30 Jun 2011

Required Solutions

10x PBS
80 g NaCl
2 g KCl
14.4 g Na2HPO4
2.4 g KH2PO4
Dissolve into 800 ml ddH2O
Adjust pH to 7.4
Qs to 1000 ml
Make a 1x working solution

Buffer C (4ºC)
20 mM Hepes-KOH pH 7.9 (from 0.5 M stock)
0.42 M KCl (from 3 M stock)
25% glycerol (from 80% stock)
0.1 mM EDTA (from 0.5 M stock pH 8.0)
5 mM MgCl2 (from 1 M stock)
0.2% NP40 (from 10% stock)
Use autoclaved stocks for all ingredients.
Store at 4ºC

Add right before use to 10 ml:
10 µl 1 M DTT  (store aliquots at -20ºC, thaw once)
50 µl 100 mM PMSF (in iso-propanol -20ºC)
1 µl 10 mg/ml Leupeptin (-20ºC)
1 µl 10 mg/ml Aprotinin (-20ºC)
10 µl 1 mg/ml Pepstatin (-20ºC)
Or, replace the last three protease inhibitors with one mini Roche protease inhibitor
Tablet (# 11 836 153001) and add DTT and PMSF as above.

For looking at phosphorylated proteins, add the following phosphatase inhibitors to
Buffer C:
Na-beta-glycerophosphate to 50 mM (stock 1 M)
NaF to 1 mM (stock 0.4 M)
Na-ortho-vanadate to 1 mM (stock 0.1 M)

Buffer D
20 mM Hepes-KOH pH 7.9
100 mM KCl
25% glycerol
0.1 mM EDTA (cold)
Add right before use, for 250 ml:
250 µl 1 M DTT
1.25 ml 100 mM PMSF

10x Ponceau S
Ponceau S 2% (w/v)
TCA 30% (w/v)
Dilute to 1x before use (can be re-used many times).

4x Laemmli Buffer
4.4 ml 0.5 M Tris (pH 6.8)
4.4 ml Glycerol
2.2 ml 20% SDS
0.5 ml 1% Bromophenol Blue
0.5 ml Beta-ME
Aliquot and store at -20ºC. Dilute to 2x before use.

10x Running Buffer
30.3 g (0.25 M) Tris Base
144 g (1.92 M) Glycine
10 g (1%) SDS or appropriate for concentrated stock
Qs 1000 ml ddH2O
Dilute 1:10 with ddH2O.  PH will be 8.3

10x Towbin’s Electrotransfer Buffer
30.3 g Tris Base
144 g Glycine
Qs 1000 ml ddH2O

1x Towbin’s
100 ml 10x stock
200 ml (20%) Methanol
Qs 1000 ml ddH2O
1 ml (0.02%) 20% SDS- optional

Lab pdf and ppts

29 Jun 2011

Bacterial Warfare

“New examine reveals how bacteria hijack our bodies' cells and outwit our immune systems--and how we can employment their own weapons counter to them”
Most bacteria are well-behaved companions. Indeed, if you are perpetually feeling lonely, remember with the purpose of the trillions of microbes living in and on the norm human being body outnumber the human being cells by a ratio of 10 to solitary. Of all the tens of thousands of acknowledged bacterial species, simply approaching 100 are renegades with the purpose of break the rules of peaceful coexistence and promote to us sick.
Collectively, individuals pathogens can cause a luck of effort. Infectious diseases are the go along with leading cause of death worldwide, and bacteria are well represented amongst the killers. Tuberculosis solitary takes almost two million lives all day, and Yersinia pestis, infamous in support of causing bubonic plague, killed approximately solitary third of Europe’s population in the 14th century. Investigators control made huge progress finished the preceding 100 years in enlightening a number of species with antibiotics, but the risky bacteria control plus found ways to resist many of individuals drugs. It is an arms compete with the purpose of humans control been trailing of late at night, in part as we control not understood our enemy very well.


Bacteria are both dangerous and helpful to the environment, and animals, including humans. The role of bacteria in disease and infection is important. Some bacteria act as pathogens and cause tetanus, typhoid fever, pneumonia, syphilis, cholera, food-born illness, leprosy, and tuberculosis (TB). Sepsis, a systemic infectious syndrome characterized by shock and massive vasodilatation, or localized infection, can be caused by bacteria such as Streptococcus, Staphylococcus, or many gram-negative bacteria. Some bacterial infections can spread throughout the host's body and become systemic. In plants, bacteria cause leaf spot, fire blight, and wilts. The mode of infection includes contact, air, food, water, and insect-borne microorganisms. The hosts infected with the pathogens may be treated with antibiotics, which can be classified as bacteriocidal and bacteriostatic, which at concentrations that can be reached in bodily fluids either kill bacteria or hamper their growth, respectively. Antiseptic measures may be taken to prevent infection by bacteria, for example, by swabbing skin with alcohol prior to piercing the skin with the needle of a syringe. Sterilization of surgical and dental instruments is done to make them sterile or pathogen-free to prevent contamination and infection by bacteria. Sanitizers and disinfectants are used to kill bacteria or other pathogens to prevent contamination and risk of infection.
In soil, microorganisms which reside in the rhizosphere (a zone that includes the root surface and the soil that adheres to the root after gentle shaking) help in the transformation of molecular dinitrogen gas as their source of nitrogen, converting it to nitrogenous compounds in a process known as nitrogen fixation. This serves to provide an easily absorbable form of nitrogen for many plants, which cannot fix nitrogen themselves. Many other bacteria are found as symbionts in humans and other organisms. For example, the presence of the gut flora in the large intestine can help prevent the growth of potentially harmful microbes.
The ability of bacteria to degrade a variety of organic compounds is remarkable. Highly specialized groups of microorganisms play important roles in the mineralization of specific classes of organic compounds. For example, the decomposition of cellulose, which is one of the most abundant constituents of plant tissues, is mainly brought about by aerobic bacteria that belong to the genus Cytophaga. This ability has also been utilized by humans in industry, waste processing, and bioremediation. Bacteria capable of digesting the hydrocarbons in petroleum are often used to clean up oil spills. Some beaches in Prince William Sound were fertilized in an attempt to facilitate the growth of such bacteria after the infamous 1989 Exxon Valdez oil spill. These efforts were effective on beaches that were not too thickly covered in oil.
Bacteria, often in combination with yeasts and molds, are used in the preparation of fermented foods such as cheese, pickles, soy sauce, sauerkraut, vinegar, wine, and yogurt. Using biotechnology techniques, bacteria can be bioengineered for the production of therapeutic drugs, such as insulin, or for the bioremediation of toxic wastes.

Restriction Enzmes

Restriction enzymes are very ordinary reagents used in the analysis and manipulation of DNA and are used in a variety of "gene technology" investigate programs and protocols. Most students now gather the aid of these enzymes lacking always learning or realizing their origins or their in vivo role. These clarification are intended to sort out solely with the intention of. Hopefully, if you understand their natural role and occurrence, you will be able to understand their attention more thoroughly.
There are several ways by which DNA is transferred from lone bacterial cell to a further (i.E., from a donor to a recipient).
1. Transduction - Phage are able to conveying viral and bacterial DNA in a species/strain point style.
2. Conjugation linking two bacteria.
3. Uptake of emancipated genetic material using crust proteins with the intention of get on to up competence systems and others.
Regardless of how DNA enters a recipient cell, once inside the cell, foreign DNA may either carry on (i.E., imitate and/or recombine with the recipient DNA) or it could be degraded. Degradation of the foreign DNA is termed restriction and is accomplished by nucleolytic cleavage. If the DNA survives, it is due to the modification endeavor of the donor cell.
Restriction Modification (RM) systems be inflicted with been found in bacteria. Whether they occur in other microbes or in eucaryotic cells is still uncertain. RM systems be inflicted with been found in Anabena, a blue-green algae (called Cyanobacteria by some). Not all bacteria be inflicted with RM systems and hence in persons with the intention of lack such systems, all genetic material expected survives.
RM systems are ordinarily named according to the bacteria in which it was found. These names are furthermore often used as their gene designations. Some ordinary examples of RM systems include:


 E. coli

5' - G|AATTC - 3'
 Ana II
5' - G|G(A/T)CC - 3'
 Hind III

5' - A|AGCTT - 3'
 Stu I
5' - AGG|CCT - 3'
 5' - N|GATC - 3'

The vertical line in the recognition site sequence is the cleavage site. N= any base
The RM systems are species/strain specific thus only allowing the survival of DNA received from a like species or strain having the same RM system.

Any native double stranded DNA can serve as a substrate for the enzymes of RM systems provided that there are specificity sequences. DNA that is modified on only one strand of the duplex DNA is still preserved from restriction. Thus, semi-conservative replication of a cells DNA will not lead to self-restricion. Soon after replication, the new strand is quickly modified prior to the next round of replication.


Restriction is accomplished by sequence specific (site specific) endonucleases. These enzymes are able to cleave the interior of DNA strands (as opposed to exonucleases which cleave from a terminus). Once terminuses are produced by the internal cleavages of the endonucleases, a large variety of exonucleases can degrade the DNA very rapidly.

Modification is accomplished by sequence specific methylase enzymes. In any cell with a RM system, both the restriction and modification enzymes have the same sequence specificity.

Generally, the methylase activity is "replication independent" and the most common site for methlyation is the extra cyclic amine group of adnine. The methyl donor for the methylases is S-adenosyl methionine (SAM). Mg++ and ATP are not needed for the activity. The methylases can compete with the restriction enzymes (since their specificity sites are the same) and thus some portions of foreign DNA may be preserved. This is termed "leakiness" of the system or "rescue". In E.Coli, up to 3% of the foreign DNA may be salvaged in this manner.

TYPE I RM was the first type discovered but is not the simplest nor the most common. In this type, the restriction modification is accomplished by a five peptide complex - R2M2S or 2:2:1 (R:M:S)
HsdR -----> R (135kd), hsdM -----> M (62kd), hsdS -----> S (55kd)
Mutants of gene hsdR are designated r-m+, etc.
Examples are EcoK and EcoB.
Type I systems recognize a bipartite asymetrical sequence:
---TGA*NnTGCT--- * = methylation sites
---ACTNnA*CGA--- n = 6 for EcoK, n = 8 for EcoB
The S peptide is the site recognition factor and may control protein to protein specific assembly of the complex. SAM must be bound to the M protein for S to recognize the site.
The cleavage site is about 1000 base pairs from the recognition site where the RMS complex is bound. This is accomplished by a "looping" mechanism and cleavage requires ATP. The strands are cleaved one at a time.
TYPE II RM systems are the simplest and most common (at least commercially). In this system, there are separate R and M proteins each able to recognize the cleavage site (no S protein needed). Each may compete directly and no complex is formed. Site recognition is via short palindromic base sequences that are 4-6 base pairs long. Cleavage is at the recognition site (but may occasionally be just adjacent to the palindromic sequence, usually within) and may produce blunt end terminuses or staggered, "sticky end" terminuses. No ATP requirements have been detected for the endonuclease activity.
TYPE III RM systems are interesting in that the M and S function is found in one protein labeled MS (hsdMS => 73-80kd in E.Coli) while the restriction is accomplished by the hsdR gene product R (hsdR => 106-110kd in Ec). The recognition site is a 5-7 bp asymmetrical sequence. Cleavage is ATP dependent 24-26 base pairs downstream from the recognition site and usually yields staggered cuts 2-4 bases apart.

Separate endonuclease & methylase proteins
Bifunctional enzyme of 3 subunits, R,M & S (2:2:1)
Bifunctional enzyme of 2 subunits, MS & R
Short palindrome 4-6 bp recog. Site, same for R & M
Recog. Site = bipartite, asymetrical sequence = 5-7bp
Recog. Site = asymetrical  sequence = 5-7bp.
Cleaves at recognition site. Yields blunt or sticky end, within or next to site.
 Cleaves 1000 bp from recognition site
Cleaves 24-26bp downstream of recognition site, staggered cut 2-4 bases apart.
R & M act separate
R & M may compete or may be exclusionary.
R & M occur simultaneously & may compete.
No ATP needed for R
Needs ATP for R
Needs ATP for R

27 Jun 2011

Clinical Value-Add for Health Information Exchange (HIE)

Jay K. Joshi

University of Chicago

Citation: J. Joshi: Clinical Value-Add for Health Information Exchange (HIE). The internet Journal of Medical Informatics. 2011 Volume 6 Number one
Key phrases: Wellness particulars trade | informatics | value-add | wellness info company

Desk associated with Material
Final results
Communication in order to

Goals: All of us clarify the actual medical utility- each good as well as negative- associated with Wellness Info Trades (HIE) to be able to determine exactly how accurate worth could be achieved with the ownership associated with this type of program. Techniques: All of us carried out the books evaluation more than several directories associated with 350 content articles regarding the subject. Following testing with regard to importance, all of us chosen 135 content articles which talk about research regarding HIE. Outcomes: All of us filled 2 furniture along with info synthesized type the actual 135 diary content articles. 1 desk targets the actual strengths associated with HIE, and also the 2nd desk targets the actual damaging facets of HIE. The concentrate would be to emphasize obvious advantages, significant questions, as well as placement all of them within juxtapose to determine a definite assessment. Findings: Although HIE possess each good as well as damaging elements, 1 should concentrate on the actual specificities from the wellness organization, such as individual quantity, demographics, as well as wellness concentrate, to be able to figure out the worthiness associated with applying this type of program.
Wellness Info Trades (HIE) tend to be quickly evolving because the next phase within enhancing individual treatment utilizing technical programs. HIE impact the essential individual treatment program through changing exactly how healthcare info is actually shipped as well as displayed. Medication in the usa is definitely the fragmented area; with the actual increasing quantity of transactional amount of individual info, the requirement to incorporate the machine inside a coherent method gets even more essential. Nevertheless, user-friendly advantages of this type of program belie natural dangers within making this type of program. Changing a sizable size HIE produces logistical as well as individual security associated difficulties. Several federal government as well as personal businesses allow us research or even simulations describing both good as well as damaging facets of HIE. Released research which show apparently obvious advantages in many cases are restricted within character as well as contingently particular. All of us execute a organized overview of the actual books as well as existing the info to acquire a thorough look at associated with exactly how genuinely efficient HIE could be. All of us concentrate the evaluation about the medical ramifications associated with HIE techniques.
In the obtainable information, all of us extrapolate crucial guidelines to create the value-added desk featuring the advantages as well as dangers or even questions of the HIE. All of us concentrate on individuals characteristics that impact individual administration, impact doctor decision-making, as well as effect each short-term as well as long-term choice looks at.
All of us carried out a good listed search on the internet while using subsequent search engines like google: PubMed, Cochrane Data source, as well as Search engines college student. The actual queries happened 06, This summer, July, as well as Oct associated with 2010. All of us started using the expression, “Health Info Trade (HIE)”, as well as extended the actual research lingo to incorporate comparable lingo related to HIE, therefore making the most of awareness. All of us examined the actual books straight dealing with research as well as studies carried out about the subject. Resources consist of impartial advisor evaluations, educational publications, as well as content articles describing educational dependent HIE endeavours. The research crafted in order to wide dependent research relevant towards the common individual populace, and never any kind of niche particular research. Even though all of us didn't differentiate arranged requirements with regard to exactly what comprises the specific exercise, all of us required is designed to tell apart the actual range of every research to higher evaluate it's applicability within highlighting common populations.
Research had been mainly obtained from educational released publications obtainable via the various search engines. Nevertheless, several personal, talking to dependent research had been contained in the research, however denoted as a result.
Information had been removed in the research, synthesized, as well as built-into detailed excerpts describing tips. All of us filled the information inside a 2 dimensional matrix featuring crucial medical guidelines. Medical guidelines and also the connected advantages incorporated are generally recognized metrics accustomed to evaluate enhancements on applying EMR/EHR. 1 The books research as well as professional evaluation in the Chi town Wellness It Local Expansion Middle (CHITREC) assisted set up the actual construction from the matrix. The actual medical guidelines consist of laboratory outcomes, medicines, medications, prior outpatient appointments, crisis appointments, present ambulatory outpatient appointments, high quality associated with information, long-term advantages, period effectiveness, as well as diagnoses. The advantages connected consist of high quality associated with treatment, impact associated with sufferers, financial savings in order to doctors, mistake decrease, as well as organizational effectiveness as well as regulating conformity. 2 parallel dimensional matrices had been produced by which 1 outlined the actual strengths associated with HIE and also the additional outlined the actual damaging elements.
The actual research had been after that categorized in line with the character from the post, and also the materials protected. All of us very first split every research in to whether qualitative or even quantitative research. Quantitative research consist of any kind of research formatted like a theory screening research, mix sectional research, or even predictive evaluation. Just about all category strategies as well as syntheses had been created in house.
All of us after that abstracted the actual appropriate information in the content articles utilizing story functionality. Within synthesizing the actual content articles, all of us examined the character from the post, and also the character from the HIE talked about. All of us recognized research upon mainly generalist dependent techniques, crisis program just techniques, as well as hardly ever niche dependent techniques. All of us discovered substantial overlap between the numerous content articles. Some qualitative, plus some quantitative, research examined the whole HIE program, a few centered on a specific element. All of us mentioned exactly what element had been outlined within the research appropriately. Numerous HIE variety within geographic as well as market range. Individuals trade facilities which were analyzed inside a restricted framework had been recognized through the ones that encompassed just about all sufferers inside the HIE.
Because some of the research had been quantitative within character, this grew to become hard to investigate all of them via a regular meta-analysis. All of us abstracted particulars within choose research as well as recognized styles inside every book. The actual styles created the foundation from the value-added chart permitting the actual ideas through every book in order to naturally circulation to the 2 dimensional desk.
You will find absolutely no financing resources to reveal from any kind of stage within our information selection.
Final results
From the almost 350 research examined, all of us incorporated 135 research. The actual fairly higher price associated with addition is basically because of the character from the evaluation as well as the help of set up evaluation research which led the books evaluation. Addition requirements incorporated a particular talking about associated with HIE as well as RHIO, or even the actual changeover associated with EHR as well as EMR in order to HIE. Additional addition requirements consists of any kind of talking about associated with HIE investigation, applicability, notable hurdles, or even natural restrictions. From the research put together which fulfilled all of the addition requirements 18% (24/135) had been quantitative within character. From the research techniques, 7% (10/135) just main studies, 28% (38/135) had been content within character, 47% (64/135) had been educational within character, 2% (3/135) carried out selection interviews, as well as 10% (14/135) had been evaluation content articles. 1 research went the simulation, as well as an additional research exemplified numerous company versions relevant to some HIE. Most stated to become wide within range and also to signify which standard individual neighborhood. Within our research, all of us discover that 73% (98/135) preserve an extensive range. The residual research possibly specific the geographic range or perhaps a specific medical concentrate.
All of us didn't differentiate in between open up as well as shut trade techniques because the majority of brand new versions thinking about implementing HIE tend to be open up versions. Nevertheless, for several instances, for example crisis sections, all of us incorporated just neighborhood dependent research, consequently taking just open up dependent techniques. This particular agreement much better suitable the study’s concentrate within building generalizations with regard to environmentally friendly aggressive benefit. From the research which fulfilled the actual addition requirements, 21% (28/135) centered on main procedures of the HIE, 3% (4/135) solely upon crisis sections within neighborhood private hospitals, 2% (2/135) about the monetary facets of HIE, 5% (6/135) upon just individual security as well as privateness, 2% (2/135) upon HIV individual security, 3% (3/135) upon long-term continuity associated with treatment, 11% (15/135) about the changeover in between digital information along with a practical trade design, 4% (6/135) upon enhancing ownership prices amongst private hospitals, 3% (4/135) upon making use of standardizations, 4% (5/135) upon enhancing price usefulness associated with HIE, as well as 2% (3/135) upon creating a nationwide size design.
Following cataloguing the research, all of us incorporated every research in to the value-added desk.

The desk shows that the actual framework through which all of us evaluate every medical parameter decides the web advantage or even drawback. The advantages reaped through arranging crisis appointments with an digital trade rapidly diminish whenever seen in the viewpoint from the doctors on their own. The actual possible advantage of documenting an infection prices across the country turn out to be substantially much more obvious since the minor dimension from the trade program reduces. The actual particulars associated with geographic range associated with size include an additional sizing which additional complicates knowing all the advantages. Whilst particular content articles be aware obvious advantages within much more shut, homogenous individual populations, it's not obvious exactly how individuals advantages convert in order to bigger wellness establishments. This isn't to express which big treatment centers don't include distinctive advantages within as well as associated with on their own. The actual set up books as well as earlier research verify the initial advantages of every program. Within our furniture, all of us make an effort to cash in about the common concepts which construe advantages as well as disadvantages within every medical parameter for each the actual vantage stage from the physicians.
Because the objective would be to know what concepts could be generalized to any or all HIE including dimension, size or even range, the factor is based on determining the actual medical guidelines within parallel. This particular under the radar structure shows which nobody parameter could be ubiquitously designated like a advantage or even drawback.
The value-added proposal suggests all of us change in the direction of a far more nuanced knowledge of a good HIE’s aggressive benefit. The environmentally friendly aggressive benefit is actually a chance to give a support towards the focus on customer in a fashion that an alternative solution choice can't. To recognize accurate advantages, 1 should know what extra advantages a good HIE might supply inside a particular framework. For example, may the prospective customer end up being federal government businesses instituting something which medical center techniques purchase in to? Or even may the prospective customer end up being scaled-down exercise organizations which are desired through bigger educational or even insurance coverage dependent techniques? These types of queries impact the actual range as well as viability associated with HIE.
Whilst most of the advantages of HIE could be countered along with genuine questions, you ought to not really low cost the actual robust possible of those techniques. Bothersome improvements, that HIE’s suggest being within today’s health care, are able to transform apparently reduced worth options in to practical choices which get to be the optimum route. Inside a area for example it trade, system externalities perform a good enormous part within making customer attention as well as growing the worthiness from the technologies. Numerous questions tend to be affected through the physician’s readiness to consider or even take the actual part associated with HIE. Elevated execution through doctors may have an optimistic rapid impact as well as offset this kind of questions.
Investigation to the area ought to be innovative as well as daring. Along with this type of complicated idea including countless parameters, it's not probably be resolved without having numerous earlier efforts. Past discovering a good optimum functional design with regard to HIE, testing may determine the reason why ideas haven't proved helpful previously.
Restrictions with this research range from the ways of selection towards the general evaluation, and also the information abstraction. All of us were not able to do the meta-analysis from the obtainable books simply because handful of all of them had been quantitative within character. Additional most of the actual research regarding this particular subject tend to be susceptible to journalistic prejudice. The actual writers, mainly frontrunners within the area, possess particular viewpoints how a good trade program ought to operate. Abstracting the story functionality using their vantage stage offers merely a part of the actual performance associated with HIE.
Furthermore, the research had been limited by the effectiveness of the actual lingo all of us employed for the books research. Terms for example “Health Info Exchange” might not properly include all of the books obtainable about them. All of us prevented this particular mistake simply through mix referencing the books evaluation along with set up books queries to make sure comprehensiveness. Nevertheless, individuals diary content articles referencing HIE along with distinctive lingo or even situation particular key phrases will probably happen to be skipped. All of us additionally restricted the research in order to English-based research as well as incorporated merely a restricted quantity of distinctive search engines like google to help the books evaluation.
We wish in order to recognize Doctor. Abel Kho from the Chi town Wellness It Local Expansion Middle (CHITREC) as well as Northwestern College for his or her factor, knowledge, as well as guidance throughout the information selection as well as meaning procedure.
Communication in order to
: The author Nited kingdom. Joshi
5807 Utes. Woodlawn Ave.
Chi town, IL 60637
Jjoshi2@chicagobooth. Edu

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