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However buy vardenafil 10 mg on line, techniques to enable efficient and highly parallel identification generic vardenafil 20 mg overnight delivery, measurement and analysis of proteins remain a bottleneck cheap vardenafil 10 mg otc. A plat- form technology that makes collection and analysis of proteomic data as accessi- ble as genomic data has yet to be developed buy vardenafil 10 mg amex. Sensitive and highly parallel technologies analogous to the nucleic acid biochip, for example, do not exist for protein analysis. New Developments in Protein Chips/Microarrays The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. Protein arrays appear as new and versatile tools in functional genomics, enabling the translation of gene expression patterns of normal and diseased tissues into protein product catalogues. Protein function, such as enzyme activity, antibody specificity or other ligand–receptor interactions and binding of nucleic acids or small molecules can be analyzed on a whole-genome level. As the array technology develops, an ever-increasing variety of formats become available (e. Various techniques are being developed for producing arrays, and robot- controlled, pin-based or inkjet printing heads are the preferred tools for manufactur- ing protein arrays. The emerg- ing future array systems will be used for high-throughput functional annotation of gene products, their involvements in molecular pathways, and their response to medical treatment and become the physician’s indispensable diagnostics tools. Protein Biochips/Microarrays for Personalized Medicine Protein biochips/microarrays are well-established tools for research and some prod- ucts for in vitro diagnostics are available commercially. Profiling proteins on bio- chips will be useful for distinguishing the proteins of normal cells from early-stage cancer cells, and from malignant metastatic cancer cells. Of all the applications of protein microarrays, molecular diagnos- tics is most clinically relevant and would fit in with the coming trend in individual- ized treatment. Universal Free E-Book Store 56 2 Molecular Diagnostics in Personalized Medicine For example, different proteins such as antibodies, antigens, and enzymes can be immobilized within protein biochips. Protein microarrays are reliable tools for detec- tion of multiple biomarkers with only a minimal quantity of sample and have enor- mous potential in applications for personalized medicine (Yu et al. Microfluidics Microfluidics is the special behavior of fluids flowing in channels the size of a human hair. Fluids in this environment show very different properties than in the macro world. This new field of technology was enabled by advances in microfabrication – the etching of silicon to create very small features. Microfluidics allows the reduction in size with a corresponding increase in the throughput of handling, processing and analyzing the sample. Other advantages of microfluidics include increased reaction rates, enhanced detection sensitivity and control of adverse events. Applications of microfluidics, in relation to molecular diagnostics, include the following: • Genomic analyses • Protein analysis • Gene expression and differential display analysis Several commercial microfluidic technologies are available and a few examples are described in the following text. Moreover, the chip test is performed within hours whereas the conventional protocol required days. The rapid detection of chromosomal mutations will increase a physician’s ability to personalize treatment strategies to target indi- vidual cancers. An important limiting factor has been the difficulty of establishing molecular assays suitable for microfab- ricated formats. The power of the lab-on-a-chip concept lies primarily in its ability to detect and manipulate at the cellular and molecular level with sufficiently high throughputs. With careful design and scaling considerations, molecular and cellular detectors (or biosensors) facilitated by controlled microfluidic separation, purification, sorting, and mixing operations are more sensitive and specific. For proteins, an acoustic cavity mixer enables an order of magnitude increase in speed of detection 3. A novel microfluidic device based on dielectrophoresis enables the detection and sorting of biological cells based on their dielectric properties. LabChip Lab-on-a-chip (PerkinElmer’s LabChip), a miniaturized and integrated liquid han- dling and biochemical-processing device, is used for computer-aided analytical laboratory procedures that can be performed automatically in seconds. This as well as the Agilent 2100 bioanalyzer is being developed in collaboration with Agilent Technologies to integrate time-consuming and costly laboratory experiments onto a miniature chip. PerkinElmer’s genotyping system is designed to integrate each stage of the com- plete experiment in a volume of 1 nanoliter, a scale 10,000 to 100,000-fold smaller than currently used technology. Fluid is moved along these pathways by capillary action and centrifugal forces generated by disc rotation, allowing the processing of many different assay types. The combination of informatics, bioassays and minia- turization are what make this “laboratory on a disc” truly revolutionary. For instance, physicians will be able to run tests for multiple strains of hepatitis all at the same time, instead of ordering them separately. The ability to identify the strain of a virus can have profound implica- tions for clinical therapy. These studies provide an opportunities to explore pathomechanism of human diseases that are unbiased by previous hypotheses or assumptions about the nature of genes that influence complex diseases. Many genetic variants identified as risk factors for dis- eases by such studies have been localized to previously unsuspected pathways, to genes without a known function. In the absence of functional information about which polymorphisms are bio- logically significant, it is desirable to test the potential effect of all polymorphisms on drug response. Genotyping and Haplotyping A genotype is the genetic constitution of an organism as defined by genetic and molecular analysis and covers the complete set of genes. Genotyping can be used for determination of relevant genetic variation in each of the two parental chromosomes in an individual. Haplotypes are gene versions that represent the genetic variations as they occur on each pair of chromosome in an individual. Haplotypes are the most precise markers possible for a given gene because they contain all the variations in a gene. Haplotyping information makes it possible to highlight the structure of the genome, notably through haploblocks which correspond to segments of chromosomes unlikely to undergo a crossing-over event. Candidate gene-based haplotype approach has been applied to the pharmacoge- netics of drug response and adverse events. Clinical trials using haplotyped indi- viduals were the first genetically personalized medical treatments. Only two genomes were completely haplotyped: the reference human genome and Craig Venter’s genome, both of which relied on Sanger sequencing and clone mapping to resolve the haplotypes, which is a labor-intensive and costly process. Although the newer sequencing technologies enabled cost reductions and higher throughput, the shorter reads are not amenable to obtaining haplotype information, which will be critical in the fields of personalized medicine and population genetics. The first method was used to determine the haplotype-resolved genome of a South Asian individual (Kitzman et al. A single fosmid library was split into a modest number of pools, each providing ∼3 % physical coverage of the diploid genome. Sequencing of each pool yielded reads overwhelmingly derived from only one homologous chromosome at any given location. This method also facilitates the analysis of structural variation, for example, to anchor novel insertions to specific locations and haplotypes. The second method used a microfluidic device capable of separating and ampli- fying homologous copies of each chromosome from a single human metaphase cell (Fan et al.

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Prions result when an abnormal prion protein binds to a normal isoform of the prion protein purchase 20mg vardenafil with visa, stimulating its conversion into the abnormal isoform discount vardenafil 10mg with mastercard. Abnormal prion isoforms have a greater proportion of β-structure and less α- helix than do normal isoforms purchase vardenafil 20mg fast delivery. The α-to-β structural transition underlies the etiology of the central nervous system degeneration 20mg vardenafil free shipping. Typically the myoclonus is provoked by startle, loud noises, or bright lights and will occur even during sleep. Brain biopsy may demonstrate spongiform degeneration and the presence of prion proteins. It is spread eas- ily person-to-person, and outbreaks in crowded conditions are common. Blood smear shows no abnormality, which is in con- trast to IgG or warm-type hemolytic anemia where spherocytes are seen. However, in older male patients with lumbar osteomyelitis, genitourinary or enteric pathogens, such as E. Pathogenesis may occur via retrograde introduction of organism into the spine via the spinal venous plexus. Polymi- crobial osteomyelitis is most often due to contiguous infection, such as a decubitus ulcer or diabetic foot infection, rather than bloodstream introductions that are more typical in the spine. Tuberculosis (Pott’s disease) is always a consideration for osteomyelitis of the spine. However, this patient’s presentation is likely too acute for tuberculosis, and the thoracic spine is a slightly more typical location than the lumbar spine. Brucellosis com- monly involves the spine, but this patient’s potential exposure to Brucella spp. Hypothetically each of the listed infections is possible, highlighting the importance of holding antibiotics before culturing the epidural space, provided that the patient does not have sepsis on original presentation. Because seroprevalence rates are high in endemic areas, subclinical infection is likely common. The disease typically presents with fever (>90% of cases), myalgias, headache, and mal- aise. Thrombocytopenia, leukopenia, and elevated aminotransaminase activity is com- mon. Adult respiratory distress syndrome, toxic shock–like syndrome, and opportunistic infections may occur, particularly in the elderly. Human granulocytotropic anaplasmosis should be considered on the differential of a flulike illness during May through December in endemic regions. Morulae, intracytoplasmic inclusions, are seen in the neutrophils of up to 80% of cases of human granulocytotropic anaplasmosis on peripheral blood smear and are diagnostic in the appropriate clinical context. This patient has high epidemio- logic risk based on his long periods of time outside in an endemic region. Human mono- cytotropic ehrlichiosis, which can be a more severe illness, has morulae in mononuclear cells (not neutrophils) in a minority of cases. Lyme disease, which may be difficult to dis- tinguish from human granulocytotropic anaplasmosis or human monocytotropic ehrlichiosis, will not cause morulae. It occurs in ~1% of patients with asthma and in up to 15% of patients with cystic fibrosis. Patients typically have wheezing that is difficult to control with usual agents, in- filtrates on chest radiographs due to mucus plugging of airways, a productive cough often with mucus casts, and bronchiectasis. In the proper clinical context, a positive skin test for Aspergillus antigen or detection of serum Aspergillus-specific IgG or IgE precipitating antibodies are supportive of the diagnosis. Patients who develop endocarditis within 2 months of valve surgery most likely have acquired their infection nosocomially as a result of intra- operative contamination of the prosthesis or of a bacteremic postoperative event. The modes of infection and typical organisms causing prosthetic valve endocarditis >12 months after surgery are similar to those in community-acquired endocarditis. Both sets of pathogens must be considered in the intermediate 2–12 months after surgery. Atovaquone is a common al- ternative that is given at the same dose for Pneumocystis prophylaxis as for therapy. Aerosolized pentamidine can be given on a monthly basis with a risk of bronchospasm and pancreatitis. Patients who de- velop Pneumocystis pneumonia while receiving aerosolized pentamidine often have up- per lobe–predominant disease. The white thickened folds on the side of the tongue can be pruritic or painful and sometimes resolve with acyclovir derivatives or topical podophyllin resin. Ultimate resolution occurs after immune reconstitution with antiretroviral therapy. Kaposi’s sarcoma is uncommon in the oropharynx and takes on a violet hue, suggesting its highly vascularized content. While more sophisticated tests have been developed, this classification scheme is still used and is of some benefit to the clinician. They will typically take 7 days or less to grow on standard media, allowing relatively fast identification and drug-resistance testing. Although many patients remain asymptomatic, malnourished persons are at particular risk for progression to symptomatic disease or kala azar, the life- threatening form. The presentation of this disease generally includes fever, cachexia, and splenomegaly. Hepatomegaly is rare compared with other tropical diseases associated with organomegaly, such as malaria, miliary tuberculosis, and schistosomiasis. Pancy- topenia is associated with severe disease, as are hypergammaglobulinemia and hypoalbu- minemia. Although active investigation is under way to determine a means of diagnosing leishmaniasis by molecular techniques, the current standard remains demonstration of the organism on a stained slide or in tissue culture of a biopsy specimen. In light of the high mortality associated with this disease, treatment should not be delayed. The mainstay of therapy is a pentavalent antimonial, but newer therapies including amphotericin and pentamidine can be indicated in certain situations. In this case it would be prudent to rule out malaria with a thick and a thin smear. In the United States, the predominant virus in up to 12% of new cases has one major geno- typic resistance mutation (patient A). Primary peritonitis is a result of longstanding ascites, usually as a result of cirrhosis. The pathogenesis is poorly understood but may involve bacteremic spread or translocation across the gut wall of usually only a single species of pathogenic bac- teria. Secondary peritonitis is due to rupture of a hollow viscous or irritation of the perito- neum due to a contiguous abscess or pyogenic infection. It typically presents with peritoneal signs and in most cases represents a surgical emergency.

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This method is commonly used in the analysis of trace elements in various samples generic 20mg vardenafil with amex. The method of production and various characteristics of radionuclides commonly used in nuclear medicine are presented in Table 5 best 10mg vardenafil. Continued g-ray Common Physical Mode of g-ray energy* abundance production Nuclide half-life delay (%) (MeV) (%) method 133Xe 5 cheap 20mg vardenafil visa. Target and Its Processing Various types of targets have been designed and used for both reactor and cyclotron irradiation purchase vardenafil 20mg without prescription. In the design of targets, primary consideration is given to heat deposition in the target by irradiation with neutrons in the reactor or charged particles in the cyclotron. In both cases, the temperature can rise to 1000°C, and if proper material is not used or a method of heat dissipa- tion is not properly designed, the target is likely to be burned or melted. For this reason, water cooling of the cyclotron probe to which the target is attached is commonly adopted. In the case of the reactor, the core cooling with heavy water is sufficient to cool the target. Most often, the targets are designed in the form of a foil to maximize heat dissipation. The common form of the target is metallic foil, for example, copper, aluminum, uranium, vanadium, and so on. Other forms of targets are oxides, carbonates, nitrates, and chlorides contained in an aluminum tubing, which is then flattened to maximize the heat loss. In some cases, compounds are deposited on the appropriate metallic foil by vacuum distillation or by electrodeposi- tion, and the plated foils are then used as targets. Equation for Production of Radionuclides While irradiating a target for the production of a radionuclide, it is essen- tial to know various parameters affecting its production, preferably in a mathematical form, to estimate how much of it would be produced for a given set of parameters. The term (1 − e−lt) is called the saturation factor and approaches unity when t is approximately 5 to 6 half-lives of the radionuclide in question. At that time, the yield of the product nuclide becomes maximum, and its rates of production and decay become equal. The intensity of the irradiating particles is measured by various physical techniques, the description of which is beyond the scope of this book; however, the values are available from the operator of the cyclotron or the reactor. The formation cross sections of various nuclides are determined by experimental methods using Eq. The number of atoms N of the target is calculated from the weight W of the material irradiated, the atomic weight Aw and natural abundance K of the target isotope, and Avogadro’s number 23 (6. These radionuclides are identified and quantitated by detecting their radiations and measuring their half-lives by the use of the NaI(Tl) or Radionuclide Generators 51 Fig. The activity produced reaches a maximum (saturation) in 5 to 6 half-lives of the radionuclide. They may also be assayed in an ionization chamber if the amount of radioactivity is high. Radionuclide Generators Radionuclide generators provide the convenient sources of short-lived radionuclides that are very useful clinically. The basic requirements for a generator are that a parent radionuclide has a longer half-life than that of the daughter, and the daughter can be easily separated from the parent. In a generator, a long-lived parent radionuclide is allowed to decay to its short- lived daughter radionuclide, and the latter is then chemically separated. The importance of radionuclide generators lies in the fact that they are easily transportable and serve as sources of short-lived radionuclides in institu- tions without cyclotron or reactor facilities. A radionuclide generator consists of a glass or plastic column fitted at the bottom with a fretted disk. The column is filled with adsorbent mater- ial such as ion exchange resin, alumina, and so forth, on which the parent nuclide is adsorbed. The parent decays to the daughter until transient or secular equilibrium is established [Eqs. Vacuum in vial B draws the eluant from vial A through adsorbent material, and the daughter is collected in vial B. After equilibrium, the daughter appears to decay with the same half-life as the parent. Because of the differences in chemical properties, the daughter activity is eluted with an appropriate solvent, leaving the parent on the column. After elution, the daughter activity builds up again and can be eluted repeatedly. The vial containing the eluant is first inverted onto needle A, and an evac- uated vial is inverted on the other needle B. The vacuum in the vial on needle B draws the eluant from the vial A through the column and elutes the daughter nuclide, leaving the parent nuclide on the column. In some commercial generators, a bottle of eluant is placed inside the housing, and aliquots of eluant are used up in each elution by the evacuated vial. The generator eluate should be free of the parent nuclide and the adsorbent material. Several radionuclide generators are available for ready supply of short-lived radionuclides: 99Mo(66hr)–99mTc(6hr); 113Sn(117 days)– 113m 68 68 82 82 In(100min); Ge(271 days)– Ga(68min); Sr(25. Of these, the Mo– Tc generator is the work- horse of nuclear pharmacy in nuclear medicine. Radionuclide Generators 53 99 99m Mo– Tc Generator The 99Mo–99mTc generator is constructed with alumina (Al O ) loaded in a 2 3 plastic or glass column. The 99Mo activity is adsorbed on alumina in the 2− chemical form MoO4 (molybdate) and in various amounts. Answer 99Mo activity on Thursday noon = 2500mCi The time from Thursday noon to 8:00 a. The 99Mo breakthrough is determined by the detection of high-energy photons (740keV and 780keV) of 99Mo in a dose 99m calibrator after stopping 140-keV photons of Tc in a lead container (6-mm thick). The presence of aluminum interferes with the preparation of 99m Tc-labeled sulfur colloid by forming larger particles, which are trapped in the lungs. Aluminum ion (Al ) is checked by the colorometric test using a paper strip impregnated with a coloring agent and comparing the intensity of the color developed by the sample solution with that by a standard test solution (10mg/ml). If 68Zn is bombarded with protons in a cyclotron and three neutrons are emitted from the nucleus, what is the product of the nuclear reaction? Calculate the 99mTc activ- ity assuming 80% yield and 87% of 99Mo decays to 99mTc. Cyclotron and positron emission tomography radiopharmaceuticals for clinical imaging. Because particulate radiations have mass and electromagnetic radi- ations do not, the latter travel through matter longer distance before losing all energy than the former of the same energy.

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It is currently in a clinical trial Universal Free E-Book Store 122 4 Pharmacogenetics in Canada discount 20 mg vardenafil mastercard, and the data from the pharmacogenomic study will help support regula- tory filings for the combined use of the test with Plavix discount vardenafil 20 mg mastercard. In patients undergoing coronary stent placement buy discount vardenafil 10 mg, a single *17 gene variant resulted in about a twofold increase in the incidence in bleeding within 30 days fol- lowing stent placement trusted 20mg vardenafil. Patients with the double *17/*17 genotype exhibited a dose-response fourfold increase in bleeding (8 %). The most striking finding was that among the patients tested, 35 % had the single *17 variant and another 5 % had the *17/*17 genotype. According to the recommendation from the American College of Cardiology on genotyping for clopidogrel, genotyping may be considered for identifying poor metabolizers in “moderate to high risk patients” as alternate therapies are available (Holmes et al. In response to this publication, cardiologists from Scripps Health as well as colleagues from Vanderbilt University and Hôpital Pitié-Salpetrière in Paris pointed out what they see as flaws in the review analysis. Moreover, the meta-anal- ysis did not test for heterogeneity among patients who underwent stenting versus those who were medically treated. It is obvious that a metal implant in a coronary artery would pose a particular vul- nerability to inadequate platelet suppression. Role of Pharmacogenetics in Statin Therapy Lowering low-density lipoprotein cholesterol with statin therapy results in substan- tial reductions in cardiovascular events, and larger reductions in cholesterol may produce larger benefits. In 10–20 % of cases, myopathy occurs in association with statin therapy, especially when the statins are administered at higher doses and with certain other medications and is a reason for discontinuation. The finding raises hope that a test could be developed to screen patients to find out who is at greatest risk for developing this adverse reaction. Although there is a statin-gene association for myopathy in the case of some statins, the usefulness of this information still needs to be proven (Giorgi et al. The problem is that it will take thousands and thousands of patients to screen in order to validate a particular marker. Because of the rarity of such events, the prospect of predicting them by genetic biomarkers is viewed as not only daunting but unlikely. The Critical Path is also linking the Association of Clinical Research Organizations with the Clinical Data Interchange Standards Consortium to form the Clinical Data Acquisition Standards Harmonization project. Areas of focus in this effort are bioinformatics and data standards, biomarkers, establishing public-private partnerships, and developing guidance and regulations. It is also partnering with researchers at Duke University to look for rare variants corre- sponding to adverse reactions to the antipsychotic drug clozapine. They also emphasize the importance of immune regulation genes, in addition to a number of well character- ized drug metabolism genes. Phenotyping can reveal defects in overall metabo- lism of a drug or drug-drug interactions but it has several disadvantages: • Requires a test drug • Testing protocol is complicated • Risk of adverse drug reactions • Errors in phenotype assignment due to co-administration of drugs • Confounding effect of the disease Universal Free E-Book Store Role of Pharmacogenetics in Pharmaceutical Industry 127 Comprehensive phenotyping is important for understanding disease mechanisms and variations in disease course and response to therapy among patients. SurroMed Inc’s phenotyping technology platform provides fundamental information about disease and enables rapid discovery of new and useful biological markers. These biological markers will have utility for better diagnosing and treating disease and developing new and improved therapeutic products. Metaprobe™ biomarkers (Phenome Sciences) offer an improved approach to identifying a patient’s phenotype. Metaprobes measure the capacity of targeted pathways that are instrumental in a disease process or metabolic pathway relevant to the activity of a pharmaceutical. Structurally, Metaprobe biomarkers are small molecules such as amino acids or other compounds that have confirmed safety pro- files and can be delivered orally, by injection, or by inhaler. Metaprobes are labeled to quantify pathway capacity by detection of release tags in breath, plasma, or urine. The rate of appearance of the release tag gives a direct and quantitative mea- surement of the in vivo activity of the targeted pathway, creating a dynamic bio- marker of phenotype. Metaprobes are available for over 120 pathways in various stages of active development. For example, metaprobes can provide very sensitive assessment of physiologic response to a known therapeutic that changes internal demand for glutathione. This presents a difficult challenge because phenotypes are numerous and diverse, and they can be observed and anno- tated at the molecular, cellular and organismal level. Efforts to develop new and efficient tech- nologies for assessing cellular phenotypes include the following: • A phenotypic map can be generated to correspond to any genotypic map. Some genes have only one corresponding phenotype whereas most genes have many corresponding phenotypes. Universal Free E-Book Store 128 4 Pharmacogenetics Genotyping Genotyping also predicts metabolic capacity but involves identification of defined genetic mutations that give rise to the specific drug metabolism phenotype. These mutations include genetic alterations that lead to overexpression (gene duplication), absence of an active protein (null allele), or production of a mutant protein with diminished catalytic capacity (inactivating allele). Genotyping, on the other hand, provides time invariant information on the individ- ual’s metabolizing capacity and it is applied in clinical and epidemiological studies. If therapeutic decisions are based on this information, 10–20 % of poor metabolizes may be wrongly classified as extensive metabolizes. Genotyping is valuable both for individual cases, particularly when a phenotype cannot be established due to concomitant therapy, and for screening of populations in clinical studies. Phenotype tests have applied successfully in some pharmacogenetics conditions such as malignant hyperthermia, porphyries and glucose-6-phosphate dehydroge- nase deficiency. It is likely that more practical genotyping tests would be used in the future and phenotypes would be predicted via genotyping. The traditional phenotype-to-genotype pharmacogenetic research paradigm is reversing direction to create a complementary genotype-to-phenotype flow of information. This relationship is frequently non- linear in nature, which poses a problem for traditional means of genetic study. These traditional methods are not well suited to accommodate the effect of quantitative trait loci or multi-dimensional genetic interactions at work in the determination of most human phenotypes. Universal Free E-Book Store Role of Pharmacogenetics in Pharmaceutical Industry 129 Table 4. This will integrate multidisciplinary research, with the goal of understanding the complex phenotypic consequences of genetic mutations at the level of the organism. Hardware and software engineers, as well as behavioral (and other) neuroscientists will co- develop test paradigms and equipment that will enable investigators to cope with the demands set by the increasing number of mutants generated by such techniques as transgenics or chemical mutagenesis. Phenomics will be a crucial approach in aca- demic, as well as industrial, research and could lead to a significant paradigm shift both in the genetic analysis of brain function and in drug development. It will be used to identify individuals who are incompatible with certain drug treatments before the drugs are prescribed and damage is done. It will be used to tease out important genetic determinants associated with complex genetic diseases, so that drugs can be developed to target these genes. Universal Free E-Book Store 130 4 Pharmacogenetics Limitations of Genotype-Phenotype Association Studies Although genotype-phenotype association studies are seemingly simple, in practice, they are prone to potential difficulties and problems. Plausible biologic context con- sistent with allele function, low P values, independent replication of an initial study, rigorous phenotypic assessment and genotyping, selection of appropriate and suffi- ciently large populations, and appropriate statistical analysis are all critical to the confidence that can be placed in a proposed association. Because such criteria are not always met, the risk of false-positive or false-negative errors is always possible. Some of the disparities between genotype and phenotype clarified by metabolomics as described in Chap. Molecular Toxicology in Relation to Personalized Medicines The term molecular toxicology covers the use of molecular diagnostic methods for studying the toxic effects of drugs.

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