Relationship between SMC1A and Chromosome Related Disease

Relationship between SMC1A and Chromosome Related Disease 1. Introduction[A1] The Structural Maintenance of Chromosomes 1 alpha (SMC1A) gene is located in Xp11.22-p11.21, consisting of 25 exons and 24 intron. SMC1A gene encoding a core subunit of the cohesin complex, which is essential to sister chromatid cohesion. SMC1, SMC3, SCC1 (also known as MDC1 and RAD21) and SCC3 (also known as SA2 and STAG2) subunits could interact with each other and form a ring-shaped cohesin complex [1-3]. As is known, central component of the cohesin and condensin complexes are required for conversion of interphase chromatin into mitotic-like condense chromosomes[4]. Structural Maintenance of Chromosomes (SMC) proteins are core component of the cohesin and condensin complex and essential for chromosome condensation during DNA replication and chromatid segregation of the genome in all organisms. They are also involved in checkpoint responses and epigenetic silencing of gene expression[5]. Cornelia de Lange syndrome is a dominantly developmental disorder with multisystem abnormalities including slow growth before and after birth, characteristic facial features, upper extremity defects, hirsutism, gastroesophageal dysfunction and cognitive retardaion. The incidence is as high as one in 10,000 to 30,000 newborns. Both sexes have the same phenotypic variability. To date, the three genes, NIPBL, SMC1A, and SMC3 involving in chromosome function, gene regulation and double-stranded DNA repair, could cause CdLS when mutated[6, 7]. Six in ten of the probands with CdLS have heterozygous mutations in NIPBL gene, whereas 5% have mutations in SMC1A and SMC3 genes [6, 8]. Eleven different SMC1A mutations in 14 unrelated patients have been reported. All patients had a mild to moderate CdLS phenotype [8-10]. In several decades, we focus on the relationship between SMC1A and chromosome related genetic disease. In recent years, we found that SMC1A may play a key role in tumorigenesis. Sun. M et al. determined that the effects of SMC1A knockdown on the cell cycle and apoptosis of lung adenocarcinoma cells. The results indicated that SMC1A is a novel oncogene, which modulates lung cancer cells in their proliferation and migration capabilities through arresting cell cycle at G0/G1 phase and promoting apoptosis [11]. The similar conclusion also was found in glioblastoma cells [12, 13]. However, SMC1A functions as a novel oncogene in human prostate cancer metastasis and progression has still not been reported. 2. Materials and Methods 2.1. Patient Samples All of the patient samples were obtained from the Urinary Surgery Department of Shanghai Changzheng Hospital, Shanghai, China. This study was approved by the Clinical Research Ethics Committee of Shanghai Changzheng Hospital, and the informed consents were acquired from all of the subjects. 2.2. Reagents and antibodies DMEM (cat no.12430-054), F12 (cat no. 21127022) and RPMI-1640(cat no. 11875-093) medium and fetal bovine serum (cat no. 10099-141) were purchased from GIO (Grand Island, NY). TRIzol Reagent was from Invitrogen (Carlsbad, CA, USA). Giemsa was from Chemicon International (Temecula, CA). M-MLV Reverse (cat no. M5301)Transcription was purchased from Promega (Madison, WI, USA). Oligo-dT(18) was synthesized by Sangon Biotech (Shanghai, China). Terraâ„ ¢ qPCR Direct SYBR ® Premix (cat no. 638318) was from Takara (Otsu, Japan). Anti-SMC1A antibody (cat no. SAB4300451) was from Sigma-Aldrich (Munich, Germany). Mouse anti-GAPDH (cat no. sc-32233), Goat anti-Mouse IgG (cat no. sc-32233) and goat anti-rabbit IgG (cat no. sc-2030) were from Santa Cruz Biotechnology (Texas, USA). All the other chemicals were of analytical grade. 2.3. Cell culture Human embryonic kidney cells 293T, Human prostate cancer cell lines PC-3, DU145, LNCap, and 22RV1 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). 293T were cultrued in DMEM containing 10%FBS. PC-3 and DU145 cells were maintained in F-12 medium supplemented with 10% FBS, 100U/ml penicillin and 100ÃŽ ¼g/ml streptomycin. 22RV1 and LNCap cells were incubated with RPMI-1640 supplemented with 10% FBS, 100U/ml penicillin and 100ÃŽ ¼g/ml streptomycin. LNCap cells were maintained in Corning Corning ® CellBIND ® Surface cell culture flasks (Corning, cat no. #3289) for a better attachment efficiency. All cells were cultured in a humidified incubator at 37oC under 5% CO2 atmosphere and used for analysis during exponential phase of growth. 2.4. RNA interference The synthesized 21-bp oligonucleotides encoding SMC1A-specific shRNA held the sequence 5’-TAGGAGGTTCTTCTGAGTACA-3’. The sequence of the negative control shRNA oligonucleotides was 5’-TTCTCCGAACGTGTCACGT-3’. The oligos were annealed and ligated into pH-L vector (Hollylab, Shanghai, China) through NheI/PacI to generate pH-Lv-shSMC1A and pH-Lv-shCon. The resulting constructs were confirmed by sequencing. 2.5. Recombinant lentivirus Transduction PC-3 or DU145 cells were plated at 5Ãâ€"104 cell/well in 6-well plates. After 24 h of culture, lentivirus recombinant encoding shRNA against SMC1A was added at a multiplicity of infection (MOI) of 50 into F-12 basic medium. After 6h incubation, the cells were added with complete growth medium replacing the basic medium containing the lentivirus. Then, 5 days post-transfection, gene reporter (EGFP) expression was examined using fluorescent microscopy (Olympus, cat no. CKX41). 2.6. Quantitative real-time RT-PCR analysis of SMC1A mRNA expression Total RNA was extracted using TRIzol reagent according to the manufacturer’s instruction. 2ÃŽ ¼g total RNA was used to synthesize the first strand of cDNA using M-MLV Reverse Transcriptase. Real-time PCR reactions using Terraâ„ ¢ qPCR Direct SYBR ® Premix were run on Takara TP800-Thermal Cycler DiceTM Real-Time System. The following primers were used: SMC1A: 5’- AGCGAAAGGCAGAGATAATGG-3’ and 5’-GGTAGTCAAGAGGCAAGAAGG-3’; ÃŽ ²-actin: 5’- GTGGACATCCGCAAAGAC-3’ and 5’-AAAGGGTGTAACGCAACTA-3’. Thermal cycling condition were 1 min at 95 °C followed by 45 cycles of 95  °C for 5 s, 60 °C for 20 s, read absorbance value at the extension stage. The data was analyzed with Takara Thermal Dice Real Time System software Ver3.0. SMC1A relative mRNA levels was calculated using the 2-ΔΔCt method with normalization to ÃŽ ²-actin. And the conditions 2.7. Western blot analysis of SMC1A protein expression Cells were washed twice with ice-cold PBS and suspended in cell lysis buffer (2% Mercaptoethanol, 20% Glycerol, 4% SDS in 100mM Tris-HCl buffer, pH 6.8), and incubated for 15 min on ice. After centrifugation at 12,000 g for 15 min at 4oC, the supernatants were collected, and the protein content were measured using BCA protein assay kit. Equal amounts of protein were subjected to SDS-PAGE. After electrophoresis, blots were transferred onto PVDF membrane using an electro-blotting apparatus (Tanon, Shanghai, China). The membrane was blocked with TBST buffer containing 5% nonfat milk at room temperature for 1 h, and incubated with the primary antibodies in the blocking solution at 4oC overnight. After 3 washes with TBST buffer, the membrane was incubated with horseradish peroxidase (HRP) -conjugated secondary antibody (1:5000) at room temperature for 1 h. The signals of detected proteins were visualized by Pierce ECL western blotting detection kit (thermo scientific, USA). GAPDH protein level was used as an internal control to verify equal protein loading. 2.8. MTT assay Cell proliferation was evaluated by MTT assay. Exponential growth phase cells were plated at a final concentration of 2000 cells/well in 96-well plates and cultured for five consecutive days. MTT (10 µl, 10mg/ml) was then added, followed by incubation for another 4 hr at 37oC under humidified 5% CO2 atmosphere. The MTT was removed and addition of 150ÃŽ ¼l DMSO. Optical density (OD) of each well was measured at 490 nm using an ELx808 Absorbance Reader (Bio-Tek Instruments, USA). 2.9. Colony formation assay Cell growth and survival ability was also determined by the plate-colony-formation assay. In brief, 200 transfected cells were plated in 6-well plates. Cells were cultured for 14 days at 37oC under humidified 5% CO2 atmosphere. Culture medium was changed at 3-day intervals. Afterward, cells were incubate in 4% paraformaldehyde for 30 min at room temperature. The colonies were stained with Giemsa for 15 min, then washed with ddH2O and air-dried. The number of colonies (>50 cells/colony) was counted. 2.10. Flowcytometry Analysis Cell cycle distribution was assessed by propidium iodide (PI) staining. Briefly, the transfectedcells were harvested by trypsinization, centrifuged at 250 g for 5 min, washed twice with ice-cold PBS, and fixed in 70% ethanol at 4oC or -20oC for at least 1 h. Cells were collected and resuspended in PBS containing 100ÃŽ ¼g/ml RNase A and 40ÃŽ ¼g/ml PI, and then incubated at 4oC for 30 min, in dark. Cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). The percentage of the cells in sub-G1, G0/G1, S, and G2/M phases were analyzed using ModFit (Verity Software House, Maine, USA) software. 2.11. Migration assay To explore the effect of SMC1A in the migration of prostate cancer cells, 24-well transwell chamber with 8.0ÃŽ ¼m pore polycarbonate filter inserts (Corning, cat no. #3422) was performed. In the upper chamber of each transwell, cells were suspended in serum-free F-12 containing 0.2% BSA. And F-12 supplemented with 10% FBS was added in each lower chamber. Then, the inserts were incubated at a 37 °C, 5% CO2/95% air incubator for overnight and the cells that had not penetrated the filters were removed. The migrated cells attached to the bottom side were fixed in 4% paraformaldehyde for 10 min and stained in0.1% crystal violet for 30 min, rinsed in PBS and examined under a bright-field microscope. 2.12. Tumorigenesis assay The influence of SMC1A silence on the tumor development of prostate tumor in vivo was examined. DU145, DU145-Lv-shCon or DU145 Lv-shSMC1A at 5 Ãâ€" 106 per mouse were injected subcutaneously into 4 weeks old Balb/c nude mice (n = 10 per group, Shanghai Laboratory Animal Center, Chinese Academy of Sciences, China). The development and growth of solid tumors were monitored by measuring tumor size using a vernier caliper in a blinded fashion every five days for a 27-days period. The tumor volume was calculated using a standard formula: tumor volume (mm3) = width (mm)2Ãâ€"length (mm)Ãâ€"0.5. At the end of the experiment, all mice were sacrificed and individual tumor weights were measured using a electronic balance. All the animal experiments were approved by the Animal Care Committee of the Second Military Medical University. 2.13. Statistical analysis The statistical analyses were performed with Graphpad Prism 5.0 software. The values are expressed as the mean of at least three different experiments  ± S.D. The results were analyzed by Student’s t-test, and P 3. Results 3.1. Expression of SMC1A in prostate cancer tissue and prostate cancer cells. To study the function of SMC1A in prostate cancer, we first analyzed its expression pattern in prostate cancer tissues. As shown in Fig 1A, SMC1A was strongly stained in prostate cancer tissue with a clear subcellular localization in the cytoplasm and nucleus of abnormal prostate epithelial cells. Then, the expression level of SMC1A was further analyzed by western blot, which showed that SMC1A was upregulated in prostate cancer tissues (Ca) compared to the adjacent normal tissues (N) (Fig 1B), implying a possible correlation between SMC1A and prostate cancer. To find a cell model for further investigation of SMC1A’s function in prostate cancer, we first analyzed the expression of SMC1A in four commonly used prostate cancer cell lines. Both WB and qPCR indicated that SMC1A expressions were elevated in PC-3, DU145 and 22RV1 cells in comparison to the androgen-sensitive LNCap cells (Fig 1C and D), which had less aggressiveness than the other three cell lines. We also found that S MC1A expression level was negatively correlated to AR expression level (Fig 1C and E), suggesting that SMC1A might be correlated with the malignancy of prostate cancer and involved in AR signaling. 3.2. Lentivirus-mediated knockdown of SMC1A in prostate cancer cells. As PC-3 and DU145 cells expressed much higher levels of SMC1A, they were used for further investigation. Both PC-3 and DU145 cells were untreated or transducated with Lv-shCon or Lv-shSMC1A. The transduction efficiencies were above 90% in both cells confirmed by fluorescent microscope (Fig 2A). WB analysis demonstrated that Lv-shSMC1A efficiently knocked down SMC1A expressions in PC-3 and Du145 cells (Fig B and C). Q-PCR results indicated that SMC1A was down-regulated more than 80% and 90% in PC-3 and DU145cells respectively. 3.3 Down-regulation of SMC1A inhibited cell proliferation in prostate cancer cells. After confirming the knocked down efficiency of SMC1A, PC-3 and DU145 cells were analyzed for cell growth rate with MTT assay. As shown in Fig 3A and E, cells transducted with Lv-shSMC1A displayed suppressed growth rate in comparison to the control or Lv-shCon transducted cells. The cells were then seeded onto 6-well plates for the analysis of colony formation ability. PC-3 and DU145 cells transducted with Lv-shSMC1A formed colonies with much smaller sizes compared to the control and Lv-shCon transducted cells (Fig 3B and F).The colonies formed in 6-well plates were photographed (Fig 3C and G), and counted (Fig 3D and H). The results suggested that both PC-3 and Du145 cells showed impaired colony formation abilities after SMC1A knockdown,indicating a pivotal role of SMC1A in regulation of prostate cancer cells proliferation.

On Education-Emerson Essay

Imaging you are the only person at a concert; now imaging yourself surrounded by other who are just as enthusiastic about the concert as you are. One may give you a certain aspect of importance, while the other could make you feel like you belong to something bigger than yourself. The situation you prefer ultimately depends on your personality, that is to say, you as an individual. Present day America has become just that, a large gathering center for individuals from all corners of the globe; the great â€Å"melting pot of the world† to say the least. With all the diversity of unique talents, ideals, beliefs, and traditions that can be found outside one’s front-door step, a few questions arise: why is individualism not sought after and praised in today’s curriculum instead of being generalized into groups as one usually is? Likewise, is our current system of education preparing young minds to be conformists while slowly killing the individual? Ralph Waldo Emerson, one of the foremost intellectuals of the nineteenth century, theorized about an education system structured around the importance of the individual as its main foundation. Emerson believed that â€Å"our modes of Education aim to expedite, to save labor; to do for the masses what cannot be done for masses, what must be done reverently, one by one: say rather, the whole world is needed for the tuition of each pupil†. To put it differently, he believed the pupil may benefit more from personalized curriculums than from an education system aimed to teach by the masses to save money, time, and labor. In my opinion, from seeing the problems with our current Education system, I feel partially inclined to agree with Emerson and his idea to distance the education system from â€Å"teaching by the masses† and focus more on the individual For one, I firmly believe that today’s education system is more focused meeting the states standards and less focused on the student itself. The amount of standards an educator has to cover over the course of the year makes it nearly impossible to make individually customized teaching plans, thus the introduction of a curriculum in which everyone learns and works at the same pace. This can come at a steep price because although exposing every student to the same lesson demonstrates fairness and indiscrimination, it may also have negative repercussions on the young and inexperienced mind. In an education system like this, the individual is not valued because he is not seen as one student but generalized and group with other, whether it is by age or grade level. In the classroom for example, we are taught the basic knowledge context that everyone is expected to know, very rarely do we see any encouragement for those who want to dive in depth into a subject or personalized assistance for those who desperately need it. From my own experienced, I have always yearned to learn more about subjects I was interested in but if one cannot do that, then going to school becomes a chore. Statistics show that 8,300 high school students drop out each day (â€Å"High†). According to Buzzfeed, an online website, one of the top 5 reasons High School Students drop out is because they start finding classes uninteresting and the same can be said for college student. When the classes get dull they start centering their life’s around their jobs and eventually drop out to go in the pursuit of money. We have statistics and the reasons for the large amount of dropout backing up the fact that there is something wrong with today’s education system, yet appropriate measures to adjust the education system aren’t being made; the personal interest and curiosity of the student are not being met to inspire ones desire for knowledge. In addition to the lack of time, the reason for why individuality is not valued is due in part to the poor teacher-to-student ratio which does not do the creative mind just. Everyone needs space to think; however, we seem to be cramming in as many students as we can into one classroom, widening the teacher-to-student ration even further. One cannot master the lesson at hand if there is still a â€Å"shaky† foundation from the previous lesson due to the lack of sufficient assistance. With the fast pace that is required to meet all the requirements set forth by the United States, educators have little or no time to teach and assist those individuals who are in desperate need of attention, while at the same time neglecting to encourage, stimulate, and challenge those who fully grasp the material. The curriculum just doesn’t allow enough room for a student to show his creativity or stand out as an individual. Is it just to teach the same material to someone who learns at a slower pace and expect him to keep up with someone who is naturally inclined to that topic? Most would say no, yet this is precisely what the education system is doing. Consequently and perhaps more importantly, by doing so we may also be pushing one student too much while holding another individual back. I am afraid that in an attempt to educate everyone, we may be putting the individual at risk. Our current education systems have failed to comprehend that every individual is different and there is no one way to teach everyone. In short, we may be better off, as Emerson believes, to leave our traditional ways of teachings and focus on the individual. Furthermore, in my opinion the current curriculum is promoting conformism by establishing certain guidelines that encourage us to stay within the â€Å"normal† knowledge one should know. This strictness towards what is taught and what is not, what is acceptable and what is not allowed may be killing the young minds creativity and curiosity for knowledge. In essence, creating a system in which â€Å"going with the flow† is acceptable, may be leading you into a lifestyle of mediocrity. One does not have to go far to find conformism being taught at a very young age. For instance, look at your local preschool center. At an early age one is taught to walk into the classroom in a line, almost military-like, sit down and face the board like everyone else, and are even encouraged to suppress ones true desires and pretend to pay attention to the instructor. At an age where creativity and imagination is in its prime, the curriculum is already teaching one to stay within the lines while they color and goes as far as to indicate what color a certain object or person should be. What happens when a student chooses to color an object a different color? More than likely he is not praised for his creativity and his decision to stand out as an individual but scolded for not following instructions. The current curriculum might be trying to teach them disciplined but It is also preaching the idea that he is more valued when he â€Å"goes with the flow† than when he stands on his own. Is it not those that defy the â€Å"norms† who create the foundation for new styles and those few who think â€Å"outside the box† who move our society forward yet that sort of thinking is not promoted in the curriculum. I take a look at myself, and my college experience and notice conformism is a real issue. I see fellow peers do the minimum required of the instructor to pass the class, with no intent to learn anything more than what is required; they have no aspiration to exceed their past grades and are perfectly comfortable being average. Very rarely does one see someone who is well-rounded in a specific subject go out of their way and learn more than what the instructor covered. Even to someone like me, who prefers to stand out as an individual, waiting till next week to learn something as a class sounds more tantalizing than researching on my own. When the thinking, as to when one will be exposed to information, is done for us there is little to motivate us to take learning into hour own hands: â€Å"people who blindly follow rules are going along with the crowd and conforming. They are doing what’s easiest and avoiding challenge and having to think† (Harrison). By not going out of our way of the normal â€Å"flow† of life and society we may be condemning ourselves to a mediocre lifestyle. James Cooper once said, â€Å"All greatness of character is dependent on individuality. The man who has no other existence than that which he partakes in common with all around him, will never have any other than existence of mediocrity†(cooper 1). Overall, I believe that by having a general curriculum dictating when and how we learn, we may be more inclined to be satisfied with mediocrity and in turn conform to the â€Å"norms† of society. In conclusion, I strongly agree with the belief that educating the masses means slighting the individual (Emerson). The current Education system was intended to teach the masses, with respectable and admirable intents, but the system may have come too far and established an environment where creativity and individualism is a rare sight to see. There are some deep concerns with â€Å"teaching the masses† that I believe should be dealt with immediately if one wishes to move along as a society and bring to the world a new era of radical and critical thinker; that is to say, people who challenge and change the way we view the world. First off, the education system should allow for a sufficient margin of time so the educator may make certain adjustment to the curriculum based on the necessity of the students at that moment. Enough time is needed so the pupil may learn his natural pace and build his knowledge on a strong foundation. As for the intellectuals in the classroom, they should be given special modifications to the curriculum that may continue to challenge and grab his interest. Secondly, in an education system where everyone is taught the same, the speed and expectations of the classroom will almost always be that of the slowest person. This may be problematic because when you live your life doing only average work, you will conform to the idea that mediocrity is acceptable and life a life of mediocrity; never realizing your true otential. With all things considered, the ideal education system is one where its main focus is not inclined towards completing the curriculum, but one where teaching for the masses can inspire creativity in the individual by collaboration and competition with fellow peers. Overall, I agree with Emerson and I find it absolutely necessary for the education system to slowly distance itself from our present day curriculum and start focusing more on the individual to promote creat ivity and individuality.

In my view the case of Dr. Sticklen’s review paper should...

In my view the case of Dr. Sticklen’s review paper should be investigate first as a violation of confidence, then as a case of presenting fake data and then as a case of plagiarism. As an inexpert in this specific field, it is not obvious for me which exact part of the paragraph is plagiarism. What is obvious is that the structure of both paragraph are rather similar. However this similarity is not enough to accuse the Dr. Sticklen of plagiarism. It also should be noted that both articles are review papers and as a common practice in scientific publication authors of such articles are ought to combine reviews of papers that are already published. Therefor Dr. Sticklen’s argument about index card mixed-up should be considered when†¦show more content†¦NRG: Although the chief editor of NRG retracted Dr. Sticklen’s article they described this incident as a paragraph being paraphrased without attribution. Considering the high prestige of Nature Review Journals among scientific community the retraction can be considered an overwhelming response to this incident. Especially when we note that this is the first ever retracted from any of the 15 Na ture Reviews journals. However the retraction and even more drastic measures are justified when it is considered as response to violation of confidence. Additionally, via this action, NRG sent a message to future manuscript reviewer and authors that such misconduct of review policies is not tolerated by NRG editors. Other possible actions: The case with details of action taken by NRG and Michigan State University could be sent to other journals. This action should not be seen as an effort to isolate and boycott an active scientist. Other editorial board should invite Dr. Sticklen as a reviewer, but they also must be aware of her medical issue and its possible effect on quality and reliability of her work as a manuscript reviewer. It is assumed that committee members that investigate this issue at Michigan State University were informed about the author’s medical problem. If this assumption is true, revealing other evidence against and in favor of Dr. Sticklen’s could provide authors,Show MoreRelatedEthics And Ethics : Ethics922 Words   |  4 Pagesand friend group to be altered. One change I was not anticipating making was my approach to ethics. Over the course of the past fifteen weeks, my knowledge of ethics as well as my approach to ethics has changed. I have become more knowledgeable about the different approaches to ethics and have gained insight as to where I stand in my approach to ethics. One thing that has changed in my approach to ethics since the beginning of the semester is I am now adamant that it is impossible to arrive at aRead MoreEthics : Ethics And Ethics Essay1578 Words   |  7 Pages†¢ Define ethics. 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How Tattoos Are Viewed - Free Essay Example

Sample details Pages: 3 Words: 931 Downloads: 7 Date added: 2019/04/26 Category Culture Essay Level High school Tags: Tattoo Essay Did you like this example? With the months going by, more teenagers are turning 18, legally able to get a tattoo, and with the popularity of tattoos rising, more and more teenagers are wanting to get one. While some get tattoos for independence, others do it for the meaning that that their tattoo holds for them. But not every 18-year-old, or adult, is able to get a tattoo be it because of religion, employment regulations, or because some are afraid of the discrimination they could face. Don’t waste time! Our writers will create an original "How Tattoos Are Viewed?" essay for you Create order Even though we are in a new age, the stereotype of tattooed people is still prevalent and is discouraging for some people. Are people who have, or want to have tattoos, discriminated against? Finally, you turn 18 and can now legally get a tattoo but the stereotypes about dirty needles is off-putting and the stereotype that tattoos are a domain for bikers, sailors, and inmates (Kaiyala para. 6). With that being said, many people, of all ages, enjoy their tattoos. Roughly 16 percent of all U.S adults have at least one tattoo with the highest number, 36 percent, of tattooed adults being 25 to 29 years of age (Kaiyala para. 6). As tattoos rise, so does the industry and one of the main concerns for both clients and artists are infections and diseases. The biggest concern is HIV for the use of needles but the biggest threat is hepatitis. Hepatitis can be transmitted through little more than a scratch with an infected needle. To combat this and any other infectious bloodborne pathogen, artists autoclave their single-service equipment (Alliance para. 3). So, while people worry about infections, all the artists equipment should be single service means that each needle and tube set is individually packaged, dated and sealed and autoclaved. An autoclave is the only acceptable means of equipment sterilization in the tattoo shop. It is a machine that uses a combination of heat, steam and pressure to kill all pathogenic microorganisms known to man (Alliance para. 8 and 9). Overall, the process of getting a tattoo is safe and people should not have a reason to worry other than choosing a design that they will be comfortable for the rest of their lives. Although only 16% of U.S adults are tattooed, it was estimated that in 1900, around 90% of American sailors were tattooed (Military Tradition para. 12). Not only did they have random tattoos but they had meaningful tattoos dictating where they sailed. Sailors with a tattoo of an anchor proved that they had sailed the Atlantic Ocean. A full-rigged ship meant he shipped around Cape Horn. A Shellback Turtle indicated the sailor crossed the equator, and a dragon meant he served on a station in or near China. Hold tattooed on the knuckles of one hand and fast on the other were said to allow the bearer to grip the rigging better. Tattoos of a pig on one foot and a rooster on the other were said to protect a seaman from drowning. It was thought since both creatures avoid the water at any chance, they would help get the sailor swiftly to shore if he fell overboard (Military Tradition para. 13). And for some, it was a meaning of what they were and what they did (Military Tradition para. 13). Although the military banned tattoos with obscene imagery, tattoos in general do not prohibit you from joining as you only need the ASVAB and a physical (Military Tradition para. 24). Even though men and women are viewed as equals in this age, Deborah Connor found discrimination when she was terminated for having a heart tattoo which, to her employer, because the company was concerned that the customers would see her and would react because a tattooed woman is seen as a prostitute, on drugs, or from a broken home ( Pechman para. 8). The same company did not require a male employee to cover up his navy tattoo, and she found it insulting and then sued the company (Pechman para. 8). With the growing rise of television and social media. More and more celebrities are getting tattoos and their influence influences many teens to get tattoos of their own or even the same tattoo. Besides the influence, some teens find tattoos as a way to woo their partner as a romantic gesture (Religion forbids tattoos para. 4). The only problem would be if the couple were to separate and the partner would have to spend their life with the image of their ex. Either that or spend time, money, and pain to get it removed and even then, it sometimes leaves a scar. Some teens however, view tattoos as a work of art, and also a way to declare their independence (Religion forbids tattoos para. 5). For many, they cannot wait to turn 18 sorely because of that reason alone. For some, they get it because it is whats trending at the moment. But the art of tattooing is not an old practice by any means as Egyptian and Libyan mummies have been found with tattoos that date back before the time of Chri st (Religions forbid tattoos para.7). Ironically as the first tattoos were uncovered, it was a picture rather than an abstract pattern, it was the picture of the Egyptian god, Bes. While the Mosiac Law forbade their followers to have tattoos and by that way, the Israelites stood out from different nations (Religions forbid tattoos para. 8). While Christians today are not under the Law of Moses, the prohibition it laid on tattooing is sobering (Ephesians 2:15; Colossians 2:14, 15). If you are a Christian, you would certainly not want to make markings on your body†even temporarily†that smack of paganism or false worship. †2 Corinthians 6:15-18 (Religions forbid tattoos para. 8).