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Remove media, snap freeze the conditioning media in liquid nitrogen (see Note 6), and proceed immediately to the next step (see Note 7). Submerge cell culture plate into the bucket lled with the liquid nitrogen, and leave it for 30 s to quench the metabolism and initiate cell lysis (see Note 9). Add 500 L per 6-cm dish of cold methanol (A20 C), scrape the cells, and transfer suspended cell pellet into 1. Anesthetize the mice with 50 L of sodium pentobarbital (65 mg/mL) by intraperitoneal injection (see Note 11). Once mice are fully unconscious (approximately 5 min follow ing sodium pentobarbital delivery), spray the abdomen with 70% of ethanol, and place mice on a surgical platform within a reservoir to contain runoff buffer and body uids. Make a transverse incision through the skin, fascia, and muscu lar layers of the lower abdomen, then a lateral sagittal incision on each side of the body, exposing the abdominal contents, and nally a second transverse incision inferiorly to the right kidney and toward the dorsal aspect of the mouse to allow the perfu sion buffer and body uids to drain from the abdomen. Expose the portal vein by gently moving the intestines laterally toward the left body wall. Cannulate the portal vein with a 24-gauge catheter needle, withdraw the needle, and reconnect the tubing with buffer to the catheter. Cut the right atrium to prevent recirculation of buffer to the liver and to terminate perfusion to the brain. Perfuse the livers with oxygenated buffer pre-warmed to 42 C (see Note 12) with a countercurrent heat-exchange circuit and a recirculating water bath (see Note 13). The standard perfusion media should contain Krebs-Henseleit bicarbonate buffer (see Note 14), 1. Maintain the buffer delivery at a rate of 8 mL/min using a peristaltic pump for a period of 1 h. Monitor the liver temperature periodically with infrared ther mometer (see Note 15). Discard the perfusate effiuent from the initial 30 min, and collect the effiuent from the nal 30 min of the procedure. Freeze-clamp the liver in a bath of liquid nitrogen at the end of the 60-min perfusion procedure. Cells, liver tissues, and conditioned media can be extracted Extraction using previously described method with modication [20, 21]. Place tubes in 12 sample carriage (if available) and repeat three times the following steps: 30 s of vortex, 1 min in liquid santanu. Repeat the following steps two times: sonicate for 5 min at 25 C and vortex for 1 min.

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Long-Term Follow-Up As with any new procedure, carefully monitored clinical trial protocols would be required for germline genome editing, with attention to monitoring off-target events as well as the efficiency and correctness of the specific edit. Unlike conventional clinical trials, germline genome editing trials would likely require long-term prospective follow-up studies across subsequent generations. This follow-up would entail study of the future children affected by the intervention, none of whom would have been party to the initial decision to participate in a research trial. Data of this type would be important for determining whether the techniques had achieved their goals (Friedmann et al. Even those who have volunteered to be research subjects cannot be compelled to participate in long-term follow-up. Experience from xenotransplantation, and some drug and device trials, shows that this encouragement can be successful. And despite the particular challenge of studying offspring rather than those who consented to the research, experience with other reproductive technologies suggests that follow-up can be carried out in numbers sufficient to permit conclusions about many possible long-term effects (Lu et al. Societal Effects Some have argued that germline editing can be justified on grounds that go beyond mere parental choice. There is a line of thought that germline modification could be used to create a level playing field for those whose traits now put their children and descendants at a disadvantage (Buchanan et al. That said, it is important to note that the history of abusive and coercive eugenics (discussed in section on human dignity and the fear of eugenics, below) is intertwined with previous, undoubtedly well-intentioned public health and hygiene movements, which is one reason why discussions of public health benefits often engender some skepticism and unease. Some contemporary transhumanists point out that the human body is flawed in that it easily becomes diseased, requires a great deal of sleep, has various cognitive limitations, and eventually dies. They suggest that it would make sense to improve the human species by making it more resistant to disease, more moral, and more intelligent (Hughes, 2004). Some, such as philosopher John Harris, say that in certain cases there is a moral obligation to enhance ourselves genetically (Harris, 2007). But these are arguments about enhancement, not the restoration or maintenance of ordinary health. Moreover, meiosis combined with fertilization creates in each individual a novel assortment of gene variants.


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It would not be associated with the involving the adrenal medulla, as they arise systemic symptoms described in this patient. Neuroblastoma is as glucosidase defciency causes a rare glycogen sociated with the N-myc oncogene, located on storage disease called Pompe disease, which chromosome 2p. It presents with cardiomegaly along with is the most common renal cancer in adults. It weakness and hypotonia in the frst six months usually presents in older adults after the ffth of life. Ornithine transcarba In the kidney, it usually involves the renal pel mylase defciency, an X-linked disorder, is the vis. Transitional cell carcinoma accounts for most common genetic defciency of the fve 5%-10% of primary renal tumors. This child and his nia, leads to hyperammonemia during the frst uncle appear to have Friedreich ataxia, a tri weeks of life and can lead to mental retarda nucleotide repeat disease. Treatment includes limiting protein in otide repeat diseases, illness occurs because take in the diet and administering compounds the unstable microsatellite regions on certain like phenylbutyrate, which help bind to amino chromosomes have triplet codons that expand, acids and promote their excretion. This disor typically worsening from generation to genera der would not present with the symptoms seen tion (and often making the age of onset earlier in this patient, nor would it normally present for each successive generation). The patient is a reich ataxia) cause a decrease in the product of young boy presenting with an abdominal mass a gene, frataxin, at the translation stage. The most common re nal tumor in young children is Wilms tumor, Answer A is incorrect. The triplet repeats in which accounts for approximately 6% of pe Friedreich ataxia typically do not affect protein diatric malignancies. Wilms tumor typically folding because the gene product does not usu presents with a palpable mass, and about 25% ally get to that stage. It is an embryonal neoplasm that Friedreich ataxia typically do not affect protein tends to occur between ages 1 and 3 years, splicing. In particular, high Friedreich ataxia typically do not cause a sub protein plus creatinine supplementation may stitution, such as that which occurs in sickle be recommended in McArdle disease, a glyco cell disease. The triplet repeats in cramps and myoglobinuria with strenuous ex Friedreich ataxia typically do not contain a ercise. This disor in the breakdown pathway for phenylala der is characterized by hepatosplenomegaly, nine. The product of this reaction is tyrosine, aseptic necrosis of the femur, and bone crisis.

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The Bowman space hydrostatic pres phrotic syndrome, with massive proteinuria sure could be increased in a patient with an and peripheral and periorbital edema. Bowman space oncotic emia and hyperlipidemia, which are also as pressure will increase, not decrease, as protein sociated with nephrotic syndrome. The im is fltered into Bowman space and thus in age shows changes typically associated with creases the protein concentration there. Hydrostatic pressures line deposits in the periphery of the glomeru are not affected in minimal change disease. Goodpasture disease is strongly associated with coronary artery dis results in a rapidly progressive glomerulone ease. It is caused by antibodies di ers are benefcial in the treatment of diabetic rected against the glomerular basement mem nephropathy. Renal biopsy with immunofuorescence would show linear deposition of IgG along the Answer A is incorrect. Acute poststreptococcal lesions, which are characteristic of diabetic glomerulonephritis is associated with recent nephropathy. In the mem with a history of pharyngitis, low-grade fever, branous glomerulonephritis pattern, biopsy swollen lymph nodes, and tonsillar exudates. It reveals wire-loop lesions with subepithelial is seen most often in children, and although it deposits. Sensitivity = true veals apple-green birefringence in patients positives / (true-positives + false-negatives). One excellent example of False-negatives signify the people with disease amyloidosis is amyloid from immunoglobulin X who will be missed by the screening test. In light chains, which is produced by cancerous this case, 100 people have the disease, and 80% plasma cells in multiple myeloma. In addition will be diagnosed correctly (80 people are the to bone pain, signs and symptoms of multiple true-positive fraction). Rearranging the equa myeloma include renal failure, elevated cal tion yields: false-negatives = (true-positives / cium, anemia, and increased vulnerability to sensitivity) true-positives or (80 /. Alport syndrome is an diagnosed with this screening test (ie, they will inherited glomerular disease caused by a muta be false-negatives). It is associ 100 people have the disease (10% prevalence) ated with sensorineural hearing loss and ocu and the test is 80% sensitive, then 80 people lar disorders. The gastroesophageal number of people who will have an incor junction is affected by gastroesophageal refux rect positive screening test result (ie, false disease, not carcinoid tumors. The pancreaticoduo there are 900 people without the disease (with denal junction is the site where pancreatic en a 10% incidence in 1000 people 100 will have docrine and exocrine secretions empty into the the disease and 900 will not).