Legionella Pneumophila Experiment

The Centers for Disease Control and Prevention has reported between 8,000 and 18,000 cases of Legionnaires' disease annually in the United States, caused by the spread of the Legionella bacteria. What can you do to protect your facility by preventing Legionella and other waterborne pathogens? Here are five practices you can implement to protect your patients, employees, and facility by preventing bacterial outbreaks in your facility water system.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

that goes into creating a clone. First, scientist remove a somatic cell from an animal that they

This process uses enzymes to cut and insert piece of DNA into a plasmid vector. This vector is then transfected into cells. The different pieces of DNA that were being inserted into this plasmid were a control and three different mutants (an alpha-5-tailless mutant, full-length alpha-5, and full-length alpha-2). These pieces of DNA code for integrins. Once these pieces of DNA were inserted into the plasmid vector, the plasmid needed to be inserted into a cell where it would be able to replicate. In these tests, rat intestinal epithelial wild-type cells (cells that would be found in nature, not cells grown in the laboratory) were used. To insert the plasmid into these cells lipofectamine plus was used. Lipofectamine plus acts like a detergent and opens the membrane of the cell so the plasmid can enter. Also, the plasmids that were transfected into cells were all treated with G-418, an antibiotic. When the cells were plated and allowed to multiply, only those containing the plasmid with the antibiotic would live. This made it possible to know which cells actually received the plasmid and properly underwent mitosis, and to eliminate those cells that never took up the plasmid in the first place.

Louis, MO). The purity of ATII cells was ~95% after adherence in culture as we determined by staining for cytokeratin CAM 5.2 [15].

Cytogenic analysis – Microscopic examination of lymphocytes to look for structural changes or changes in number of chromosomes.

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

The results concluded that β-phellandrene effected level of DNA but no damage to chromosome level, comet and Ames showed genetic level damage. I felt as though the results and conclusion supported the hypothesis that they were testing by concluding DNA and chromosome levels had no damage, but damage did occur to genetic levels. The next step that should be taken is to determine the certain genetic toxicity that β-phellandrene contains. I thought that their hypothesis made sense because they have been previous tests done but none really saying if β-phellandrene the extent of genetic toxicity. I did not have any issues with their methods or results because from the methods and materials they used they got solid results and the experiments appeared to have worked well, however there was no certainty on the genetic toxicity of β-phellandrene, so I felt as if the results were not fully concluded by their experiment/methods and that they should have gone deeper into Comet assay testing and or Ames testing, so they could have gotten more information on genetic

Endocytosis is the capacity to disguise material from outside the cell is critical for a few cell forms including the ingestion of fundamental supplements, expulsion of dead or harmed cells from the body, and guard against microorganisms. Eukaryotic cells disguise liquid, extensive and little particles, and even different cells from their surroundings by a procedure called endocytosis. Amid endocytosis, the plasma layer of the cell frames a pocket around the material to be disguised. The pocket closes and afterward isolates from within surface of the plasma layer to shape a film encased air pocket, or vesicle, containing the ingested

Legionnaires’ disease can be treated with antibiotics and can be very successful if started early on. The sooner antibiotic treatment is administered, the smaller the chance of serious complications. In most cases, treatment of a Legionella pneumophila infection involves hospitalization. An antibiotic such as a broad-spectrum cephalosporin along with a macrolide or a quinolone is the recommended treatment for those hospitalized with Legionnaires’ disease. Other effective antibiotics include tetracyclines and ketolides. The antibiotics used most frequently have been erythromycin, levofloxacin, doxycycline, and azithromycin. These antibiotics are the most effective because they have good intracellular penetration into Legionella-infected

Because of this, they are often the targets of many bacterial pathogens of eukaryotes. By doing so the bacteria are able to create protective barriers for themselves inside the host cell. L. pneumophila targets vesicles that are destined for the ER. They convert an endosome that was originally from the ER into what is called the Legionella containing vacuole (LCV).9 The LCV allows the bacteria to avoid degradation and to instead replicate and thrive inside it far away from the host defense.9 A large amount of effector proteins are required for this process and are secreted into the host cell via the T4SS, which is specifically called the Dot/icm pathway in Legionella.9 One important effector protein in this process is SidM, which gathers rab proteins at the LCV that are essential for regulating the pathway.7 They are modified by this protein and stay at the LCV as they are rendered permanently active and resistant to being shut down.7 This consequently inhibits the removal of the LCV from the cell.7 These new rab proteins can now help to bring more vesicles to the LCV to camouflage it from the host cell’s immune response.7

Legionnaires’ disease is a form of pneumonia which is caused by Legionella bacteria. Legionella bacteria was first identified in 1976 as a cause of pneumonia. This bacteria can account for 2-15% of total number of pneumonia cases requiring hospitalization in the United States. There are around 40 types of Legionella. Around 10 to 40 thousand people in the U.S. develop Legionnaires’ disease every year. People over 50 years of age are more likely to become ill from this disease. A greater risk is involved in people who suffer from certain health conditions such as diabetes, kidney disease, lung disease and or malignancy. Studies have shown people with AIDs and HIV infection do not seem to contract this disease.

I found the paper fascinating because it talks about a medically relevant topic which is of interest to me. The paper is discussing a very relevant topic that has several commercial and medicinal applications. The paper had several strong points such as supporting research done by other scientists. For example, their claim that the cell has other mechanisms to fight toxicity other than glutathione accumulation was supported by a study done by Trotter. Also, the paper had strength in the data that it was consistent with what they hypothesized. However, there are a few weaknesses in the study. First, they used acrolein to select for the strain that overproduce glutathione but later acknowledged that the effectiveness of strain selection using acrolein seems to be strain dependent bringing their methods into question. Also, the study made inferences without any supporting work like other claims they made which means those are not really facts they could be the result of chance or bias by the researchers, requiring replication and further studying if they are true and to understand the mechanism that causes them.

Exosomes are membranous vesicles enclosed in lipid bilayers with diameter of 30-100nm and are responsible in carrying cytoplasmic proteins such as Rab proteins and actin-binding proteins and signal transduction molecules such as G-proteins and protein kinases, as well as some heat-shock proteins like Hsp70 and tetraspanins like CD9 [5-30]. Exosomal proteins can directly represent the specific profiles of their cells of origin, for

1. Only cDNA cloning is covered in this video. What other methods are there for