1

Focused on Focusing and Other Mysterious Stories

Pier Giorgio Righetti

Prof. Emeritus, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20131 Milano, Italy. E-mail: [email protected]

Figure 1. Wanted? Not any more, I suspect!

FAMILY AFFAIRS

I was born in April 1941, in the second year of World War II, almost one year after that disgraceful tenth of June 1940, when Mussolini, from the balcony of Palazzo Venezia in Rome, entered the war as an ally of Hitler, paving the way to total disaster for the entire Italian population. The first one occurred already in November of the same year when this genius of war, second only to Napoleon or Julius Caesar (so he thought!) declared war to 2

Greece claiming that we would have broken the back bones to this population in no time at all. In fact, after barely a week, at the border between Albany and Greece, in a desolate village called Perati, the Greek army broke Mussolini’s back and cut to pieces the most glorious and well-trained Division Julia, the glory of the alpine troops. His second ”Beau Geste” was the invasion and total dismembering of Yugoslavia, which started on 8th of April 1941 and was over in a few days. In fact, he was no winner in this miserable and treacherous affair: It was Hitler who, after signing a peace treaty with the Yugoslavian newly-appointed government, on a vile surprise air attack destroyed completely Beograd and took over the nation. Mussolini simply moved in and claimed part of the spoils, annexing Croatia and few extra territories. Two weeks after this Mussolini’s superb exploit I was born in Forlì, close to the Adriatic Sea shore. Things began to rapidly deteriorate. Towards the end of 1941, Mussolini joined again Hitler’s army in the invasion of Russia and sent one hundred thousand of our best-trained alpine troops to fight on the steppes of the river Don (there were no mountains there, why sending mountain troops?). These soldiers were poorly equipped and had no proper clothing: in the winter of 1942, in the icy steppes reaching -30 to -50°C, most of them died as frozen statues planted in the snow. Less than 10% managed to return home. Curiously, even in such hard war days, my parents kept generating children (not the midnight children as per the famous novel by Salman Rushdie, born to be free in the evening of India’s independence) bound for war misery. Seven of us were born in rapid succession. Years after, when thinking of it, it did not look like a wise choice, so why did they do it? For one reason, Mussolini had been encouraging large families, giving prizes to the larger ones. As an additional reason, both my parents were strictly educated in the catholic religion, which preached sex only for the purpose of generating new life, pure sex for sex being a mortal sin. One has to add to it that, in those war days, there were no newspapers worth reading (all the press being thoroughly controlled by the regime), no radio worth listening to (again, due to tight regime control) and, of course, no TV. As odd as it looks, my mother had a miscarriage at the first pregnancy, and she lost the child, called Nazareno. Would you believe it? Although she had to take care of another seven children (and she did it heroically) she still kept complaining the loss of poor Nazareno. Why should it be so? I guess that, due her strict religious upbringing, she was mourning the loss of Jesus Christ (in Italy nicknamed Nazareno, he who was born in Nazareth).

Things kept getting worse, of course, as war progressed. Towards the end of 1943, as the US army, that has crossed over from Sicily into Calabria, began the move northbound 3 while weeding away the Nazi’s army from the South of the Stivale (Italy is shaped as a boot), the entire nation began to disintegrate. As they reached the Gothic defense line (similar to the Maginot line in France), the Italian army began to melt away. Our family moved from Forlì running away in front of the front of the US army (my father was an officer in Mussolini’s army). We ultimately landed in the suburbs of Vigevano and got a lodging in wooden barracks, no heating, no running water (except from the roof when it was raining). We simply did no have enough food and my early memories were the constant fights with my brothers for breadcrumbs at the end of our poor meals, even scraping them from the floor.

A FIRST ENCOUNTER WITH FOOD CHEMISTRY

When I was six years old, my father got a job as a schoolteacher in a rural village, where we moved to live in. We had lodging in a medieval farm, which in those days was built like a four-sided stronghold, surrounded by a large plaza where, at the end of a work day, all carriages, horses, animals and agricultural machines would be stored and the entrance gates closed at night. It might sound strange to still keep these medieval customs in modern times, but it should be remembered that Italy had a disastrous outcome in World War II, since we not only lost the war but we were criminals too as we had sided with the Nazis. The entire nation was poverty stricken and plenty of criminals and desperados were on the loose to rob anything they could get their hands on. At least we had a handkerchief of land to till, so my mother kept a small garden to grow vegetables, tomatoes, cabbages, salads, pumpkins and the like, enough to get some extra food for survival. There I had my first (traumatic) experience with food chemistry. Come October and November, the farmers used to sacrifice the biggest pigs, so as to produce salami and prosciuttos and fat for the coming cold winter. So, one day I saw a gallows (not like those erected in US courthouses in eighteen hundred, such as the one you can see in the courthouse of Tombstone, AZ, for hanging bandits) rising in the middle of the courtyard. Late in the afternoon, the farmers brought in a pig, the largest animal I had ever seen, and started hanging it up, not by the neck, like criminals, but by the hind legs. The animal was so large and fatty that they had to use pulleys to lift it up. Knowing what was coming, my mother had locked us inside the house and closed the window shades, so that we would not witness the slaughter to come. As the pig was hanging head down, the poor animal started screaming in a frightening manner, the tremendous sound bouncing along the walls of the 4 quadrilateral, thus being amplified. Somehow I managed to peep through a hole in the window shades and what I saw got printed in my retina for years afterward. A farmer approached the pig with a sharp knife and slashed its jugular vein, with blood spurting down copiously and filling a large basin held in situ by a circle of other farmers. A horrifying view for the young chap I was. Yet, come winter, we appreciated a lot the outcome. The collected blood, after clotting, had been boiled with aromatic herbs and garlic and made into sausages, called “sanguinacci” (blood sausages). Well, they were nutritious and tasty and were a blessing to survive the harsh winter. I had to witness, unfortunately, more slaughter, albeit on a smaller scale. Occasionally, as they grew up, my mother would sacrifice a chicken from our small chicken coop. She knew very well how to do it, since she grew up in a farm: wring the neck of the poor animal with a sharp stroke, plunge it for a couple of minutes in boiling water, and pluck all feathers out (a facile operation thanks to the hot water bath). I had to assist her and that did not make me happy at all. Yet, when I think that today this is done on a giant scale, killing millions of those animals, grown in miserable conditions in overcrowded cages, I think we were somewhat more merciful, since at least our chicken grew happily while pasturing freely in the fields.

THE FIRST WHIFF OF VOLATILE CHEMICALS

At the age of eight we finally moved to the town of Vigevano (35 Km south of Milano), a metropolis for us (albeit not so dreadful as the Metropolis by Fritz Lang). We were living in a house in whose courtyard stood the stables and carriages for the horses of a small firm delivering goods, parcels and furniture around town. In those days, it was cheaper to do that by horse-drawn carts than by trucks, due to the general poverty of the nation. Needless to say, swarms of flies kept colonizing our kitchen. There was this most powerful insecticide, whose acronym was DDT (1,1,1-trichloro-2,2-di(4-chlorophenyl) ethane), but that we had nicknamed “flit”. It was its onomatopoeic attribute, due to the fact that, for spraying it, we used a kind of a bicycle pump with a small reservoir containing the poison. At each stroke, a cloud of fine mist would issue exhaling this “flit” sound. We kids were instructed to go into the kitchen, close the window and spray it generously around while avoiding breathing it and closing our eyes. I guess we did it, but I still remember the acrid smell of DDT and I suspect we inhaled it no matter what. After running out of the kitchen, we could witness the miracle that, within a few minutes, all flies were laying dead on the floor. Relief only lasted for a short time, though, since as we had to aerate the kitchen with 5 clean air, the flies would occupy again the territory in no time at all. So, this was my first encounter with organic chemistry and the first intoxication too! And, for that matter, it was not the only one. Vigevano had acquired a special status in Italy and abroad as the “capitale della scarpa” (capital of shoes production) since the entire economy of the town was based on fabrication of shoes sold in the country and abroad all over Europe. The economy was booming, not so much for the workers, who still received minimal wages, but certainly for all factory owners. It became a custom all over town that, in order to raise more money, all workers in the factories would bring home plenty of partially assembled shoes, which had to be glued at home, up to late evening, by their wives. All houses had robust supplies of this strong glue, which we kids called “tenacio” (tenacious, since this glue was most powerful in binding the various parts of the shoes). When I visited the houses of these ladies, with my mother who wanted to chat with them while observing their work, I felt I was in paradise: the air was permeated by a most pleasant aromatic smell, that impregnated even furniture, clothes, everything around. I inhaled it with full lungs. Only when I went to university and started studying chemistry I learned I was inhaling xylene, the special solvent of this type of glues. I am lucky I am still alive, but years later epidemiologists, while studying the incidence of various deadly diseases, noticed that in Vigevano there was a much higher rate of deaths due to tumours, likely due to breathing in high doses over the years of the deadly xylene. I confess, this experience was a far cry from the famous song of Woody Guthrie:

Take a whiff, take a whiff, take a whiff on me

Everybody take a whiff on me

Hey-hey, baby, take a whiff on me.

As a lucky event, we managed to move from the “horse’s stables” to a house just outside the medieval enceinte surrounding the town, indeed built against the wall of the castle and next to the main entrance gate to town. In those days we had no access to the magnificent castle built by the Visconti and then by the Sforza families, since it was occupied by military troops, which we called “carristi”, i.e. a tank’s division. On a few occasions, invited by the children of these military corps, we could sneak inside and play with them by even jumping inside the tanks and feigning an attack by aiming with the turret cannons. As odd as it sounds, the tanks were housed in the horse’s stables, designed by the master architect Donato Bramante, perhaps due to the fact that the “carristi” had substituted, over 6 the years, the division of cavalry, the beloved assault division by Napoleon. Unfortunately I was too young to appreciate the architecture; all I remember was the smell of lubricants for the gears of the tanks and of petrol filling the tanks, although I do not recall having ever seen them parading through the streets on June 2nd, the day of the birth of the Republic (up to the end of World War II Italy was a monarchy, although the king was not the weak Emanuele III, but Mussolini in person). So, this was my third experience on sniffing chemistry.

Although my father had a permanent position as a schoolteacher, the salary was not enough to sustain the entire family (nine people, as you might recall). So, my mother resorted to another survival expedient: under the roof of the house (among the shallow beams) she started a chicken coop, the poor animals living in this secluded place with little light filtering through the wooden beams. Well, every now and then a disoriented chick, drawn by the dim light impinging from the roof edges, would follow it and fall down in the street. Amazed passers-by would wonder if this was a gift from heaven and would have run away with the booty, had it not been for my diligent mother rushing us down to the sidewalk to recover the prey. But we, the three musketeers (together with my elder brother Pier Luigi and the younger one Pier Paolo) lived and dreamed on the roof terrace, leaning against the castle wall and fantasizing of adventures while discovering the outer word by reading adventure romances (the most popular ones being those written by Emilio Salgari, on Caribbean and Malaysian pirates). Not disdaining too those taking place in the Four Corner area of USA, as illustrated in different cartoon strips. Meanwhile dreaming of exploring the secret passageway our terrace was leaning against, an escape route, so went the legend, which would have taken the Visconti or Sforza families, in case of a siege, all the way to the Ticino River, some five miles away from town. This part of the secret passage was above ground, but it would have then continued underground to the river. There was a hole in the wall and we were often peering through it, but it was too dark inside and we could not distinguish anything, except dreaming of hearing the hoofs of the Sforza’s horses running to a hideaway.

In real life, things were much different: we had to become strict catholic and, towards the end of each month, go to church and fiddle with our rosaries, asking God to preserve father’s life and let him survive till that magic 27th of each month, the salary day. This was the day much coveted by millions of state employees, rushing to collect their monthly salary. A salary that would instantly evaporate like mist on a rising sun. The custom among 7 most families was to buy “on credit” in any shop. The shopkeepers would annotate, on a daily basis, anything bought there and collect the due bills precisely on the 28th of each month. My father would make a pilgrimage to all these shops and, come evening, he and we were just as penniless as before. Had he known about it, he might have wanted to rob a horse and gallop to the Ticino river; there, he could have rolled the last Lira (the Italian currency before the Euro) and thrown it into the water, like Johnny Cash did in his lyric:

Lake Michigan waves hit the beach

And I stand and let them wash at my feet

And then I throw it just as far as I can

Into the chill my wrinkled, crinkled wadded dollar bill

Somehow help was at hand. Two of us, Pier Paolo and myself, won an entrance in boarding houses (two different ones) run by the state to help poor families. It was a lifesaver. Away from the overcrowded quarters we lived in, we grew in a highly disciplined environment, daily life scanned regularly like a Swiss clock, meals, recreation, studying hours, bedtime; one could not escape this routine. But it shaped our characters and feelings for duty, while learning social contacts and equilibria with the mates around us. Six years we spent in this environment, missing though family life and contacts with our brothers and sisters. Yet, the two of us were the only ones who were later successful: Pier Paolo as Professor of Organic Chemistry at the University of Pavia, myself as Professor of Biochemistry at the University of Milano.

You know, it is a shame that I lived in Vigevano and did not appreciate the beauty of this magnificent town, although I went-by daily through the enchanting “Piazza Ducale” (Plaza of the Dukes, the fiefdom of the Visconti and Sforza family). In the summer of 2014 I visited the premises again, this time with a more robust background on art and history. I was amazed by the beauty of the renaissance plaza, among the most celebrated of Italy (see figure 2) with the porticos on three sides, the fourth one closing the quadrilateral with the gently arching façade of the baroque cathedral. On the left side stands superbly the tower designed by Filarete, the same architect that built its twin in Milano’s castle. Well, some twenty years ago the town hall had managed to dislodge the tank division; the castle is now almost completely refurbished and open to the public. For the first time I could enter it and climb the Filarete’s tower, wherefrom I could enjoy a superb aerial view over the 8 medieval and renaissance roofs. Even more, now the horse’s stables, masterpieces of Bramante, had been liberated from the tanks and refurbished too: you will see in figure 3

Figure 2. Piazza Ducale in Vigevano the interior of one of them (there were no less than three, probably the Visconti and Sforza had a herd of horses, since they would often go into hunting expeditions in the woods that surrounded Vigevano and extended to the Ticino River). It is a rich interior, looking almost like a church composed by three naves, with sumptuous marble columns. Well if there is a resurrection into a second life, I do not want to be a reborn Christian, nor, as per the Hindu religion, be reborn as a cow, but I rather be reborn as a horse if I will be assigned lodging into Bramante’s stables! Much to my surprise, also the secret passageway leading to the Ticino River had been opened to the public, so I could finally see what I had dreamed about in my youth from the terrace of our lodging. It was almost as large as a highway; ten horses could easily prance side by side. The passageway went down into a slow incline to 9

Figure 3. Bramante’s horse stables in the Castle of Vigevano the ground level and there terminated. Experts told me that the underground secret tunnel had collapsed, denying any access.

UNIVERSITY AT LAST!

At the age of eighteen, almost towards the end of senior high school, we moved to Milano, the business capital of Italy (Rome being the “moral capital”, although at the time of this writing it turned out to be “immoral”, as it was found fully colonized by Mafiosi, almost like in the Godfather novel by Mario Puzo, and it has been dubbed “Mafia Capitale”). Around 1960 Milano was a burgeoning town, rich, full of activities, plenty of business opportunities. We were looking around for a suitable place to enroll in university. Of course Milano had excellent universities, yet the problem was still how to concentrate in studies while living in an overcrowded environment, as our small apartment was. In fact it was like a campsite: 10 we had to set up beds on the floor in the evening and clear them up next morning. We focused on a neighbouring town, Pavia, some 40 Km east of Milano. It was like a little

Figure 4. Galeazzo Visconti’s medallion at Pavia’s University corner of Vietnam (luckily we were not waging war against this country) since it was surrounded by very wide extensions of rice paddies. The AlmaTicinensis Universitas (the Ticino River cut through town) was listed among the oldest ones in Italy, second only to Bologna and Padua, since it was funded in 1361 thanks to the efforts and liberality of Galeazzo II Visconti, whom you can see in the sculpture of figure 4. The official bull was signed by the Emperor Charles IV in Nuremberg and approved by Pope Bonifacius IX. It was one of the ONLY two universities in Italy to be granted a Nobel Prize: Camillo Golgi, awarded in 1906 together with the Spanish scientist Santiago Ramón y Cajal for their studies on the central nervous system (I am pleased to state that the other university is the Politecnico di Milano, whom I am proud to belong to, the prize being given to Giulio Natta in 1963 for his work on isotactic polypropylene). Sadly, pride deflates immediately when one notices that, over >110 years, the entire system of universities in Italy expressed ONLY these two Nobel winners. Most appealing was the fact that Pavia housed some of the top university colleges, such as the Ghislieri, funded in 1567 by Pope Pio V, and the 11 no less prestigious Borromeo, funded in 1561 by Saint Carlo Borromeo, in those days archbishop of Milano. Winning a place there was like winning a lottery, and competition for being enrolled (through entrance exams) was fierce. I failed at the Ghislieri (but my brother Pier Paolo won a place there the following year) and of course I was much disheartened. Luckily, there was an extra chance, a college called Cairoli, for the peons like me. Although not as sumptuous a palace as the other two, it was a noble building. During the Austrian domination of Lombardy, the vice queen Marie Therese embellished Milano with novel, prestigious buildings, among which stood the most famous opera theater La Scala. In the fierce competition for the project, it was the architect Piermarini who won. However there was an official architect she had brought to Milano from Vienna, Leopold Pollack, who was not quite happy with this choice. To keep him busy, Marie Therese bestowed to him the construction of college Cairoli, for which he did draw an excellent plan; the palace, called Austro-Hungarian College, was terminated in 1781 and was meant for training and lodging the officers of the Austrian empire. Perhaps it was an omen, since at the end of my studies in university I had to enrol in the army and became an officer in Mountain Artillery. I won a place at the Cairoli and decided to take a Ph. D. in chemistry. My choice was not vocational; I simply wanted to do something totally unrelated to my father’s activities. He was a humanist, a poet too and a musician as well, but as a schoolteacher, was penniless all his life. Better choose something more robust than a career in humanities. Chemistry in Italy in the sixties was among the top ones in Europe, so occupational chances in this field were quite high. These were five years of intense labour, since, to keep the place in the college, we had to maintain high grades throughout (on a scale from zero to thirty the global average of exams had to be 27/30 and no less than 24/30 in any single exam). Failure often came in the third year, studded with a lot of hard examinations. I must say that the school was good and the teachers up to the expectations, although maybe a bit on the old fashion side of chemistry. An important turn came in the third year when, as a teacher of organic chemistry, a prof. (Paolo Grunanger) from Milano joined the faculty. Grunanger was a brilliant chemist and brought to us fresh, modern ideas, such as all reaction mechanisms, which other teachers did not quite know. I am happy to say that, as I learned recently, he also graduated from the Politecnico of Milano and came from the very department I worked in; quite a coincidence! We received a good theoretical training, but the practical courses were short of expectations: we had only old instrumentation and none of the modern separation methods already circulating and well exploited in foreign universities. Additionally, the chemistry labs did not have a minimum of security for 12 students, including luck of fume hoods. So, we got intoxicated by vapours of HCl, sulphuric acid, organic solvents and had to work, even during deep winter, with open windows and, from time to time, rush out in the garden to breathe fresh, non-toxic air. In the last year I enrolled in a lab for the experimental work for my thesis; I went to a biochemistry lab and had as a project the study on the degradation pathway of UDP-xylose in the cartilage of new-born rabbits. We did discover an enzyme responsible for its transformation, which we called UDP-xylose decarboxylase. In was my first acquaintance with modern electrophoretic equipment and enzyme assays. My supervisor, Prof. Alessandro Castellani (who later on became Rector of the university) wrote up the paper, which was published in the Italian J. of Biochemistry. Later on, as I started working as a post doc in USA, I met an American professor who had obtained the very same results and published them in JBC (Journal of Biological Chemistry, among the top ones in those days). Well, he laughed at me and stated that he was the real discoverer of this pathway. Lesson No. 1: never publish good data in regional and obscure journals! You might as well store them in your circular filing cabinet (also called waste basket).

A quite different experience, during my university years, was the compulsory draft into military service. I could postpone it, but in the last year I choose to join the army, while leaving the last few exams behind, so as to have a chance to enjoy a few permits (in general a week each time) to go back home for each exam. Well, I was sent to a course in Mountain Artillery, first in the Umbria region for six months of training, to be sent later on north into the Dolomites. I had my first dour training in biology, since we had mules that would carry the howitzers up the mountains and had to learn how to take care of them. Learning how to handle them properly was a tough job. On rotation, we had to spend a week on night guards in the stables, rushing to clean their ejections, least be severely punished by the sergeant in duty. After such a week, our clothes smelled of mules and their urines. When sent north, in South Tyrol (Bolzano), we had to prepare for a winter camp. We had to conquer a high peak in the Dolomites and descent on the opposite site with the entire company, consisting of 36 mules, six howitzers and 52 soldiers and officers. The mule is an intelligent animal, sturdy and highly reliable. Yet, it had a strong phobia: it would never set the hoof into something not solid, in our particular case on snow. Since the track to the high pass was snow bound (more than one meter deep) we had to shovel it out by opening a path one meter wide up to the summit. A titanic effort that took one week of hard labour. In one occasion, I had to climb this path to the top three times in a day, to bring to the foot of the mountain exhausted alpine soldiers. The last day, when we 13 were ready for the ascent, a high avalanche risk was issued and we had to be transported to the other side of the mountain by trucks. What a failure! Even more curious was the incident that occurred in the summer camp. The terrain was clear, the ascent to the top uneventful, till we started the descent on the other side of the peak (the northern slopes). Just beneath us there was a narrow canal with an icy, long slab in the middle. The mules were stubborn and simply refused to proceed. Each howitzer was disassembled into six blocks, which were loaded on the saddle of as many mules. We had to download the cargo, bridle the mules on heavy ropes and slide them down one by one on the icy slope. I always wondered if the mules enjoyed skidding on the ice, but they certainly had their long ears standing at attention and looked terribly worried!

There was a grand finale to it. I had passed the few remaining exams and discussed the thesis. Prior to ending the military service, I decided also to take a national exam required for enrolment in the professional register. Three professors were to examine me on just about all I had learned in the five years of chemistry. However, after 15 months of hard duty in the military service, I realized my chemical knowledge had almost evaporated in the high Dolomite peaks. So, the day of the exam I appeared in front of the examination committee in my shiny lieutenant uniform, proudly wearing my alpine hat with the black eagle feather. The committee lost no time in understanding how rusting my chemical memories were so, wisely, they asked me to talk about my military adventures. Needless to say, I passed the exams with high grades, thanks also to the tales of the adventurous events passed in company of our friendly mules, a genuine and most valuable asset for surviving in the harsh alpine environment.

FLYING OVER ELLIS ISLAND

Roughly between 1880 and 1910 more than two million inhabitants of poverty-stricken Italy migrated to the States in overcrowded ships, confined to poor and crumpled quarters, with little food and miserable hygienic conditions. They were not the only ones; Irish, Russian Jews escaping pogroms and many other nationalities ran away from misery and life- threatening conditions. A port of entry was set in Ellis Island, on the doorsteps of New York, and immigrants were checked for health conditions. Not as bad as the Charlie check point in divided Berlin, though; only sick persons were sent back, most of them made it to a new life. As soon as I graduated I got married and we left right away for USA, the mythical land 14 of opportunity. It was on October 1966 and in those days one did not have to go through Ellis check point any longer: We flew over it on the way to Boston. During military service I had negotiated an appointment with a professor (George Wolf) at MIT, just across the Charles River, a stone throw from Harvard. For three years I walked across the Charles bridge and entered with awe at 77 Mass. Ave., the impressive entrance gate of MIT, looking like a Greek temple with massive Doric style columns. Although this institution was a polytechnic school, a biology building had been set up, with a Department of Nutrition and Food Science on the third floor. The first few months were a nightmare: I had exaggerated with my boss by asserting I knew English language. In fact I had studied only French and my knowledge of English was almost non-existent. No wonder that Wolf was annoyed with me and I had to plunge into its study full time. How to do it as quickly as possible? By watching commercials in TV and taking notes on anything they would offer for sale: I learned everything on house tools, furniture, gadgets, and any possible item for sale. To integrate these lessons, I started reading comic strips and became an aficionado of Peanuts. Up to this day, I admit that my language is, in reality, “Peanuts English”. Things did not go well either in my research projects. In those days, the army powers had deeply penetrated in most US campuses with a program called ROTC (Reserve Officers Training Corps) aimed at encouraging educated youngsters to enrol in the army. Among their activities was to dole out research money for projects of strong interest for improving US military power. My boss had won a grant on a most curious project: it consisted in developing “cold-adapted” soldiers, i.e. able to stand the icy winters in Alaska in case of a Russian invasion. What for? Well, to stand in the snow ambushing the invaders with steady hands, not trembling due to the harsh cold and thus keeping them under unshaken aim. We had placed rats in the cold room and kept feeding them large doses of carnitine: the idea was that the excess of this chemical would greatly facilitate transport of extra fat into the mitochondrion, thus producing more energy and abolishing the need of shaking and trembling for production of mechanical energy to keep the body warm. It turned out that these “cold-adapted rats” after a month of this treatment would cease shaking; but whether it would function on humans remained a mystery and in the long run the project was abandoned.

The magic event occurred towards the end of my third year at MIT: a Japanese scientist visited us and gave a lecture on isoelectric focusing (IEF), a just launched separation method by an obscure Swedish firm, LKB Produkter AB. It was love at first sight: as the lecture progressed and we appreciated the outstanding results, I knew I would embrace 15 this methodology and grow up with it in my scientific career. IEF, as developed and commercialized, was a rather cumbersome and complex technique. It was born as a preparative separation method meant to purify even grams of proteins into glass columns of 110 or 440 mL, i.e. medium and large scale approaches. The column had to be filled with a sucrose density gradient, to prevent decantation of focused, dense protein zones in the gravitational field. As the electric field was applied, a second gradient, co-linear with the first one, would develop, a pH gradient against which proteins would migrate till they reach the isoelectric point (pI), which is the pH region where their net charge was zero. This focuses the analyte in a narrow region along the strip. It was a superb technique, perhaps the first one able to counteract entropy, the archenemy of any separation method, since entropy causesdilution of the sample zone. In was not the only method able to accomplish that: almost simultaneously, isopicnic centrifugation was developed, based very much on the same principle, namely a contrast between an external force field able to drive the sample zone to its “isoperichoron” and an internal non-isocratic environment created by the external applied force. The balancing between these two opposing fields kept each band in very sharp conditions. On a similar mechanism would work, as developed a few years later, isotachophoresis (ITP), whose basic principle was exploited in the initial phases of disc (for discontinuous, not for the disk- or ring-shape assumed by analytes) electrophoresis to sort out and concentrate (by up to four orders of magnitude!) each protein zone present in the applied sample. In a way, also pore-gradient electrophoresis would considerably sharpen the macromolecular analyte, but via a different mechanism, i.e. a progressive slowing down of the movement as the limiting pore size in the polyacrylamide matrix would be approached. I realized immediately that this exquisite method would become extremely popular if simplified to improve user- friendlyness. In that period I left MIT and moved down town to Professor James Drysdale’s lab at the Harvard Medical School. Drysdale is an ingenious Scotsman that encourages new ideas. While he worked on his pet project, ferritin purification and analysis, he directed me to work on IEF and, since I had full experience on disc electrophoresis (in those days run in glass tubes) we tried the most obvious approach, namely to polymerize the gel as a single little sausage containing just the monomers, no standard buffers but the mixture of carrier ampholytes (CA), i.e. that multitude of amphoteric buffers (of largely unknown chemical composition, but stated to contain at least 600 different species) that would create and maintain the pH gradients under a strong electric field. It worked very well, as we could see by monitoring the red bands of haemoglobins (Hb) admixed to the gelling 16 mixture and reaching steady-state into very sharp, “focused” zones. After working out all aspects of this analytical IEF version, we published the data, thinking they would make a hit.

Figure 5. IEF of partially and fully oxidized human haemoglobins (Annals NY Acad. Sci. 209, 1973, 163-186).

Indeed a flurry of papers appeared simultaneously on this very same topic, so we lost priority.

Lesson No. 2: new ideas spread like prairie fire all around. If you have robust data, hurry to publish!

Figure 5 gives the IEF of native and progressively oxidized human Hbs. Disc electrophoresis could easily separate the reduced form (lower band, pI 7.0) as well as the fully oxidized species (upper band, pI 7.4) and the 50% oxidized band, half a way in between. Here we had a serious problem, though: the intermediate bands were two, not one, and this sent shivers along the spine of those Solon haematologists sitting in the editorial board of their pet journals. The manuscript was pluri-rejected, although we had suggested and demonstrated that, even though the formal global charge of the 50% oxidized species was the same, it did matter whether the oxidized irons were on the β- as 17 opposed to the α- chains. It turned out that IEF was a probe of conformational transitions, thus able to pick up minute pI differences (close to the resolution limit of 0.01-pH units between two adjacent species). We continued the work on ferritin and, again via IEF, we could demonstrate that the 24 subunits composing the macromolecules were not identical but consisted into at least two different populations of slightly diverging pIs. Boldly, we even explored the possibility of focusing also nucleic acids, and published a paper in Biochemistry on the IEF of tRNAs. Although most of us think that DNAs and RNAs are very acidic, non-amphoteric molecules, indeed they have a pI, since below pH 4 three out of the four bases get protonated. However, it turned out that most of their pI values were below pH 3, thus outside the proper working range of IEF; additionally, most of their pIs, above a 100 bp length, would be essentially identical.

Globally, the four years spent by us in USA (1966-1970) were most productive and formative. My wife Adriana Bianchi Bosisio had an appointment at the Tuft University at the New England Medical Center located downtown in Boston. She did top work on alkaline phosphatase purified from the bile duct of normal and pathological rats. She published very good papers in the field of enzymology together with her supervisor, Dr. Marshall Kaplan. For both of us, it was the first job appointment, the first experience of living in a rented house, the first car and, most importantly, the first baby, our daughter Sabina, the only US citizen in the family. For me, it was also the first driver license although, needless to say, I failed the first exam! And it was also a political training ground. In 1968 protest spread among US campuses against Vietnam War, which had peaked in killing and brutality, like the massive use of Agent Orange for forest defoliation and setting in fire hamlets of peasants as well, all suspected to be supporters of the Vietcong. We had folk singers (Joni Mitchell, Joan Baez, Phil Ochs, Judy Collins, Bob Dylan) visiting MIT and chanting all together ballads against the war. With a lady friend, we visited on a regular basis the North End (where most Italian Immigrants lived) and helped youngers, whose army recruiting was approaching, to escape to Canada: better be a draft-dodger than lose your skin in Vietnam fighting an insane war (see, e.g., Tim O’Brien, Going After Cacciato, Bantam Double Day Dell, NY, 1975)! For those having negative opinions about this, it might be worth reading Mark Kurlansky, 1968 the Year that Rocked the Word, Random House, London, 2004. In November 1969 I also attended the “Moratorium to End the War in Vietnam” in Washington DC, stated to have reached more than 500,000 participants (and many more around the country). We paraded down Pennsylvania Avenue to the White House. Hour after hour, many of us walked in single file, each bearing a placard with 18 the name of a dead American soldier or a destroyed Vietnamese village. The marches finished in front of the Capitol building, where the placards were placed in coffins. We were lead by Pete Seeger who kept singing “Give Peace a Chance”, a John Lennon’s classic. One of the most picturesque slogans was against Gen. Westmoreland, whose name had been transformed, aptly, into Gen. Waste More Land. Back in Boston, I used to listen on a free radio channel to the great folk singer Pete Seeger chanting (among other top hits):

This land is your land, this land is my land, From California to the New York Island, From the Redwood Forest to the Gulf Stream waters, This land was made for you and me.

Unfortunately it was not our land: regulation for immigrants allowed only four years of staying in USA; thus, at the expiration of this period (and driven by home-sickness) we went back to Italy.

AN ITINERANT IEF PRIEST

Back home in Italy, I obtained an appointment as a research fellow (a permanent position, equivalent to an assistant professor level, since we had teaching duties) at the State University of Milano. Although it was not my field, the appointment was at the Faculty of Agriculture, since my job was attached to the chair of biochemistry in the same area. Although I was supposed to work on the purification and biochemical properties of succinate dehydrogenase extracted from beef heart (on each purification cycle we had to homogenize two Kg of those large size hearts, minced in five-six litres of buffer. At the end of the process, the walls of the cold room was tinged with blood) I soon abandoned this field (after publishing a couple of papers on the structural subunits of this enzyme) to go back to my American dream, IEF, of course. A move that my supervisor did not appreciate, since those appointed as research fellows attached to a chair were treated like slave labour by the system, largely based on what we nicknamed the “University Barons”, a kind of a Mafia system by which, no matter how smart or lousy you were, if you wanted to progress in your career, you simply had to be the lackey of your boss. My first encounter with the high education system of Italy, thus, did not go so well since I had been trained in USA and could not understand this oppressive and suffocating system. 19

In this gloomy landscape a life-saver turned out to be an offer from LKB Produkter AB, the Swedish company that held the IEF patent and was marketing it the world over. They hired me as a consultant on a friendly basis, i.e. no fees, but offering me travel and all expenses for seminars around Italy, plus donation of the precious carrier ampholyte bottles (rather costly) for my lab research. I became an IEF itinerant priest: for years I travelled all over Italy spreading the gospel of IEF, in daylong seminars coupled to a lab course. In those days none in Italy knew about modern electrokinetic methodologies, never mind IEF, but even disc electrophoresis as well as SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). I believe an entire generation of biologists/biochemists was trained by my associates and me during the decade 1971-1980. The courses were highly appreciated and the lecture rooms were filled to the brim. As time went buy, we enlarged the seminars to the entire Europe: it was a feast meeting so many young and enthusiastic scientists, an intellectual reward much superior to any consultation fee!

Back in the lab, I worked systematically on improving the IEF methodology: we proposed new, different synthetic routes for the carrier ampholyte (CA) buffers, as it turned out that, no matter how many chemical species composed this mysterious concoction, there was a minimum of buffering capacity and conductivity in the pH 6-7 region, something that the inventor of the technique, Harry Svensson-Rilbe, had predicted in his theoretical papers. Somehow nature had been stingy in the physiological pH region (I recall here that even proteins are poor buffers around pH 7 with a scarce buffering contribution only via their histidine residues). Concomitantly, I kept working on biological/biomedical projects, with analysis of my pet protein, haemoglobin (Hb). As we used to spend the summer vacations in the island of Sardinia, I met there a haematologist, Dr. Gianfranco Cossu, who was practicing in a small town outside Sassari, Ozieri. He was studying thalassaemia, still highly spread in the island, and trying to eradicate this disease (deadly in the homozygous condition). We set-up IEF of umbilical cord blood and, via our modified IEF technique, we could detect hetero- and homozygous conditions at a glance in the gel slabs (meanwhile the little gel sausages had been substituted by flat-bed IEF, allowing simultaneous analysis of >20 samples, with easy comparison from lane to lane). Figure 6 shows a portion of the gel slab, stained with bromophenol blue (in acidic conditions, the adduct with Hb took an intense purple colouring). This is not an ordinary IEF in the pH 6-8 region (the narrow-cut ranges spanned two pH units): at birth, there are three Hb populations, of which the fetal (F) represents 70%, the adult (A) 20% and the acetylated fetal (Fac) 10%. 20

These last two species had almost identical pI values and could not be separated at all. We had to resort to non-linear pH gradients by adding large amounts of a “poor” ampholyte (6-amino caproic acid) flattening the gradient in the pI region of A and Fac, thus obtaining a good separation between them. Proper quantitation by densitometry was thus possible. Over several years, we must have run close to 10 thousands gel slabs, screening all the new-born population; thalassaemia was essentially eradicated thanks to the combined efforts of Cossu and my group.

Figure 6. Focusing of cord blood haemoglobins. The hetero- and homo-thalassemic tracks are marked, as well as fetal (F), adult (A) and acetylated (Fac) Hb zones (Amer. J. Hematol. 13, 1982, 149-157).

Another important project we could solve was the separation between two fetal globin chains, called Gγ and Aγ, the products of two genes expressing either Gly or Ala in position 136 of the polypeptide, which we did in collaboration with Prof. Sergio Ottolenghi. The assessment of the Gγ/Aγ ratio was important for diagnosis of diseases such as hereditary persistence of fetal Hb, thalassaemia and other haematological disorders. It could only be obtained via long and cumbersome separations in chromatographic columns in presence of urea and 2-mercapthoethanol (2-Me), a volatile compound that impregnated with his stingy smell the entire Ottolenghi’s lab, so that we had to wear gas masks when visiting him (the columns were constantly leaking). So we tried to set up a gel-slab IEF alternative without any success, though. Reducing the Hb chains with 2-Me in presence of 8 M urea gave only the three expected globin chains, α, β and γ, but the latter resisted further splitting. One day, out of desperation, I added 3% of a surfactant (Nonidet 21

P-40) to sample and gel and, miracle, now the Gγ and Aγ chains were fully resolved (Figure 7, right side)! That occurred in 1979, when I attended a meeting in USA and I met an associate editor of Blood, who invited me to visit her at the Albert Einstein College in NY and give a seminar on our data on the elusive Gγ/Aγ ratio. I was invited to submit my results to Blood, which I did. Much to my surprise, the paper was rejected, only to appear a few months later with her name and those of her associates!

Lesson No. 3: trusting your scientist colleagues is good, distrusting them is even better!

Figure 7. Separation of gamma globin fetal chains by IEF in 3% surfactants (Biochem. Biophys. Res. Commun. 87, 1979, 1-8).

Other collaborations led to quite unexpected results. A colleague of mine, Prof. Francesco Chillemi, was working on the synthesis of the active sequence of the human growth hormone, via the (by then) classical Merrifield process, which consists in elongating the growing chain not in solution, as done up to his report, but bound to a resin, much like in vivo proteins grow attached to an mRNA and ribosomes. Very smart approach, indeed, for which Merrifield was awarded the Noble Prize in 1984. Poor Chillemi could not find any hormone activity after the chain grew progressively in length to the point at which, when reaching the final length of 54 amino acids, the growth activity of the synthetic polypeptide had entirely dissolved. We hypothesized that, as the chain was elongated, since at each addition of one amino acid the yield was, at best, 98%, failed and truncated sequences would accumulate in the final product, leading to loss of biological activity. Proving it was not that easy, also because Svensson-Rilbe had predicted that IEF would work only for macromolecules and would give meagre results with peptides. Indeed, as shown in Figure 8, we fully succeeded on that, by exploiting a micellar preparation of Coomassie Brilliant 22

Blue (in a leuco form in strongly acidic conditions), by which the temporarily precipitated peptides into the gel fibres would be cross-linked within the gel meshwork by the sulphonated dye molecules, able to form large size aggregates via their acidic groups. As one can appreciate in the figure, as the peptides grew in length above 10 amino acid residues, more and more spurious bands began to appear, among which it was impossible to sort out the desired, biologically active sequence (the peptides loaded in the gel ranged

Figure 8. IEF separation in the pH 3-10 range of growth hormone fragments obtained via the Merrifield approach (J. Chromatogr. 157, 1978, 243-251). from a decapeptide to as long as 54 residues). In 1984 I was visiting again NY and was invited to give a lecture at the Rockefeller University, in the chemistry department. It was the very year in which Merrifield had been granted the Nobel medal and the university was displaying banners and flags in his honour. By sheer luck, Merrifield happened to be present at my lecture and did not quite like our data. Yet, after a private discussion, he agreed that our results were solid and that his method had some holes, after all. There was a happy ending to this story: within a short time, all synthetic chemists stopped performing the Merrifield elongation to the very end of the chain. The winning strategy was to synthesize rather short peptides (max 5-6 amino acids in length) and then release them one by one to obtain the right sequence by a progressive splicing of the short segments. 23

THE KING IS DEAD, LONG LIVE THE KING!

Towards the end of the seventies it became apparent that IEF in CA buffers was showing the crippling diseases of age: the pH gradient would decay in time, the CA buffers were totally unknown chemical concoctions (in 2005 we finally reported as many as 3000 different chemical entities in one commercial preparation) and complex formation among polymers and CAs was suspected. In 1980 we joined forces with LKB (Bengt Bjellqvist) and German scientists (Angelika Goerg and Rainer Westermeier) to work out a novel IEF system, based on immobilization of the buffering species onto the polyacrylamide gel. LKB Produkter AB had synthesized six of these chemicals, commercialized as Immobiline (to recall the concept of Ampholine for conventional IEF; abbreviated in the acronym IPG, immobilized pH gradients), indeed acrylamide weak acids and bases, not any longer amphoteric by bifunctional: at one extreme they contained an acrylic double bond, at the other extreme a weak protolytic group. By admixing them in ratios calculated via the Henderson-Hasselbalch (H-H) equation, one could generate a one-pH-unit gradient (or narrower, but not wider!). Such gradients had two major advantages: they would be stable forever in the electric field and they would permit recovery of proteins isoelectric and isoionic, in that the recovered species would not be contaminated by chemical leaching out from the matrix.

We presented these data at an international conference on IEF and ITP organized by Dimitri Stathakos in April 1982 in Athens: our data went unnoticed, no one perceived their unique novelty. In a way they were right: as we could produce ONLY 1-pH-unit gradients, their use was quite limited. Back home, my lab engaged in two quite difficult tasks: on the one hand, synthesize more buffering species (at the end we described no less than 15) and on the other hand to try to generate extended pH gradients. Here we had tremendous difficulties: nobody knew how to generate such gradients, since the H-H equation was based on a “tandem” principle: only one buffering ion and one titrant. How to mix dozens of buffering species and titrate them in any extended pH gradient was “terra incognita”. We joined forces with mathematicians and physicists (notably Prof. Fabrizio Celentano and Dr. Giulio Dossi) to solve these problems, which took years of efforts to be completed. Finally we could tabulate any possible extended pH gradient, from 2-pH up to 10-pH-wide formulations; such recipes are still the ones in use today. Instrumental to achieving this aim was the work of Dr. Elisabetta Gianazza, at that time a post doc in my lab, who spent 24 tremendous energies and hard work in this projects (success also came thanks to 3 or 4 books written later on by Westermeier on all electrophoretic methodologies and proteomics, rendered precious by superb illustrations that he produced in endless nights at the computer, worth reading by all means). With our recipes and due to the fact that, with time, a few companies started producing and commercializing ready to use strips (home- made IPGs required plenty of lab work and good theoretical knowledge), towards the end of the next decade (nineties and up to the year 2000) IPGs became extremely popular. Today they have completely supplanted conventional CA-IEF. Indeed IPG are the only focusing process adopted for generating two-dimensional (2D) maps, the technique in large use for any proteomics study up to the discovery of 2D chromatography, which took

Figure 9. IPG separation between Gϒ and Aϒ native HB fetal tetramers in ultra-

shallow pH gradients (Electrophoresis 7, 1986, 213-216). over also due to the fact that it is fully instrumental vs. the hard labour required by electrokinetic 2D maps. I will shorten this chapter by giving just two examples: one is

Figure 9, a unique separation between Gϒ and Aϒ, this time performed not on globin chains but on the intact tetramer of Hb (the bands are not stained, they are in the natural red colour of Hb). It was a hard separation: we generated an extremely shallow (0.1 pH unit) pH gradient over a 10 cm distance (thus with a slope of 0.01 pH/cm) and had to drive the proteins to the pI with an extremely high (in those days) voltage, namely 10,000 V. The resolution between the two bands is close to the limit of IPGs, namely 0.001-pH unit, one order of magnitude greater than that of CA-IEF. Here too, the separation must have been 25 driven by minute conformational transitions between the Ala vs. the Gly species, since in principle there should be no charge but at best only minor hydrophobic differences, since Ala is 35% more hydrophobic than Gly. Curiously, though, such a separation could be achieved via IPGs and not via chromatography. I am proud to say that this figure appears in the classic book Unified Separation Science by J. Calvin Giddings (Wiley, NY, 1991, Fig. 8.8, p. 179). In addition to the exquisite resolution as visible in Figure 9, there was another important advantage of IPGs: even on an analytical scale, they could afford much higher protein loads than in CA-IEF, to the point at which mini-preparative runs could be easily performed: as shown in Figure 10, when running ovalbumin in the pH 5-6 range, there was no sign of overloading (in a 0.5 mm thin gel slab) from 0.1 to 10 mg load.

Figure 10. Mini-preparative IPG runs of ovalbumin in 0.5 mm thick IPG gel slabs. Load: from 0.1 to 10 mg protein (J. Biochem. Biophys. Methods 8, 1983, 134-155).

Of course, at higher protein levels, one could see many more impurity (or isoform) bands that were not visible at the 0.1 mg load. During this decade, my life was full of events: with Gianazza, Goerg and Westermeier we made a travelling circus throughout Europe, spreading now the gospel of IPGs, this greatly contributing to its adoption by many labs. I also was invited to several lecturing tours in the Far East, notably China and Japan. Visiting Beijing in 1984, when foreigners were “rara avis”, was a magic experience. It was April and the Forbidden City was an awesome sight: one could enter all imperial buildings, no cue, very few visitors, whereas today millions are milling around. The Temple of Heaven (or of the Good Harvest) was almost like Heaven, with blue tiles impressing your retina and a grand park in blossom; unforgettable! 26

Perhaps the most incredible event, though, was an invitation, in 1985, to present my work at the 5th international HPLC meeting in Toronto. Prof. Milton T. Hearn kept calling me from Australia, but I adamantly refused: I was scared, I had nothing to do with them and my experience on chromatography was essentially non-existent. I was given the closing lecture and I was glad by thinking that almost every attendee would have gone home by then. In fact the whole 500 of them remained just for hearing my stories. I felt that I had to invent something in addition to science to keep them happy. And I did: I narrated the story of the “Blue Finger Society”, a secret society whom only hard practicing electrophoreticists could belong to. How did we recognize its adepts? When meeting around in airports or elsewhere we would look at their hands: if their thumbs and indexes had a blue tinge, then we knew they were bona fide practitioners, since in those days we did not have all the safety gadgets of modern labs, no gloves, no masks. We would plunge our hands in the Coomassie Blue staining solution and pick up the 2D slabs anxious to see the results; naturally our fingers were constantly blue. One member of this society, though, was also identified by a shopkeeper in Milano, in the Corso Buenos Aires shopping district. While I was around with Goerg helping her to choose elegant shoes, Westermeier was visiting alone another shop. When he finally made his choice and offered to the owner large Lire bills to pay, the shop owner, alarmed by the blue fingers, thought he had forged the money and called the police. I arrived just in time to rescue him, although I had to explain to the police, who also wanted to arrest me, why the hell I had blue fingers too! As luck goes, the Gold Finger movie made a great success the world over, so nobody cared any longer about our Blue Fingers.

As a last picture, I would like to show the covers of my two books: the first one on CA- IEF and the second one on IPGs: perhaps these were the two manuals than made me largely known in the field (Figure 11). Some people believe that they represent the Old and New Testament books; surely they contain just about any “revelation” in the field. In reality there should be yet another cover, as I discovered when I was invited by the Russian Academy of Sciences to Moscow, at the Lomonosov University, in February 1987 (the year after the Chernobyl nuclear disaster). The Russian had pirated my first IEF book of 1983 and made a non-authorized translation. It was printed on poor paper, indeed and it probably sold for a few Roubles. Probably, feeling guilty about that, they invited me to repair this affair. Well, it was the best experience in my life! The book had been printed with a green cover and at the end of the weeklong seminars/lab courses, there was a long queue of young scientists and old professors patiently waiting on line for my autograph. 27

That made my day! I was elated like Chairman Mao: Chinese people parading around with Mao’s red booklet and Russians waving the flag of Righetti’s green booklet. Matter of fact, the first one, although covering a very narrow field, sold close to 9000 copies. The second one would have likely had the same success, were it not for Pharmacia that, in 1990, acquired LKB Produkter in one dark night (for me). Those chaps were so infuriated with the LKB scientists and their tremendous success in the field that they wiped out completely the company by beheading all their directors throughout Europe and the world. Struck by the NIH syndrome (Not Invented Here) they committed the crime of wiping out IPGs by stopping selling the chemicals and not advertising it any longer. A gigantic bad move: when in the year 2000 they realized that the technique had become popular, no

Figure 11. Covers of the two manuals on CA-IEF (left) and on immobilized pH gradients (right). matter what, they could not cash much on it since the patent had expired and now other companies had entered the arena.

Lesson No. 4: it does not matter whether or not you can make money on you inventions or on your book sales. It is the appreciation of your fellow scientists that counts most!

28

FANTASTIC VOYAGE

Yes, we all know that this is the title of a science fiction book written by Isaac Asimov in 1966 (rewritten then in a second version in 1987). A group of brave scientists have to remove a cloth from the brain of an important personage that has vital information for the future of USA. Since surgery cannot be performed from without it has to be done from within, so these scientists, on board to a submarine, are miniaturized to the size of a cell and injected into the carotid of the patient. They have barely one hour to navigate through arteries and capillaries to reach the brain and remove the cloth, prior to being blown up to the former size. Plenty of traps along the route, but miraculously they succeed. This fiction became a reality when in 1983 J.W. Jorgenson and K.D. Lukacs published a paper in Science by the title Capillary Zone Electrophoresis (CZE), a truly miniaturized system by which all molecules, including macromolecules such as proteins and nucleic acids, could navigate in narrow bore channels with inner diameters as small as 10 µm. Growth was sluggish, though, since commercial instrumentation was not available and inertial laws predominated. An important upturn took place in 1989 when Barry Karger, boldly, called the first international conference in Boston. Next year he did it again this time in San Francisco and the attendance was large, also because by that time a few companies had produced instrumentation and the pool of users had grown in size. This was the first time I was invited to attend (luckily I was present at many more editions after this one), but to present IPGs, since I had still not entered the arena. It was quite a shock to me. Although this was a genuine electrokinetic methodology, the fauna had totally changed: no one of my fellow electrophoreticists was there, only chromatographers. Curiously, they were the first ones to pick it up and to apply to CZE all the concepts and methods they had developed in their chromatographic approaches. CZE grew mostly through their efforts. I remember Karger regretting the complete absence of electrophoresis people (I guess the lucky event of my attendance to the Toronto meeting in 1985 helped me a lot to mingle with chromatographers who, by that time, knew me quite well). Berthold Radola, the chief editor of Electrophoresis, peeped into this methodology only after 1993 (although today this journal is almost solely devoted to CZE!). Back home a couple of companies helped me to set up CE in my lab and soon we had four instruments to run. Yet, growth was slow also because I had been trained into “orthodox” electrophoresis, which preached that anolytes would move to the anode and catholytes, of course, to the cathode. But this technique run against these moral laws and we soon learned that, given a proper and swift 29 electroendo-osmotic flow, all analytes would be swept to the cathode (were the detector was placed).

Here too some events that occurred earlier in my career were an important help. In the earlier eighties I had been moonlighting and taking summers off to go to work at the Red Stone Arsenal, in Huntsville, AL, were NASA had local headquarters. My boss, Robert Snyder, a physicist, was supposed to prepare electrophoretic experiments in micro-gravity, and I spent few summers with him working on these projects. Not to mention the summers spent also in Milan Bier lab in Tucson, AZ, who was also generously supported by NASA. He was a giant in the field and had edited two books Electrophoresis (Theory, Methods and Applications), Academic Press, NY, 1959 and its sequel (vol. II) in 1967. With him we run hot projects on IPGs, which included full separation of 20 free dansylated amino acids in very acidic IPG gradients, spanning a pH 3.0-4.0 interval, an almost forbidden electrokinetic region (Electrophoresis 7, 1986, 128-133). Back home, I was hired as a consultant in the same field by ESA (European Space Agency; later on I also consulted for ASI, Italian Space Agency, which supported me with generous grants), whose headquarters in Paris I frequently visited. There I met a Russian couple that presented a micro-gravity project on polyacrylamide gel polymerization, claiming that at the earth gravity a faulty gel was produced due to sedimentation of growing chains caused by the gravity pull. I had never seen this phenomenon and I regret I rejected their project. Yet we became friends and, in 1991, Tatyana Lyubimova invited me to a conference on “mass and heat transfer in microgravity”, something that again frightened me as the congress was solely populated by theoreticians. The conference lasted 11 days as we navigated from Perm (in the Urals) towards Moscow along four rivers (enchanting though: lectures in the morning, afternoons mooring on the river banks and visiting old monasteries that were slowly coming back to life and being restored after 60 years of hard communism and atheism). In the last days, panic erupted on board: our cabins (we were some 30 or so foreign scientists, mostly Europeans and a few Americans) were visited by a constant flow of pilgrims, asking refugee status in our countries. Well, those were the last few months of the reign of Michail Gorbačëv, after which Russia disintegrated. In the total chaos that ensued, everyone was trying to find an escape route. Back home, via generous support from ESA, from the Ministry of Foreign Affairs of Italy and other granting agencies, I was able to offer some jobs to a few Russian scientists: first came Michael Bello (from St. Petersburg) and then Sergey Ermakov and Alexander Stoyanov from Moscow. With the help and dedication of this troika, we worked out important theories in the CZE field and 30 many other methodological innovations (I am glad to state that, after working a couple of years in my lab, they could publish several papers that granted them a job in USA, where they currently live). As a further lucky event, I was visited a few times by Mikhail Yu. Zhukov, a brilliant theoretician who, together with V.G. Babskii and V.I. Yudovich (all of them from Kiev), had written in 1988 a most important book Mathematical Theory of Electrophoresis (translated in English the year after but not so well known as it was tremendously dour to follow). Zhukov also helped a lot and, together with Stoyanov, the three of us wrote a book The Proteome Revisited, published in 2001 by Elsevier (of scarce success, because I had cheated considerably by offering a book with such a title; in reality the first 119 pages were devoted, again, to the theory of IEF and IPGs). There was a sorrowful event, though. Just prior to the micro-gravity meeting in Perm, Babskii was supposed to join us and, after the meeting, fly with me to Milano, as I had found a grant to support him for one year. It was a hot day in July and he had gone down to the Dnieper River to take a swim. He never touched shore. His corpse was recovered a few miles down stream. A dreadful event, as he had two children ready to go to university. Back home I wrote an obituary (Electrophoresis 12, 1991, 1063-1064) but I could hardly see what I was jotting down, as I was crying all throughout. We had met the year before at a meeting in Germany and had become very good friends. We spent one afternoon going from shop to shop searching for top china ink to purchase for his daughter, who was studying architecture. In those days drawings were made by hand, no AutoCAD, and in Russia the quality of the ink was rather poor. I loved to see how much he cared for his children.

The year 1990 marked the beginning of my third decade in research: the first one dedicated mostly to CA-IEF, the second one to IPGs and now the new adventure in CZE. At the beginning we started playing around with metabolites, since small molecules would not stick to the silica wall. But when we approached separations of peptides and proteins the game became though, since these species would adhere to ionized silanols even when the capillary was coated by different means (mostly by covalently affixing hydrophilic, neutral polymers such as celluloses). Thus we turned to separation of DNA fragments for screening of genetic defects. Nucleic acids, due to their strong negative charge, would be repelled by the silica wall essentially at all pH values. We teamed up with Maurizio Ferrari and Laura Cremonesi, expert geneticists working at the S. Raffaele Institute in Milano. Plenty of interesting results were obtained: in one instance we could achieve a simultaneous detection of ΔF508, G542X, N1303K and 1717-lG!A mutations in cystic 31 fibrosis. In another project, CZE in fluidified polyacrylamide chains produced with modified monomers was adopted for CAG triplet analysis in families with androgen insensitivity syndrome. In a more ambitious project, we could achieve simultaneous resolution of 18 dystrophin gene exons in Duchenne and Becker muscular dystrophy patients. The validity of our approach was proven by repeating the screening of 75 Italian families with 21- hydroxylase deficiency (already genotyped by allele-specific oligonucleotide hybridization or direct sequencing) via CZE. Whereas dot-blot is time consuming due the high number of hybridizations with radioactive probes, our protocol was found to be more rapid, giving sufficient separation on CZE after PCR reactions without pre-concentration or desalting of samples. Moreover, LIF (laser induced fluorescence) detection provided very high sensitivity. For detection of point mutations we even described a novel CZE methodology by which temporal thermal gradients, reaching as high as 90°C, were created within the capillary lumen by voltage ramping and not via an outer recirculating coolant/heater. Notwithstanding the considerable advantages of the CZE technique as opposed to those currently used in geneticist’s labs I am afraid CZE was scarcely adopted by them, here too due to inertial laws.

Although we run many more successful projects, I would just like to focus onto a unique coating we developed in collaboration with Profs. Roberto Sebastiano and Attilio Citterio here in my department. I believe that hundreds of papers have appeared describing special silica wall coatings for abolishing adsorption of proteins. Most of them were faulty

Figure 12. Structure and mechanism of action of the silica wall coating quaternarized salt 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (J. Chromatogr. A 924, 2001, 71-81). 32 since their robustness was checked by running only four, well behaved protein standards. When injecting an entire proteome (e.g. sera, human body fluid etc.) disaster ensued. Additionally, since plenty of such coatings were not covalently bound to the silica surface but physically adsorbed, they would constantly leach out and plague the mass spectrometer in hyphenated CZE-MS instrumentation. Figure 12 shows the chemical formula of this agent as well as the mechanism of wall binding. We nicknamed it “skorpio”, in that it has two chelae that allow docking to the silica surface via hydrogen and ionic bonding. This transient adsorption is made perennial via the tail of the skorpio, which “stings” the wall by forming a covalent bond via reaction of the silanols with the iodinated tail. This reaction is spontaneous at alkaline pH values and thus the chemical modification of the surface occurs at no efforts at all, it is the most simple and most effective chemical deactivation of silica. Curiously, though, this reaction has tremendously low kinetics and would never occur as here described. Not by divine intervention, the miracle occurred by simply by switching on the current for a few minutes. We named this phenomenon as “electrically-engendered chemical reaction”. The reason? The forward reaction was greatly favored by the local removal of the free iodine via the electric current. As an extra bonus the coating is indefinitely stable and does not leach out, so that hyphenation with MS instrumentation is highly successful. Needless to say, we patented the chemical and offered it to one leading company in the field. With a curious result: they took it, locked it into a drawer and threw away the key. The reason? Our chemical would have ruined the sales of their (I admit rather poorly and in constant need of replacement) coated capillaries!

Lesson No 5: companies will occasionally favour their clients and potential collaborators but most of the times they will only patronize the fattening of their wallet.

In the middle of this decade a dramatic event occurred that jeopardized my life as a scientist. A full professorship can only be obtained not locally but via a national contest, which in those days (but I suspect still today) was largely orchestrated by the Baron’s (i.e. those full professors able to bend the will of the majority) Mafia, who had already jotted down a list of agreed-upon winners years in advance of the competition. In fact my applications in the previous 15 years had been simply rejected, via a mathematical process that eliminated the outliers (i.e. all outstanding candidates, whose curricula were burned in the fire, assimilated to heretics). In 1994, though, a pool of judges in Milano had started taking to court corrupted politicians (and there were too many indeed) starting a 33 nation-wide process called “Mani Pulite” (clean hands). The members of the professorship committee cleansed their dirty hands and, surprise, promoted me as the first one in the list of winners. With a “comma 22”, though: by sending me to the deep south, Mafia-land tout court, i.e. the region of Calabria, with a one way ticket, hoping I would die there or resign into pension. My life became pure hell: I lost my precious lab in Milano and, down south, I had to go to a university called Arcavata di Rende, a place that I had tremendous difficulties in locating in the map of Italy. This place was indeed a large territory owned by the Baron of Rende, a God forsaken village in the middle of grazing land for cows and sheep at the foothills of the Sila Mountains. The Baron, with the help of smart politicians, converted this pasture land, worth pennies as an agricultural land, into a terrain for settlements, that was sold for a huge price to the Ministry of Public Education for building a local university (I bet the booty was split among him and various politicians).

There was nothing there: the few departments were hosted in a large bunker of World War II, perhaps built in Mussolini’s era. The first time I visited it, I was wondering if I had to wear a helmet and my lieutenant’s uniform and perhaps borrow a couple of mules from my former unit of Mountain Artillery (grazing for them would be free). I fell into depression and started seriously thinking of resigning, as I had no lab there, no office, not even a chair and a desk. What saved my life was a nicely equipped lab of a physicist, Prof. Roberto Bartolino, who had won good grants from the EC (European Community) and had bought modern equipment, including an atomic force microscope (AFM). I moved to his lab and we started doing advanced work on the chemistry and structure of capillaries: we would cut them lengthwise and explore the surface via AFM to monitor, e.g., adsorption of large proteins (such as ferritin, as it could be seen by our probe) and other surface structures. In those days, there had been some reports suggesting that the inner capillary surface was strewn with craters, pits, ravines (had it been bombed by soldiers in Vietnam?); a rather faulty territory, then, responsible for adsorption of analytes, devious liquid flows and the like. Our AFM exploration indeed revealed a rather smooth surface, free of all faults previously reported. We submitted the manuscript to Electrophoresis (16, 1995, 1445- 1450) and, in a bold move, to drive home our thesis, I quoted the famous verses of Cyrano de Bergerac (la tirade du nez, regarding the jokes that his comrade cadets de Gascoigne were making about his giant nose):

“C’est un roc, c’est un pic! C’est un cap!

Que dis-je, c’est un cap? … C’est une péninsule! …. 34

L’animal seul, monsieur, qu’Aristophane

Appelle Hippocampelephantocamélos

Dut avoir sous le front tant de chair sur tant d’os”

(It is a rock, it is a peak! It is a cape! What do I say, it is a cape? … It is a peninsula! The only animal, sir, that Aristophanes calls Hippocampelephantocamélos had to have under the forehead so much flesh on so many bones) thus equating a funnel shaped hole in the silica to a protuberance. Few weeks later I received a phone call from Radola (at the helm of the journal) and I could hear laughter roaring in his room. “Pier Giorgio”, he exploded, “the science is great, as usual, but I hope you will appreciate that Cyrano belongs to theater and literature and can hardly enter the realm of science”. I understood. Poor Cyrano, it was a no no.

In my pilgrimages around the Sila Mountains, I chanced upon a hermitage, comprising a church and a convent erected by a local saint (Bruno) in the village of Serra San Bruno, quite popular among the natives, who visited the place asking for miracles. As I paced around the woods that surrounded the sanctuary, I started mumbling “Sanctus Bruno, ora pro me”, repeating it over and over like a mantra. Help DID NOT come from heaven ….

AN ITINERANT ROMEO WITOUTH A GIULIETTA AT THE BALCONY … It did come from earth, though. During my yearly journeys attending the CZE meetings around the world, I had met a scientist from the University of Verona, Prof. Franco Tagliaro, at the Department of Forensic Medicine, also working with CZE in order to assess drug abuse in athletes and report his data in court cases. He was using a cute process: pick a few hairs from the suspect, cut them lengthwise, elute the chemicals therein and analyse them by CZE. This would have permitted a time-long study of drug uptake, although I had always been wondering how well he would have done with bold individuals, like the famous actor Telly Savalas. We had become good friends and, hearing my sorrows, he pleaded my case with the rector of Verona’s university, Prof. Mario Marigo, who happened to be his boss. Marigo, also much involved in CZE work, thought I would be a good acquisition for this young university, made a pilgrimage to Rome and stole away a chair for me. That saved my life. After two years of exile (in what had been supposed to be a life sentence), like the Count of Montecristo, I dug a 600-mile long tunnel and surfaced in the fresh air of the Adige River in Verona. It was really an escape because, in order not to 35 alert the Mafia in the field of biochemistry, who would have made a fierce opposition to my call, the transfer and my nomination were made during August, when all of Italy was in vacation. Resurrected not after three days, but after two years. The department of Agricultural and Industrial Biotechnology there had been opened only two years earlier and was largely unpopulated. Slowly I began colonizing the top floor and, as I won just about all the grants I applied for, opened a few labs that were soon filled by several enthusiastic post docs. So, as I gently started abandoning CZE, come the year 2000 I returned in full forces to proteomics, which had become the rising star in the field of biology/medicine as the human genome sequencing had just about terminated. Unfortunately we did not have a mass spectrometer, so we had hard times in analysing all the polypeptide spots eluted from our large 2D maps, a situation that in the long run would have lead us to disaster. Just cross the highway, though, Glaxo Smith Kline had built one of the largest drug research centres in Europe, well equipped with an entire laboratory dedicated to mass spectrometry (MS). I started a fertile collaboration with the head of this lab, Dr. Mahmud Hamdan, which continued for all the eight years I spent in Verona. Mahmoud was my teacher from whom I learned the first MS rudiments and was generous in supporting our common projects, which generated many publications in top journals on proteomics and interaction of proteins with drugs and other chemicals. One of the main lines we explored was in fact the generation of chemical adducts of a variety of acrylamide monomers with proteins, since earlier it had been discovered that this chemical was a toxic agent. We indeed reported via MS analyses that it would form a stable chemical bond with the –SH groups of Cys as well as, at appropriate pH, with terminal –NH2 residues. Toxicological reports had demonstrated that plenty of Chinese workers in giant acrylamide production plants (where not even minimal safety was provided for the labourers) had developed neurological damages. We could establish a scale of reactivity, and thus toxicity, for any possible monomer and cross linker, not only the neutral ones, but also all the Immobiline chemicals, i.e., as here reported in previous sections, the acrylamide weak acids and bases that would permit the creation of IPGs. Needless to say, it turned out that the acidic ones had the least toxicity, whereas the basic ones had much higher toxicity, increasing at progressively higher pK values. The explanation was at hand, of course: for proper reactivity, the –SH groups of Cys have to be partially ionized, so their negative charge would have repelled the acidic Immobilines and attracted their basic counterparts. During these several years of friendly collaboration, we decided to write a book in proteomics and we did it, although it took plenty of work: M. 36

Hamdan and P.G. Righetti Proteomics Today (Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis and Microarray Technology; Wiley-Interscience, Hoboken, 2005). I am happy to state that it became quite popular in the field. During these 8 years my lab became a kind of pilgrimage centre for many visitors from all over Europe and as far as Australia (don’t get me wrong, though: the real Mecca of Separation Science was Uppsala, that generated two Nobel laureates and a missing third one, Svensson-Rilbe, not to mention giants such as Porath and Hjertén). In fact Ben Herbert from Sydney visited me on different occasions and spent several months in my lab. With him and Hamdan we developed a project that deeply changed the standard routine of 2D maps. For about a decade, the basic rule for sample treatment was to reduce the –S- S- bridges prior to the IPG run, but alkylation of the free –SH residues was only performed when interfacing the first to the second dimension, when the IPG strip was briefly equilibrated in the SDS buffer. That did not make much sense to us. In fact, after extended work, we found that this protocol led to disaster: in the first dimension, at any pH above 7.0, all alkaline proteins began to form again disulphide bridges, not only between homo- but also between hetero-chains. This generated plenty of spurious spots and other unexpected disasters, like β-elimination and even, upon prolonged focusing, rupture of peptides bonds with production of large fragments from the original intact proteins. We reported all these data in different papers, which received wide attention from the user’s community. But not from the leading company in the field, selling chemicals and instrumentation for electrophoresis, that adamantly defended their protocol and stated that our data were faulty (our strict recommendations were: reduction and alkylation had to be performed prior to entering the electric field, thus prior to the IPG step and NOT in between the first and second dimension, when disaster had already occurred). In those days I had been invited to all the Siena meetings on proteomics (10 of them, in the long run, for a period of 20 years, since they were run every other year starting from 1996). They had become a classic in the field and were truly overcrowded. At the fourth, 2002 edition, when I presented our data, the chaps of this company attacked me stating that their wrong protocol was the right one and that my right protocol was wrong. Hell broke lose at the evening reception in a medieval cloister: a verbal clash ensued and I, in a hungry mood, started shouting at them. The attendants were startled and, slowly and silently, they started abandoning the battlefield mumbling the refrain “libera nos Domine” (God free us [from this quarrel]). I would have not been surprised if, in order to pacify the 37 duellists, they would have forced us to kneel down and humbly sing the famous motet of Tom Lehrer from the Vatican Rag: First you get down on your knees, Fiddle with your rosaries, Bow your head with great respect, And genuflect, genuflect, genuflect though I suspect none knew it (needles to say, the year after the same company fully adopted our protocol but by stating that they invented it, see Lesson No. 5). Other important projects we developed were in collaboration with the neurological department at the Borgo Roma Hospital on prion proteins and other brain disorders such as Creutzfeldt–Jakob Disease, Alzheimer, senile dementia and others. I am glad here to recall Profs. Salvatore Monaco and Gianluigi Zanusso, whom with I run top and successful projects. Prior to leaving, I would also like to mention some of the young ladies who collaborated with me and got jobs at the University: Annalisa Castagna, Daniela Cecconi and Natascia Campostrini (all the other ones were immediately hired by GSK after graduation but most lost their job as GSK shut down its research centre in Verona).

THE RETURN OF ULYSSES TO ITHACA It took 10 years to Odysseus to return to his island, just as many as it took to me to get back to my beloved Milano. Life was tough since, after commuting in the first couple of years, I had to rent an apartment in Verona and spend all week days there, seeing my family and my two daughters (Sabina, the US citizen, and Monica, who was graduating in architecture at the Politecnico; I missed them and family relations) only on week ends. There was no way to get an appointment at the State university in my field (biochemistry) since the Mafia was still terribly upset for my escape from Arcavata di Rende. Help came from my friend Citterio at the Dept. of Chemistry at the Politecnico, with whom I had been collaborating for several years. He managed to collect 100 thousand Euros to transfer my chair from Verona to Milano (those were the rules set by the Ministry). Once there I set up a proteomics lab, an innovative experience in any politecnico in Italy, where the fauna was exclusively composed by engineers and architects (not so around Europe, where many polytechnic schools had proteomics labs). Needless to say, the lab flourished again and I spent my last six years there in a flurry of projects and publications in top journals in the field. As this story is becoming too long, I will only mention here some top projects. I teamed up with Egisto Boschetti, former vice-president of Ciphergen Biosystems and then 38 consultant for Bio Rad, to apply the revolutionary technology he had invented and patented (Combinatorial Peptide Ligand Libraries, CPLL) to real-life proteomics projects. Figure 13 offers a pictorial representation of the library and its mechanism of action. Figure 14 gives some unique results obtained when analysing the cytoplasmic proteome of the red blood cell (RBC) in collaboration with Dr. Bernard Monsarrat in Toulouse, France, and Prof. Alberto Zanella here in Milano. Whereas haematologists knew barely maybe 50-60 such proteins, we described no less than 1578 species thus sending shivers (again!) along their spines. When Zanella encouraged me to submit an abstract for an oral presentation at their congress in San Francisco, I boldly sent one by the title of “fishing on a red pond”. It was immediately rejected both as oral and poster presentation (I wonder if they suspected me of being a communist). Come my seventieth birthday, tough, due the strict national laws, I had to resign for age limits and, instantly, I become a “sans papier”, excommunicated as an illegal alien in the EC.

Figure 13: pictorial representation and mechanism of action of the combinatorial peptide ligand library developed for amplifying the signal of the low-abundance proteome (E. Boschetti, P.G. Righetti Low Abundance Proteome Discovery, Elsevier, Amsterdam, 2013).

39

Figure 14. 2D maps of a control (left side) and of species eluted from CPLLs of the proteins present in the red blood cell proteome (Mol. Cell. Proteomics 7, 2008, 2254-2269).

HOW TO APPEAR IN NATIONAL TV WITHOUT REALLY TRYING Life as a pensioner, though, might not be so bad. On the first of April of 2014 (April’s fool?) I was invited to “Uno Mattina” a most popular morning show on the No. 1 national TV in Italy. I could not believe on this invitation, but it was real. Well, finally all my work as a scientist got full recognition! No way. It all started one month earlier, when one morning a lady, Prof. of physiology at the John Hopkins University in Baltimore, while navigating in Internet, found a paper I had published in J. Proteomics (75, 2012, 914-920) by the title: Harry Belafonte and the secret proteome of coconut milk. Today most journals ask, as additional material, a TOC (table of contents), in reality an image that will appear only on the list of articles published in any given issue and not in the paper itself. As I had dedicated the manuscript to Belafonte, a staunch defender of civil rights, and quoted also some verses of the celebre song “Coconut Woman” (the story was in fact on the tribulations of this poor lady wearing down her feet in villages and beaches trying to sell a few coconuts for a few dollars), I wanted to offer her redemption into a better life and chose as a TOC the image of a lady in a bar offering coconut milk to the clients. She had a bare chest but the mammary glands were fully covered by two half coconuts; nothing exciting, simply an enticing image. This prof. in Baltimore must have been a devout Muslim and immediately wrote to the Rector of the politecnico and to the head of my department asking my skin as a sex offender. She also posted it on Facebook. The news spread like prairie fire and poor Righetti became a dirty sexist in no time at all, who used 40 sex images to enhance the visibility of his science work. She (and many others) also wrote to Elsevier asking that not only the TOC be removed, but also my scientific article be deleted from the face of earth (note that NONE had read and appreciated the science in it!). Elsevier people removed the TOC (available upon request) and suspended me “a divinis” for three months (I am associate editor of J. Proteomics). I will make it brief: I appeared in many journals around the world; luckily, conscientious newspaper men interviewed me and, when hearing the real story of the Coconut Woman and of the scientific achievements in my career, took a strong defence of my image. Remember the famous verses by Gioacchino Rossini in Il Barbiere di Siviglia:

La calunnia è un venticello, un'auretta assai gentile che insensibile, sottile, leggermente, dolcemente incomincia a sussurrar?

(The calumny is a breeze, a gentle breeze that insensitively, thinly, softly, sweetly and slightly begins to whisper) Well, this time the wind of calumny (and Facebook is an excellent vehicle for it, since nobody checks the facts and it is simply a Babel, demential confusion) turned against the promoters and this invitation came from the National TV only because I had originated this (pseudo) scandal. They gave me 13 min of TV time; it was like being judged by a tribunal, but it was huge fun and the verdict was that I was a great communicator (video available on request). I do hope that, given another chance, I will be invited for something more serious, although, I confess, it was the greatest enjoyment of my life!

IS THERE LIFE AFTER RETIREMENT? May be there is. Below you will find the cover of my first novel on World War II, to be published in a month by Reverdito Editore from Trento. I hope you will be able to read its English translation soon.

41

A volte i fantasmi tornano e quando lo fanno hanno storie incredibili da raccontare. Il fantasma “…Ivan, noi siamo costruttori di morte, ma non abbia- di Ivan trova una distorsione spazio-temporale per mo scelta se vogliamo continuare a vivere…” emergere dall’Ade a narrare le sue avventure militari. Il racconto di Ivan si snoda sull’arco di sette anni “… altissime fiammate illuminavano il teatro delle ope- (1938-1945), poco più della metà passata in camicia razioni. Si vedevano marinai, ridotti a torce umane, che nera, nella Regia Marina Italiana, per l’adempimento si gettavano in acqua, proiettili di cannone che scop- del servizio di leva, e poi come chiamata alle armi obbligatoria in quel nefasto 10 giugno 1940 in cui piavano e altri fusti di benzina che esplodevano…” Mussolini si assunse l’arbitrio di dichiarare guerra agli Alleati, proprio nello stesso giorno in cui Ivan in- “…È rimasta la leggenda dei bottoni di Napoleone, vece aveva maturato il suo congedo. […] nella campagna di Russia del 1812 i bottoni di sta- Così Ivan trascorre altri due anni a bordo dell’in- gno delle uniformi si disgregarono al gelo dell’inverno crociatore leggero Giovanni dalle Bande Nere fno a russo, impedendo ai soldati d’imbracciare il fucile. […] quando questo vascello viene affondato dalla mari- na inglese, seguendo la sorte degli altri tre membri Pier Giorgio Righetti si è laureato in Chimica a Pavia se Napoleone potesse avere un’altra chance rifarebbe i della famiglia denominata Classe Grandi Condottieri. nel 1965. Ha lavorato come ricercatore al Massachusetts bottoni con denti e zanne d’elefante” Institute of Technology (MIT) per tre anni e un quarto anno Rientrato in licenza nella sua natia Croazia, a Rijeka a Harvard (Cambridge, Mass, USA) dal 1966 al 1970. È (Fiume, Croazia), è stavolta arruolato forzatamente stato ricercatore e poi Professore Associato alla Statale di tra i partigiani di Tito e trascorre gli altri tre anni della Milano dal 1971 al 1994. Nominato Professore Ordinario, sua vita sotto l’insegna della stella rossa. ha speso due anni all’Università della Calabria, poi otto “…Certo, Alessandro aveva vinto ancora una volta, cre- Ivan annoda i fli di storie dimenticate, in un in- anni all’università di Verona e infne sei anni al Politecnico ando un Commonwealth greco secoli prima di quello treccio sorprendente per l’agilità del racconto, spa- di Milano, al Dipartimento di Chimica Industriale, dove ha britannico. Ma le sue truppe non ne volevano più sape- ziando tra eventi bellici della Grande Storia che van- creato una forente scuola di proteomica. no da Alessandro il Macedone su fno ad episodi di È stato consulente della NASA (National Aeronautics re di ulteriori battaglie ed erano tornate sui loro passi guerre romane, epici scontri dei popoli slavi fno a and Space Administration), dell’ESA (European Space alla reggia di Babilonia …” Agency) e dell’ASI (Agenzia Spaziale Italiana) per allestire Napoleone. Questo suo spaccato della Seconda Guerra Mon- esperimenti di elettroforesi e crescita di cristalli di proteine “… È stato fucilato dagli italiani. Un giorno sono entrati in micro-gravità. diale rappresenta in realtà la maturazione di una gio- Ha pubblicato più di 500 articoli su giornali scientifci nel mio paese. Ci hanno fatto uscire dalle case. Tutti ventù che a 18-20 anni si è trovata intrisa di cultura internazionali e scritto otto libri nel settore della biochimi- piangevano disperati ma mia mamma era quella che fascista inculcata fn dai primi anni di scuola ma che ca e della proteomica, alcuni adottati come libri di testo piangeva di più. […]. Hanno fucilato dodici uomini; tra proprio a causa dei tremendi eventi bellici è riuscita nelle università americane. Ha vinto diversi premi scientif- questi c’era anche mio padre. […]. Poi ci hanno brucia- a liberarsi del nero orbace e trovare la via della liber- ci a livello internazionale, tra cui il Beckman award (2012), to la casa e ci hanno portati verso l’internamento”. tà e del riscatto. Che qui, in particolare, è il riscatto lo HuPO (Human Proteome Organization) award (2014) e del popolo croato e di tutta la Jugoslavia smembrata l’American Electrophoresis Society Award (2014). dall’invasione nazi-fascista dell’aprile del 1941, che, Nel 2003 ha pubblicato un libro di racconti tramite L’Autore Libri, Firenze, dal titolo South West e Dintorni nel corso di quattro durissimi anni di lotta partigiana, (ambientato in Arizona e nel deserto di Sonora). tra stenti e sofferenze indicibili, si è di nuovo liberata Dal 2014 è stato nominato Professore Onorario al Po- dal giogo nemico. litecnico di Milano.

Figure 15. Cover of my novel Black Shirt and Red Star on World War II.

Narration terminated in this day of Our Lord 28th of September 2015 in Milano.