued, while sensibility lacked ideological coherence, its capaciousness and elasticity only served to enhance its cultural appeal across the eighteenth century.Answer to Gregory must be understood in this context. As the earlier quotation demonstrates, Bell contrasted the emotional experience of surgery with Gregory's merely rhetorical appeal to feeling. While this might itself be interpreted as a rhetorical device (and of course it was), it is also indicative of Bell's wider conception of surgery. Throughout his oeuvre there is a powerfully embodied quality to the descriptions of operative surgery which transcend the functional requirements of surgical instruction and embrace, instead, the visceral, affective dimensions of surgical experience. Take, for example, the following passage from his Discourses on the Nature and Cure of Wounds (1795), in which he describes an operation for the relief of an arterial aneurism:You are to . . . expect that the moment you cut the tumour, the blood will rush upon you with a terrifying violence: nor should you ever expect to clean the great cavity with sponges or cloths, for the artery will fill the cavity with blood, faster than you can throw it out, till the patient breathes his last. Instead of this, you draw the knife deliberately and fairly over the tumour, so as to lay it open. The skin being thus divided, the great livid bag of the aneurism surrounded with its strong fascia, rises into view. Next you push your lancet into the bag, and then do all that remains in your operation with great boldness; run your bistory upwards and downwards so as to slit up the tumour quickly; plunge your hand suddenly down towards the bottom; turn out the great clots of blood with your hand and fingers, till having reached the bottom entirely, you begin to feel the warm jet of blood, and directed by that, clap your finger upon the wounded point of the artery, as it is but a point, your finger will cover it fairly, and your feeling the beating of the artery, assures you that all is now safe.One of the consequences of this ‘politicisation of sensibility’ was that some began to place increasing emphasis on the sincerity and authenticity of emotional experience as opposed to the mannered artifice of social performance. Indeed, as a number of scholars following Lionel Trilling have observed, emotional sincerity and authenticity were among the key markers of an emergent culture of Romanticism, and its peculiar inflection of sensibility.Answer to Gregory:We pretend no finer feelings than nature has implanted in every common breast ‘but we are accustomed to see people, once the gay and the happy, sunk in deep, retired distress: sometimes devoted to a certain, but painful and lingering death; sometimes struggling with bodily anguish, or the still fiercer tortures of the mind’. We venerate the miseries of our fellow creatures, and the profession that brings relief.For Bell it was precisely the embodied nature of his work, and his exposure to the extremes of human pain, suffering, and endurance, which rendered the surgeon uniquely sympathetic and emotionally astute. ‘Wherever we turn, it is towards objects of compassion’, he wrote in his Additional Memorial Gregory reiterated his distaste for surgery and ridiculed Bell's description of the affective qualities of surgical experience, writing ‘Whether I am or am not subject to hysterics, and accustomed to exhibit the horrid grimaces thus beautifully described by Mr John Bell, is a question of no moment.’Had you but called your metaphysics to your aid, and trusted rather to your reason than your feelings, you might have conjectured, that in this simple description all must be right; that what you imagined to be merely cunning, was but a simple unostentatious description of emotions very natural to those not altogether void of humanity. I do indeed believe, Sir, that if you ever again pretend to express excessive sensibility, it will be . . . ‘in HORRIBLE GRIMACES;’ and it would no doubt be painful to see you disordered by a feeling, or the affectation of a feeling, as foreign to your physical and spiritual frame, as ‘Hysterics’ are to robusteous [sic] health. Be advised, Sir, and write no more on sensibility and charity!As we have seen, Gregory expressed what Bell referred to as ‘a perfect horror . . . at the unnecessary presence of surgeons’ and claimed to have not attended an operation in over twenty years of practice.What is remarkable about this aspect of Bell's argument is that it turned conventional professional and social prejudices on their head. Gregory, like many physicians, deprecated surgeons because the manual nature of their work rendered them closer to tradesman and inferior to the intellectual labour of the physician. Traditionally, too, sensibility was held to be a quality of nervous refinement more marked in the gentleman and lady than in the horny‐handed working man or woman. By contrast, what Bell was suggesting was that the embodied emotional experience of surgery had a morally improving quality which was lacking from the conventional practice of the physician. As such, it was the surgeon, rather than his supposed social superior, who was the truly authentic man of feeling, and it was this feeling that gave the surgeon his sense of professional self‐worth. As he later wrote, ‘I have spent much of my life in services which could only arise from those feelings, in useful and active duty, which you would disdain.’expertise derived from a scientific education (of the kind Bell provided) and not a product of mere habit or routine:There is all that difference which reflection should lead us to expect, betwixt a man learning BY EXPERIENCE to become an operator, and one taught BY SCIENCE, and prepared by every study that may contribute to success. The one is supported by conscious knowledge and skill, in a manly, steady, dignified state of mind; the other, fluttering with fear and anxiety, and greedy of applause.It is not hard to see the political implications of this argument. As we have heard, Gregory's principal objection to the established system of surgical rotation at the Infirmary was that it allowed inexperienced surgeons to practise on poor patients. More generally, Gregory thought that a few genteel surgeons were preferable to an army of low‐status ‘drones’. In opposition to this fundamentally aristocratic understanding of professional and social relations, Bell advanced what we might call a democratic model of surgical identity. This vision was in keeping with an inchoate conception of the medical profession that I have explored elsewhere.It is the factitious character that opens the way to public employment and professional honours: In what does it consist? in suavity of manners, a specious carriage, an agreeable person, a pleasing address, a facetious conversation, a thorough knowledge of the politics and courtliness of high life. A splendid establishment, a gaudy carriage, family connections, and the solicitation of friends . . .Even if the surgeon derived his authority from scientific training rather than from individual character, this is not to say that character was unimportant. However, this character was not of the kind that Gregory admired, namely the social graces of the gentleman. It was not what Bell dismissed as ‘the mere accomplishments, mere ornaments’ of ‘gaudy trivial beings, all fluttered with metaphysics, philosophy [and] literature’. This was ‘the finery’ with which, according to Bell, Gregory had ‘strutted most insignificantly through life’. Instead, surgical character was of a moral kind, shared by all surgeons. It was simple, honest and ‘manly’, the product of authentic emotional experience, not social privilege or polite manners. Asking what kind of character defined men like Gregory, Bell replied:And will the Memorialist, a philosopher, and a liberal one, speak of these as specific qualities, which ascertain a man's professional skill? We hope, for the credit of bare unsophisticated nature, that the honest and feeling heart, the thinking head, and the steady hand! the open liberal hand, which drops its alms while it is assuaging pain! is not more frequent in the gilded chariot, than in the humble walks of life; where men drag along the burden of their duties, and crawl even on their lower extremities in the pristine manner.Recalling Gregory's allusion to the ambulatory surgeon, Bell continued:Looking back on the conflict that had convulsed the Edinburgh medical community in the summer of 1800, Henry Cockburn claimed that Bell ‘had the best both of the argument and of the clever writing; but the public sided with the best laughter; and so Gregory was generally held to have the victory’.However, while Bell's career was frustrated by his dispute with Gregory, in another sense his reputation and influence were considerably enhanced. He continued to be a significant intellectual presence in Edinburgh surgery. For example, in 1808 Thomson trialled what he claimed was a new method of cutting for the stone at the Royal Infirmary. The technique was unsuccessful and led, in at least one case, to a patient's death. Bell published a scathing attack on the operative skill of a man who ‘after a life of busy, restless intrigue, in collusion with you [Gregory] and your worst associates, had procured himself to be elected surgeon to the Infirmary’.Principles of Surgery (1801–6). An ‘undying book’, in Struthers’ words, it outlined a comprehensive system of surgical disease and practice with a particular emphasis upon the science of surgery.character and identity. The first volume opened with a preliminary discourse on the ‘education and duties of a surgeon’ in which Bell not only emphasized the indispensability of a thorough anatomical education, but also advised that:When a man enters upon such duties as these, he should be diligent, watchful, and laborious; humane, friendly, and self‐denying; full of unceasing anxiety for others, and a noble disregard of himself. We will not define the chief qualities of a surgeon, in this cold‐hearted way, of personal and showy accomplishments; mercy and tenderness towards his patients, and every kind of charity, are the chief virtues and most becoming ornaments of a surgeon.Bell's posthumous reputation is evident in the surgeon John Struthers's mid‐nineteenth‐century account of the ‘Edinburgh Anatomical School’ in which he claimed that John Bell was ‘the reformer of Surgery in Edinburgh, or rather the father of it’. Moreover, Bell's legacy stretched far beyond the walls of the Old Town. During the period of his dispute with Gregory, which lasted from 1800 until around 1810, he published some of his most influential works. The most celebrated of these was his Letters on Professional Character and Manners: On The Education of a Surgeon, and the Duties and Qualifications of a Physician, consciously referenced his father's Lectures on the Duties and Qualifications of a Physician (1772). In this text, Bell presented his most well‐rounded vision of the surgeon as man of sensibility: as compassionate, sympathetic and selfless, always placing his patient's interests before his own concern for reputation or fame. He was particularly concerned to deprecate the notion that operative flair, courage or daring should be regarded as admirable traits of surgical identity, arguing instead for an unostentatious and scientific character characterized by a dignified and considered deportment:A man of science never proceeds without due reflection: The whole plan of his operation is perfect in his own mind: He commutes with his assistant rather by signs than words, and his manner commands that stillness which is due to a moment of suffering, and essential to his self‐possession and success: He is formed by education, and qualified, from the first moment in which he takes those public duties upon him, to give impressive lessons to the younger members of the profession: They are awe‐struck with the first horrors of incisions and blood, but depart with gratified feelings, when they see the scene closed with entire relief to the sufferer, and happy prospect of success; and they learn to love and respect their profession, and to study it with emulation.As can be seen, then, the emotional qualities of the surgeon continued to be one of the most characteristic features of Bell's writing and he expanded considerably upon these observations in 1810 with a book that, despite the intervening years, was explicitly addressed to James Gregory and whose title, Principles of Surgery was dedicated to the surgeons of London, including John Abernethy and Astley Cooper, and it is clear that he was as admired as much in England's capital as he was in Scotland's. Moreover, while it would be extravagant to claim that he was the inventor of what we might call the ‘Romantic model’ of the operative surgeon, a model in which Cooper and Abernethy were, in their own ways, equally formed, he was certainly its most passionate and articulate advocate. And this model, with its rejection of rashness and cruelty and its embrace of selflessness and compassion, can be said to have opened the door, rhetorically speaking, to surgery's transition from a craft to a modern scientific profession.Bell's vision of the surgeon as calm, compassionate and composed would resonate across the succeeding decades. His


HDAC inhibitors (HDACIs) induce irreversible cell cycle arrest and senescence in mouse embryonic fibroblasts transformed with E1A and c-Ha-Ras oncogenes (E1A+Ras cell line). The aging rate has been associated with the production of high levels of Reactive Oxygen Species (ROS). Specific increases of ROS level have been demonstrated as potentially critical for induction and maintenance of cell senescence process. It's known that HDACs regulate the ROS-dependent FoxO factors, which are responsible for cell growth, proliferation, and longevity. The characteristic ROS increase during aging may be responsible for the decreased HDAC activity, which facilitates the senescent-like phenotype. The objective of this study was to investigate the impact of FoxO transcription factors on HDACIs-induced senescence of E1A+Ras oncogenes transformed cells. This study shows the specific time-dependent effect of HDACI sodium butyrate treatment on FoxO proteins in E1A+Ras cells. Indeed, short-term treatment with NaB results in FoxO activation, which takes place through nuclear translocation, and accompanied by accumulation of such ROS scavengers as MnSOD and SOD2. However, prolonged treatment leads to extensive FoxO degradation and increased intracellular levels of ROS. This degradation is connected with NaB-induced activation of Akt kinase. All of these findings establish that one of the possible mechanism involved in NaB-induced senescence of transformed cells is mediated through down-regulation of FoxO transcription factors and ROS accumulation. This diversity arises from multi-level regulation, which in turn is greatly dependent on the cell context. That makes regulatory mechanisms of FoxO proteins intricate and to some extent contradictory.Acetylation, like the rest of post-transcriptional modifications, is an essential part of FoxO regulation. FoxO proteins bind to coactivators and corepressors causing changes in FoxO acetylation level. It is worth noting that many acetylation sites are positioned within DNA-binding region, therefore acetylation in most cases suppress DNA-binding HDACs was furthermore reported to recover FoxO activity under the stress conditions, since oxidative stress ultimately results in FoxO inhibition by acetylation. Oxidative stress initially leads to FoxO phosphorylation and subsequent translocation to the nucleus, but then activated acetylases repress nuclear activity of FoxO The study has been performed on cells transformed by E1A and Ras, both of which are involved in FoxO signaling pathways. E1A stabilizes FoxO by preventing its ubiquitin-dependent proteolysis It should be also mentioned that NaB affects E1A+Ras-transformed cells in a special way. Sodium butyrate, like another HDACIs, is commonly known as apoptosis-inducing agent. This effect of NaB has been demonstrated on a wide range of cell lines, for example nasopharyngeal carcinoma cells The objective of this study was to investigate the impact of FoxO transcription factors on HDACIs-induced senescence of E1A+Ras oncogenes transformed cells.The paper reveals the FoxO transcription factors role in antiproliferative effects of HDACI sodium butyrate in E1A+Ras-transformed cells. We established time-dependent regulation of FoxO expression by NaB, which was accompanied time-dependent ROS accumulation. The overall aim of the study is to reveal specificity of treatment response in E1A+Ras-transformed cells, constituting a model of cancer cells with overactivation of Ras/PI3K/Akt, which are one of the most difficult to treat. Knowing molecular features of this response can lead us to better treatment solutions.2.2.1.E1A and cHa-Ras (line E1A+Ras). HEK-293 cells were kindly provided by Dr. van der Eb. Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) with the addition of 10% fetal bovine serum and antibiotics. Cells were treated with 4 mM sodium butyrate (NaB) or 2.5 µM suberoylanilide hydroxamic acid (SAHA) for 24–72 hours (Sigma-Aldrich).The study has been produced with the use of the stably transformed cells, derived from mouse embryonic fibroblasts by the transfer of complementing oncogenes 2.2.Cells were washed in PBS and then lysed. The protein concentration of each cell lysate was determined and equated. Prepared samples were boiled in sample buffer at 100 °C for 5 minutes. Samples were loaded in SDS-PAGE gel and the gel ran at 140–160 V. After electrophoresis proteins were transferred to the PVDF membrane. Membranes were blocked for 1.5 h at room temperature using blocking buffer (5% nonfat dry milk) and incubated with primary antibodies for 1 h at room temperature or overnight at +4 °C. Then membranes were incubated with HRP-conjugated secondary antibodies diluted in blocking buffer for 1h at room temperature. As primary antibodies, we used antibodies to Foxo1 #2880, Foxo3a #2497, phospho-Foxo3a #9466, Akt #9272, phospho-Akt #4060 .2.3.RNA was isolated from E1A+Ras cells using a TRIzol-based method. Amount of RNA used for generating of cDNA template was 3 mkg. RT-PCR has been performed using primers (100 ng) to the following genes: SOD2 5′-GGCCAAGGGAGATGTTACAAC-3′/5′-CTGAAGGTAGTAAGCGTGCTCC-3′; MnSOD 5′-GCACTGAAGTTCAATGGTGG-3′/5′-ACTGAAGGTAGTAAGCGTGCTC-3′. Quantification of gene expression has been made using the relative standard-curve method and all data was normalized to the absolute control group and subsequently normalized to the gene expression.2.4.Cells were grown on cover glasses, and then fixed in 3.7% formalin for 15 minutes at room temperature, washed in PBS for 10 minutes and in 0.15 M glycine for 15 minutes. After fixation cells were permeabilized in 0.2% Triton X-100 solution and blocked in 3% BSA/PBS for 1.5 hours. Cells were incubated in diluted in the blocking buffer primary antibodies to FoxO1 (#2880P) and FoxO3 (#2497S) overnight at 4 °C and then stained with the anti-rabbit secondary antibodies, conjugated with Alexa Fluor (488) for 1 hour at room temperature. The coverslips were mounted with ProLong Gold Antifade with DAPI.Images were analyzed and images captured using the Confocal Laser Scanning Microscope Olympus FV3000 .2.5.The cell membrane permeant agent 2′,7′-dichlorofluorescin diacetate (DCFDA) was used for detection of reactive oxygen species (ROS). After diffusion into a cell DCFDA undergoes acetylation forming non-fluorescent chemical compound, which can be subsequently oxidized to 2′,7′-dichlorofluorescin (DCF). DCF is a fluorescent compound that can be detected using the method of fluorescent spectroscopy. Cells were washed in warm PBS and incubated for 20 minutes at 37 °С in 5 mkM DCFDA/PBS. After incubation cells were washed in warm PBS and analyzed on flow cytometer Coulter Epicks XL  using an excitation wavelength of 488 nm.3.3.1.The accumulation of ROS has been shown to be in inverse proportion to the level of FoxO1 protein. Etoposide is a well-known cytotoxic agent that causes DNA-damages through an inhibition of topoisomerase II activity It should be mentioned, that NaB alone is also capable to affect the ROS level in time-dependent manner. Short-term treatment with NaB didn't lead to any significant increase in the ROS level . HoweverOther class of HDAC inhibitor such as SAHA have been used to demonstrate the specificity of NaB-dependent inhibition of FoxO expression. We have observed the same effect of SAHA on FoxO1 expression . Our res3.2.foxo transcription and total FoxO content in cells treated with NaB for 24–72 hours were measured. The study shows that prolonged NaB treatment causes only a slight decrease in foxO1 mRNA level  initially have lower FoxO1 protein level. In the absence of E1A constitutive Ras-induced Akt activation leads to intense FoxO degradation, which explains the difference in basal FoxO1 level. However, we observed straight accumulation of FoxO1 protein under NaB treatment in time dependent manner in HCT-116 and A-549 cancer cells with Ras mutation . The obt3.3.According to the immunoblotting data performed on nuclear and cytoplasmic lysates, FoxO1 is evenly distributed between the nucleus and the cytoplasm of control untreated cells . ProlongThe immunobloting data performed with nuclear, cytoplasmic and total cell extracts, reveal that a decrease of the total FoxO3a protein quantity under NaB treatment is accompanied by the redistribution of FoxO3a into the nucleus . The immThe expression of FoxO-target genes coding for ROS scavengers, providing protection against oxidative stress, has been shown to increase under NaB treatment .3.4.PKB/Akt kinase is a key mediator of Ras-induced ROS. There are several lines of evidence supporting that FoxO inhibition is important for Akt-induced ROS accumulation. As it was mentioned above, PKB/Akt kinase is constitutively active in E1A+Ras-transformed cells due to expression of cHa-ras oncogene 4.The study has revealed that initially sodium butyrate has stimulating effect on FoxO1, but by prolonged exposure it causes degradation of FoxO through additional phosphorylation of PKB/Akt. This initial activation of FoxO1 can be presumably explained by a slight increase in ROS-level, triggering expression of the scavengers of ROS. The PCR results are also support this assumption. In some researches MnSOD was shown to be regulated directly through FoxO3a Regulation of FoxO stability takes place through phosphorylation of FoxO by PKB/Akt kinase. PKB/Akt is a key mediator of Ras-induced ROS 5.To summarize, our data show that NaB regulates FoxO transcription factors on the post-transcriptional level, in time-dependent manner, activating FoxOs and triggering their nuclear translocation after short-term treatment. But the general pattern of FoxO activation is being eroded by concomitant activation of PKB/Akt, which induces FoxO degradation at longer treatment. There are several lines of evidence supporting that FoxO inhibition is important for Akt-induced ROS accumulation and senescence Click here for additional data file.


Neuropilin-1 (Nrp1) mRNA was upregulated in layer 5 pyramidal neurons in the motor cortex 14 days after SCI, when the pruning occurred. Nrp1 knockdown using adeno-associated virus (AAV) vector encoding Nrp1 shRNA in the hindlimb motor area impaired the pruning of collaterals after SCI. Nrp1 knockout by injecting AAV vector encoding Cre recombinase into Nrp1 floxed mice also suppressed axonal pruning. Propriospinal neurons, interneurons that connect CST and motoneurons, expressed Semaphorin 3A (Sema3A), the ligand of Nrp1. Furthermore, the genetic deletion of Nrp1 specifically in the hindlimb motor area suppressed the recovery of skilled movement at 21 and 28 days after SCI. The present findings demonstrate that the pruning of collaterals mediated by Nrp1 is required for motor recovery after SCI, and suggest that refinement of the neuronal network facilitates motor recovery.Following incomplete spinal cord injury (SCI), reorganization of the corticospinal tract (CST) contributes to spontaneous motor recovery. Axotomized CST fibers form collaterals and make synapses with interneurons, followed by pruning of excess fibers. Although axonal pruning is involved in refinement of neural circuits, its molecular mechanisms and functional roles remain poorly understood. To address these questions, we performed dorsal hemisections of mouse thoracic spinal cord. We observed that  Recent studies suggest that motor recovery after SCI is attributable to the reorganization of damaged or undamaged descending pathways3, particularly the corticospinal tract (CST), which regulates voluntary movement4. In the course of CST reorganization, axotomized CST fibers form collaterals and make synapses with interneurons, such as propriospinal neurons above the injured site to construct compensatory neural pathways for bypassing the lesion8. Subsequently, excess collaterals are eliminated, a process called axonal pruning8. Although many studies have shown that axonal sprouting is beneficial for motor recovery9, the functional significance of pruning in motor recovery after SCI remains poorly understood. Pruning is widely observed in the developing brain, and is considered essential for activity-dependent elaboration of neural circuits11. Therefore, we investigated if axonal pruning was also necessary for motor recovery after SCI.While complete spinal cord injury (SCI) often leads to permanent motor, sensory, and autonomic disorders, partial recovery of motor function is occasionally observed after incomplete SCIHere, we identified Neuropilin-1 (Nrp1) as a molecule responsible for axonal pruning. We further revealed that the pruning of collaterals was required for motor recovery after SCI. These findings may lead to the development of novel treatment for SCI. Specifically, the present study suggests that promoting the pruning of collaterals could facilitate motor recovery after SCI through the refinement of compensatory neural circuits.n = 12). As sham controls, we conducted only laminectomy at T8 of mice  . We confirmed its knockdown efficiency both in vitro and in vivo by transfection and infection of Nrp1 shRNA, respectively , in which Cre-mediated recombination induced site-specific and neuron-specific knockout of Nrp119, and the expression of tdTomato. Then, we evaluated the number of tdTomato-labeled collaterals at 10 days and 28 days after SCI according to the same time course with knockdown experiments . Nrp1 is a member of the semaphorin-plexin family that regulates neural circuit formation21. Nrp1 is suggested to be associated with axonal pruning in the developing hippocampus14. These findings prompted us to investigate the role of Nrp1 in the pruning of collaterals. As a result, we found that suppression and depletion of Nrp1 expression in the motor cortex suppressed the pruning of collaterals , in which exon 2 of the Nrp1 allele was flanked by loxP sequences bilaterally18, and Ai14 female mice 26Sortm14(CAG-tdTomato)Hze/J from the Jackson Laboratory) were used in this study. All experimental procedures were approved by the Institutional Ethics Committee of Osaka University and complied with the Osaka University Medical School Guidelines for the Care and Use of Laboratory Animals.Adult C57BL/6J female mice , Nrp1 floxed female mice -Nrp112. Briefly, mice were anesthetized with a mixture of 0.5 mg/mL Vetorphale (Meiji Seika Pharma), 0.4 mg/mL Dormicum (Roche), and 0.03 mg/mL Domitor (Orion Pharma). Thoracic level 8 (T8) laminectomy was performed, and a dorsal hemisection of the spinal cord at a depth of 1.0 mm was conducted with a surgical microknife.The surgical procedures were slightly modified from a previous study31. For the rotarod test, animals were placed on a rotating rod (diameter 30 mm) that accelerated from 0 to 50 r.p.m. within 5 min. Mice were trained three times a day for 3 days before SCI. The total time was recorded until the mouse fell off the rod or gripped and spun around two times as described previously32. The baseline value was scored as the mean of three trials 1 day before SCI (third training session).The rotarod was used to assess motor recovery in rodents after SCI27. The animals were placed on a 1-m runway comprising randomly spaced metal rungs. Foot faults for each hindlimb were counted during continuous steps. The setup of rungs was changed every week to rule out learning effects. The baseline value was assessed 1 day before SCI.The ladder walk test was used to assess skilled walking, precise hindlimb placing, and interlimb coordination33. Briefly, Nrp1 and control shRNAs, tRFP, Cre recombinase, and EGFP cDNAs were cloned into plasmid pAAV-MCS (Stratagene). AAV 293cells were cultured in sixty 10-cm diameter dishes in Dulbecco’s modified Eagle medium  with 10% fetal bovine serum . After cells were 70–80% confluent, 300 µg of Rep/Cap plasmid, 600 µg of Adenovirus-helper plasmid, and 300 µg of pAAV-MCS were transfected into cells. Five days after transfection, cells were washed in phosphate-buffered saline (PBS) and the cell suspensions were collected. After five freeze/thaw cycles of the cell suspensions, Benzonase Nuclease (Sigma-Aldrich) was added into the suspensions and centrifuged at 8000 rpm, 4 °C for 40 min. Ammonium sulfate was then added into the supernatants, which were centrifuged at 8000 rpm, 4 °C for 40 min, and filtered with 0.45 µm filter (Merck) to collect the fraction containing AAV. After establishing a gradient of AAV solution by OptiPrep (Axis-Shield), the gradient was ultracentrifuged at 36,000 rpm, 16 °C for 16 h. AAV solution was then concentrated with PBS by Amicon ultra-15 (Merck) until the volume was ~250 μL.The procedures were slightly modified from a previous study12. Briefly, the animals were anesthetized and placed in a stereotaxic frame. The corresponding skull area was exposed. BDA was injected at 10 days before the animals were perfused. 0.8 µL/site BDA was bilaterally infused into the hindlimb motor area of wild-type mice . AAV1-H1-shRNA-CMV-tRFP or AAV9-CAMKII-Cre was injected at 28 days before the animals were perfused. 0.4 µL/site AAV1-H1-shRNA-CMV-tRFP was infused into the right hindlimb motor area of wild-type mice . 0.4 µL/site AAV9-CAMKII-Cre was bilaterally infused into the hindlimb motor area of Ai14 mice or Nrp1 floxed mice crossed with Ai14 mice . These tracers were infused using a glass capillary attached to a microsyringe (Ito). After injection, the pipette tip was left at the injection site for 2 min before withdrawal to prevent tracer backflow.The descending CST fibers were labeled with the anterograde tracer, biotinylated dextran amine , AAV1-H1-shRNA-CMV-tRFP, or AAV9-CAMKII-Cre. Tracer injection procedures were performed as described previouslyTo genetically deplete Nrp1 expression in neurons, 0.4 µL/site AAV9-CAMKII-Cre or AAV9-CAMKII-EGFP (cotrol) was bilaterally infused into the hindlimb motor area of Nrp1 floxed mice  at 1 day after SCI.34. Briefly, the animals were anesthetized and placed in a stereotaxic frame. To label the LPSNs and SPSNs, 0.6 µL/site or 0.4 µL/site Fluoro-Ruby was bilaterally infused into the spinal cords at vertebral level T12 or C7 , respectively.The long propriospinal neurons (LPSNs) and short propriospinal neurons (SPSNs) were labeled with the retrograde tracer Fluoro-Ruby  2 weeks before the animals were perfused. Tracer injection procedures were modified slightly from a previous study35 was used as the primary antibody. The sections were subsequently washed three times with PBS, followed by incubation with a secondary antibody for 1 h at room temperature in the dark. Donkey-anti-goat IgG Alexa 488  was used as the secondary antibody. For BDA labeling, the sections were immersed three times in PBS and incubated in 0.3% Triton X-100/PBS for 2 h, followed by incubation with Alexa Fluor 488-conjugated streptavidin  for 1 h at room temperature. All images were acquired with a confocal laser-scanning microscope (Olympus FV1200).The animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Following perfusion, the brain and spinal cord were postfixed in 4% PFA at 4 °C overnight and subsequently transferred to 30% sucrose in PBS. The tissues were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek). The spinal cords were cut into 30-µm-thick coronal sections using a cryostat and placed on MAS-coated glass slides (Matsunami). For immunohistochemistry, the sections were immersed three times in PBS and blocked with 5% bovine serum albumin/0.1% Triton X-100/10% normal donkey serum/PBS for 1 h. Subsequently, the sections were incubated with primary antibody overnight at 4 °C. Goat anti-semaphorin-3A (SEMA3A) 36. The primer sequences for Nrp1 probe were as follows: forward, 5′-AATGAATGGCTCCAAGTG GAC-3′; reverse, 5′-GATCCAGGGTCTTAAGCACAT-3′. All images were acquired with a fluorescence microscope (Olympus IX83).In situ hybridization and histology were performed as previously described37. After electroporation using a Nucleofector kit (Lonza), the cells were immediately mixed with 500 μL of DMEM/F-12 containing 10% FBS, and the cells were plated onto poly-l-lysine-coated dishes and cultured at 37 °C in the presence of 5% CO2. The medium was exchanged with DMEM/F-12 (Thermo Fisher Scientific) containing 2% B27 supplement (Thermo Fisher Scientific) 12 h later.E16 mouse cortices were digested in 0.25% trypsin (Thermo Fisher Scientific) supplemented with DNase  for 15 min at 37 °C. Cells were dissociated in DMEM/F12 (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific). Cells were then washed and resuspended in 100 μL of Mouse Neuron Nucleofector Solution (Lonza) containing 3 μg of Nrp1 shRNA or control shRNA (points-mutated Nrp1 shRNA) vector. The shRNA sequences were as follows: Nrp1, 5′-AGAGAAGCCAACCATTATA-3′; control, 5′-AGAGAAGACAGCCCTTATA-3′Fresh brains were immediately frozen on dry ice 28 days after bilateral injections of AAV vector. The tissues were then cut into 10 μm-thick sections and mounted on glass slides with Poly(p-phenylene sulfide) (PPS) foil (Leica Microsystems). After toluidine blue staining, the motor area was dissected bilaterally with an LMD 7000 (Leica Microsystems) and transferred into microcentrifuge tube caps filled with 50 μL of TRIzol reagent (Thermo Fisher Scientific).Nrp1 were as described previously38. Real-time polymerase chain reaction (PCR) analysis was conducted using SYBR Green Master Mix (Thermo Fisher Scientific). The expression of Nrp1 was normalized to Mrpl3238. The reaction and subsequent quantification were conducted using QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems).Cells and the motor cortex of mice were dissected and homogenized in TRIzol reagent (Thermo Fisher Scientific). Isolated total RNA was purified with an RNeasy Micro kit (QIAGEN). The complementary DNA (cDNA) was synthesized with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Primer sequences for 6. Briefly, to assess the number of sprouted collaterals from hindlimb CST, collaterals entering the gray matter from the dorsal funiculus in the cervical cord (segments C4 to C7) were counted in 30 transverse sections. We then divided the total number of collaterals by the number of fibers in dorsal CST at C3 level to normalize tracing differences among individual animals. The number of fibers was determined with ImageJ software (US NIH).The quantification method was slightly modified from a previous study34. Briefly, to calculate the ratio of long and short propriospinal neurons expressing Sema3A, retrogradely-labeled neurons located in C4 to C7 were counted until the number of cells reached 30 per mouse. Results were expressed as a ratio of the number of double-labeled neurons in 30 assessed neurons.The quantification method was slightly modified from a previous studyt-tests, Welch’s t-test, one-way ANOVA followed by Tukey-Kramer tests, and two-way ANOVA followed by Bonferroni tests. p values of <0.05 were considered statistically significant.All data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). Results were analyzed using Student’s


Drosophila melanogaster, to evaluate the dynamics of FOXO gene targeting during aging. Intriguingly, the number of FOXO-bound genes dramatically decreases with age (from 2617 to 224). Consistent to the reduction of FOXO binding activity, many genes targeted by FOXO in young flies are transcriptionally altered with age, either up-regulated (FOXO-repressing genes) or down-regulated (FOXO-activating genes) in adult head tissue. In addition, we show that many FOXO-bound genes in wild-type flies are unique from those in insulin receptor substrate chico mutants. Distinct from chico mutants, FOXO targets specific cellular processes  and signaling pathways  in young wild-type female flies. FOXO targeting on these pathways decreases with age. Interestingly, FOXO targets in aged flies are enriched in cellular processes like chromatin organization and nucleosome assembly. Furthermore, FOXO binding to core histone genes is well maintained at aged flies. Together, our findings provide new insights into dynamic FOXO targeting under normal aging and highlight the diverse and understudied regulatory mechanisms for FOXO transcriptional activity.FOXO transcription factors have long been associated with longevity control and tissue homeostasis. Although the transcriptional regulation of FOXO have been previously characterized , how normal aging impacts the transcriptional activity of FOXO is poorly understood. Here, we conducted a chromatin immunoprecipitation sequencing (ChIP-Seq) analysis in both young (2-week-old) and aged (5-week-old) wild-type female fruit flies,  Caenorhabditis elegans where insulin-like receptor mutant daf-2 extends lifespan via FOXO homolog daf-16 -dependent phosphorylation of mammalian FOXO4 and its nuclear translocation. FOXO proteins can also be activated and phosphorylated by mammalian Sterile 20-like kinase 1 (MST1), to extend lifespan . In resposophila .Drosophila. Intriguingly, we found that the number of FOXO-bound regions sharply decrease with age. The age-related decrease in FOXO binding is correlated with either the transcriptional activation of FOXO-repressing genes, or the downregulation of FOXO-activating genes during normal aging. Furthermore, we observed strong FOXO nuclear localization in well-fed wild-type flies, while FOXO targets distinct sets of genes between wild-type and insulin mutants. Taken together, our findings provide new evidence linking age-dependent FOXO transcriptional activity to its role in longevity control and tissue maintenance.Although insulin/IGF signaling is well-known aging regulators, how insulin/IGF signaling is altered during normal aging remains largely unclear. It is generally believed that insulin/IGF signaling declines with age. This is primarily based on age-dependent decrease in the expression of FOXO target genes . HoweverDrosophila FOXO activity changes with aging, we first performed immunofluorescent staining using a polyclonal antibody against Drosophila FOXO to monitor the FOXO nuclear localization in female wild-type flies (Ryw) at two different ages, 2-week-old (young flies) and 5-week-old (aged flies). Female flies were used in the present study is because most of the previous Drosophila aging studies used females and large amount of genomic data are available for female flies. To capture molecular events associated with early onset aging phenotypes, 5-week-old flies was used. This is because the epigenetic and transcriptional changes have been previously observed in 5-week-old flies (To examine whether ld flies . Besidesld flies .Ryw), where insulin/IGF signaling is presumably active (Oregon R (OreR)  . FOXO prR (OreR) . CompareR (OreR) . In addiR (OreR) . Thus, tR (OreR) .Drosophila reference genome  and aged (5-week) female wild-type flies. Using Illumina high-throughput sequencing, we obtained a total of 261 million reads from FOXO ChIP and input DNA samples at two ages. On average, 90.08% of unique reads were mapped to annotated e genome . Intrigue genome . There we genome . About 1e genome , while te genome . The rede genome .Atg3, Atg17, Tor, wdb, Pten), which is consistent to previous known functions of FOXO in autophagy and tissue homeostasis , while the FOXO enrichment at two newly identified target genes, jim (C2H2 zinc finger transcription factor) and dlg1 (a key factor for the formation of septate junctions and synaptic junctions), decreased dramatically at advance ages (from 80∼90-fold to 3∼8-fold) (The age-dependent changes in FOXO binding activity were verified by quantitative PCR (ChIP-qPCR). The FOXO binding to the promoters of two known target genes, insulin receptor analysis . FOXO shnce ages . On the with age . We also∼8-fold) . Thus, oDrosophila tissues, such as fat body and head tissue. Out of 2447 FOXO target genes (uniquely bound by FOXO at young ages), 408 of them were differentially expressed in aging fat body   , while 8gulated) . Interesgulated) , 2016. Ggulated) . On the gulated) .foxo null mutants (c431foxo), a site-specific deletion mutant generated by CRISPR/Cas9 , a GAGA-factor that also found in previous ChIP-Seq data from C. elegans  was detected with low significance (p < 10-4)  . TogetheDrosophila aging. Intriguingly, genome-wide FOXO-binding underwent an immense reduction during normal aging. Consistently, genes that are negatively regulated by FOXO showed an increased expression with age, whereas the FOXO-activating genes were downregulated in aged flies. Thus, age-associated decrease in FOXO binding is tightly linked to the transcriptional alterations of FOXO target genes in aged flies. In addition, we found that FOXO targets distinct sets of genes between wild-type and insulin/IGF mutants across species, suggesting a conserved insulin/IGF-independent transcriptional regulation by FOXO transcription factors.As a key player in longevity control, FOXO transcription factors and their direct targets have been well characterized in many model systems . HoweverC. elegans, FoxA/PHA-4 exhibits differential binding patterns at different stages of development to regulate organogenesis  and signaling pathways  are uniquely enriched in young wild-type, but not in  pathway . MAPK si pathway , and is utophagy . EGFR reutophagy . Our ChIutophagy . In adulutophagy . Thus, HC. elegans  was used as a control genotype for foxo mutants in RNA-Seq analysis. Female flies were collected and sorted 1–2 days after eclosion. To age flies, vials contained 25–30 flies were transferred to fresh food every 3 days.Flies were maintained at 25°C with 12 h light/dark cycle, 60% humidity on agar-based diet with 0.8% cornmeal, 10% sugar, and 2.5% yeast. wR flies  were usefoxo deletion lines were generated through CRISPR/Cas9 mutagenesis as previously described , forty heads from female flies were dissected and homogenized in a 1.5 ml tube containing 1 ml of Trizol Reagent . Three biological replicates were performed for each age and genotype. Total RNA was extracted following manufacturer instruction. TURBO DNA-free kit was used to remove genomic DNA contamination . About 1 μg of total RNA was used for sequencing library preparation. PolyA-tailed RNAs were enriched by NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs (NEB), Ipswich, MA, United States Catalog number: E7490S). RNA-Seq library was prepared using NEBNext Ultra RNA library Prep Kit for Illumina . The libraries were pooled together in equimolar amounts to a final 2 nM concentration. The normalized libraries were denatured with 0.1 M NaOH (Sigma) and sequenced on the Illumina Miseq or Hiseq 2500 platforms  .For transcriptomic analysis on the head tissues of aged flies and Chromatin immunoprecipitation (ChIP) protocol was performed and modified from DNA size selection and library prep were done using NEBNext Ultra II DNA library prep kit and indexed using NEBNext multiplex oligos for Illumina (Primer set 1) . DNA from either ChIP or input samples was mixed with AMPure XP beads  to select for a final library size ranging from 280 to 345 bp. Following are the bioanalyzer results from four ChIP samples: Young ChIP DNA_1: 345.7 ± 38.4 bp; Young ChIP DNA_2: 306.3 ± 16.4 bp; Aged ChIP DNA_1: 305.8 ± 19.5 bp; Aged ChIP DNA_2: 281.8 ± 14.7 bp. Samples were then diluted to a final concentration of 2 nM for Illumina sequencing on Illumina HiSeq 3000  .2foldchange| > 1, adj p < 0.01).RNA-Seq reads were first mapped to the reference genome Dm6 with STAR_2.5.3a by default parameter. The read counts for each gene were calculated by HTSeq-0.5.4e. The count files were used as inputs to R package DESeq for normalization. The differential expression genes were computed based on normalized counts from three biological replicates  was set for a peak detection cutoff of 0.05 and did not build the shifting model. The MFOLD for the model was set from 10 to 50 to detect fold-enrichment. Peak-calling was set to identify peaks 300 bp in length, and no peaks could exceed 10 Kb in size. After MACS2 peak identification, peak regions were expanded 2 kb (1 kb upstream and 1 kb downstream) and assigned to nearby and overlapping genes using BEDTools/intersect4 with Dm6.16 genome annotation file . All non-protein coding identified targets were removed from the data set manually based on annotation symbol.For ChIP-Seq, raw FASTQ reads were merged using mergePeaks (Homer suite) then uploaded into Galaxy5. For cross species comparisons, gene ID’s were converted to fly ID’s using DIOPT6. Genes that were the best possible match for each ortholog were selected for gene list comparison.Venn diagram were created using the Bioinformatics and Evolutionary Genomics Venn calculator at UgentQuantitative PCR was run on QuantStudio 3  with above ChIP and input library samples. PCR reaction was conducted using PowerUp SYBR Green Master Mix . FOXO binding enrichment was determined based on the fold-change between ChIP samples vs. Input samples. The FOXO binding to Actin5C locus was used as a negative control. Two biological and two technical replicates were performed for each age. Primers are listed in 7, String8 and DAVID9. All three methods were used to obtain a more complete picture of shared regulation between datasets. KEGG pathway maps were obtained through KEGG Pathway10.Pathway and gene ontology analysis was conducted using PantherfindMotifsGenome script11 to compare peak regions with Dm6.01 FASTA data from UCSC.Motif analysis was conducted using Homer’s Drosophila chico heterozygotes), GSE15567 (Daf-16 ChIP-Seq of wild-type C. elegans), Daf-16 ChIP-Seq of C. elegans Daf-2 mutants from ChIP-Seq datasets: GSE44686 (FOXO ChIP-Seq of Drosophila aging fat body), GSE81100 (Drosophila aging head tissue). The expression data from the original studies were used in the present study. Since no direct comparison was performed between our RNA-Seq and previously published datasets, there is no normalization issue between different sequencing platforms and bioinformatics packages.RNA-Seq datasets: GSE62580 (Flies were anesthetized with FlyNap and dissected in 1X PBS. Fly tissues (muscle or fat body) were then fixed in 4% paraformaldehyde for 20 minutes at room temperature. Tissue was washed in 1X PBST (0.1% Triton X) and blocked with 5% normal goat serum (NGS) for 1 h at room temperature. Fly tissues were stained with anti-FOXO antibody in 1X PBST at a dilution of 1:1000 for 16 h at 4°C on a rotator. Tissues were placed in secondary anti-body goat-anti-rabbit conjugate Alexa Fluor 488  at a Dilution of 1:250 and kept in the dark at room temperature for 2 h. The nucleus was stained using SlowFade with DAPI. Images were captured using an epifluorescence-equipped BX51WI microscope . Image deconvolution was conducted using CellSens software , and compiled using ImageJ Fiji.t-test or one-way ANOVA was performed using Dunnett’s test for multiple comparison.GraphPad Prism  was used for statistical analysis and to generate Boxplot. To compare the mean value of treatment groups versus that of control, either student AB, XW, NL, and HB conceived and designed the experiments and wrote the manuscript. AB and XW performed the experiments. AB, XW, MT, NL, and HB analyzed the data. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Virtual reality (VR) and augmented reality (AR) are two novel graphics immersive techniques (GIT) that, in the last decade, have been attracting the attention of many researchers, especially in psychological research. VR can provide 3D real-life synthetic environments in which controllers allow human interaction. AR overlays synthetic elements to the real world and the human gaze to target allow hand gesture to act with synthetic elements. Both techniques are providing more ecologically environments than traditional methods, and most of the previous researches, on one side, have more focused on the use of VR for treatment and assessment showing positive effectiveness results. On the other, AR has been proving for the treatment of specific disorders but there are no studies that investigated the feasibility and effectiveness of AR in the neuropsychological assessment. Starting from these premises, the present study aimed to compare the performance and sense of presence using both techniques during an ecological task, such as cooking. The study included 50 cognitively healthy subjects. The cooking task consisted of four levels that increased in difficulty. As the level increased, additional activities appeared. The order of presentation of each exposure condition (AR and VR) was counterbalanced for each participant. The VR-cooking task has been performed through “HTC/VIVE” and AR through “Microsoft HoloLens.” Furthermore, the study recorded and compared the psychophysiological changes [heart rate and skin conductance response (SCR)] during the cooking task in both conditions. To measure the sense of presence occurring during the two exposure conditions, subjects completed the Slater-Usoh-Steed Questionnaire (SUSQ) and the ITC-Sense of Presence Inventory (ITC-SOPI) immediately after each condition. The behavioral results showed that times are always lower in VR than in AR, increasing constantly in accordance with the difficulty of the tasks. Regarding physiological responses, the findings showed that AR condition produced more individual excitement and activation than VR. Finally, VR was able to produce higher levels of sense of presence than AR condition. The overall results support that VR currently represents the GIT with greater usability and feasibility compared to AR, probably due to the differences in the human–computer interaction between the two techniques. Virtual reality (VR) and augmented reality (AR) are two novel graphics immersive techniques (GIT) that, in the last decade, have been attracting the attention of many researchers, especially in the fields of psychology and education . On one According to this, at present, both techniques are providing advantages along with traditional scientific research procedures, providing accurate real-simulated stimuli control and behavior measurement of reactions times and scores and allowing researcher to address issues that would simply be difficult to pose in natural environments . In psycIn order to improve similarity between tests and real-life activities, several VR environments have been developed such as virtual mall/supermarket , and claDespite the opportunities, that VR has been providing in psychological assessment, to our knowledge no previous studies have investigated the differences in behavioral responses to ecological tasks presented through AR compared to other methods – particularly VR.Finally, both systems are also compatible with other neuroscientific tools such as wrist devices able to measure changes in electrodermal activity (EDA) and heart rate variability (HRV) . EDA andStarting from these premises, the first aim of this study was to analyze and compare behavioral and physiological data collected before, during and after performing a cooking task in VR and AR environments. Second, the study aimed to determine the degree of presence, or the feeling of “being there,” that produced VR through the “HTC Vive” and AR through “Microsoft HoloLens.”The experimental sample included 50 healthy individuals . Participants were recruited through local advertisement among college students and workers of the Polytechnic University of Valencia. The mean age was 25.96 ± 6.51. To be included in the study, participants were required to have a score higher than 24 in the “Mini-Mental State Examination” (MMSE) . Before Before the experimental session, the following questionnaires were administered to each participant:•Attentional Control Scale (ACS)  is used •Barratt Impulsiveness Scale (BIS-11)  is a mea•Cognitive Flexibility Scale (CFS)  consistsFurthermore, participants completed a total of 5 standard tasks (ST): Dot Probe Task (DOT) version published by After each exposure condition, the following presence questionnaires were administered to each participant:•post hoc test consists of three questions that are evaluated on a scale of 7 points. The items evaluate the sensation of being in the environment, the extent to which the medium becomes the dominant reality and the magnitude in which it is remembered as a “place.”Slater-Usoh-Steed Questionnaire (SUSQ) : This po•ITC-SOPI : This teDescriptive data on presence are reported in At the beginning and during the experimental session, skin conductance response (SCR) and HRV were recorded to obtain subjects’ physiological responses to VR and AR cooking task. SCR and HRV are considered indexes of arousal responses . The phy1) and through two hand controllers, and the augmented cooking task using Microsoft HoloLens2. The AR experience was performed in a real kitchen in which the augmented synthetic objects appeared in front of the subject according to the subjective human gaze. The interaction in AR was ensured by various sensors integrated into the headband, like cameras that, through the human gaze to target, allow the real hands’ interaction with the synthetic elements.The virtual and augmented system was developed using Unity 5.5.1f1 software, applying c# programing language using the Visual Studio tool. Participants performed the virtual cooking task wearing an HMD device , one for each technology, in order to learn the main body movements and hands’ interactions useful to perform the virtual cooking task. The tutorial consisted of a simulated task, similar to the virtual cooking task. In both conditions, body movements were real in the physical space and hands’ interaction in the VR was performed through the use of two controllers and in AR, participant interacted with objects with their own hands. Participants could train for as long as necessary, according to the needs of each one. When they felt confident about body and hand movements and interactions, a button pulsed to start the virtual cooking task.The virtual cooking task consisted of four levels of difficulty, involving the abilities to pay attention, planning, and shifting. All were based on cooking a series of food in a set time, avoiding burning (in which the ingredient was in the fire more than the set time) or cooling them (switch off the glass-ceramic switch or remove the food from the pan during cooking). As the level increased, additional activities appeared . In the Participants exceeded the following level when they have cooked all the foods, completing the level. Before each level, instructions, explaining what activities participants had to be carried out, what time they had to do it, times for each food and remembering to cook foods without burning and letting them cool, have been showed .The order of presentation of each exposure condition (AR and VR) was counterbalanced for each participant. Before the beginning of the experiment, participants were administered the MMSE and standard questionnaires  and tasks . Once this first phase was completed, we recorded 3-min of EDA and HRV baseline, asking to participants to stay completely relaxed during the recording. Once the physiological baseline was recorded, the experimental session started, and EDA and HRV were continuously recorded until the end of the experiment. To measure the sense of presence occurring during the two exposure conditions, subjects completed the SUSQ and the ITC-SOPI immediately after each condition.The analyses were performed using SPSS version 22.0  for PC. The biosignals’ processing and computation were analyzed using MATLAB and Ledalab programs. First, we verified the assumptions of normality applying Kolmogorov–Smirnov and the internal consistency of the scales was assessed via Cronbach’s alpha.Second, it has been verified the normal cognitive functioning and the physiological health  of the subjects.t-tests were conducted to compare behavioral, physiological data (SCR and HR), and sense of presence responses in AR and VR conditions. The level of significance was set at α = 0.05.Next, four paired p > 0.05) and the internal consistency of the self-report scales has been measured .The assumption of normality was confirmed  at baseline and during the tasks are in the normal range of 60–100 beats/min. Also, SDNN values indicate that participants are not in danger of suffering from any cardiac episode since the data is greater than 100 ms, while the rMSSD are also in the normal range (greater than 25 ms)  Table 4Table 4.Regarding performance, t-test was conducted to compare behavioral responses in AR and VR conditions. There were significant differences in the scores for the total four levels’ time between AR  and VR  conditions; t(49) = 7.75, p = 0.00, as well in the total time of level 1 in AR  and VR  conditions; t(49) = 3.08, p = 0.00 and in the level 2 in AR  and VR  conditions; t(49) = −3.08, p = 0.00 , VR ; t(49) = 3.08 p = 0.00], level 2  and level 3 . Finally, significant differences on burning times have been found between conditions at level 2 , level 3 , and level 4   and -post-task  conditions; t(49) = −5.16, p = 0.00, as well for VR-pre  and -post-tasks  conditions; t(49) = −4.22. Another significant difference in the scores for EDA for AR-post  and VR-post-task  conditions; t(49) = −2.95, p = 0.00 has been found. Finally, there was a significant difference in the scores for number of peaks in the first level task between AR  and VR  conditions; t(49) = 2.01, p = 0.05  and -post  task; t(49) = −1.97, p = 0.05  and VR  conditions; p = 0.00. The ITC-SOPI showed significant differences between AR and VR in the four dimension of presence: SP ; E ; EV ; and NE  (A paired = 0.51)] .The first aim of this study was to analyze and compare behavioral and physiological data collected before, during and after performing the cooking task in VR and AR environments. The results on the behavioral data comparison showed that times are always lower in VR than AR, both for the times’ means of the four levels and in the specific levels. This may be because the interaction with VR is usually simpler; however, the VR and AR descriptive data showed that AR levels times decrease, while in VR levels increase . The resCoherent with the behavioral data are the physiological results, showing that both conditions produced individual activation with higher values in AR than VR . Higher Regarding the second aim on the sense of presence, scores between conditions showed that VR always produces a higher sense of presence than AR . SpecifiAlthough the results are interesting for their possible applications in neuropsychological assessment, our study has some limitations that could affect the generalizability of the results or that may have influenced the findings. The main issues are related to the small sample size and the specific sample of healthy subjects included in this study. At the technological level, FOV and user interaction differences between the two technological systems can have generated the variability of the test scores. Futures studied are needed to investigate differences in behavioral responses comparing clinical populations and healthy subjects, as well as comparing AR and VR with other condition, such as the real condition. Furthermore, to overpass possible differences between the technological systems results, in the test design it would be important to focus more on the accuracy of the responses rather than in reaction times and also implement an individual baseline on the same or another measure using the different systems before the experimental task . In thisThis research represents a step toward better understanding the differences between AR and VR and opens up several new venues for future research works. In particular, we conclude that future test designs took into consideration some changes in the experimental design – adding an individual technological baseline and considering also the responses accuracy – and in the self-report scale to measure presence in AR.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Universitat Politècnica de València (ID: P3_02_05_18). The patients/participants provided their written informed consent to participate in this study.IC and MA designed and directed the project. IC developed the theoretical framework and wrote the manuscript. CB carried out the experiment. IC and CB analyzed data. MA reviewed the final version of the manuscript. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.