Top 50 U.S. MBA Schools * U.S. News and World Report 2007
1 - Harvard
2 - Stanford
3 - University of Pennsylvania (Wharton)
4 - Sloan (MIT)
5 - Northwestern (Kellog)
6 - Kellogg
7 - Columbia
8 - Duke, Fuqua
9 - University of California Berkely
10 - Chicago
11 - Dartmouth, Tuck
12 - Darden
13 - Stern
14 - Michigan-Ann Arbour
15 - UCLA, Anderson
16 - Yale
17 - Cornell, Johnson
18 - Carnegie Mellon
19 - Texas, McCombs
20 - Ohio, Fisher
21 - Southern Calif, Marshall
22 - Emory, Goizueta
23 - North Carolina, Kenan-Flagler
24 - Indiana, Kelley
25 - Georgetown, McDonough
26 - Purdue, Krannert
27 - Minnesota, Carlson
28 - Rice, Jones
29 - Florida, Warrington
30 - Brigham Young, Marriott
31 - Iowa, Tippie
32 - Notre Dame, Mendoza
33 - Washington, Olin
34 - Penn State, Smeal
35 - Illinois-Urbana
36 - University of Calif - Davis
37 - University of Washington
38 - Arizona State, Carey
39 - Michigan State, Broad
40 - Rochester, Simon
41 - Wake Forest, Babcock
42 - Boston, Carroll
43 - Tulane, Freeman
44 - Georgia, Terry
45 - Maryland, Smith
46 - Vanderbilt, Owen
47 - Irvive
48 - Wisconsin-Madison
49 - Babson
50 - Arizona, Eller
51 - Pittsburgh, Katz
Top 25 Asian Business Schools
1 - Chinese University of Hong Kong
2 - IIM Ahmedebad
3 - National University Singapore
4 - Hong Kong University of Science and Technology
5 - Asia Institute of Management, Philippines
6 - Aust Grad School of Management
7 - University of Queensland
8 - University of Hong Kong
9 - IIM Bangalore
10 - Nanyang Technology University Singapore
11 - Melbourne Business School
12 - IIM Calcutta
13 - CEIBS
14 - Chulalongkorn University Thailand, Sasin
15 - Chicago GBS Singapore
16 - City University Hong Kong
17 - Yonsai University South Korea
18 - IIM Lucknow
19 - Monash
20 - International University Japan
21 - Macquarie
22 - Asia Institute of Technology Thailand
23 - Peking University
24 - National Institute of Development and Administration Thailand
25 - Lahore University of Management
2007年12月22日星期六
point of sale software
Keystroke POS Point Of Sale Software System - Home PageKeystroke POS Point Of Sale Software System - Home Page Of The Specialized Business Solutions Web Site.
POS Software & Systems Group - Point Of Sale Software, Hardware ...Provides pos software and systems to retail, restaurant, and rental businesses.
Retail Plus POS Software - Affordable Point of Sales & Inventory ...Point of sale software with inventory control, sales tracking, invoicing, purchase orders, client database and support for various POS devices. [Win 95/98/NT/DOS]
Point of Sale Hardware and Software - pointofsale.comPOSitive Software Company: POS software solutions for your business.
Inventory Control / Point of Sale SoftwareAtrex Inventory Control/Point of Sale for Win 95/98/NT/2000/XP.
Welcome to Microbiz.com, Point of Sale software and hardware for ...Popular Windows based POS system targeted towards smaller hardgoods retailers.
Point of Sale Software: pos software, pos sales system, retail and ...The Capterra Point of Sale Software Directory helps IT buyers find, compare, and research Point of Sale software solutions.
Point of Sale (POS) Software Page 1The software in this section is primarily for the business user. Home users will also find much that is useful. Included in this section are Personal Information Managers, Accounting software, Investor's tools and much more.
Plexis POS Software / Point Of Sale SoftwareOffers software for Windows 98, NT, and Windows 2000. Includes support faq, downloadable demonstration and manual.
Retail/Wholesale Software SolutionThai company offers point of sale management software and restaurant management software solutions.
POS Software & Systems Group - Point Of Sale Software, Hardware ...Provides pos software and systems to retail, restaurant, and rental businesses.
Retail Plus POS Software - Affordable Point of Sales & Inventory ...Point of sale software with inventory control, sales tracking, invoicing, purchase orders, client database and support for various POS devices. [Win 95/98/NT/DOS]
Point of Sale Hardware and Software - pointofsale.comPOSitive Software Company: POS software solutions for your business.
Inventory Control / Point of Sale SoftwareAtrex Inventory Control/Point of Sale for Win 95/98/NT/2000/XP.
Welcome to Microbiz.com, Point of Sale software and hardware for ...Popular Windows based POS system targeted towards smaller hardgoods retailers.
Point of Sale Software: pos software, pos sales system, retail and ...The Capterra Point of Sale Software Directory helps IT buyers find, compare, and research Point of Sale software solutions.
Point of Sale (POS) Software Page 1The software in this section is primarily for the business user. Home users will also find much that is useful. Included in this section are Personal Information Managers, Accounting software, Investor's tools and much more.
Plexis POS Software / Point Of Sale SoftwareOffers software for Windows 98, NT, and Windows 2000. Includes support faq, downloadable demonstration and manual.
Retail/Wholesale Software SolutionThai company offers point of sale management software and restaurant management software solutions.
mesothelioma doctor
With early diagnosis and experienced mesothelioma doctor it is possible to cope with asbestosis and other disordrers related to exposure to asbestos. Cancer is a disease, which is accompanied by abnormal and uncontrolled growth of cells penetrating and affecting nearby areas. There is still much uncertainty about its nature, but it is well known, that smoking and asbestos exposure are conductive to the development of malignant tumors. With early diagnosis and experienced mesothelioma doctor it is possible to cope with asbestosis and other disordrers related to exposure to asbestos.
Just select from the Research Topics on the adjacent gray column for easy viewing of important articles. If this is your first visit, click here for a brief summary of this website.
Attorney Seattle, California Lawyers, Hendry County Attorney, Kentucky Attorney, Louisiana Lawyer, Lung, Lung Cancer, Pennsylvania Lawyer, St Louis Attorney, Tennessee Lawyer.
Mesothelioma Doctor -&<60;&<60;&<60;asbestosis, asbestos
This particular type of cancer develops either in pleura, a protective membrane covering the lungs and the walls of the chest cavity, or in the peritoneum, the same type of membrane covering abdominal organs and the walls of the abdominal cavity. Pleural cancer is much more common than peritoneal cancer. With early diagnosis and experienced mesothelioma doctor it is possible to cope with asbestosis and other disordrers related to exposure to asbestos.
Just select from the Research Topics on the adjacent gray column for easy viewing of important articles. If this is your first visit, click here for a brief summary of this website.
Attorney Seattle, California Lawyers, Hendry County Attorney, Kentucky Attorney, Louisiana Lawyer, Lung, Lung Cancer, Pennsylvania Lawyer, St Louis Attorney, Tennessee Lawyer.
Mesothelioma Doctor -&<60;&<60;&<60;asbestosis, asbestos
This particular type of cancer develops either in pleura, a protective membrane covering the lungs and the walls of the chest cavity, or in the peritoneum, the same type of membrane covering abdominal organs and the walls of the abdominal cavity. Pleural cancer is much more common than peritoneal cancer. With early diagnosis and experienced mesothelioma doctor it is possible to cope with asbestosis and other disordrers related to exposure to asbestos.
mesothelioma lawyers info
mesothelioma lawyers(是一种病)Choosing the right mesothelioma lawyer for your case is a very important decision for you and your family. You do not want to make a mistake by not choosing an experienced, successful, honest and qualified attorney. We are here to help. We have have already prescreened and prequalified mesothelioma lawyers who are willing to handle cases from across the United States. Once you contact us we will put you in touch with a law firm that is qualified to handle your case. Our screening process requires that the lawyer has represented a client in court that has been awarded a single verdict of at least $10 million, been licensed to practice for at least 15 years, has no disciplinary violations with any state licensing authority, and is willing to represent clients in your geographic location. Our prescreened and prequalified attorneys are willing to evaluate your mesothelioma claim at no obligation to you. Contact us today at 1-800-926-9654 or by filling out the contact information form. We will put you in touch with the right lawyer for your case today.
After you have compiled a short list of mesothelioma lawyers you have done most of the hard work. Narrowing your list down to the lawyer that you choose to represent you shouldn't be difficult at this stage. It is important to call and speak with each of the potential firms / lawyers so that you get a feel for not only their practising style, but also so you can see if you feel comfortable with them. You'll be spending a lot of time with this person and the outcome of your case will depend on them.
Mesothelioma settlements
The value of a mesothelioma settlement can vary quite drastically, anywhere from thousands to hundreds of millions. The average settlement for a mesothelioma case was 1 million US in 2001. If the case went to trial, the average amount awarded to the plaintiff was 6 million. These amounts were 3 times the average that was awarded just 2 years before in 1999. As you can see from the amounts awarded in these types of cases, when looking for a lawyer they should take a great deal of time to discuss their strategy and experience in this area and also answer any questions that you may have.
Required documentation
When considering starting a mesothelioma case, it is important that you have various documentation. Medical records showing asbestos-caused medical conditions (particularly mesothelioma, which can only be caused by asbestos) are necessary. There is a statute of limitations on mesothelioma cases so make sure that you file within a couple years of diagnosis. If left too long you may not be entitled to any compensation. It is also useful to have documents showing employement by the company that you worked for at a specific time and place.
After you have compiled a short list of mesothelioma lawyers you have done most of the hard work. Narrowing your list down to the lawyer that you choose to represent you shouldn't be difficult at this stage. It is important to call and speak with each of the potential firms / lawyers so that you get a feel for not only their practising style, but also so you can see if you feel comfortable with them. You'll be spending a lot of time with this person and the outcome of your case will depend on them.
Mesothelioma settlements
The value of a mesothelioma settlement can vary quite drastically, anywhere from thousands to hundreds of millions. The average settlement for a mesothelioma case was 1 million US in 2001. If the case went to trial, the average amount awarded to the plaintiff was 6 million. These amounts were 3 times the average that was awarded just 2 years before in 1999. As you can see from the amounts awarded in these types of cases, when looking for a lawyer they should take a great deal of time to discuss their strategy and experience in this area and also answer any questions that you may have.
Required documentation
When considering starting a mesothelioma case, it is important that you have various documentation. Medical records showing asbestos-caused medical conditions (particularly mesothelioma, which can only be caused by asbestos) are necessary. There is a statute of limitations on mesothelioma cases so make sure that you file within a couple years of diagnosis. If left too long you may not be entitled to any compensation. It is also useful to have documents showing employement by the company that you worked for at a specific time and place.
2007年11月30日星期五
Peritoneal mesothelioma (peritoneal mesothelioma) in the primary peritoneal mesothelial epithelium and the tumor, clinical rarely see. Pathology can be categorized as adenomatous mesothelioma (adenomatoid mesothelioma), cystic mesothelioma (cystic mesothelioma), and malignant mesothelioma (peritone al malignant mesothelioma, PMM). The first two are benign. Cystic Mesothelioma more common in women, cause unknown, occurs in the pelvic or around the annex, was single or multiple cystic masses; Patients often palpable abdominal mass and treatment. SILVER malignant mesothelioma about 30%; Its incidence is also closely linked with exposure to asbestos. About 5% of patients with history of exposure [1]; Asbestos fibers oral intake, translocation through the intestinal wall into the peritoneal and pathogenicity. From exposure to asbestos that were diagnosed, the disease incubation period could be as long as 25 ~ 40 years. But domestic 1951 ~ 1993 20 literature analyzing 161 cases only one cases with asbestos exposure history. In the absence of a history of exposure to asbestos population, the incidence rate of about 1 / 1 million a year, may be some infection and genetic factors [2,3,4]. Abroad have reported a case of analyzing patients 40 years ago, I contacted glial thorium dioxide (Thorotrast) [ 5]. SILVER often occurred in men over the age of 40. Visceral or parietal peritoneum and may happen; Tumors can be a direct violation of abdominal, pelvic organ; 50% ~ 70% of patients with lymphatic and (or) hematogenous metastasis distant as liver, kidney, adrenal gland, lung, bone and lymph node, and so on. Clinical manifestations of this lack of specificity, and may have abdominal pain, constipation, swelling, weight loss and other obstruction performance [6]. Physical examination can be found ascites or abdominal mass. Ascites exudate part of gutsy. The disease often misdiagnosed as tuberculosis peritonitis and recurrent spontaneous bacterial peritonitis, mesenteric inflammation or peritoneal metastasis of cancer, and so on. Ascites hyaluronic acid increased significantly, "0.8 g / L were only found in analyzing. Ascites cytology has a certain value, but often difficult to judge. Serum carbohydrate antigen 125 (CA125) increased help diagnosis of the disease [6,7]. Ultrasound and CT performance varied, the typical person irregularly thickened peritoneum, omentum was Caky adhesions, tissue samples were mesenteric; CT may also show increased Peripancreatic large mass, or intraperitoneal substantive diffuse large mass, and bowel and mesenteric violations; or peritoneal nodules or showed a cystic mass; more with different degree of ascites [8]. Ultrasound or CT-guided biopsy is a definite value. Laparoscopy is analyzing the diagnosis of simple and effective method of endoscopic see peritoneum, omentum and diffuse nodular plaque, may look biopsy pathology examination. We had given one 83-year-old male patient laparoscopy, peritoneal biopsy report mesothelial cell hyperplasia, After immunohistochemical tests confirmed analyzing side. Butchart and others will be divided into four SILVER : I, confined to the peritoneal tumor; Phase II, intraperitoneal tumor invasion lymph nodes; Phase III, to the peritoneal tumor metastasis outside; Ⅳ, distant metastasis. These classifications will help choose the method of treatment. SILVER so far no effective standard treatment programs. The prognosis is poor, survival after diagnosis for a period, the median survival of more than two years of less than 20%. Mainly died for cachexia, or obstruction, and the cause of death rarely distant metastasis of the tumor.
Treatment
Surgical excision of cystic mass effect is very good and without postoperative deaths reported. But 25% of patients with local recurrence. Van der Klooster reports on one cases with multiple cystic mesothelioma patients. by suction and occlusion treatment were ineffective, a tumor recurrence five years times, the last to switch to vaginal intubation continuous drainage, eventually cure. Surgical excision of cystic mass effect is very good and without postoperative deaths reported. But 25% of patients with local recurrence. With laparoscopy can complete resection of giant cystic peritoneal mesothelioma.
Right I, Phase II analyzing the preferred surgical treatment. Operation include the reduction of tumor surgery (cytoreductive surgery), possible removal can see the tumor tissue. But because of the difficulty in surgery, diffuse lesions, the complete resection difficult to achieve the objective. Right recurrence can be re-operation. Ileus is feasible right palliative surgery, obstructive symptoms.
SILVER less sensitive to radiotherapy, radiotherapy result is not as good as pleural mesothelioma. But the surgical resection of the lesions has not been fully or not surgery, radiotherapy is nonetheless an important therapy. Including external irradiation and (or) irradiation. External irradiation generally choose 60 or 186KV Co. as X-ray sources, depending on the scope of diseases to choose partial or whole abdominal irradiation. Medicine tumor patients to the hospital whole abdominal irradiation, six-seven weeks of radiation doses up to 24 gray (Gy). Results of local recurrence rate to 11.4%, three-year survival rate increased to 66.7%. Radiotherapy can cause abdominal radiation, radioactive radiation myelitis and hepatitis. Intraperitoneal injection of radionuclide P as 32 or 198 Au, through radiation and mesothelioma organizations and small vascular sclerosis. and the anti-free ascites tumor cells, a short-term illness mitigation. But this method will need equipment, and the price is very expensive, and can inhibit the bone marrow, has been seldom used.
Drug analyzing moderately sensitive to chemotherapy. Pre-operative chemotherapy, surgery and post-operative adjuvant chemotherapy can significantly reduce tumor recurrence, three years to improve the survival rate.
SILVER certainly result in the treatment of chemical drugs doxorubicin, cisplatin and carboplatin, bleomycin and a new anticancer drug Eiemene other. Vincristine, fluorouracil, cyclophosphamide, mitomycin also worth a try.
Doxorubicin (Adriamycin, ADM) for each adult 30 ~ routinely. Every three weeks one time, intravenous or intraperitoneal injection of a total dose of not more than 550mg/m2. Adverse reaction to a cardiac toxicity, there is accumulation, with the total dose; Followed by bone marrow suppression. gastrointestinal reactions and alopecia.
PDD (cisplatin, DDP, cisplatin) for each adult 80 ~ 120mg/m2, every three times a week; or 20 mg/m2 for 5 days every 3 weeks for a course of treatment, intravenous injection. Adverse reactions are nephrotoxicity and ototoxicity, neurotoxicity, gastrointestinal reactions and bone marrow suppression. Plus mannitol to reduce its accumulated in the tubular cells. The drug is commonly used in the intraperitoneal injection, the specific method described below.Carboplatin (Carboplatin, CBP) Adult 300 ~ 400mg/m2 each. by adding 5% glucose solution or saline, diluting the concentration of the solution 0.5mg/ml, intravenous drip, Each three-four weeks duplication; or 100 mg / d, to 5% glucose solution 500ml intravenous drip for 5 days; 3 ~ 4 times a week to repeat. Can be used for each 300 ~ 500 mg intraperitoneal injection once a week.
Bleomycin (hopefully, the BLM) Adult use 15 ~ 30mg, dissolved in saline or modest 5% glucose solution deep intramuscular, or intravenous injection two times a week; also, under the circumstances, to a time / d or several times a week. Can also be dissolved after 60 mg intraperitoneal injection of slow. Stey with BLM intraperitoneal injection, treatment one cases analyzing patient outcome ascites disappeared, the withdrawal did not arise again. survived for more than three years. But BLM large dose intraperitoneal injection can cause pneumonia-like symptoms, or even pulmonary fibrosis; In addition, fever, gastrointestinal reactions more common. Patients allergic individual.
Treatment
Surgical excision of cystic mass effect is very good and without postoperative deaths reported. But 25% of patients with local recurrence. Van der Klooster reports on one cases with multiple cystic mesothelioma patients. by suction and occlusion treatment were ineffective, a tumor recurrence five years times, the last to switch to vaginal intubation continuous drainage, eventually cure. Surgical excision of cystic mass effect is very good and without postoperative deaths reported. But 25% of patients with local recurrence. With laparoscopy can complete resection of giant cystic peritoneal mesothelioma.
Right I, Phase II analyzing the preferred surgical treatment. Operation include the reduction of tumor surgery (cytoreductive surgery), possible removal can see the tumor tissue. But because of the difficulty in surgery, diffuse lesions, the complete resection difficult to achieve the objective. Right recurrence can be re-operation. Ileus is feasible right palliative surgery, obstructive symptoms.
SILVER less sensitive to radiotherapy, radiotherapy result is not as good as pleural mesothelioma. But the surgical resection of the lesions has not been fully or not surgery, radiotherapy is nonetheless an important therapy. Including external irradiation and (or) irradiation. External irradiation generally choose 60 or 186KV Co. as X-ray sources, depending on the scope of diseases to choose partial or whole abdominal irradiation. Medicine tumor patients to the hospital whole abdominal irradiation, six-seven weeks of radiation doses up to 24 gray (Gy). Results of local recurrence rate to 11.4%, three-year survival rate increased to 66.7%. Radiotherapy can cause abdominal radiation, radioactive radiation myelitis and hepatitis. Intraperitoneal injection of radionuclide P as 32 or 198 Au, through radiation and mesothelioma organizations and small vascular sclerosis. and the anti-free ascites tumor cells, a short-term illness mitigation. But this method will need equipment, and the price is very expensive, and can inhibit the bone marrow, has been seldom used.
Drug analyzing moderately sensitive to chemotherapy. Pre-operative chemotherapy, surgery and post-operative adjuvant chemotherapy can significantly reduce tumor recurrence, three years to improve the survival rate.
SILVER certainly result in the treatment of chemical drugs doxorubicin, cisplatin and carboplatin, bleomycin and a new anticancer drug Eiemene other. Vincristine, fluorouracil, cyclophosphamide, mitomycin also worth a try.
Doxorubicin (Adriamycin, ADM) for each adult 30 ~ routinely. Every three weeks one time, intravenous or intraperitoneal injection of a total dose of not more than 550mg/m2. Adverse reaction to a cardiac toxicity, there is accumulation, with the total dose; Followed by bone marrow suppression. gastrointestinal reactions and alopecia.
PDD (cisplatin, DDP, cisplatin) for each adult 80 ~ 120mg/m2, every three times a week; or 20 mg/m2 for 5 days every 3 weeks for a course of treatment, intravenous injection. Adverse reactions are nephrotoxicity and ototoxicity, neurotoxicity, gastrointestinal reactions and bone marrow suppression. Plus mannitol to reduce its accumulated in the tubular cells. The drug is commonly used in the intraperitoneal injection, the specific method described below.Carboplatin (Carboplatin, CBP) Adult 300 ~ 400mg/m2 each. by adding 5% glucose solution or saline, diluting the concentration of the solution 0.5mg/ml, intravenous drip, Each three-four weeks duplication; or 100 mg / d, to 5% glucose solution 500ml intravenous drip for 5 days; 3 ~ 4 times a week to repeat. Can be used for each 300 ~ 500 mg intraperitoneal injection once a week.
Bleomycin (hopefully, the BLM) Adult use 15 ~ 30mg, dissolved in saline or modest 5% glucose solution deep intramuscular, or intravenous injection two times a week; also, under the circumstances, to a time / d or several times a week. Can also be dissolved after 60 mg intraperitoneal injection of slow. Stey with BLM intraperitoneal injection, treatment one cases analyzing patient outcome ascites disappeared, the withdrawal did not arise again. survived for more than three years. But BLM large dose intraperitoneal injection can cause pneumonia-like symptoms, or even pulmonary fibrosis; In addition, fever, gastrointestinal reactions more common. Patients allergic individual.
2007年11月17日星期六
《环球科学》杂志:组装生命的生物工厂
对于以电子工程为模式的生物技术,生物组件是它的基础
撰文 生物工厂研究小组*(Bio Fab Group)
合成DNA
如果说单个的晶体管是电子电路的基本组件,那么在生物学中,与之对应的便是基因(有序的DNA片段)。为了给高级的生物装置构建基因电路(genetic circuit),我们就需要一种快速可靠、价格合理的DNA片段合成法。
20年前,在前人的工作基础上,美国科罗拉多大学博尔德分校(University of Colorado at Boulder)的马文·H·卡拉瑟斯(Marvin H.Caruthers),利用DNA自身的化学性质,研发出一种单链DNA合成法。DNA由4种核苷酸组成,而每种核苷酸又含有一个相应的碱基,分别是腺嘌呤(adenine,A)、胞嘧啶(cytosine,C)、鸟嘌呤(guanine,G)和胸腺嘧啶(thymine,T)。碱基之间的亲和力使它们两两配对(A-T配对,G-C配对),形成梯状双链DNA分子中的梯级。化学键不仅在碱基对之间形成,在邻近的核苷酸之间也会形成。
卡拉瑟斯所使用的方法被称为固相亚磷酰胺法,这是目前大多数商业DNA合成法的基础。合成反应起始于一个单核苷酸,这可不是一个普通的核苷酸,它附着在悬浮于酸性液体中的固相支持物(如聚苯乙烯颗粒)上,并承担着发起合成反应的重任。当碰见“新来”的核苷酸,两者便会通过形成化学键相连。如果不断加入核苷酸,反应就会持续进行,核苷酸链就会不断延长。这样,就可以合成任何想要的核苷酸序列,并且不易出错(出错几率约为1%)。
但很多时候,生物工程师想要的基因片段,远远超出了该方法的合成能力。一个简单的基因网络也许就有数千碱基对;就算像细菌这样的微小生物,基因组也可达数百万碱基对。因而,我们如果想找到高产出、低误差的合成法,就只能寄希望于从自然界中获得一些提示了。
在生物体中,像酶(如聚合酶)这样的生物机器,能以高达每秒500个碱基的速度,合成和修复DNA分子,而错误率仅为十亿分之一!这就意味着,即便是最好的DNA合成机器(每300秒合成1个碱基),产出率(输出量/错误率)也不及聚合酶的万亿分之一。更有甚者,在细菌体内,当复制像基因组那样的长链DNA时,多个聚合酶会同时运作,在20分钟内就能合成含有500万个碱基的DNA!
于是,丘奇开始仿效细菌聚合酶的这种平行作业方式,以适应现有的基因芯片技术。基因芯片其实就是特殊的玻璃片,在它的表面上,繁星般地点缀着长为50~70个碱基的寡核苷酸(oligonucleotide/oligo,短小的核苷酸链)。通过亚磷酰胺反应法,寡核苷酸被同时合成于基因芯片的表面,并以格状排列,密度高达100万点/平方厘米。在传统技术的基础上,我们又在这些寡核苷酸上加上可剪切的连接子,以便特定的寡核苷酸能够从基因芯片上释放出来。在我们的实验性基因芯片上,每个点约为30微米宽,含有大约1000万个寡核苷酸分子。
通常,基因芯片上的核苷酸链被称为构建性寡核苷酸(construction oligo),因为它们的部分序列是相互重叠的,通过重叠的序列,可将它们组装成更长的DNA结构(如整个基因)。但是,任何含有错误序列的寡核苷酸都必须被清除。为此,我们采用了两种不同的纠错方法。
第一种是选择性寡核苷酸(selection oligo)法。合成选择性寡核苷酸的方法与制造基因芯片的方法相同,只是在核苷酸序列上,前者有特殊的要求:与构建性寡核苷酸的序列互补。合成之后,便对连接子进行剪切,从玻璃片上释放选择性寡核苷酸,并使它们流过构建性寡核苷酸的芯片。按照碱基配对的原则,选择性寡核苷酸就会与互补的构建性寡核苷酸结合(杂交,hybridize),形成双链DNA。这样,任何不能配对,或者含有错误序列、配对不完全的构建性寡核苷酸,都无法逃过我们的“法眼”,也就无法继续在芯片上“滥竽充数”。与制造基因芯片一样,在合成时,选择性寡核苷酸的序列也会出错。但是,构建性与选择性寡核苷酸的错误序列很难完全互补。因此,利用一组寡核苷酸对另一组进行校对是一种有效的纠错方法。利用这种方法,我们合成寡核苷酸的平均错误率可以低至1/1,300。
正如人们所料,生物体系非常注重自身复制的精确度。我们的第二种纠错方法就来源于自然界。十年前,莫德里奇首先发现了生物体系复制时的详细纠错过程,并将这个过程命名为“MutS,L,H”。当两条DNA链的碱基不能完全配对时,那么在错配区,便不能形成双螺旋结构。MutS是一种天然存在的蛋白,它会识别这种缺陷,并与之结合。随后,它招集“同伴”—— MutL和MutH,共同完成修正任务。利用该方法,雅各布森与美国麻省理工学院的彼得·卡尔(Peter Carr),已经将DNA合成的错误率降到1/10,000。对于生产小型基因网络,这样的保真度足矣。
在可释放性平行合成技术和纠错技术的支持下,长链DNA的合成速度更快、成本更低、精确度更高。这些技术将是生物工厂的基础,随着时间的流逝,它们会像半导体芯片光刻技术那样,不断进步。先进的技术是宝贵的财富,能解放我们的思想,让我们思考更多:在生物工厂里,我们可以做些什么呢?
基因工程药物
利用生物工厂的平台探索征服疾病的新方法,是我们最早的目标之一。基斯林和贝克的研究对象是疟疾和艾滋病——两种困扰人类多年的恶疾,他们致力于开发对付这两种疾病的药物。尽管我们所研究的治疗方法与他们的不尽相同,但在很大程度上,两个小组的研究都依赖于精确合成长链DNA的能力。我们的研究只是工厂化的一个代表,工厂化拥有强大的力量,必将颠覆传统的新药开发模式!
以疟疾为例,已有药物能将感染者体内的致病寄生虫彻底消灭,从而根治疟疾。这是一种小分子药物,叫做C-15倍半萜(sesquiterpene),俗名青蒿素(artemisinin),是由青蒿植物(sweet wormwood,多发现于中国华北地区)合成的天然化合物。但在植物中,天然合成的青蒿素过少,造成青蒿素的价格极其昂贵,无法推广。因此,在过去五年中,基斯林的研究小组努力克隆合成青蒿素的遗传途径,并将它插入酵母菌中,让酵母菌大量合成青蒿素。
在酵母菌中,我们还能对遗传途径进行改良,使青蒿素的合成效率大幅提高。青蒿素的合成途径叫作甲羟戊酸途径(mevalonate pathway),目前,我们已经能够对途径中的关键基因进行重新设计。较之细菌中的原始途径,关键基因的改变可使青蒿酸(amorphadiene,青蒿素的前体)的产量提高10万倍!但这仍然不够,要使青蒿素得到广泛应用,必须进一步提高产量。这就需要我们对整个青蒿素途径进行综合重建。
整条遗传途径由九个基因构成,每个基因的平均长度约为1,500个核苷酸。因此,我们所构建的每个新途径大约包含13,000个核苷酸。另外,还需要制造每个基因的突变型,再对不同基因的突变型进行组合,挑选出最优组合。假若为每个基因制造两种突变型,那么,我们就得合成29即512条遗传途径,共约600万个核苷酸!对于传统DNA合成技术,这项任务无疑难于登天;而对于基因芯片合成技术,这不过是小事一桩。
工厂化技术不仅可以用于大规模合成基因网络,还可以用于创造新型蛋白,例如化学合成反应或治理环境污染所使用的新型催化剂,以及用于基因疗法或杀灭病原体的高特异性酶。贝克的研究小组正在开发一种计算机设计法,用于设计新型蛋白结构。他们已设计出两种新型蛋白,可以模拟人类免疫缺陷病毒(HIV)表面的重要特征。目前,这两种蛋白已作为候选疫苗,并进入了测试阶段。
计算机辅助设计法也有局限性:不够先进,不能保证设计出来的所有蛋白都具有期望中的功能。但计算机可以设计出成百上千的、有希望的候选蛋白,供我们试验。如果把这些蛋白结构转化为相应的基因序列,那就需要合成上百万的核苷酸。对于现今的技术,这是一个困难而昂贵的方案,但利用工厂化技术,可以毫不费力地完成任务。
上述针对疟疾和HIV的DNA、蛋白质合成研究表明,这种以生物工厂技术为基础的方法,可用于对付更多的疾病,包括新出现的疾病。比如,将高效而廉价的DNA测序方法[参见《环球科学》2006年第2期P.14乔治·M·丘奇《1000美元测出你的基因组》一文]与工厂合成能力相结合,我们就可以快速鉴定新型病毒(如SARS病毒)或新型流感病毒,然后再以现有技术难以企及的速度,制备相应的蛋白疫苗。
当然,生物工厂并非只是一个高速合成技术的集合体,而是一种方法:不仅对现有生物机器进行思索,而且借用工程学的语言和方法,构建新型生物机器。
撰文 生物工厂研究小组*(Bio Fab Group)
合成DNA
如果说单个的晶体管是电子电路的基本组件,那么在生物学中,与之对应的便是基因(有序的DNA片段)。为了给高级的生物装置构建基因电路(genetic circuit),我们就需要一种快速可靠、价格合理的DNA片段合成法。
20年前,在前人的工作基础上,美国科罗拉多大学博尔德分校(University of Colorado at Boulder)的马文·H·卡拉瑟斯(Marvin H.Caruthers),利用DNA自身的化学性质,研发出一种单链DNA合成法。DNA由4种核苷酸组成,而每种核苷酸又含有一个相应的碱基,分别是腺嘌呤(adenine,A)、胞嘧啶(cytosine,C)、鸟嘌呤(guanine,G)和胸腺嘧啶(thymine,T)。碱基之间的亲和力使它们两两配对(A-T配对,G-C配对),形成梯状双链DNA分子中的梯级。化学键不仅在碱基对之间形成,在邻近的核苷酸之间也会形成。
卡拉瑟斯所使用的方法被称为固相亚磷酰胺法,这是目前大多数商业DNA合成法的基础。合成反应起始于一个单核苷酸,这可不是一个普通的核苷酸,它附着在悬浮于酸性液体中的固相支持物(如聚苯乙烯颗粒)上,并承担着发起合成反应的重任。当碰见“新来”的核苷酸,两者便会通过形成化学键相连。如果不断加入核苷酸,反应就会持续进行,核苷酸链就会不断延长。这样,就可以合成任何想要的核苷酸序列,并且不易出错(出错几率约为1%)。
但很多时候,生物工程师想要的基因片段,远远超出了该方法的合成能力。一个简单的基因网络也许就有数千碱基对;就算像细菌这样的微小生物,基因组也可达数百万碱基对。因而,我们如果想找到高产出、低误差的合成法,就只能寄希望于从自然界中获得一些提示了。
在生物体中,像酶(如聚合酶)这样的生物机器,能以高达每秒500个碱基的速度,合成和修复DNA分子,而错误率仅为十亿分之一!这就意味着,即便是最好的DNA合成机器(每300秒合成1个碱基),产出率(输出量/错误率)也不及聚合酶的万亿分之一。更有甚者,在细菌体内,当复制像基因组那样的长链DNA时,多个聚合酶会同时运作,在20分钟内就能合成含有500万个碱基的DNA!
于是,丘奇开始仿效细菌聚合酶的这种平行作业方式,以适应现有的基因芯片技术。基因芯片其实就是特殊的玻璃片,在它的表面上,繁星般地点缀着长为50~70个碱基的寡核苷酸(oligonucleotide/oligo,短小的核苷酸链)。通过亚磷酰胺反应法,寡核苷酸被同时合成于基因芯片的表面,并以格状排列,密度高达100万点/平方厘米。在传统技术的基础上,我们又在这些寡核苷酸上加上可剪切的连接子,以便特定的寡核苷酸能够从基因芯片上释放出来。在我们的实验性基因芯片上,每个点约为30微米宽,含有大约1000万个寡核苷酸分子。
通常,基因芯片上的核苷酸链被称为构建性寡核苷酸(construction oligo),因为它们的部分序列是相互重叠的,通过重叠的序列,可将它们组装成更长的DNA结构(如整个基因)。但是,任何含有错误序列的寡核苷酸都必须被清除。为此,我们采用了两种不同的纠错方法。
第一种是选择性寡核苷酸(selection oligo)法。合成选择性寡核苷酸的方法与制造基因芯片的方法相同,只是在核苷酸序列上,前者有特殊的要求:与构建性寡核苷酸的序列互补。合成之后,便对连接子进行剪切,从玻璃片上释放选择性寡核苷酸,并使它们流过构建性寡核苷酸的芯片。按照碱基配对的原则,选择性寡核苷酸就会与互补的构建性寡核苷酸结合(杂交,hybridize),形成双链DNA。这样,任何不能配对,或者含有错误序列、配对不完全的构建性寡核苷酸,都无法逃过我们的“法眼”,也就无法继续在芯片上“滥竽充数”。与制造基因芯片一样,在合成时,选择性寡核苷酸的序列也会出错。但是,构建性与选择性寡核苷酸的错误序列很难完全互补。因此,利用一组寡核苷酸对另一组进行校对是一种有效的纠错方法。利用这种方法,我们合成寡核苷酸的平均错误率可以低至1/1,300。
正如人们所料,生物体系非常注重自身复制的精确度。我们的第二种纠错方法就来源于自然界。十年前,莫德里奇首先发现了生物体系复制时的详细纠错过程,并将这个过程命名为“MutS,L,H”。当两条DNA链的碱基不能完全配对时,那么在错配区,便不能形成双螺旋结构。MutS是一种天然存在的蛋白,它会识别这种缺陷,并与之结合。随后,它招集“同伴”—— MutL和MutH,共同完成修正任务。利用该方法,雅各布森与美国麻省理工学院的彼得·卡尔(Peter Carr),已经将DNA合成的错误率降到1/10,000。对于生产小型基因网络,这样的保真度足矣。
在可释放性平行合成技术和纠错技术的支持下,长链DNA的合成速度更快、成本更低、精确度更高。这些技术将是生物工厂的基础,随着时间的流逝,它们会像半导体芯片光刻技术那样,不断进步。先进的技术是宝贵的财富,能解放我们的思想,让我们思考更多:在生物工厂里,我们可以做些什么呢?
基因工程药物
利用生物工厂的平台探索征服疾病的新方法,是我们最早的目标之一。基斯林和贝克的研究对象是疟疾和艾滋病——两种困扰人类多年的恶疾,他们致力于开发对付这两种疾病的药物。尽管我们所研究的治疗方法与他们的不尽相同,但在很大程度上,两个小组的研究都依赖于精确合成长链DNA的能力。我们的研究只是工厂化的一个代表,工厂化拥有强大的力量,必将颠覆传统的新药开发模式!
以疟疾为例,已有药物能将感染者体内的致病寄生虫彻底消灭,从而根治疟疾。这是一种小分子药物,叫做C-15倍半萜(sesquiterpene),俗名青蒿素(artemisinin),是由青蒿植物(sweet wormwood,多发现于中国华北地区)合成的天然化合物。但在植物中,天然合成的青蒿素过少,造成青蒿素的价格极其昂贵,无法推广。因此,在过去五年中,基斯林的研究小组努力克隆合成青蒿素的遗传途径,并将它插入酵母菌中,让酵母菌大量合成青蒿素。
在酵母菌中,我们还能对遗传途径进行改良,使青蒿素的合成效率大幅提高。青蒿素的合成途径叫作甲羟戊酸途径(mevalonate pathway),目前,我们已经能够对途径中的关键基因进行重新设计。较之细菌中的原始途径,关键基因的改变可使青蒿酸(amorphadiene,青蒿素的前体)的产量提高10万倍!但这仍然不够,要使青蒿素得到广泛应用,必须进一步提高产量。这就需要我们对整个青蒿素途径进行综合重建。
整条遗传途径由九个基因构成,每个基因的平均长度约为1,500个核苷酸。因此,我们所构建的每个新途径大约包含13,000个核苷酸。另外,还需要制造每个基因的突变型,再对不同基因的突变型进行组合,挑选出最优组合。假若为每个基因制造两种突变型,那么,我们就得合成29即512条遗传途径,共约600万个核苷酸!对于传统DNA合成技术,这项任务无疑难于登天;而对于基因芯片合成技术,这不过是小事一桩。
工厂化技术不仅可以用于大规模合成基因网络,还可以用于创造新型蛋白,例如化学合成反应或治理环境污染所使用的新型催化剂,以及用于基因疗法或杀灭病原体的高特异性酶。贝克的研究小组正在开发一种计算机设计法,用于设计新型蛋白结构。他们已设计出两种新型蛋白,可以模拟人类免疫缺陷病毒(HIV)表面的重要特征。目前,这两种蛋白已作为候选疫苗,并进入了测试阶段。
计算机辅助设计法也有局限性:不够先进,不能保证设计出来的所有蛋白都具有期望中的功能。但计算机可以设计出成百上千的、有希望的候选蛋白,供我们试验。如果把这些蛋白结构转化为相应的基因序列,那就需要合成上百万的核苷酸。对于现今的技术,这是一个困难而昂贵的方案,但利用工厂化技术,可以毫不费力地完成任务。
上述针对疟疾和HIV的DNA、蛋白质合成研究表明,这种以生物工厂技术为基础的方法,可用于对付更多的疾病,包括新出现的疾病。比如,将高效而廉价的DNA测序方法[参见《环球科学》2006年第2期P.14乔治·M·丘奇《1000美元测出你的基因组》一文]与工厂合成能力相结合,我们就可以快速鉴定新型病毒(如SARS病毒)或新型流感病毒,然后再以现有技术难以企及的速度,制备相应的蛋白疫苗。
当然,生物工厂并非只是一个高速合成技术的集合体,而是一种方法:不仅对现有生物机器进行思索,而且借用工程学的语言和方法,构建新型生物机器。
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