Friday
Oct112013

Using confocal microscopy for fluorescence imaging

Confocal microscopy is an essential optical tool in biological research which can be used to study cellular structures as well as sub-cellular components. The main advantages of confocal are the elimination of out-of-focus glare that leads increased resolution and the ability to collect serial optical sections (z-sections) from thick specimens. Hence this method generates better (and certainly prettier!) images than regular widefield microscopy (transmitted light or epifluorescence).

My research work in the Hooker lab, is focused on activity based small molecule fluorescent probes for histone deacetylase (HDAC) imaging. Confocal microscopy has become one of the very useful techniques that I use for HDAC-probe imaging in HeLa-cervical cancer cells. It allows a more precise view of cellular components and thus obtained images permit the development of an advanced understanding of the probe/enzyme of interest.

I have been using the Zeiss LSM510 laser scanning confocal microscope at the Ragon Institute and it’s an enormous help in taking my research project further. If any of you are interested in using a confocal microscope or the microscopy facility, I encouraged you go ahead and do so.

Here’s the link to the Ragon center microscopy core.

http://www2.massgeneral.org/aids/microscopy/introduction.htm

I hope this would be a useful bit of info! After all, by using all the technology and tools available to us, we are unraveling the mysteries of biology and taking science one step further.

- Himashinie

 

Wednesday
Oct092013

Editorial, "Mind how you go" in Nature

I came across an editorial in Nature (thanks RAH) that points to a shift in the focus of treatments for mental illnesss.  The article mentions a shift toward genetics and I would augment that with epigenetics (see blog by Al below).  I am glad that companies like Novartis are re-energized to solve or at least help patients suffering from mental illnesses.  Imaging biomarkers of mental illness are going to be critical since the populations that suffer are so heterogeneous.  A lot has been learned by imaging neurotransmitters, but there are no robust biomarkers for any psychiatric illness.  With time, we'd like to change that. 

Tuesday
Oct082013

What doesn’t kill you makes you smarter: apoptosis and cognitive enhancement by HDAC inhibitors.

Histone deacetylase (HDAC) inhibition has been a recurring topic at the leading edge of preclinical drug design.  Beyond approved utility for cancer treatment – inducing death in cancer cells - HDAC inhibitor drugs may be useful in therapeutic development for addiction, mood-related disorders and cognitive deficits.  

HDACs are a family of enzymes that function in part to control the acetylation modification of histone proteins which organize DNA packaging within the nucleus of a cell.  Dysfunction in HDAC enzyme expression or activity is thought to be an important facet of a number of brain diseases.  Blocking the function of HDACs with small molecule inhibitors may help treat these diseases – potentially by correcting aberrant gene expression.   

For new drugs that might alter brain circuitry, a lot depends on the target tissue exposure (how much drug gets in the brain?) and residence time (how long does it stick around?)

Until recently, these basic questions were largely addressed in literature on HDAC inhibitors.

The two major classes of HDAC inhibitors, hydroxamates and benzamides, feature differences in the number of HDAC family members they inhibit, but differ moreso in their BINDING KINETICS.  Hydroxamates bind their targets very quickly (on the time scale of minutes) and benzamides are s-l-o-w…only maximally binding HDAC targets after a few hours time. 

Binding HDACs over time: Lauffer et al. JBC, 2013

How do these differences impact histone acetylation, gene expression and biology?  Using a cancer cell model, Lauffer and collegues - J.Biol.Chem.  ‘Histone deacetylase inhibitor kinetic rate constants correlate with histone acetylation but not transcription and cell viability’ July, 2013 -  recently outlined the time-dependent effects of hydroxamate and benzamide exposure on histone acetylation.  Logically, the fast-binding hydroxamates induced histone acetylation quickly, whereas benzamides took time.  This histone acetylation was also long lasting for benzamides, in line with slow dissociation kinetics & long residence times of compounds in this class.

Interestingly, for both hydroxamates and benzamides, gene expression profiling experiments and monitoring of cell death indicated that a refined set of genes were altered by both drug classes and that drug exposure time was linked to a large number of the changes. The authors suggest that genes sensitive to the onset kinetics of either hydroxamates or benzamides leading to downstream changes in gene expression and, over extended exposure, inducing cell death.

We understand from Lauffer’s work that differential outcomes (intermediate gene expression changes vs. cell death) can result from HDAC inhibitor residence times.   To understand the biology induced by HDAC inhibition, it is insufficient to focus on histone acetylation and we should instead focus on biological changes in the brain, including behavior as the net output of these changes.

Impacting the HDACs in brain: Hanson et al. PlosOne 2013.

Cognitive deficits associated with neurodegenerative disease and normal aging have been reported to be alleviated in animal models by HDAC inhibitor treatment, including by the well-known hydroxamate, SAHA.   In a report nearly parallel to Lauffer’s work, Hanson et al (Plos One, July 2013, Vol.8 (7)) demonstrated that the hydroxamate HDAC inhibitor, SAHA, indeed suppress HDAC activity and neural activity, consistent with its role as a potential cognitive enhancer.  However, in experiments in mice, almost NO SAHA could be detected in brain, even when very high doses were given.  This was explained in part by the identification that SAHA is actively pumped OUT of the brain by specialized proteins.  However, Hanson’s work underscores the importance of identifying HDAC inhibitors with good brain exposure in order to best understand the biology of behavioral and neurochemical changes.

Advice to consider:

  1. Don’t use histone acetylation as a metric for biological / behavioral change.
  2. Do design experiments to understand dynamic impact of HDAC inhibition in your system.
  3. Don’t use behavioral change as an indicator of drug presence in brain.
  4. Do investigate brain exposure and residence times early in evaluating novel inhibitors.

-Al Schroeder

Tuesday
Sep102013

My overall Hooker Lab experience/personal updates

Since I've been back home I've had to undergo a number of immediate changes to adjust to being back in Mississippi. It felt great to be able to get in my car and drive to a given location in 10-15 minutes versus taking hours to commute using the T. However, I do miss the weather in Boston as it is unbelievably hot and humid here. Before I left Jacob and I had a discussion on coffee and caffeine and why it seemed to have the effects of making me tired. To help me answer this question I decided to conduct an experiment on myself. I've tried different types of coffee and analyzed the effects I experienced from caffeine. I believe I have found why I seemed to be tired after drinking coffee. I found that sugary coffee drinks make me extremely sleepy, while sugar free coffee gets me wired and has increased my ability to focus. I have even taken quantitative data (blood glucose levels, blood pressure, and pulse) to try to find when I am at my highest mental functionality while studying. Doing this has helped me tremendously in more ways than one.

Working with Jacob and Emily this summer has truly opened my mind and I've noticed how differently I think about things in the academic setting. I've found myself asking more questions from my professors, and truly analyzing the concepts I'm learning in my classes. My Biochemistry professor even asked me to give the class a lecture on Epigenetics and talk about some of the work I did this summer! I feel much more confident going forward and hopefully I will get into a good graduate school where I can continue to push myself mentally. Overall, I'm extremely grateful and humbled by this experience. I've learned so much and have made great friends in the process. Me and Emily still keep in contact with updates on everything we have going on with research and personal endeavours. I'm really thankful to have had the opportunity to work with her this summer as she is highly intelligent, a great teacher, and a not so bad poker player. So, I would like to end by saying thank you to Jacob and Emily so much for pushing me. This summer I have learned so much and it has truly made a difference in my studies and in my life overall. 

-William B. Taylor

Tuesday
Aug132013

Smashing atoms and taking pictures: my experiences at the Martinos Center Radiopharmacy.

I’m Chris Moseley and I am a former radiopharmacy technician and founding member of the Martinos Center for Biomedical Imaging PET production facility. I joined MGH in 2011, just prior to the tracer facility becoming operational. Like many fresh college graduates (or students for that matter) I had few, if any, appreciable skills. But I did have an expensive piece of paper and a dream to build something more for my future self. I contacted Dr. Jacob Hooker looking for a position in his lab and was pleasantly surprised to get a response. Despite him freely agreeing to the aforementioned assessment of my utility, he conceded to my enthusiasm and determination and offered me a job. Two years later I had written and coauthored over half a dozen publications including a first authorship for a chapter in a textbook, worked with some of the world's finest physicians and scientists, and realized my wish to enter medical school. The following is an account of what life was like for me at the radiopharmacy and of what Dr. Hooker was able to do for me as an employer, mentor, and person.

I was ecstatic to join the juggernaut collaboration between Massachusetts General Hospital, Harvard University, and Massachusetts Institute of Technology. The Martinos Center has an abundance of material and intellectual resources, uniting to create an environment that fosters innovation and makes definitive progress. Whereas I had been accustomed to associating labs with mediocre equipment repurposed until it became more duct tape than machine, I now worked in a facility outfitted with cutting edge and occasionally "prototype stage" technology. On any given day I operated a particle accelerator, chemical synthesis machinery, a full quality control lab, and blood analysis robot. Using these tools, I was able to participate in the development of novel molecules. It was remarkable to watch the evolution of a compound synthesized by Dr. Hooker's chemists as it developed from cold to "hot" chemistry and to the eventual injection into a living organism. I cannot emphasize enough the uniqueness of this opportunity. I was literally offered a chance to take part in every piece of the research effort including drug development, equipment engineering, imaging analysis, pharmacokinetic modeling, and more things I can't recall. Leading me in each of these ventures were some of the most gifted and world renowned scientists in fields including chemistry, biology, neuroscience, radiology/imaging, engineering, biophysics, computational sciences, and so on. Furthermore, I was encouraged to propose my own research projects, with the full resources of the center at my disposal.

Despite the fantastic research environment my ultimate goal was to attend medical school. Having unsuccessfully applied to medical school in the past, Jacob helped me to formulate and execute a plan to improve my application and put me in touch with prominent physicians. It was a boon to interface with physician scientists, of which MGH has no short supply. I began a project with a neuro-oncologist to assess treatment outcomes for malignant brain tumors by using a radioactive form of a 'standard-of-care' chemotherapeutic. For my part in this collaboration, I was tasked with producing the radioactive compound, which was no trivial matter. Along with the very talented post-docs in Dr. Hooker's lab, I improved the synthesis of this drug, and I wrote my first publication as a first author. As an added bonus, I was able to shadow the neuro-oncologist in the clinic, where I met fascinating patients and learned a tremendous amount about people coping with terminal diseases. Professionally, this meant I could add another letter of recommendation to my application package as well as plenty to write about for my admissions essays. Personally, I was able to experience medicine in a fashion that I had been otherwise unable to explore and ultimately this understanding galvanized my desire to become a physician. I believe this was apparent during my medical school interviews, when I recounted these events, and was instrumental to me gaining acceptance to multiple medical colleges and tens of thousands in scholarships.

To give a fair warning to those interested in attaining a graduate education, the fun doesn't stop once medical school starts. In the first week students are coached on how to prepare for the next step in medical training, residency – a placement that is four years away. However, the exposure to medicine I gained at MGH has already given me an idea of the specialties that I wish to pursue and so I am able to join interest groups and begin networking to advance my career. My research experiences have already opened up a few doors regarding interning at summer programs for medical students to supplement their resume with additional research projects. On a day to day basis, the skills I learned during my time at MGH have been extremely useful during medical school. My experience in an academic hospital has prepared me for the medical school environment as there is an interlacing of academic work and patient care during all hours. Furthermore, I find that some assignments are easier as I am used to rapidly acquiring and digesting scientific literature. However, I am most amused at how the little abilities that you acquire become surprisingly beneficial later in life. Attending the Hookerlab presentations has made me a Microsoft office pro and I've already been nominated as a tech whiz in my problem based learning group. Working at the Martinos Center has benefitted my medical education in more ways than I could have imagined.

Finally, I asked myself what it was like to work with Dr. Jacob Hooker and unexpectedly found myself more appreciative than I would have anticipated. He has helped several of my colleagues to achieve their goals of graduate school, medical school, and professional placement. It is apparent to everyone in the field that he is the rare type of primary investigator; a rising star in science that constantly publishes material, undertakes new studies, wins grants, and builds his research team. However, the following is my honest assessment of my boss for two years. Keep in mind that he is no longer paying me and so I am not obligated in any way to express any of the following. Simply put, Jacob is the only individual that I have ever met and considered to be truly gifted. On top of being renowned as a chemist, I have seen him show unrelenting enthusiasm and proficiency when working in all manner of endeavors including: engineering, mathematics, teaching, speaking, etc... and even athleticism (he has finished multiple Ironman competitions). Yet despite his talents, he is a humble, compassionate, and empathetic man. On numerous occasions, he took significant time from his day to listen to me vent personal matters, always offering me advice and encouragement. I have seen him do the same for several other employees and associates. I admire him professionally, respect him as a person, and I consider it an honor to work alongside him. That being said, good luck to all of the future lab members and associates. I hope you find your time with the Hooker group as rewarding and gratifying as I did.