LAB METHODS: Esterase staining

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

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"

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

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

LAB METHODS: Muscle fiber damage by Evans blue dye staining

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

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

ARTICLES: link to Coletti et al. Cytometry 2007

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.

CLASSES, LECTURES ETC: Microscopy and histological analises

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

Italian Court Rejects Prime Minister’s Immunity

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..

CLASSES, LECTURES ETC: Blood & Hematopoiesis pt IV

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

CLASSES, LECTURES ETC: Blood & Hematopoiesis pt III

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

CLASSES, LECTURES ETC: Blood & hematopoiesis pt II

For students of the School of Dentistry. In Italian.

CLASSES, LECTURES ETC: Blood & Hematopoiesis pt I

For students of the School of Dentistry. In italian.

LAB METHODS: Antibodies available in the lab

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

LAB METHODS: Cell culture conditions

Quick review of the culture conditions for the main cell lines and primary cultures currently used in the lab.

LAB METHODS: Tunel assay on cryosections

LAB METHODS: myeloperoxidase enzymatic assay

LAB METHODS: H&E staining

LAB METHODS: automatic counting of muscle fibers on cryosections

EXPERIMENTAL MODELS: breaking and building the skeletal muscle

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

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... However, be aware of significant differences in running ability among different mice strain, recently reported in Strain screen and haplotype association mapping of wheel running in inbred mouse strains. Lightfoot JT, Leamy L, Pomp D, Turner MJ, Fodor AA, Knab A, Bowen RS, Ferguson D, Moore-Harrison T, Hamilton A. J Appl Physiol. 2010 Sep;109(3):623-34. Epub 2010 Jun 10. Freely available in PubMed Central.

We currently use several substrain of BALB/c mice, with different behaviors for what concerns physical activity (see also the post entitled "EXPERIMENTAL MODELS: BALB/c substrains & running behavior")

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

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

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

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

ARTICLES: link to Moresi et al Stem Cells 2008

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

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.

CLASSES, LECTURES ETC: Introduction to tissue engineering

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

fellow lab members

here is a slide show with past and present lab members

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

blog launch on Sept the 16th

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.