Dario Coletti's science cellar

postdoctoral fellow position available

2011-12-21T16:50:00.001+01:00

We are searching a postdoctoral fellow willing to join us in the frame of the founded project UPMC EMERGENCE 2011 on cardiac wasting. The location is Paris (at the UNiversity Pierre et Maire Curie) in 2012. Please, refer to the online flyer for additional information.

EXPERIMENTAL MODELS AVAILABLE IN THE LAB (2011)

2011-12-21T11:16:00.001+01:00

A quick overview of the experimental models currently available in the lab to study the hormonal control of muscle differentiation and homostasis

LAB METHODS: Assessing cell number with a counting chamber

2011-12-21T10:13:00.000+01:00

A Chamber with a View...each one has a different one. Supposedly trivial, estimating the number of cells in a cell suspension is matter of never-ending debates in our labs, that's why we finally wrote instructions for either a Thoma or a Malassez chamber, two widely used options in the lab.

LAB METHODS: Cardiac Stem Cell Isolation

2011-12-16T16:05:00.000+01:00

Claudia Serradifalco, a PhD student from our collaborators' laboratory at the University of Palermo, has established in Paris their simple and elegant method to obtain Cardiac Stem Cells from adult rat hearts (link to the original paper by Di Felice et al.) by adapting the procedure to our laboratory. Here is the modified protocol...

Blind tasting session at the lab

2011-12-16T15:19:00.002+01:00

To welcome a new PhD student in the lab, we organized a tasting session of charcuterie. This included a celebrated home-made fois gras and several salami from different countries. The latter were married to Chardonnay-based wines, a Mersault 1er cru 2008 A. Bouteller and a Chablis VV 2010 Vaucher & fils. For the fois gras I proposed an Alsace Gewurztraminer 2006, moelleux and traditional, by Schueller.A blind testing session concerned four salami and four sausages from the following countries and regions:A) Spain, Cataluna; B) France, Auvergne; C) France, Aveyron; D) Italie, Lombardia (Varzi) and Lazio (Cassino)

Most loved:Category “saucisson (big salami)”: region Avyron (France), producer Linard;Category “saucisse (small sausage)”: region cataluna (Spain)

ARTICLES: Teodori et al. Chimica e Industria 2011

2011-12-07T09:29:00.001+01:00

THE ADVANCEMENT OF SCIENCE: SHARING OR EXCLUDING? THE “NEW BIOTECHNOLOGY DIVIDE”: AN ALARMING PERSPECTIVE OF SCIENTIFIC DUAL USE Linked to the title above, you can find the full version of our paper on biotech divide, an emerging issue related to recent advances in information technology (IT).The role of scientists is of paramount importance in understanding and predicting the impact of their research in issues related to thethreat of conflicts inherent in a polarized society. They must increase their own awareness of these issues and better informthe political community by advising and helping to assess programs of cooperation that will lead to more equitable access to benefitsand reduce inequalities driven by the technology divide. This article will focus on the use, distribution, and accessibility of researchoutcomes in one particular area of biotechnology, i.e. technology related to health care. We have identified some biotechnological barriers, given some specific examples of positive action in the field of our expertise in bridging such a divide and highlighted the direction we believe should be followed.

LESSONS: REGENERATIVE MEDICINE

2011-10-27T11:34:00.000+02:00

Linked here you can find a presentation dealing with regenerative medicine (in French/ oui, en Français!) for the master students in "Molecules and therapeutic targets". The presentation consists of three parts: 1) stem cells and their therapeutic use 2) what is tissue engineering 3) strategies of the regenerative medicine: in situ regeneration, stem cell transplantation, transplantation of pre-assembled organs. A similar lesson, more focused on tissue engineering and in Englligh is visible here.Learning about the outstanding capacity of regeneration shown by the newt will allow the full regeneration of human organs? Hopefully better than what we are currently doing.

IS THIS BLOG GOING TO BE SHOT DOWN?

2011-10-05T13:33:00.000+02:00

What is going on in Italy? Today, the Italian Wikipedia is ON STRIKE and one has only access to this sober communicate (screen image from the Wikipedia homepage http://it.wikipedia.org/wiki/Wikipedia:Comunicato_4_ottobre_2011 )

Unfortunately, this communicate is in italian, so I will try to summarize it below.The Italian parliament is discussing a bill to strongly limit the publication of texts deriving from phone call tapping used in trials. Wikipedia cites the text of the bill and remarks that a modification of the bill proposed this morning will force the responsible of a web site to rectify within 48h a given information that is possibly considered incorrect by anyone who finds this information as negatively affecting his image. All this by default, without any third party judging the dispute. It is feared that the easiness of the censoring action will discourage everybody from saying just anything on anyone, in sharp contrast with the article 27 of the Universal Declaration of Human Rights.All this happens while the bill to help the agonizing Italian economy is postponed. If you have recently read the content of hot conversations between our Prime Minister and his friends reported worldwide by media, you may imagine the reason why such bill is more urgent than boosting the economy.Indeed, I find reasonable to foresee the risk that such a proposed bill will affect any web site posting free contributions related to whatever subject, including this blog which has hosted in the past political opinions and analyses (concerning science in most of the cases). Shall I shoot it down rather than checking every 48 h not to have received an injunction of rectification?

ARTICLES: Perniconi et al. Biomaterials 2011

2011-07-22T18:29:00.005+02:00

In this paper in press we show that one can transplant ghosts of tissues to obtain again the corresponding tissue in mice. Our "ghosts" are acellular scaffolds derived by whole organ decellularization (in 1% SDS) of skeletal muscles (the EDL and the TA). Therefore, they represent the extracellular matrix voided of the cellular component. We show that this biomaterial has niche properties, since it is able to support neomyogenesis once transplanted to replace the matching muscle.

LAB METHODS: transplantation of an acellular scaffold to replace the corresponding muscle

2011-07-16T16:23:00.011+02:00

We are about to publish a paper where we characterize the in vivo response to a graft composed by an acellular scaffold obtained by a previously decellularized skeletal muscle. The grafting procedure is now available as a ppt - link embedded in the title of this post. The corresponding video on how to replace a TA with the corresponding acellular scaffold(iPod version) is available through the link in parentheses. For an alternative format, try to click here (avi version). The video is supplemented as Additional materilas to the Biomaterials article.

LAB METHODS: Toluidine blue staining

2011-07-16T16:05:00.007+02:00

There is no staining method as fast and informative (two for the price of one!) as the Toluidine blue staining. We use it while cryosectioning or while doing semithin sections to monitor sample quality and orientation. Toluidine specifically stains some cell and ECM features. Linked to the title of this post, you'll find our method for Toluidine staining, with references and additional examples. Fig. legend: Toluidine-stained skeletal muscle cryosections.

Research fundings: an update...

2011-07-16T15:14:00.008+02:00

Well...I was too pessimistic. The fundings for the Fiscal Year 2009 ("PRIN 2009") has been released by the Italian Ministry of University and Research , with a delay of only three years and not four years, as I was foreseeing.
That's good news, worth at least a bottle of Prosecco di Valdobbiadene Giustino B. by Ruggeri!
That is also a good chance to have a look at what the USA are doing. Linked to the title is the analysis of the current presidential plan for R&D in that country. President Obama requested $ 147,696 bilion for research in the current Fiscal Year. With this rate they will DOUBLE the fundings in 11 years. Linked to the title, please find the full text of the analysis of this plan.

Left:
Research & Develoment funding path in the USA

Source:
Federal Research end Development Funding - FY 2011
JF Sargent jr., coordinator, specialist in Science and Technology Policy
June 10, 2011

Blind tasting session at the lab

2011-06-20T18:46:00.008+02:00

To celebrate a few recent events (the UPMC Emergence 2011 grant, the Mol Endocrinol paper) and to welcome a new student in the lab, we have tasted five Bordeaux 2006 wines, from different appellations characterized by marked nuances of their terroirs and specific grape assembly. Given that the different wineyards are only about 50 Km from each other, the differences were outstanding.

Results of the blind tasting (panel : laboratory members):

1st Château-Haut Maurac, Médoc Cru Bourgeois (60 % Cabernet sauvignon, 40 % Merlot)
2nd Château Musset Chevalier , Saint Emillon Grand cru (50 % Merlot noir / 45 % Cabernet-Franc / 5 % Cabernet-Sauvignon )
3rd Les Hauts du Tertre, Margaux (55 % Cabernet sauvignon, 20 % Merlot, 20 % Cabernet franc, 5 % Petit verdot)
4th Château Prieuré-les-Tours, Graves.

We liked the winner for its intense bouquet of red fruits and its full body, with mature tannins and a long lasting aftertaste. One more cru Borgeois showing the great quality/price ratio of this category. From the color to the marked tannins it expressed the Medoc pretty well. However, I preferred the Margaux of Les Hauts de Tertre, a second wine produced by Château du Tertre, for its elegance and its more floreal bouquet. Margaux came out in the good balance between tannins, acidity and alcoolic warmth. The superb roundness of the Libournais St Emillon and the acidity of the Graves (Alas! - in such a poor interpretation) came out as well, but nobody guessed the crus for all the wines.

ARTICLES: Toschi et al. Mol Endocrinol 2011

2011-06-20T12:11:00.009+02:00

In this paper, entitled "SKELETAL MUSCLE REGENERATION IN MICE IS STIMULATED BY LOCAL OVEREXPRESSION OF
V1a-VASOPRESSIN RECEPTOR", we identify skeletal muscle as a physiological target of hormones of the vasopressin (AVP) family and show a novel in vivo role for vasopressin-dependent pathways. FIG LEGEND In red Myc (i.e. overexpressed V1a-R) immunolocalization in skeletal muscle fibers highlighted by laminin staining in green.

In the last 10 years, we have characterized in detail AVP signaling pathways in myogenic cells in vitro. Also, we have reported that the muscle specific, V1a, AVP-receptor is modulated during myogenic differentiation in vivo, which suggest a role in muscle development. Consistently, we have shown that AVP intramuscular injection enhances muscle regeneration, a process which recapitulates muscle development in the adult.
With the last paper by Toschi et al. we formally demonstrate the biological role of AVP on skeletal muscle homeostasis and we pinpoint some molecular mechanisms underlying this effect, including calcineurin-mediated IL-4 production in the musculature in response to AVP.

FIG LEGEND role of Calcineurin-dependent effects of V1a-R overexpression on muscle regeneration.Further links to the press which cited the article: ANSA and Corriere della Sera

Against cuts in cultural funding

2011-06-20T11:12:00.008+02:00

Again a non scientific, still relevant post insomuch as politics affect culture, research and education.

Cuts on culture and arts.
We have recently celebrated the 150th anniversary of Italian unification. As reported by the New York Times, a very intense moment occurred when Riccardo Muti conducted the "Va pensiero" at the premiere of Verdi's “Nabucco” at the Teatro dell’Opera in Rome in March, in the presence of the Prime Minister and the Mayor of the capital.
The issue was the heavy cut plan on cultural founding performed by the current government. The event had its climax at Muti's brief statements against this plan while introducing an exceptional bis of the "Va pensiero". Linked to the title of this post there is the touching video on youtube.

Cuts and management of university funding.
University budget cuts represent the other branch of the current harmful intervention on state budget, in a country which spending on university is already very low as compared to most other countries, as reported by the BBC last year. However, it is not only a matter of budget. What is even worse is the total incertitude for the CURRENT available fundings: in 2011 we are still waiting for the results of a major funding call of the Italian Ministry for the University and Research (MIUR) which is called PRIN 2009 and was released in 2010! In 2012, if and when some groups will receive the grants to which they applied three years before, what will remain to be accomplished of the proposed research projects? Won't the latter be born already aged and out to date?
A lucid analysis on the inceritude which reigns on italian university has been published a few months ago on the web pages of lavoce.info (in Italian).

LESSONS: Mechanisms controlling skeletal muscle homeostasis

2011-04-29T09:22:00.009+02:00

Linked to the title there is a lesson for Master students (in English) on the mechanisms controlling skeletal muscle homeostasis.

OVERVIEW:

SKELETAL MUSCLE HOMEOSTASIS, HYPERTROPHY AND ATROPHY
The skeletal muscle tissue accounts for the majority of our body mass, nonetheless, the amount of skeletal muscle can vary significantly throughout life. There are specific mechanisms finely tuning the exact amount of muscle that we have at a given time.These are apparent in conditions far from homeostasis, i.e. when we have an excessive growth (hypertrophy) or reduction (atrophy) of muscle fibers. Throughout the presentation, I also try to state the case that not only muscle protein metabolism is important for controlling muscle homeostasis but also muscle stem cells support a "flow" of myogenic cells contributing to the maintenance of muscle fibers.

EXPERIMENTAL MODELS FOR STUDYING SKELETAL MUSCLE HOMEOSTASIS
Where I presents different approaches to study the regulation of muscle differentiation, growth and repair in vitro and in vivo.

MUSCLE ATROPHY, WASTING, CACHEXIA
Where I present different forms of muscle fiber atrophy and present in detail the features of the most severe form of muscle wasting, the syndrome of cachexia.

ENDURANCE EXERCISE & PROTEIN METABOLISM
Where I present some experimental data on exercise effects on muscle metabolism and homeostasis in physiological and pathological conditions.

MUSCLE REGENERATION IN PATHOLOGICAL CONDITIONS
Where I presents mechanisms whereby skeletal muscle regeneration is affected in cachexia, ultimately providing the molecular explanation for an important deficit in muscle regenerative capacity accounting for loss of muscle mass.

SUGGESTED READINGS

Glass D. 2003 Molecular mechanisms modulating muscle mass

Moseri V. 2010 Myogenin and calss II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases

Musaro` A. 2004 Stem cell mediated muscle regeneration is enhanced by local isoform of Insulin-like Growth Factor 1

Zhou X. 2010 Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival

The Real Face of Death

2011-02-15T15:49:00.004+01:00

A creative, fanny interpretation of a real TEM image representing an eucariotic cell in culture undergoing apoptosis (programmed cell death). Apoptosis major features are represented in this photomicrograph: loss of cell attachment, but maintenance of cell integrity, cell shrinkage, nuclear fragmentation and chromatin condensation.

ARTICLES: The Problem of Subjective/Objective Genitive in Matters of Heart

2010-11-29T16:57:00.004+01:00

Following recent discoveries of cardiac wasting in cachexia of both cardiac and non-cardiac origin, it appears that the heart can be both the trigger and the target of cachexia. By analogy to muscle cachexia the use of “cardiac cachexia” may arise in a double subjective and objective sense inducing misleading interpretations. We recently got the following comment published on Cell online.

Comment on:
Cell, 20 August 2010, Volume 142, Issue 4, 531 - 543
doi:10.1016/j.cell.2010.07.011
Article
Reversal of Cancer Cachexia and Muscle Wasting by ActRIIB Antagonism Leads to Prolonged Survival
Xiaolan Zhou, Jin Lin Wang, John Lu, Yanping Song, Keith S. Kwak, Qingsheng Jiao, Robert Rosenfeld, Qing Chen, Thomas Boone, W. Scott Simonet, David L. Lacey, Alfred L. Goldberg, and H.Q. Han

by:
Dario Coletti
Barbara Perniconi, Sergio Adamo, Zhenlin Li, Denise Paulin, Mathias Mericskay
19 novembre 2010
10:21:04 HNEC
Affiliation:University Pierre et Marie Curie Paris 6, France & Sapienza University of Rome, Italy

The Problem of Subjective/Objective Genitive in Matters of Heart

Zhou et al. uncovered a previously unappreciated loss of heart mass in cachectic mice for which, from our point of view, they correctly used the expression “atrophy of the heart”. This study is likely to generate a new line of research, which is distinct from cardiac cachexia, i.e. the atrophy of the skeletal muscle induced by cardiac pathologies. We urge to clarify the terminology to describe these phenomenons, since we foresee the risk of misleading use of related expressions, e.g. cardiac cachexia and cardiac atrophy, sounding alike but very different de facto one from the other. In particular, “cardiac cachexia” poses a problem of ambiguity, thus its use might be abandoned.
The old problem of the subjective/objective genitive case.
Does amor patris (father's love) mean that the father (pater) loves his children (subjective genitive) or that the children love their father (objective genitive)? The father can be either the subject or the object of the action of loving. There is no difference in form between the subjective and the objective genitive. Only context can make a final determination. In addition, English does not typically mark nouns for a genitive case morphologically. Rather, it uses the Saxon genitive “ 's” for people or the preposition “of” like in “Molecular Biology of the Cell”. Biology of the cell can also be referred to as “Cell biology” by using the noun as adjective.
What is cardiac cachexia, then?
About 200 papers to date referred to cardiac cachexia as a syndrome of skeletal muscle wasting associated to a specific pathology, i.e. Chronic Heart Failure (CHF). Cardiac cachexia is not meant as cachexia of the heart, i.e. cardiac atrophy. However, the existence of a cardiac component of cardiac cachexia has also been reported (Florea et al., 2002). Several papers demonstrate the growing use of “muscle cachexia” referred to as skeletal muscle wasting associated to a chronic disease, including CHF (Pajak et al., 2008). Following the recent discoveries of cardiac wasting in cachexia of both cardiac and non-cardiac origin (Florea et al., 2002, Zhou et al., 2010), it appears that the heart can be both the trigger and the target of cachexia. We are concerned that, by analogy to muscle cachexia, the use of “cardiac cachexia” may arise in a double, subjective/objective sense.
Different names for cardiac muscle wasting: PROs and CONs.
The following are alternative ways to refer to the phenomenon of cardiac muscle wasting.
1) Atrophy of the heart. This expression is unambiguous, even though relatively long. It is very general and implies the need to clarify the context in which the atrophy arises, e.g. cancer-induced atrophy of the heart to specify that the latter is triggered by cancer.
2) Heart atrophy, which is formally ambiguous in terms of subjective/objective genitive.
3) Cardiac atrophy, which has the same risk and sounds dangerously similar to cardiac cachexia.
4) Cachectic heart, a more holistic expression not entirely described to date.
5) Cardiac wasting, which has a nuance toward pathology and appears as a specific, easily discernible expression. Therefore, we are in its favor.
Highlighting the atrophy of the heart in the definition of cachexia.
The current consensus definition of cachexia, “Cachexia is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle [...]” (Evans et al., 2008), does not explicitly refers to the loss of cardiac muscle. We think opportune to refer to both skeletal and cardiac musculature when reporting about muscle wasting in cachexia.
References
Evans, W.J. et al. (2008). Clinical nutrition (Edinburgh, Scotland) 27, 793-799
Florea, V.G. et al. (2002). American heart journal 144, 45-50
Pajak, B. et al. (2008). J Physiol Pharmacol 59 Suppl 9, 251-264

Published online on 11/19/2010
http://www.cell.com/comments/S0092-8674(10)00780-4

LAB METHODS: isolation of skeletal muscle fibers

2010-08-31T17:28:00.003+02:00

The procedure after Dr. Peter Zammitt's...linked here are the figures of our version of the method

LAB METHODS: the simplest mycoplasma test

2010-08-31T17:11:00.007+02:00

Mycoplasma is a common contaminant of cell cultures throughout the wolrd. It affects experimental results and it perturbs cell behaviour. Mycoplasma is a genus of bacteria that lack a cell wall.Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. It is very difficult to get rid of mycoplasma: the best is to screen regularly for contamination and to throw out the contaminated cells! Here is the method for the screening....in italian

RELOCATING IN PARIS - UPMC

2010-08-31T16:44:00.004+02:00

NEWS! Effective September the 1st I am at the University Paris VI Pierre et Marie Curie. I do teaching and research as Maître de Conférences. When I left Italy for the USA I had in my luggage a bottle of Barolo Granbussia 1982 by Aldo Conterno. It was a great experience - both the wine and postdoc! This time, coming to Paris, I put in my bag a Barolo Vigneto Arborina 2005 by Elio Altare - thanks Elio! I got to teach something to these guys...

ARTICLES: link to Aulino et al. BMC Cancer 2010

2010-07-30T08:45:00.009+02:00

In the paper linked above we perform an extensive molecular, cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse. This widely diffesed murin model of cancer and cancer associated cachexia was originally developed by Corbett et al. in 1975, during an effort to establish an animal colon tumor model for biological and chemotherapy studies. Colon tumors were induced and transplanted in different inbred mouse strains. Four tumors survived the first transplant, which displayed a variety of histological and malignancy features. These four tumors included the colon tumor 26, described as an undifferentiated Grade IV carcinoma.
Since then, the model spread around the world and was exploited for a pletora of studies in the absence of an organic description of its main features.This gave rise to misunderstandings and mistakes, such as describing the C26 tumor as an adenocarcinoma without showing histological images.Today, the communities of scientists exploiting the C26 model to study either cancer or cachexia are not fully aware of each other’s works (as shown by cross-reference analysis on PubMed) and this may have deleterious consequences for the progress of integrative medicine applied to a complex syndrome associated with underlying illness. We suggest that “C26” be included among the keywords whenever work is conducted on this experimental model to provide adequate visibility.

Among the NOVEL DATA that we shown in this paper, I'd like to stress the functional analysis of cachectic muscles. We found that while the drop in muscle force is a hallmark of cachexia, it is simply due to muscle atrophy. In fact, no differences exist between normalized force (i.e. specific force) of control and cachectic animals. On the other hand, murine cachectic muscles are characterized by fatigue, which is in agreement with clinical observations. Left: C26 H&E staining. The measures shown below refer to the EDL muscle of ctr and C26-bearing mice.

LESSONS: Materials for the students of Tissue engineering School of Dentistry

2010-06-30T12:03:00.006+02:00

Attached to the link there is a summary (in Italian) of basic concepts of Tissue engineering for the sutudents of the School of Dentistry.
IN allegato - link nel titolo - una dispensa con i concetti base di ingegneria tissutale presentati nell'ADE per gli studenti di OPD.
Linked to this text there are several articles on this topic.

A review...

LAB METHODS: 3D acellular scaffold from skeletal muscle

2010-06-24T13:56:00.006+02:00

The method linked here is a simplified procedure to obtain an acellular scaffold suitable for cell culture of myoblasts into a 3D scaffold.

Cachexia, sarcopenia, inactivity: the three Fates (Moirae) of muscle atrophy

2010-05-26T15:06:00.008+02:00

A common question is: what is the difference, if any, between muscle atrophy conditions which look all similar? What happens to our muscles when one lays in bed with a brokne leg, as opposed to getting cancer or happily ageing? Dr. Evans, one of the wolrd experts on muscle atrophy, simply and concisely pinpoints the differences in this review...

from:
Am J Clin Nutr. 2010 Apr;91(4):1123S-1127S. Epub 2010 Feb 17.
Skeletal muscle loss: cachexia, sarcopenia, and inactivity.
by Evans WJ.
Division of Geriatrics, Department of Medicine, Duke University Medical Center, Durham, NC 27709, USA. william.j.evans@gsk.com

LAB METHODS: CMFDA cell tracker

2010-05-25T17:14:00.003+02:00

The protocol linked here is a method to label live cells and assess whether they are VITAL and/or STRESSED. The dye in fact accumulates inside the cells due to a chemical modification that makes it non-cell permeant. This esterase reaction requires glutathione. Therefore a shift toward low fluorescence characterizes a cell population with depleted levels of glutathione, i.e. that have been subjected to oxidative stress - GSH being the principal buffer of the redox state.

caveat for the use of the Molecular Probes anti-Mouse Alexa Fluor 350 Ab

2010-05-24T18:12:00.004+02:00

Linked here is the comparison between two Molecular Probes secondary antibodies: anti-M AF350-conjugated vs the classical AF488-conjugated. Thanks Paola for trying...
Looking at something in blue is cool but rather blues!

Il caso e la necessità, Le hasard et la nécessité, Chance and Necessity

2010-05-24T10:49:00.002+02:00

"Tutto ciò che siste nell'universo è il frutto del caso e della necessità"
"Tout ce qui existe dans l'univers est le fruit du hasard et de la nècessité"
"Everything in the universe is the fruit of chance and necessity"
Democritus, circa 460-370 BC
IN: Jacques Monod, Le Hasard et al nécessité: Assai sur la phyolosophie naturelle.

Léon-Alexandre Delhomme (1841-1895)
Description
Democritus che medita sull'anima, bronzo, 1868.
Démocrite méditant sur le siège de l'âme, bronze, 1868.
Democritus meditating on the seat of the soul, bronze, 1868.

LESSONS: Regenerative Medicine & Tissue Engineering

2010-05-19T19:27:00.008+02:00

Here is the link to the ENGLISH VERSION of an introductory lecture to tissue engineering. The principles of the latter are summarized, and various strategies for regenerative medicine are presented through discussion of the most recent outbreaking reports in the field. For the 1st y medical students and biotechnology students. Talking about students, I have to acknowledge the great work done in the lab by Barbara Perniconi and Alessandra Costa (some of their data on acellular skeletal muscle are shown). A similar, broader lesson on regenerative medicine in FRENCH has been published here.

Figure legend: confocal image of a 20 micron tick cryosection of a murine acellular skeletal muscle matrix (laminin staining, red)

A flu dealed with a secret treatment - article by Nerina Dirindin

2010-01-08T09:39:00.003+01:00

Linked to the tile is an article, published by our great online magazine Lavoce.info, on the incredible management of the flu crisis by the Italian Minister of Health. The article is in Italian, but I wish to summarize it in English asap. Below is a little taste of the content...

About 4% of the Italian population has been vaccinated against the flu virus, which has recenlty given rise to pandemic emergency. On the other hand, the Italian government has bought from Novartis viruses for 40% of the population. This is not the real issue, though (except for singificant stocking and expiration problems). What is unbeliavable, is that the Ministry of Health has purchased the vaccines by using a procedure allowed by the Prime Minister (ordinanza n. 3275 del presidente del Consiglio del 2003) for cases of risk of terrorist attack. This procedure is secret, direct and charges almost all the burden on the government rather than on the company - the criticism comes from our Corte dei Conti (the top organ of suveillance of public expenses). For istance, the government will take care for all the expenses due to adverese effects or aother complications in the population underging vaccination. To date, it is not clear the cost of such operation.

LAB METHODS: Esterase staining

2009-12-18T11:46:00.003+01:00

For aspecific esterase staining (detecting macrophages and neuromuscular junctions), we follow the method linked to this title and published by NEUROMUSCULAR DISEASE CENTER Washington University, St. Louis, MO.
A typical result depicting macrophages invading a necrotic muscle fiber is shown

LAB METHODS: Caspase activity staining

2009-12-16T14:35:00.007+01:00

Linked here there is an in situ method - IN ITALIAN, TO BE TRANSLATED ASAP! - to detect caspase activity on tissue cryosections, originally developed by our group. Validation in Supplemental material of Moresi et al. Stem Cells. 2008 Apr;26(4):997-1008 (PMID: 18258721)

The future of Italian university and the last "reform"

2009-12-16T10:25:00.013+01:00

We are having a hot debate on the whole university system in Italy nowadays. Lavoce. info, the best online magazine in our country, is covering the issue -the last article on this topic is by Daniele Checci and Tullio Jappelli.
ANPRI, a National Association of Researchers has organzied a workshop in Rome last year, entitled "A Future for the Italian Public Research: Autonomy, Evaluation, Resources". Linked to the title of this post there is the presentation which represents my contribution. Below, the English version of the corresponding text. FIGURE LEGEND. Yearly impact factor of my publications plotted over the months I spent abroad (as compared to Italy) in the same year.

ANRPI WORKSHOP: Rome, Nov 24 2008
STAYING OR LEAVING (AGAIN): A HARD CHOICE

The following is a witness. As a scientist who does research in Italy and abroad I am able to highlight some data on my activity in the two environments. I hope this rationalization process can be useful to me for strategic choices in the next future and to others for discussion. I present my personal experience. In a scientific report, the following would be a case report rather than an epidemiologic study.
I am male, 37 year old, I am an Italian citizen, married with 1 child. Following the MS in Biology, summa cum laude, at the Sapienza University of Rome (1995) I did my PhD in Cell Science and Morphogenesis at the same University (2000). During the doctorate, I was visiting scholar at Stanford University, CA. My postdoctoral training was done at the Mount Sinai Hospital, NY (2000-2003). Back in Italy (2004) with the brain storming program “Rientro dei Cervelli”, I was contract professor. In 2007 I was invited researcher at the University of Paris VI-PMC. From 2005 I have a tenured position as research associate at the Sapienza University of Rome. I am responsible of the laboratories of electron microscopy and calcium imaging, I teach at the School of Dentistry, I am member of the editorial board of Basic and Applied Myology, member of scientific societies and author of articles published in peer-reviewed international journals. In brief, I do research in an international context.

When did I perform the best researches of my career? To answer this question, I evaluated my scientific production by mean of the Impact Factor (IF), an empiric evaluation factor calculated by the Institute for Scientific Information. The IF mainly refers to the frequency of citations the articles published in a given journal generate, assuming that this fact mirrors the “impact” of the articles on the scientific community. This being said, the IF is often used as a mean to assess the quality of the scientific production of a given researcher, on the basis of the assumption that any article published in journals with high IF is also relevant.

The figure (bar graph)depicts the kinetics of two variables of my professional life: the months/year abroad (red bars) and the total IF/year (blue bars) of my publications. To read the graph it is important to take into account a right-shift (i.e. temporal shift) of the IF bars, since a researcher first does the experiments and then the results are published. In other words there is a delay in the outcome (blue bars) of one’s work (red bars). This graph suggests the following considerations: 1) my IF is growing with time, possibly due to increased productivity thanks to growing experience; 2) a correlation exists between staying in a foreign laboratory and IF; 3) I was able to publish decently while in Italy (also thanks to collaborations with foreign groups); 4) a period abroad, even a short time, boosts my IF. The conclusion is that abroad I have been more productive. Why?

The American research system has two main features: it is attractive and it is productive. USA is an attractive country, on a professional point of view, thanks to the financial investment (worth noting, the greatest financing agency in the US is NIH, thus a public granting agency). US investment in research is not particularly big, around 3% of the Industry Gross Product. However, this is a significant amount of money, since the USA IGP is big. Such investment is sufficient to make the USA a leader country in research. For young people aiming at a career in research it is mandatory a significant experience in a leading country - a sort of grand tour of the modern era. Young PhD from around the World plan to work in the USA, thus, the America laboratories can be picky in selecting candidates. The final result is that the initial investment attracts the crème of world researchers. These people move to the States at the top of their youth and energy, between the end of their studies and the beginning of their career as independent investigators. Most of these people are very determined to exploit their stay at the best, which means maximal production (of scientific results) in the shortest time possible. The process described above produces a plethora of scientific articles, patents and other “products”, including the future generation of scientific leaders for American companies and universities. Thus, the most dynamic population of international researcher is captured by the American system. The remaining population goes back to the countries of origin, adding burden to their welfare system. On the contrary, the international population of young researchers had just a minor impact on the welfare of the USA.

Italy barely invests 1% of its IGP in research, it is the Cinderella of research and it is characterized by its scarce ability to attract scholars from abroad. In our laboratory we always had Italian students and postdocs. I remember a single episode of interest from abroad: we were contacted by and Indian student, who then preferred to move to a Korean laboratory. Our system is characterized by: 1) poor resources, thus, 2) creative ways to solve problems (e.g. finding the cheapest way to perform an experiment). This, in turn, implies 3) high personal effort (e.g. I work longer than what is allowed by my contract, since the same experiments require a longer effort here than in the States). Also, unfavourable are 4) the high costs (e.g. the same product is cheaper in the USA than here, also thanks to their vast market), in spite of 5) grants that are small(er and smaller) and often handled in mysterious ways.

The conclusion is that the hubris of the low Italian investment in research – way down as compared to USA or to what recommended by the EU – determines a vicious circle leading to a great personal and economical disadvantage to perform research in Italy. The economical side should be stressed, also keeping in mind that research institutions are somewhat similar to business companies: research and teaching activity determines additional working positions, investments and expenses which have a great impact on the national market. Thus, it is OK to reason with business logics: there is need of major investments, assessment of quality to relocate these resources, and autonomy in their management. Our laboratory relies mostly on non-national funding (French charities, EU, etc.), and we go though international calls, based on peer-review judgments (the criticisms to a research project, performed by a colleague who is an expert in the filed- the anonymous comments are sent by the granting agency to the group who presented the project, so they know why they have been granted or rejected). We never knew why our projects were granted or not by the Italian Ministry of Research.

I would like to end by sharing a few questions that I am facing now. I think these questions are not just a personal matter. Rather, they are central in relation to the debate of the future of Italian research.
- in which institution I have, now, the greatest chances to perform at best my research and teaching activities?
- in which Country my career will have the best perspective of programming and growth?
- where will I have the best access to resources needed for my job?
- how can mobility and competition be increased within and among Italian universities and research institutes?
- why these institutions contribute to a rich and complex R&D mix abroad?
- can research institutions be a target for interventions aimed at stimulating economic growth?
- how to make the distribution of resources efficient, avoiding a generalized micro-financing on one hand and the generation of exclusive clubs and lobbies on the other hand?

Methodological papers: my favourite hits

2009-12-15T18:25:00.012+01:00

FIGURE LEGEND: Cross-sectional view of a Tibialis Anterior injected with GFP DNA (green), electroporated in our Roman lab following Donà et al., and immunostained for laminin (red).

Some times a scientific article is outstanding because of its breakthrough findings, some times it is particularly elegant in its demonstrations, some other times an article is just so useful! The following are some of my favourite methodological papers.

For people working on gene delivery by electroporation to skeletal muscle tissue, Donà et al. carefully address which are the best settings for the square wave electroporator. The efficiency of this approach can be pretty high, as shown by this article and by the image above. We often perform electroporation-mediated delivery of two different plasmids with the aim to obtain co-expression. Which is the correct ratio between the two constructs? For instance, if we want to co-express Green Fluorescent Protein (GFP) as an expression marker, which amount of the plasmid of interest must we combine with the GFP to be sure that it is expressed by the fibers that turn out green? Rana et al. have found that co-expression rate between BFP and GFP in skeletal muscle is about 100% in their experimental settings.

A caveat on the use of GFP comes from this paper by Goodell and co-workers: they explain why skeletal muscle fiber-specific green autofluorescence can generate potential artifacts and show how to discriminate between autofluorescence and GFP signal. There is a (fake) green side and a dark side of GFP, i.e. the fact that its expression interferes with polyubiquitination. Those working on proteasome-mediated protein degradation should be very careful when ectopically expressing GFP, as shown by Baens et al.

To assess cell damage it is possible to exploit Evans Blue Dye, which is cell-impermeant unless the plasma membrane is damaged. A complete characterization of its use to label muscle fiber damage is described by Hamer et al. They tell you everything you wanted to know on EBD and you never dared to ask.

YACs, BACs, PACSs…are they Dr. Seuss’ creatures or carriers for successful generation of transgenic mice when transfer of large fragments of cloned genomic DNA is needed? Giraldo and Motoliu will tell you.

When we deal with myogenic cell cultures, we often obtain a mixed population of myotubes that have differentiated in the presence of a residual population of unfused myoblasts, i.e. syncitia vs single cells. The paper by Kitzmann et al. is not only a great JCB paper on cell cycle-dependent regulation of MyoD and Myf5 but also provides a trick to separate myotubes from myoblasts.

It makes no sense that we produce data if we do not know how to handle or to communicate them. I was struck by the news that about 50% of scientific articles contain errors of data analysis or reporting. C.H. Olsen reviews the use of statistics in articles published – yes, published – in Infection and Immunity and discusses the most common mistakes.

LAB METHODS:vital blood collection from mice

2009-12-15T13:37:00.000+01:00

LAB METHODS: Muscle fiber damage by Evans blue dye staining

2009-12-15T13:31:00.004+01:00

Linked here is our method for labeling muscle fibers suffering of plasma membrane damage, e,g, following freeze injury. Based on the great methodological paper produced by the group of Miranda Grounds.

LAB METHODS: A brief guide to suturing material

2009-11-24T16:48:00.005+01:00

How to read the label of a needle (i.e. what does EP1USP5/0CM45TT17 mean?!?), why do we use silk, what is needle curvature etc.
For surgery procedures, see great tutorials at
http://cal.vet.upenn.edu/index.php?page=principles-of-surgery

LAB METHODS: IF analysis of BrdU incorporation in regenerating muscle fibers

2009-10-29T17:00:00.001+01:00

ARTICLES: link to Coletti et al. Cytometry 2007

2009-10-09T16:30:00.002+02:00

In this paper we present a novel biological effect of Static Magnetic Fileds (SMFs), i.e. enhancement of myoblast allignment and fusion in vitro, that can be exploited for biotechnological applications as well as a model to study mechanisms underlying SMF biological effects.

LESSONS: Microscopy and histological analises

2009-10-09T16:25:00.003+02:00

Introductory lesson to microscopy and histological analysis, for undergraduate student at the School of Dentistry. In Italian.

Italian Court Rejects Prime Minister’s Immunity

2009-10-09T13:52:00.003+02:00

This has nothing to do with science, but I propose to celebrate what ruled by our "supreme court" on wednesday with a bottle of Amarone Bertani (mythical bottles from the '60s) - a wine as strong and long lasting as our Constitution, which states citizens equality under the law.

More details on the NYT..

LESSONS: Blood & Hematopoiesis pt IV

2009-10-07T10:03:00.002+02:00

For the students of the School of Dentistry. In Italian.

LESSONS: Blood & Hematopoiesis pt III

2009-10-07T09:53:00.003+02:00

For the students of the School of Dentistry. In Italian.

LESSONS: Blood & hematopoiesis pt II

2009-10-07T09:51:00.002+02:00

For students of the School of Dentistry. In Italian.

LESSONS: Blood & Hematopoiesis pt I

2009-10-07T09:47:00.003+02:00

For students of the School of Dentistry. In italian.

LAB METHODS: Antibodies available in the lab

2009-10-06T14:43:00.004+02:00

This slides illustrates the species of I and II Abs currently avaiable in the lab and how they can be combined for IF experiments

LAB METHODS: Freezing muscle samples

2009-10-01T18:41:00.003+02:00

LAB METHODS: Cell culture conditions

2009-10-01T14:24:00.002+02:00

LAB METHODS: Tunel assay on cryosections

2009-09-29T19:07:00.001+02:00

LAB METHODS: myeloperoxidase enzymatic assay

2009-09-29T19:05:00.001+02:00

LAB METHODS: H&E staining

2009-09-29T19:03:00.001+02:00

LAB METHODS: automatic counting of muscle fibers on cryosections

2009-09-29T18:59:00.002+02:00

EXPERIMENTAL MODELS: breaking and building the skeletal muscle

2009-09-29T00:05:00.004+02:00

CLICK ON THE POST TITLE FOR A VISUAL ABSTRACT OF THE MAIN EXPERIMENTAL APPROACHES USED IN OUR LABORATORY. We study mechanisms controlling muscle regeneration and homeostasis by focusing on the murine musculature, which is possibly made transgenic by electroporation-mediated gene delivery. We also exploit cell cultures of myotubes for tissue engineering applications and to study regulation of muscle differentiation.

EXPERIMENTAL MODELS: wheel running

2009-09-24T22:56:00.004+02:00

Below is shown how we obtain free exercise in (control and) tumor-bearing mice. The mice are hosted in wheel-equipped cages and the features of physical activity are individually recorded by a computerized revolution counter.

How much does a mouse run (following 2 wks of training)?
10 Km/day, running for about 4 h/day at an average speed of about 2.5 Km/h
Mice LOVE running! And I wonder whether these lazy, obese mice we host in the standard cages mean anything physiologically relevant...

2009-09-24T10:08:00.000+02:00

ARTICLES: link to Schwarzkopf et al. Basic Appl Myol 2008

2009-09-21T11:02:00.002+02:00

The picture depicts primary cultures of myogenic cells purified from wt (+/+), p53 knock out(-/-) and p53 mutant (+/mt) mice and induced to differentiate in vitro. Myotubes expressing MHC (red) are sincitia (nuclei in blue) and p53 is required for PW1 expression (green) .

This is an original study on p53 role in stem cell biology in cachexia. The article was awarded the journal cover (see image).

Basic and Applied Myology, European Journal of Translational Myology is an open access journal edited at the University of Padua, Italy.

ARTICLES: link to Perniconi et al. Basic Appl Myol 2008

2009-09-21T11:00:00.003+02:00

This is a meta-analysis on the effects of exercise on cachectic patients.

Basic and Applied Myology, European Journal of Translational Myology is an open access journal edited at the University of Padua, Italy

ARTICLES: link to Coletti et al. Basic Appl Myol 2008

2009-09-21T10:57:00.001+02:00

This is a review article on highlights from the IV Cachexia Conference, Tampa, FL, 2007

Basic and Applied Myology, European Journal of Translational Myology is an open access journal edited at the University of Padua, Italy

ARTICLES: link to Berardi et al. Neurol Res 2008

2009-09-21T10:55:00.002+02:00

ARTICLES: link to Moresi et al Stem Cells 2008

2009-09-18T14:50:00.001+02:00

This article was awarded the journal cover (see image).
The picture represents interstitial stem cells infiltrating tumor necrosis factor-α-treated, regenerating skeletal muscle. Stem cells exhibit active caspases (red) and rarely coexpress activated caspase-3 (green). Hoechst (blue) stained nuclei; orange stained laminin.

ARTICLES: link to Moresi et al PLoS ONE 2009

2009-09-18T14:40:00.001+02:00

This work highlights the role of Heat Shock Protein 70 at the crosstalk between pro- and anti-myogenic cues for skeletal muscle regeneration. Amelioration of muscle regeneration by vasopressing treatment or Hsp70 overexpression results in fast and complete muscle functional recovery following damage.

LESSONS: Introduction to tissue engineering

2009-09-17T15:08:00.001+02:00

The link leads to a slide show (in Italian) illustrating the basic concepts of tissue engineering.

fellow lab members

2009-09-17T13:31:00.001+02:00

here is a slide show with past and present lab members

LAB METHODS: flow cytometric analysis of M-CAD+ cell cycle

2009-09-17T13:22:00.001+02:00

blog launch on Sept the 16th

2009-09-16T16:06:00.000+02:00

to celebrate the launch of the blog a bottle of Champagne is needed: Krug Champagne 1985 ! An outstanding, masculine, still raffinate champagne, which leaves an aftertaste as long as its ability to remain vibrant after decades.

2009-09-16T14:51:00.001+02:00