Here is the rough copy of the article…hot before the press!
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Top psychological reasons people desire plastic surgery, namely females :

1. Insecurity – Insecurity goes hand in hand with self consciousness and is almost the blanket term or concern for the following other three reasons of why women will undergo plastic surgery. Many women growing up will sometimes have shallow beliefs about aesthetics regarding looks as a major role playing factor in how they are perceived, harsh as this may sound, this is assessed as relatively minor when opting the nip and tuck; whereas on the other end of the spectrum exists the possibilities of deep rooted psychological issues stemming from heavy paternal criticisms during their early years.
2. Self Esteem – Women can be very nervous in social situations. This is a branch of insecurity where women feel they don’t belong to a social group or to be talking with a man because they feel, for lack of better words, their social counterparts are physically more attractive than them. By undergoing the knife they can relieve themselves of the fear as having plastic surgery done has been known to be a major factor in the boosting of self esteem and helps give women the courage to not only engage in healthy social contact but even approach men to spark a conversation, something they would never have dreamed of doing before.
3. Relationships – Many women already in relationships, regardless of whether or not the relationship is of marriage or has only been going on for a few weeks. They see their partner in social situations or know they are going out where there are other women perhaps they think are more beautiful than they themselves. In order for them to feel they need to assure their partner’s loyalty they may go to the measures of plastic surgery to repress these insecurities of their relationship.
Another factor here is non-relationships. In what is now socially known as ‘peacocking’, many individuals without a partner and worrying about their appearance will see a plastic surgeon for the nip and tuck so they can catch a mate in whatever venue they are seeking a partner.
4. Abnormal Psychological Diseases – One of the most common examples of this is body dysmorphic disorder, a rare psychological issue where many women have strong fears that certain elements of their body are very imperfect or not line. To be more descriptive an example of this could be if a women has eyes that they feel are not spaced properly apart from what they believe to be the norm (usually the images one would see in magazines or on TV of models); what is interesting about this disorder is that the eyes could be spaced perhaps less than half a centimeter from what the standards they have set for themselves, yet the effect of what seems so insignificant and minor to most is a heap of tremendous suffering for them.

Note : I did not write this article, I only provided insight from what I learned from Dr. Rose at Plastic Surgery Houston.

Dyschromia is basically any type of disorder of pigmentation to the hair or the epidermis.  This also applies to discoloration of the nails.  This is actually because the nails are surprisingly comprised of the same the fibers that make up the hair.  Ever wonder why your hair or nails grow?  That is partly why.  The other part being are the dermal papillae which are actually the true reason for their formation.  It is these biological sites that cells continually reproduce and transform.

Information about the limelight system :

The Limelight creation, by Cutera, is the latest in technology and is a programmable IPL device and core physician favorite, with vast improvements from past models making it the most superior and effective machine for its applications. The device excellent for treating dyschromia including vascular conditions such as rosacea, diffuse redness and telangiectasia with Cutera’s LimeLight device. LimeLight’s multiple wavelength technology delivers three pre-set modes with a tailored wavelength spectrum, pulse width, real time callibration and adjustable cooling for superior clinical outcomes. “Low contrast pigmentation is a clinical challenge and while most IPLs can treat high contrast pigmentation, LimeLight effectively treats both. Further, LimeLight addresses dyschromia including solar lentigines and a range of vascular conditions such as telangiectasia, diffuse redness and rosacea.” A versatile and powerful device for treating vascular and pigmented lesions.
•    Three distinct pre designed programs create a version of combined elements with excellent hemoglobin and melanin absorption for uniform treatments and ease of use.
•    Adaptability technology permits treatment of multiple indications, including both high and lower contrasts of pigmentation.
•    Features such as the industry’s fastest treatment speed and adjustable climate control provide clinical versatility.
•    The only machine with real-time calibration (ensures accurate and consistent power output during treatment that factor the patient’s safety as well as effective results

Programs and their functions A | 520 | provides the latest to filter Fine telangiectasia, diffuse redness B | 560 | provides the latest to filter Telangiectasia, diffuse redness, low and high contrast pigment C | 580 | provides the latest to filter Solar lentigines

FDA Clearance: LimeLight is cleared for a variety of indications including benign pigmented lesions and vascular lesions.

I’ve heard the price tag of this machine. Ehhhh, not really safe for your pockets.  (note the plural for emphasis) . They are available in high-end day spas such as Utopia Med Spa from association  Plastic Surgery Houston

One of the most important steps before a patient decides to have plastic surgery is research.  By reading this article you are well on to your first step.  This article recommends links to other websites  which will further broaden your understanding of the plastic surgery processes.  However the most important source for a patients full understanding would be their doctor or plastic surgeon; so a well recommended and esteemed surgeon is key for procedures and is definitely a person you would need to talk to well in depth before making important, life altering decisions, well at least in regards of the physical perspective.

There are a multitude of cosmetic options that can be done in the forehead and eye region for both males and females. The main scientific terms for forehead and eyelid procedures respectively is the endobrowlift and blepharoplasty.

From quick glances to extended scrutiny under observation of subjects, the region of the eyes and forehead is a major benchmark of aesthetics in both sexes.  The same goes for males though the chin is also considered a major factor in evaluating aesthetically pleasing features.

With the impending signs of aging diminishing your attractiveness, one can look and feel older than they actually are. That is why more and more people are seeking rejuvenation through brow lift plastic surgery to restore some semblance of their once youthful appearance to improve their overall self-esteem and quality of life. Brow lifts are aimed at the forehead and eyebrow. It helps to soften and improve horizontal forehead wrinkles, vertical frown lines between the eyes, crow’s feet, and drooping brows. All procedures may involve tightening loose skin and excising extra skin.

This page is dedicated to informing you about available brow lift procedures available, the length of procedure, anesthesia involved, and whether it is performed as an in or out patient operation.

More information regarding forehead cosmetic procedures, blepharoplasty and brow lifts can be found from Dr. Franklin Rose’s website on the section called Houston brow lifts.

Image of Blepharoplasty. The lower image represents the post operative procedure.

There is both a multitude of procedures that can be done for both these areas as well as reasons for getting these procedures.  While the most common reason is due to aging and stress which causes wrinkles and droopy eyelids there is also a factor of many young people who will also get this surgery as they may be unsatisfied with how their eyes are spaced or leveled and reshaping the brow area can bring out a better facial presentation to help boost their confidence and have a more impressing look.  This region of the face as well as the mouth is a major way of conveying emotion and communicating through body language so during social conversations this area is often highly focused upon.

For facial plastic surgery this is a very popular option, especially for those who want to achieve a younger look as this facial region is the biggest hint of one’s presumed age.

For the brow many prospective plastic surgery patients will be enticed by its benefits but the effects of the operation are of great importance as well.

For the coronal brow lift these effects include an instantaneous new  appeal at the edge of the eye brows where it has its lift.

The procedure itself is one of the earliest facial plastic procedures and involves a great deal of knowledge before one jumps into the procedure.  It is key to understand the patient has a scalp incision across the entire eyeline from one ear to the other.  The anesthetics which are always local and sometimes general as well are going to last about one to two hours for the procedure to take place.  The procedure ends when the surgeon creates the lift and sews back the parts of the forehead.  Bandages are generally applied after the procedure and are not to be removed until informed by the surgeon.

Many patients are nervous about such an invasive procedure and can opt for a more mild change with their brow lift and opt for an endoscopic brow lift. This relatively new procedure is involves the superseding coronal brow lift but uses smaller incisions and has a much faster recovery with less  lower side effects and faster recovery.

This procedure coupled with a lateral brow lift can give the appearance of an arched eye brow lift.  Many people who want to have a famous face in plastic surgery or would like to know what this look resembles a good celebrity example it is akin to would be that of Catherine Zeta Jones.  Houston Plastic Surgeon Dr. Franklin Rose is known for appearances on the MTV show “I want a famous face”.

Generally high end models and celebrities are a good starting off point when selecting a face even when one does not want to look exactly like the celebrity in the show (though this is a pop-culture fascination I will write about much later in my articles as this is a fascinating phenomenon – so stay tuned to my articles if you are interested ).  A good example of this is a patient can select the face, in this case Catherine Zeta Jones, and can if they are interested in the appearance of her beautiful appearance around the eyes, they can select the arch, which is the main factor in the lateral eye brow lift, of determining how the brow lift will appear on their face.

By using advanced computer technology and 3D imaging a patient can see exactly how the brow lift will appear on their face and can choose between a mild arch or a high arch in order to get an effective representation of what look they would like to appear.

Plastic Surgeons are almost like image consultants with their study in the field juxtaposed with their experience and taste in giving excellent advise and recommendations on which type of look would be best, especially when a patient, which the majority, select multiple plastic surgery procedures on the face and body.

The latter being particularly important with how close the patient would like to resemble the model.  For example if the patient wanted to resemble a look of certain models and celebrities, for example, the eyes of Catherine Zeta Jones yet with the body of Ali Larter.  They could choose to have an exaggerated arch with the brow lift and have their body trimmed in a way using liposuction and breast implants or a breast lift.  Since the models have different breast sizes it would be more than likely a women would have to select the exact breast size she would want, images and more information regarding the breast implants would be found on the section of the website here : Houston Breast Augmentation.  And here is a link to the History of Breast Augmentation where its main roots developed in Houston.

Breast Augmentation procedure by Dr. Franklin Rose

For more information of breast augmentation there are prior articles written by this article with a wealth of information on the subject.

The concern of appearance.

The alteration of appearance.

Appearances dictate many results from an encounter or situation as well as consist of many facets.

For the latter appearances concern genetic aesthetics, taste of clothing, and quality of clothing.

One’s appearance is a major factor when contemplating whether or not to undergo the knife.  Therefor it is essential one must fully take into account the many impacts an appearance in an encounter.

This blog post is a general overview of appearances, encounters, reactions, results, and impressions.

For males, the xy chromosome carrier, who has utter determination to plant his seed prolifically must face other males in which appearance is a key factor.  The size of one’s pectoral area is generally universal in the animal kingdom as a bearing appearance that can both frighten and intimidate another male in an encounter as well attract the opposite sex.  Though it is important to note that hormones play a role in the latter on a universal level as well.

In this sense for the male, the first impression is key in either dominating a business relationship or establishing one’s presence or their territory in respect to other competing males.

For the female the appearance and gait serves as a lure to the male.  The honing of such preening behavior establishes non-verbal seduction to the opposite sex.

These basic root instincts are what cover a major deep-rooted psychological factor that many human’s must come face to face with on an emotional level as they grow up and as they enter the years post their heyday.

Dissatisfaction with the genetic or developed appearance in both females and males of the human species are a significant factor in determining whether or not they will choose to alter their body, which can be done through exercise, diet, and due to scientific breakthroughs of the 20th century – plastic surgery.

Many centers in the US provide breast reconstructive procedures and some national programs offer health care to alleviate the financials of the procedure. However one charity group in particular however has provided completely free plastic surgery.  This charity resides in Houston and is headed by Houston plastic surgeon, Dr. Franklin Rose who provides the free breast reconstruction for those who are victims of breast cancer and have deformed or removed breasts due cancer spreading (cancer multiplies as it spreads which is why a highly noticeable deformation of the breast is common). The breast reconstruction charity run by Dr. Rose and his wife is called The Rose Ribbon Foundation.

Physical disfigurement is always followed by emotional distress. Dr. Rose is very adept at performing reconstructive surgery for those in need of the procedure and whom are located in Houston or surrounding areas. Dr. Rose delivers only the most professional results and takes care of his clients from pre-surgery all the way through the rehabilitation stages.

Texas is known both for its copious amounts of breast implant surgical procedures as well as having one of the highest populations where women (and sometimes men) undergo breast augmentation and breast reduction procedures.  Houston, and Texas as well,  is very reputable for the development of breast implant procedures.

Obtaining breast augmentation or breast reduction will allow a female to be more in tune or comfortable  with her body not to mention her appearance as well.  Plastic Surgery Houston offices actually were the first to bring about silicone breast implants.  A popular procedure developed from advancement in Houston however declined as the more popular type of breast augmentation procedures utilizes saline as this reduces much of the risks and some of the complications.

Breast Reduction Surgery Information

Breast reduction surgery can assist females with their look and help them feel much more balanced. Over-sized breasts can be very cumbersome and  look awkward or unnatural.   This is usually the case if the female has a small body and large breasts.  Of course this last fact translates visa versa with breast augmentation procedures.  Over-sized breasts can also make fun walks and simple exercises a daunting task.

Deciding whether or not this surgery is right for you?

Undergoing breast reduction surgery is not a decision you should take lightly. If you are contemplating mammoplasty, it is crucial you are fully informed about the procedure, consequences and potential risks.

Special thanks to Dr. Rose for explaining the surgical details of surgical breast procedures. Read more information here to see the range of plastic surgery procedures performable by Houston plastic surgeon, Dr. Rose in Houston, Texas.

Popular forms of media TV and the internet have no longer condoned media advertising, as it causes women to feel unhappy with their bodies.  This of course is a contradiction in the sense that the media is calling itself out on its own actions.  Even on blogs like this one the hypocrisy is prevalent as there Google AdSense campaigns, [well this blog does not have a Google AdSense campaign, but for those that do] they sometimes unintentionally, humorously recommend plastic surgery even when the  article of the blog in case is focused against plastic surgery and depicts it in heinous matter as I saw the other day.  Of course I can not link to this blog as the AdSense words have changed as of since.

The contradiction indicates that the media is merely going for rankings as much of the audience and viewers who already by large agree with the networks sentiment will follow suit on the story in case.  These same networks however advertise the ‘subliminal’ perfect female on the beach drinking whatever product is being sponsored.   How can the media, especially news networks, manage to criticize itself when its advertisements pressure women into feeling fat and unwanted.

In a clear outlook, from a social perspective this is evidently mockable as this serves both a contradiction and hypocrisy, all in the name of ratings, furthermore being a lower standard in that essense theres a thousand more stories which could potentially be covered of more value.

From a cultural perspective we can use Asian areas, such as small populated areas in Thailand where women who have a very fat figure are very well admired by their male counterparts as this indicates a high substance of wealth as well as nobility and tradition.

Also men, even in American culture, as well as throughout Europe and globally from now and centuries past have had fetishes for chubby or fat women.

Of course this causes many females to turn to liposuction.  The often unsaid worry child of the media in its depiction as it is such a common procedure and if a TV network were to indignity it, the catch-22 would set in where it would lose its ratings and potentially an audience area.

This boils down to choice as if a woman prefers to be fat and not have to undergo all the trouble and time of losing weight or getting lipo then she will not be well suited for her social environment

The female figure is continually objectified by the media and when the media tries to call itself out on this action it ironically is just objectively seen as childish or even more ironically would only catch the interest of someone who is intellectually childish.

Construction of Ligation Independent Cloning Vectors for Genetic Manipulation in Clostridium difficile

An Honors Thesis submitted in partial fulfillment
of the requirements for Honors Studies in
Biology

___________________

Spring 2009
Biology

Acknowledgements
First and foremost, this project would have been completely impossible without the help and guidance of Dr. __________ His vast knowledge in the field of molecular biology paled in comparison to the immeasurable amount of patience he showed to me over the course of my undergraduate research. Thanks to his personal attention and open-door policy, I felt like I always had someone I could turn to in matter of academia.
I would also like to thank Dr. _______ and Mrs. ____________. Without Dr. ______’s General Microbiology course, many of the key concepts involved in this project would be intimidating and foreign; while the challenges presented were still formidable, consulting the notes accrued from his class assisted in this endeavor tremendously. Important as well was the comprehension and application of General and Organic Chemistry principles, and without Mrs. _______ I would be lost on both accounts. From plating techniques to stoichiometry, both instructors helped lay the foundation for my undergraduate research project.
A debt of gratitude is also owed to Dr. ___________, who I believe was my fourth(!) assigned Honors Council Representative, and no doubt had to move his schedule around to accommodate my thesis defense.
Lastly, I would like to thank the Biological Sciences Department for their dedication to undergraduate research and the many interesting courses I have had the privilege to take over the past four years.

Table of Contents
Acknowledgements……………………………………………………………………………………………………2

Abstract……………………………………………………………………………………………………………………..4

Introduction……………………………………………………………………………………………………………….5

Materials………………………………………………………………………………………………………………….16

Methods…………………………………………………………………………………………………………………..18

Results and Discussion………………………………………………………………………………………………21

References……………………………………………………………………………………………………………….27

Abstract
Every year, the hospital stays of countless patients are further complicated by the onset of symptoms ranging from severe diarrhea, fever, and abdominal pain to toxic megacolon and potential death in particularly susceptible patients. These nosocomial infections are caused nearly exclusively by Clostridium difficile, a gram-positive bacterium which proliferates in hospital settings due to its spore-forming, antibiotic-resistant nature. The disrupted gut flora of patients undergoing antimicrobial treatment is the primary location of proliferation of C. difficile, as the naturally competitive environment of the intestines is instead transformed into a hospitable environment for rapid colonization. As it begins to spread, C. difficile produces two toxins, A and B, which cause its virulence by damaging tissues in order to increase adhesion and facilitate widespread infection. The protein complex that is thought to facilitate the spread of the bacterium is the oligopeptide permease (Opp) system, which is instrumental in both peptide uptake rates as well as adhesion to intestinal mucosa. Efforts to effectively study this system are complicated by the notoriously uncooperative nature of the C. difficile genome, the consequences of which include the fact that no treatment has been developed that can either fully prevent C. difficile infection, nor completely ensure that the infection will not reoccur. The purpose of this research was to facilitate the study of the Opp system in C. difficile. Using a variety of polymerase chain reaction(PCR)-based cloning procedures, it was my hope to construct a vector suitable for ligation-independent cloning, which could then be used to study various aspects of the opp system in C. difficile.
Introduction
Consistent with its name, the bacterium Clostridium difficile has long been a difficult and recurring problem for healthcare institutions around the world. Prevalent in hospital settings especially, C. difficile and the intestinal infections it is responsible for have become increasingly costly for nations around the world, with conservative estimates of treatment costs exceeding $1.1 billion dollars each year in the United States alone (1). While mild C. difficile-associated diarrhea (CDAD) in young healthy patients can result in moderate to severe diarrhea, those at both ends of the age spectrum are often in danger of developing severe complications, such as pseudomembranous colitis (PMC) or even colon perforation in the most extreme cases (2).
A study conducted in 2002 by Kyne et al. found that 15% of observed patients suffered from some form of C. difficile¬-associated infections, with an average increase of 54% in cost of care and 3.6 days in length of stay (1). A more recent study conducted in Pennsylvania from 2002-2006 found that the costs and length of stay for patients suffering from CDAD was more than double those that underwent similar procedures and did not acquire CDAD (3). The Kyne study also found that 48% of patients that developed C. difficile complications died, compared to a 22% mortality rate among those that did not develop CDAD (1). The emergence of particularly virulent and resistant strains of C. difficile such as the NAP1 strain that caused an epidemic of CDAD in Quebec hospitals from 2003-2004 can cause even higher costs and mortality rates than those typically seen in healthcare institutions (4).
Though C. difficile was first isolated from infant fecal samples by Hall and O’Toole in 1935, it would be considerably longer before the “difficult clostridium” was realized to be nearly the sole cause of PMC (5, 6). C. difficile was categorized as a Gram-positive, obligate anaerobe bacillus with the capability of forming spores and later germinating back to the vegetative state. After its initial characterization, and perhaps because it was isolated from healthy infants, C. difficile was relegated to the shadows of microbiology until studies began in earnest chronicling the relationship between antimicrobial treatment and symptoms ranging from diarrhea to severe colitis (6). After researchers in the 1970’s noticed the presence of cytopathic toxins in fecal filtrates of those patients suffering from PMC, antimicrobial studies were conducted on hamsters that led to the association of C. difficile with hospital-acquired intestinal infections (7).
The primary cause for increased levels of C. difficile in the human gut is the administering of antibiotics or chemotherapeutic agents to those already in a hospital setting (8). Due to the extensive natural bacterial flora found in the human colon, C. difficile bacteria are normally unable to establish a large enough foothold in the competitive environment by which to produce harmful levels of toxin (2, 8). Therefore it is not uncommon to find C. difficile in the lower intestinal tracts of healthy individuals, allowing them to be carriers of the bacterium (9). The spread of C. difficile is accomplished via the fecal-oral route, as the spore-forming ability of the bacterium allows it to undergo the severe changes in pH, temperature, and gaseous environment that occur during progression through the digestive system (2). It is after these spores have reached the ideal conditions of the human colon that C. difficile’s extensive antibiotic resistances allow it to flourish when its host is administered antimicrobials (7).
Once a host has been administered antibiotics, the various symptoms of CDAD may soon develop, including abdominal pains, cramping, and various degrees of colitis (9). Left untreated, these symptoms, especially in the case of more virulent strains of C. difficile, can quickly lead to more serious infections with intense pain and diarrhea, with the possibility for such life-threatening conditions such as PMC or toxic megacolon to develop in those with weakened immune systems (2, 10). The possibility of developing CDAD is particularly alarming for those undergoing antimicrobial treatments in hospitals, as a primary means for acquiring C. difficile spores is from the hospital environment, where hearty spores can survive for weeks, resisting the antiseptic techniques commonly used in most healthcare institutions (8). Furthermore, the predominant number of C. difficile infections occur in the elderly; it is thought that this is due to numerous factors, such as already lengthy hospital stays, increased antibiotic exposure, and an already reduced barrier effect of elder patients’ natural gut flora (7).
The first step in the process of treating CDAD involves the need for a quick and accurate diagnosis. This usually entails a cytotoxin culture assay for one of C. difficile’s two toxins, but this standard is being increasingly replaced by faster enzyme immunoassay testing for the toxins and the antigen (4). Once a patient is diagnosed with CDAD, there are a couple commonly followed procedures used to fight infection. The first step is the cessation of all antibiotic treatments that the patient was being administered at the time of infection (2). Those broad-spectrum antibiotics that have been found to induce disease more commonly through their flora disrupting action include clindamycin, cephalosporins, and many types of penicillin (8). For those suffering from their first episode of CDAD, the antimicrobial metronidazole is typically administered orally or by the parenteral route (2). Vancomycin, another antimicrobial, was once considered the drug of choice, but its high cost, requirement for oral administration, and potential for selection of vancomycin-resistant Enterococcus have resulted in it becoming a second-line drug for CDAD treatment (10, 13). After successful treatment, the recovering patient is still at risk for the development of recurrent CDAD, as 10 to 20 percent of patients have been found to relapse after the discontinuation of initial treatment (2). It is thought that these relapses are due to a failure to fully cleanse the colon of C. difficile, or to the reuptake of spores from the hospital environment (2).
Complicating the treatment picture and increasing healthcare costs is the possibility for hypervirulent strains to emerge and devastate a hospital population. The NAP1 strain that was first noticed in Quebec in 2003 is particularly alarming, as the development of CDAD was found in typically low-risk populations, such as healthy young individuals and those that had neither been administered antibiotics nor received in-patient treatment in the previous three months (9). This strain is characterized by significantly higher levels of toxin production, as well as fluoroquinolone resistance (9, 11). The underlying reason for the increased levels of toxin produced by this strain is thought to be a partial deletion of the tcdC gene, which possibly downregulates toxin production (9). Furthermore, a sequencing of the genome of strain 630 of C. difficile confirmed that there are numerous mobile genetic elements, such as conjugative transposons, that help the bacterium develop resistance to antimicrobials, which serves as a reminder that prescription drug options for the treatment of CDAD may be narrowed even further in the future (12).
There are several known or suspected virulence factors of C. difficile but the most studied and well-characterized are two toxins, labeled A and B. Both toxins are high molecular weight proteins, and are believed to be coordinately regulated, as strains that produce high levels of toxin A have also been found to produce high levels of toxin B (14). Toxin A, a potent enterotoxin, was initially believed to be the toxin more responsible for the infectious nature of virulent C. difficile strains, but recent studies have found that strains that have defective toxin A production are still pathogenic (15). This combined with the findings of a study conducted by Riegler et al. that show that toxin B, a cytotoxin, is actually ten times more potent in intestinal human mucosa suggest that toxin B is the toxin that contributes more to the infectious nature of the bacterium (16). Despite this, it is still believed that both toxins acting synergistically result in optimal levels of mucosal adherence and the fast spread of the pathogen (14).
The genes that code for the production of toxin A and toxin B are located next to each other on the C. difficile genome. Located in between these genes is a small open reading frame (ORF) and the 19.6 kb toxigenic region of the genome also contains three more small ORFs. Contained within this pathogenicity locus are the toxin genes, tcdA and tcdB, as well as tcdC, a negative regulator, and tcdD, as positive regulator. Immediately upstream of the tcdB gene and its promoter is the txeR gene, which has been found to have positive regulatory effects on both toxins A and B (17, 31). Strains of C. difficile that have mutations in their toxin genes that result in a lack of toxin production are not pathogenic (2).
The mechanism of toxin action on intestinal mucosa has been relatively well-characterized, and both toxins are thought to be important in the adhesion process (8). The first step involves binding of toxin to specific cellular receptors on the colonic epithelium, after which they are transported into the cytoplasm. The mode of action for both toxins following cellular entry involves adding glucose molecules to a specific threonine on Rho proteins, which deactivates them. The Rho family of proteins are GTP-binding and small in nature, and are critical to various cellular processes such as movement, cytoskeletal integrity, and actin polymerization. Deactivation of Rho quickly results in the loss of structural integrity in the cell, followed by cell death (10). Production of both toxins is growth-dependent, which is why disruption of the normal colonic flora allows the bacterium to obtain more growth substrates from its environment, produce toxin, and spread (11). Toxin A is believed to be the more active protein early in the infection process, penetrating the mucosa and allowing the more potent toxin B to act on deeper cellular structures (8). Both toxins activate cytokine release from human monocytes, resulting in inflammatory effects that are associated with the development of severe colitis (18).
Besides both the previously discussed toxins, C. difficile has several other virulence factors that assist in cellular adhesion. Among these are the flagella, the cell wall protein labeled Cwp66, and the high-molecular weight surface layer protein (HMW-SLP) (19). Following toxin production, hydrolytic enzymes are also used to assist the degradation of epithelial cells in the gut. These degradation products are frequently used as nutrients by virulent strains of C. difficile. Once the C. difficile bacterium has managed to adhere to the colonic mucosa, it rapidly begins to accumulate the substrates that allow it to grow, produce more toxins, and ultimately divide (20).
Because of the antimicrobial-resistant nature of C. difficile and the increasing healthcare costs associated with nosocomial CDAD, much research is being conducted to determine if a method could be devised to prevent successful colonization of the human gut. While a vaccine has been created that was found to induce immunity to C. difficile in hamsters, a human vaccine will no doubt cost millions of dollars to develop, and the emergence of hypervirulent strains that affect populations that are not normally considered at-risk would complicate decisions regarding which patients to vaccinate (21). Another strategy that warrants consideration would involve the suppression of growth substrate uptake, effectively shutting down the pathogen’s toxin production system and preventing mucosal colonization. The benefit of this approach would be the need to only administer therapy, assumedly in the form of a prescription drug, to those that are suffering from CDAD or have a significant chance of contracting it (22).
Previously conducted studies have established that peptides are the preferred growth substrate of C. difficile (23). Similar to many other microorganisms, the mechanism for bringing these key nutrients into the cell involves a transporter of the ATP binding cassette (ABC) superfamily. ABC transporters are ubiquitous, being present in members of every phyla of both the prokaryotic and eukaryotic domains, and function to transport substrates across cellular membranes (24). These substrates can range from small inorganic ions to large proteins, depending on the specific transporter (25). The transporter itself is characterized by two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Substrate specificity is dictated by the TMDs, where multiple α-helices span the phospholipid bilayer (24, 25). The exact mechanism by which ABC transporters work has yet to be elucidated, but it is thought that the NBDs function to bind and hydrolyze ATP, inducing a conformational change in the TMDs and forming a channel through the membrane (24).
Central to the research conducted in the Ivey Lab thus far is the ABC transporter that C. difficile uses to import peptides into its cytoplasm. This transporter, the oligopeptide permease (Opp) system, consists of four subunits in a complex, labeled A, B, D, and F (22). During and after colonization, the Opp system energetically fuels the cell by bringing in the crucial nutrients that allow for increased toxin levels that better facilitate adhesion. Because the Opp system is so important to virulent strains of C. difficile, it stands to reason that developing a means to control or even cripple its capabilities would achieve the goal of thwarting toxin production. It is believed that the a possible method by which to do this would be to control the transcription of permease system genes, thereby creating a barrier to messenger RNA production, and ultimately keeping the Opp proteins from being translated outside the nucleus (22).
What makes the Opp system unique and worthy of study lies in the bidirectional nature of the genetic locus encoding the complex. Whereas genetic loci for most transporters of the ABC superfamily contain a single promoter followed unidirectionally by the genes coding for that transporter, the opp genes have evolved in a different fashion (22, 26). In one direction on the genome are the oppA and oppB genes which code for their respective subunits, while in the other direction there are the oppD and oppF genes. No control mechanism has been specifically determined, nor has a competitive advantage to having the operons oriented in a bidirectional manner been elucidated. It is due to this relative lack of information concerning the Opp system that the Ivey Lab has chosen the opp genes for study (22).
Previous research endeavors have focused on measuring transcription levels of the opp genes by using a dual luciferase system that was designed to flank the locus of interest. The construction and implementation of this system resulted in the creation of Escherichia coli plasmid pUA442, which contains two different genes coding for different forms of bioluminescent firefly luciferase on either side of the opp locus (22). By measuring the levels of light emitted after exposing cells to luciferin, levels of gene expression can be determined, and the functionality of the Opp proteins can be recorded based on varying conditions such as temperature, pH, and growth substrate (22, 27).
Unfortunately, thus far the only means by which to study the activity of opp gene promoters has been in E. coli cells that contain them. The problem with studying opp gene expression in C. difficile directly is the uncooperative nature of the bacterium’s genome. The conjugative transfer that would be required to study the previously mentioned dual luciferase system in actual C. difficile cells would require circumvention of restriction/methylation system that protects the bacterium from the intrusion of foreign DNA (28). While other research labs have had some success in bypassing this barrier, to date the successful integration of opp genes from an E. coli donor to a clostridial host has not occurred (29).
The object of this research project was to construct a shuttle vector capable of being transferred from gram-negative E. coli cells to their gram-positive target, C. difficile. A hybrid E. coli plasmid, labeled pCTC1, was singled out as a promising candidate for the purpose of this research. First constructed by Ross et. al in 1989, pCTC1 was designed to be a high-copy-number vector capable of being transferred via conjugation from E. coli to Clostridium acetobutylicumi, a gram-positive, spore-forming bacterium related to C. difficile. Its conjugative ability is due to its possession of certain replication regions as well as the origin of transcription (oriT) gene, which is the site where plasmid DNA is nicked, allowing for the transfer of a single strand of DNA from the donor cell to the recipient (30).
Unfortunately, the pCTC1 plasmid as constituted does not allow for ligation independent cloning (LIC). LIC is an efficient method of creating recombinant clones between polymerase chain reaction (PCR) products and amplified plasmid vector (32). The LIC procedure relies on the creation of 12-18 bp “sticky ends” in both the vector of interest and the DNA sequence that is to be inserted into the vector. These sticky ends are complementary to each other, eliminating the need for restriction enzymes or DNA ligase (32, 33). The resulting recircularization of the recombinant plasmid occurs with minimal background (undesired) DNA being formed, making it an ideal method for any molecular biology laboratory (32).
The objective of this research project was ultimately to construct a desired shuttle vector that was capable of undergoing LIC. The modification strategy pursued to accomplish this goal entailed the attempt to integrate a region of DNA into the pCTC1 plasmid that allows for relatively easier cloning. A desired region of LIC-capable DNA was determined to be present in the pDG148-Stu plasmid (34). The stuI gene specifically was targeted for, and standard PCR reactions allowed for the isolation and amplification of this locus, after which primers that created 12-18 bp overhands at either side of the desired region were used. Following this, attempts at ligating this PCR product with a vector digestion of pCTC1 were performed, followed by efforts to integrate this complex into competent cells.
Methods
Growth of Bacterial Strains
The first step conducted involved taking a loop of E. coli cells that contained the plasmid of interest, pCTC1 (these cells containing pCTC1 were renamed UA470 for use in the Ivey Lab), and then streaking them onto a plate that consisted of Luria-Bertani (LB) broth medium. This plate was then placed in a 37° C incubator overnight. Following this step, a single UA470 colony was isolated from the streak plate and inoculated into a tube that contained 50 µL of ampicillin and 50 mL of liquid LB. This was done to select only for those cells that carried the functional gene for ampicillin resistance.
Plasmid DNA Purification
Plasmids were isolated from UA470 Luria-Bertani (LB) broth cultures according to the QIAGEN QIAprep Spin Miniprep Kit procedure, with the accompanying reagents being used during the process.
Agarose Gel Electrophoresis
The results of various plasmid purifications and PCR reactions were evaluated using the Ivey Lab agarose gel electrophoresis recipe and procedure. The procedure itself entails comparing the size of product samples against molecular ladders of known size. Initially an agarose gel of 0.7% is made by mixing 0.7 grams (g) of agarose with 30 milliliters (mL) of 1X tris-acetate-EDTA buffer, then adding 1.5 microliters (µL) ethidium bromide solution. The TAE was first measured into a 150 mL Erlenmeyer flask, and then had the weighed amount of agarose added to it. This mixture was microwaved and allowed to barely reach a boil (~1 minute), after which it was removed and cooled for 7 minutes. After this cooling period, ethidium bromide solution was added, and the mixture was poured into an assembled gel tray, after which a “comb” was placed over the gel tray, creating small slits where the mixture was displaced. The comb allowed for small slits to be created during the subsequent 15 minute setting period, after which the comb was removed and the loading slots it created made apparent. This hardened gel was then submerged in an Embi Tec RunOne™ Electrophoresis Cell until the level of TAE above the gel’s surface reached 1-3 millimeters, keeping the loading slots from drying out. A single microliter of either Supercoiled or 1 kb Ladder that was to be subsequently loaded via pipette into a slot of this gel was first diluted with 10 µL of deionized (DI) water, then mixed with 2 µL of 6X Blue/Orange Loading Dye. When loading a PCR or vector sample, 5 µL of the sample were mixed with 2 µL of the same dye, then pipetted into the gel.
After correctly loading the samples of interest, 50 volts of electric current were run through the gel tray for about 40 minutes. During this time, negative electric current from the anode flows from the top of the gel tray to the cathode at the bottom, effectively “pushing” any negatively charged particles with it as the current moves. Since DNA’s phosphodiester backbone is negatively charged, it migrates and a steady pace through the molecules present in the gel. Smaller pieces of DNA will correspondingly navigate this molecular maze faster than pieces of DNA that are larger and more obstructed by the agarose gel. The resultant “bands” that designate size would normally be invisible to the eye, but the loading dye as well as ethidium bromide solution allows for the bands to be displayed prominently, especially under ultraviolet light. A Kodak DC290 Camera associated with a UV light source was used to digitally photograph gels of interest, which were then further analyzed using the Kodak 1D Limited Edition 3.5 Imaging System.
The aforementioned procedure was used several times over the course of this research, each time using the same equipment and concentrations, the only variance being in whether Supercoiled or 1 kb DNA Ladder was used.
Results and Discussion
PCR Product
After the initial plasmid purification of UA470 was determined to be of the correct size, work went into creating a PCR product that could successfully be integrated into the plasmid. The PCR reaction recipe that was followed consisted of 0.5 µL each of pDG148-Stu, pStu-F, and pStu-R, which were mixed with 10 µL of Premix I and 8 µL of DI water. After this mixture had been created in a 0.5 mL PCR tube, 0.5 µL of highly enzymatic NovaTaq DNA Polymerase was added, and the tube was placed into the Techne Toughgene Gradient Thermocycler. The program used involved a 5 minute initial denaturation at 94°C, followed by thirty cycles of 30 seconds at 94°C, 30 seconds at 50°C, and 150 seconds at 68°C. After the completion of thermocycling, the PCR product was analyzed using the previously mentioned gel electrophoresis procedure. Unfortunately, this attempt and several further attempts to get the pStu-F and pStu-R primers to work resulted in either no products visible on the gel or products of incorrect size. After this the same PCR reaction was conducted using pDG-F and pDG-R, two primers that have similar activities but act at different locations on the plasmid. The PCR product created using these primers was analyzed and the band corresponding to the desired insert was determined to be of the correct size, allowing for subsequent steps to proceed.
Vector Digestion
In order to effectively “open up” the CTC1 plasmid in order to integrate the stuI site into it, a vector digestion was conducted. The recipe consisted of 2 µL each of UA470, NEB 2, and 10x BSA, as well as 13.5 µL of DI water and 0.5 µL of StuI. This mixture was pipetted into a 0.5 µL PCR tube and then placed in a 37° C Precision 280 Digital Water Bath for one hour. Following this, 0.2 µL of Shrimp Alkaline Phosphatase was added, and the tube was then placed in a 30° C water bath for 30 minutes. Finally, the tube was heated in the thermocycler at 70° C for 20 minutes, after which it was measured using agarose gel electrophoresis, confirmed as the desired product, and stored at 4° C.
Preparing PCR Product for Ligation
In order to make the PCR product capable of integration into pUA470, the product had to first be phosphorylated. The restriction endonuclease DpnI was added initially, followed by placing the tube in a 37° water bath for one hour. The actual phosphorylation was then accomplished by conducting the QIAGEN QIAQuick Spin Prep on the PCR product, then adding 5 µL of DNA Ligase Buffer, 3 µL of ATP, and finally 1 µL of PNK. This mixture was then held at room temperature for an hour, followed by 20 minutes of 70° C in the thermocycler.
Ligation
Following preparation of both the pCTC1 vector digest as well as the PCR product, the process of ligating the two together began, and 4 µL each of the vector digest and PCR insert were mixed with 1 µL of T4 DNA Ligase 10x Buffer and 1 µL of DNA Ligase, a modifying enzyme. This ligation complex was allowed to sit at room temperature on a laboratory workbench over the course of a weekend, as the ligation process is time consuming.
Electroporation
Following ligation, 1 µL of the ligation complex was added to a microtube containing 100 µL of iced electrocompetent (ZAP) cells that had been stored at -80° C. The ligation complex was then transformed through electroporation in the Bio-Rad Gene Pulser set at a resistance of 200 ohms, a capacitance of 25 µF, and a voltage of 1.5 kV.
Plating
Electroporated cells were collected with 1 mL LB broth, incubated for one hour in the orbital shaker, and then plated on LB/ampicillin/erythromycin plates for selection. Plates were allowed to incubate at 37° overnight. Eight isolated colonies were selected for further analysis, and were then inoculated into 50 mL of LB broth and shaken overnight. Plasmid minipreps followed by gel electrophoresis revealed the presence of plasmid DNA in five of the eight cultures.
Determination of LIC-capable Vector
These five plasmids were then used as template for PCR reactions using the original primers pDG-R and pDG-F. The original pDG148-Stu plasmid was also subjected to this PCR reaction, in order to determine which PCR reactions had resulted in the integration of the appropriately sized LIC-capable insert into pCTC1. Agarose gel electrophoresis of the PCR samples revealed that Sample 3, located in the third lane of Figure 4, contained the appropriate recombinant plasmid, and the strain was subsequently named UA543.
Discussion of the Construction Process and Future Usefulness of pUA543
Through the use of recombinant DNA technologies, the goal of creating a LIC-capable vector for potential genetic manipulation in C. difficile was achieved. The first indication of the pursuit of a sound recombination strategy involved the use of the pDG-R and pDG-F primers, which succeeded where other primers had failed. Following this, the successful ligation of the vector digest with PCR products was another pivotal moment in the course of this project. The careful examination of the final agarose gel was key to determining the overall result of the project, as the presence of both a plasmid that was slightly larger in size as well as the correct insert in the same PCR reaction was required for the construct creation to be considered a success.
The benefits of this accomplishment should be seen in future research conducted in the Ivey Lab, as pUA543 is tested for the ability to reliably undergo LIC with actual C. difficile genomes. Although it is beyond the scope of this project, future undergraduate researchers will have the opportunity to potentially measure the activity of the opp promoter using the aforementioned dual luciferase system that has already been studied in E. coli cells. Further study could determine what growth conditions allow for optimum opp gene expression, which could have enormous implications in the fight against nosocomial CDAD. If a method for regulating opp gene transcription could be determined using transformed C. difficile cells, or if drugs could be designed to inhibit utilization of peptides, the persistent threat of the “difficult clostridium” could be rather easily controlled or eradicated.

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Undergoing a mammoplasty surgery is not a decision you should take lightly. If you are thinking of getting breast reduction learn the full information regarding the process.

Why Undergo a Breast Reduction?

Women tend have breast reduction surgery because their large breasts are a burden, such breasts can cause back pain and bad posture.

Information regarding breast reduction surgery from a Houston plastic surgery office I am linked with.

“Aside from those looking to solve physical problems associated with large breasts, many women may look into breast reduction surgery for purely aesthetic reasons. If this is the case then insurance policies are unlikely to cover the cost of the procedure. Particularly large breasts on a small frame can look disproportionate and may be the reason for undergoing breast reduction surgery. Having disproportionate breasts can be especially alarming for teenagers who are still having problems dealing with their changing bodies. It can also be hard to find clothing that fits properly if you have an unusual shaped body (due to oversized breasts). Some women may have to resort to customised clothes which can be extremely expensive. All of these reasons can lead a woman to be displeased with her breasts.”

These are many valid factors that should be considered when undergoing the knife to reduce the size of breasts.  Consider the danger of the procedure itself, requiring anesthesia as well as factoring in the recovery period which can take up to a month.

The major argument in favor of undergoing breast reduction surgery is if the mammary glands are so excessive in size in comparison to the body that the female may be mentally pressured into taking prescription painkillers which can cause addiction.

Dangers of Rhinoplasty
These types of surgery are not known to have very many risks, but do sometimes have complications. These complications depend on how are bodies are built, quick nosebleeds are a potential complications have been infection not to mention a potential bad reactions to the anesthesia
The nasal bump. They typically desire a conservative change, creating a nose that is in consistent harmony in respect to the full area of facial features and proportions.
The twisted nose is majorily daunting task in rhinoplasty, as it requisites an highly experienced and skillful rhinoplasty surgeon. Reputation and years in the field bode well for this area, so in the hands of an such well esteemed and precise surgeon, significant improvement is of great expectation and can go without any hitch, obtaining a completely straight nose.

The majority of plastic surgery procedures where necrosis is assumed to occur tends to fall under the categories of facelifts, tummy tucks, as well as breast reductions.  It occurs in several other procedures to.  The reason of this being is because of the blood supply is being shortened or cut off for a period of time, usually when flaps of skin are separated from the other structures.  This is very, very rare and that is why it is important to use a trusted, certifiable plastic surgeon with many years of experience before undertaking such a procedure.
Usually, there is ample blood circulating flowing to the tissue carrying caplets, white blood cells, to supply the tissue with a high degree of consistency regarding the required nutrients.   However in certain patients who live unhealthy lifestyles that may include overeating, smoking, excessive drinking, and drug abuse this risk is over four-fold.

There are many signs of necrosis which all involve a slight discoloration of the skin.  The skin may develop a bluish or purple tint in color and later on can become darker to a gray or even black in the most serious of cases as seen in the picture above.  This is palpable infection of the tissue, if necrosis is not realized at an early stage than serious problems could occur such as nerve damage (signalled by numbness, tingling).

Nerve damage could take up to half a year in order to heal on average.  Some cases have been reported to have lasted four years.

Paralysis is a major result that can potentially be caused by nerve damage, however usually the case is just a weakness of the movement in the muscle category.  If paralysis occurs the patient will never re-heal, this is permanent, unless reconstructive surgery is undergone to take action.
It is necessary to reiterate that necrosis and nerve damage are incredibly during plastic surgery.  Later in this blog a statistic report from case studies will reveal close estimates to how low this percentage truly is.  The current hypothesis of the author of this blog and his colleagues is between .35 and .38%