Diagnosis final notes, Exercises of Diagnostics

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Diagnosis final notes Lecture 1 Q1: miRNA biogenesis in animal cell -miRNA is transcribed from a non-coding gene via RNA polymerase || -Then a stemlooped structure containing a poly A tail and a 5 cap to be protected called PrimiRNA is formed then a protein called DGCR8 binds to Pri-miRNA once this protein is attached it permits for Drosha enzyme to cut Pri-miRNA into Pre-miRNA -Then PremiRNA enters the cytoplasm via a protein carrier called Exportin-5 then Dicer -Enzyme cuts Pre-miRNA into ds-miRNA then Ago2 converts ds-miRNA into ssmiRNA -Then RISC protein complex binds to ss-miRNA to form fully activated miRNA -If the sequence of miRNA is completely complementary to mRNA it will cut mRNA into 2 portions and if the sequence of miRNA is partially complementary to target mRNA it will prevent translation without cutting target mRNA in a process called translational repression Q2: miRNA biogenesis in plant cell -miRNA is transcribed from a non-coding gene via RNA polymerase || -Then a stemlooped structure containing a poly A tail and a 5 cap called Pri-miRNA is formed -Then Dicer-like 1 enzyme converts Pri-miRNA into ds-miRNA then ds-miRNA enters -The cytoplasm via a protein carrier called HST1 then Ago1 converts ds-miRNA into ss- miRNA then RISC protein complex binds to ss-miRNA to form fully activated -miRNA if the sequence of miRNA is completely complementary to target mRNA it will cut miRNA into 2 portions and if the sequence of miRNA is partially complementary to target mRNA it will prevent translation without cutting mRNA in a process called translational repression Q3: Differences between miRNA biogenesis in plant and animal cell In animal cell: DGCR8 binds to Pri-miRNA once this protein is attached it permits for Drosha enzyme to cut Pri-miRNA into Pre-miRNA Pre-miRNA enters the cytoplasm via a protein carrier called Exportin-5 Dicer enzyme cuts Pre-miRNA into ds-miRNA (inside the cytoplasm ) Ago2 converts ds-miRNA into ss-miRNA In plant cell: Dicer-like 1 enzyme converts Pri-miRNA into ds-miRNA (inside the nucleus) ds-miRNA enters the cytoplasm via a protein carrier called HST1 Ago1 converts ds-miRNA into ss-miRNA Q4: short notes about Circulating miRNA -Present in blood where they located in plasma, erythrocytes, platelets, and nucleated blood cells -Plasma miRNAs are stable under extreme conditions such as low or high PH, high temperature, long-time storage, and multiple freeze-thaw cycles however, when synthetic miRNAs are added exogenously they are degraded by the high level of RNase activity in the plasma this means endogenous plasma miRNAs are protected -To prevent their degardation as miRNAs can be packaged in microparticles such as exosomes or associated with RNA-binding proteins such as Ago2 or lipoprotein complexes to prevent their degradation the presence of miRNAs in microparticles suggests that miRNAs may function as mediators of cell-cell communication Q5: sort notes about Oncomirs -miRNAs that cause cancer by down-regulating tumor supressor genes by both translational repression and mRNA destabilization mechanisms (miR21 down regulating the tumor suppressor PDCD4) Q6: TS-miRNAs -Fight cancer by down-regulating oncogenes by both translational repression and -mRNA destabilization mechanisms (miRNA let-7 down regulating the expression of MYC and RAS) Q7: Viral miRNA and role in cancer -Could regulate cellular mechanisms and viral replication (early and late stage replication) -Examples of viruses that have miRNAs HIV 1 and HSV1 (3) Fetal trauma (4) Chorioamnionitis Chorionic villus sampling Performed in the first trimester A sample of cells is taken from placental chorionic villus under ultrasound guidance then cells are cultured for chromosomal and biochemical studies Time frame: 9.5-12.5 weeks of gestation Advantages: (1) First trimester diagnosis (2) Results usually obtained in 5-7 days (3) Fetal loss rate is low Disadvantages: (1) Can’t measure AFP levels (2) Only collect cells not fluid (3) Looks only at extraembryonic material (4) Confined placental mosaicism may be a problem Lecture 3 Q11: Types of Infectious diseases A. Bacterial infections: Bacteria is responsible for a broad variety of infectious diseases, from strep throat and urinary tract infections to meningitis and tuberculosis. Many skin rashes are also caused by bacteria. B. Bone infections: An infection that affects a bone is referred to as osteomyelitis. It is most commonly caused by bacteria that enter the body through a deep wound or surgical incision, but fungi may also be to blame for a bone infection. C. Fungal infections: There are roughly 300 types of fungi known to cause infectious diseases. Commonly occurring fungal infections include ringworm, pneumocystis pneumonia, blastomycosis, and histoplasmosis. D. Joint infections: Bacterial joint inflammation, or bacterial/septic arthritis, most frequently affects the knee joint. Most cases are caused by the bacterium Staphylococcus aureus (staph), although viral or fungal infections can also cause joint infections E. Tropical diseases: Experienced largely by the world’s poorest populations, the most common tropical diseases include elephantiasis (lymphatic filariasis), river blindness (onchocerciasis), snail fever (schistosomiasis or bilharzia), and trachoma. F. Viral infections: Like bacteria, viruses can cause many different infectious diseases like the Flu, Mononucleosis, Smallpox, Ebola, COVID-19 and HIV/AIDS. G. Parasitic infections: The majority of parasitic infections affect countries in the tropics and subtropics. Prevalent parasitic infections include Malaria, Chagas disease, and Toxocariasis. Q12: The action of pathogen in infectious process  The pathogenicity of pathogen is related to : 1. Invasiveness. 2. Virulence. 3. Number of pathogen. 4. Mutation (variability). Q13: The Epidemic process and epidemic factors of infectious disease  Source of infection (Patients or Infected animal).  Route of transmission. Contact transmission (direct and indirect), Air-borne, Food water fly borne, Insects borne, Blood borne, Soil borne.  Susceptibility of population.  Factors of influencing epidemic process (nature factors or social factors). Q14: Specimen Selection, Collection, and Processing The quantity material must be adequate Specimens are selected on the basis of signs and symptoms, should be representative of the disease process Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful. Meaningful specimens to diagnose bacterial infections must be secured before antimicrobial drugs are administered. Q15: Biochemical Reaction "Memorize 4 or 5" Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface H2S production: Depends on production H2S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Fibrinogen: This protein is a precursor to fibrin, which is an essential part of a blood clot. Decreased fibrinogen results in an increased bleeding tendency. D-dimer: this is a product of clot breakdown, and is increased in conditions of increased clotting activity in the body. Urinalysis A urinalysis is a test of your urine. A urinalysis is used to detect and manage a wide range of disorders, such as urinary tract infections, kidney disease and diabetes. Many disorders may be detected in their early stages by identifying substances that are not normally present in the urine and/or by measuring abnormal levels of certain substances. Some examples include glucose, protein, bilirubin, red blood cells, white blood cells, crystals, and bacteria. A complete urinalysis consists of three distinct testing phases: Visual examination, which evaluates the urine's color and clarity Chemical examination, which tests chemically for about 9 substances that provide valuable information about health and disease and determines the concentration of the urine Microscopic examination, which identifies and counts the type of cells, casts, crystals, and other components such as bacteria and mucus that can be present in urine Types of urine crystals Uric acid crystals Calcium oxalate crystals Q16: Parasitology Unit Semen Analysis Sperm count can be important if you’re trying to conceive a child. An abnormal sperm count may also indicate an underlying health condition. A normal sperm count ranges from 15 million sperm to more than 200 million sperm per milliliter (mL) of semen. Anything less than 15 million sperm per milliliter, or 39 million sperm per ejaculate, is considered low. A low sperm count is often referred to as oligospermia. A high, or above average, sperm count is over 200 million sperm per millimeter. Collection instructions and methods Semen collection include masturbation, condom collection, and epididymal extraction. The sample should never be obtained through coitus interruptus as some portion of the ejaculate could be lost, bacterial contamination could occur, or the acidic vaginal pH could be detrimental for sperm motility. The optimal sexual abstinence for semen sampling is 2 to 7 days. The most common way to obtain a semen sample is through masturbation without chemical substances such as lubricants and the best place to obtain it is in the clinic where the analysis will take place in order to avoid temperature changes during the transportation. Once the sample is obtained, it must be put directly into a sterile plastic receptacle. Sperms Analysis Motility The motility of sperm are divided into four different grades: Grade a: Sperm with progressive motility. These are the strongest and swim fast in a straight line. Grade b: (non-linear motility): These also move forward but tend to travel in a curved or crooked motion. Grade c: These have non-progressive motility because they do not move forward despite the fact that they move their tails. Grade d: These are immotile and fail to move at all. Sperms Morphology Sperms Abnormalities Aspermia: absence of semen. Azoospermia: absence of sperm. Hypospermia: low semen volume. Hyperspermia: high semen volume. Oligozoospermia: Very low sperm count. Postcoital test The postcoital test (PCT) is a test in the evaluation of infertility. The test examines interaction between sperm and mucus of the cervix. The PCT examines sperm survival in cervical mucus and determines whether sperm are migrating into the female reproductive system. It does not predict whether pregnancy can occur. Collection instructions and methods The PCT is scheduled close to ovulation when mucus is abundant, and the infertile couple is asked to have sexual intercourse, preferably in early hours of morning. Several hours later (usually 2), the woman is examined by the physician. The mucus is aspirated from cervical canal and spread on a glass slide. Smear from posterior fornix is used as control. 10-50 motile sperms per high power field are considered normal. Rotatory or shaky motion of sperms indicates presence of Sandwich ELISA Direct: antibody coated wells are present then sample containing the tested antigen is added then antibody conjugated with enzyme that binds to the tested antigen is added then a colorless substrate is added it changes color upon reaction with conjugated enzyme then spectrometer is used for color strength Indirect: antibody coated wells are present then sample containing the tested antigen is added then primary antibody that binds to the antigen is added then secondary antibody conjugated with enzyme is added then a colorless substrate is added it changes color upon reaction with conjugated enzyme then spectrometer is used for color strength Competitive ELISA The sample antigen competes with refrence antigen for binding to a specific amount of labeled antibodies as refrence antigen coated wells are present and the sample containing the tested antigen is incubated with a specific amount of labeled antibodies then added to the wells depending on the amount of the antigen in the sample the more or less free antibodies will be available to bind to the refrence antigen so that the more the antigen in the sample the less refrence antigen will be detected and the weaker the signal Q21: Give short notes about Westernblotting Add SDS-treated protein mixture to the well of the gel then electrophorese in SDSPolyacrylamide gel to separate proteins according to their molecular weight then proteins in the gel are transferred into a sheet of nylon or nitrocellulose by electrotransfer then blocking then primary antibody is added then secondary antibody conjugated with enzyme is added then the position of interest is visualized by the mean of ELISA Q22: Compare between following Immunohistochemistry A technique allows the visualization of a specific antigen in a sample of fixed tissue sections by binding a primary antibody to the antigen then binding secondary antibody conjugated with enzyme to the primary antibody this enzyme gives a color reaction when reacts with a colorless substrate Immunocytochemistry A technique allows the visualization of a specific antigen in a sample of fixed cells by binding of primary antibody that binds to the specific antigen then binding of secondary antibody conjugated with enzyme to the primary antibody this enzyme gives a color reaction when reacts with a colorless substrate Immunofluorescence Is a technique allows the visuallization of a specific antigen in cells under fluorescence microscope by bidning of antibody conjugated with fluorophore such as fluorescein isothiocyanate that makes it glows apple-green under ultraviolet color Lecture 6 Q23: Case Study "V.I.P" RT-PCR equations RQ = 2 -∆∆ CT ∆ CT(Cont. Sample) = CT(TG) - CT(RG) ∆ CT(Target Sample) = CT(TG) - CT(RG) ∆ ∆ CT = ∆ CT(TG) - ∆ CT(RG) RQ > 1 upregulated RQ < 1 downregulated Q24: Give short notes about Advantages of following Melting curve (1) To detect primer dimer (2) To detect genomic DNA contamination (3) To detect handling problems Trouble shooting