1、Medical English,Lecture 2: English Communication in a Laboratory,Zhang Ning Research Center of Basic Medical Sciences Tianjin Medical University ,Improve Your Spoken English,Everyday English only uses about 2000 words, you dont need to remember a dictionary to speak English Watch news in English eve

2、ryday Watch a TV series, such as Friends Try to talk to your mentor in English Try to discuss science in English with your colleagues,Improve Your English Writing,The difference between writing in English and in Chinese Writing in English is Easy: make it simple and clear Make your idea clear, topic

3、 sentences, one point in one paragraph, Expand and support your argument, a few frequently used words: suggest, indicate, show, demonstrate, required, sufficient, essential, contribute to. Summary and Speculations, taken together, in sum, Revise, Revise, and Revise,Recommended Readings for English i

4、n General,Barack Obama Dreams of My Father Winston Churchill My Early Life Bertrand Russell Autobiography of Bertrand Russell,Acquire Basic Knowledge,Go to seminars Participate a journal club Read Reviews Read papers, read a lot of papers Who did what at where, and how? Generate your own ideas,Readi

5、ng a Paper,Who did What at Where,Reading a Paper,Read Abstract First,Read a Paper,Followed by Figures and Results,Reading a Paper,Then, Discussion,Participate a Journal Club,Presentation, in ppt format, one paper/talk, background, results in details, other related works Listen to a journal club, cri

6、tical thinking, ask questions,Scientific Ideas,The difference between science and technology Find question and develop your own hypothesis The hypothesis driven research versus fishing expedition The hypothesis need to be novel and testable,Case Study,What has been known,Metastasis, the major cause

7、of morbidity and mortality in many cancers, . is mediated by chemotaxis, the directional movement of a cell following an extracellular chemical gradient. Mechanistic studies have shown that chemotaxis of cancer cells is mediated by both G protein coupled receptors (GPCR) and receptor tyrosine kinase

8、s (RTK). Previous reports suggest that these two types of chemotactic receptors share common downstream signaling components. My long-term goal is to identify these shared components and examine whether these molecules can be used as targets of novel therapies to inhibit cancer cell metastasis. The

9、protein, phosphatase and tensin homolog deleted on chromosome ten (PTEN), a tumor suppressor, regulates cell proliferation and apoptosis by hydrolyzing a critical secondary messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3). ,Propose a Hypothesis,PTEN plays a non-redundant role in both CXCR4

10、 and EGF receptor-mediated chemotaxis and metastasis.,Test the Hypothesis,Test the hypothesis that depletion of PTEN inhibits CXCR4-mediated chemotaxis. We will examine the effects of PTEN depletion on CXCL12-induced chemotaxis, cell adhesion, chemokinesis, and activation of downstream signaling com

11、ponents. We also plan to test more human breast cancer cells to evaluate the role of PTEN in chemotaxis Test the hypothesis that depletion of PTEN elevates basal activities of downstream signaling molecules and renders cells unresponsive to EGF stimulation, resulting in impairments in chemotaxis. We

12、 will examine the activities of signaling molecules downstream of PTEN in the absence or presence of EGF, the spatial distribution of these signaling molecules, and the effects of PTEN depletion on EGF-induced chemokinesis. Test the hypothesis that PTEN plays a non-redundant role in metastasis of hu

13、man breast cancer cells We will use a tail vein injection model to evaluate extravasation capacity of PTEN depleted human cancer cells by immunohistochemical staining of GFP on mouse lung slices and RT-PCR detecting human hypoxanthine phosphoribosyltransferase (HPRT) in SCID mouse lung lysates,Talk

14、to Your Mentor,Make an appointment Organize your thoughts Plot the data and bring with you the raw data Draw your own conclusion first,A Good Example,Chemotaxis after Akt2 rescue in C97 (0.03ug and 0.09ug plasmid for 60 mm dish),Although the expression of Akt2 was rescued, the chemotaxis didnt impro

15、ve much. This experiment need to be repeated.,A Bad Example,MDA Control 7 8 9 10 11 13 14 15,MDA Control 7 8 9 10 11 13 14,MDA control control 13 13 15 15 17 17,MDA control 1 4 5 MDA control 1 4 5,Start a Project,Dont treat yourself as a technician! Think thoroughly about the project before start it

16、 (more than 70% of projects are doom to fail). Plan a strategy Keep up with literatures and always think about the projects. Design each experiment carefully, positive controls and negative controls Study the protocols, follow them, improve them.,Study the Protocol,Miniprep Protocol Inoculate 3ml LB

17、 medium (containing antibiotic) with a bacterial clone, culture with vigorous shaking at 37 degree for 14-16 hrs. For carbamicillin add 3ul carbamicillin (50mg/ml) to 3ml LB broth. 2. Aliquot 2 x 750 ul culture into microcentrifuge tube, harvest bacteria by spinning at 12000rpm (11000g) for 1 min. A

18、spirate supernatant. Add an additional 750 ul culture media, respin and aspirate supernatant. 3. Resuspend bacterial pellet by complete vortexing in 110ml resuspension buffer (100 ul solution A (25mM Tris-HCl, pH8.0, 10mM EDTA) + 10ul RnaseA). The bacteria should be completely resuspended - no clump

19、s should be visible. Solution A is stored in the refrigerator. RNAse is in the -30C freezer.,4. Add 100ul freshly prepared lysis buffer (50ul 400mM NaOH, 50ul 2% SDS) and mix gently by inverting 5-6 times at room temperature. To make lysis buffer mix equal volumes of 800mM NaOH and 4% SDS solutions.

20、 The mixture should appear translucent and mucous-like. 5. Add 120ul solution K (neutralization buffer)(5M potassium acetate, pH5.5) and mix gently by inverting 5-6 times, incubate at room temperature for 3 min. This solution is kept in the refrigerator. The mixture should contain flocculent white p

21、recipitate at this point. 6. Remove bacterial debris by centrifugation at 12000rpm for 2 min, transfer supernatant to a fresh microcentrifuge tube. The precipitate is sticky. To transfer use a 1 ml pipet tip, depress pipet plunger, move tip to bottom of tube and aspirate supernatant. When transfering to new tube, do not touch outside