The Pickman report part 2

Methods: The following methods were used for the analyses of the blood sample [translators note: most of this seems to be taken from biochemistry laboratory modern theory and techniques by Rodney F Boyer and makes up the bulk of the report. So I will try and keep it short]
Paper and Thin-Layer Chromatography [used for Basic metabolic panel]
 Because of the similarities in the theory and practice of these two procedures, they will be considered together. Both are examples of partition chromatography. In paper chromatography, the cellulose support is extensively hydrated, so distribution of the analyte occurs between the immobilized water (sorbent) and the mobile developing solvent. The initial stationary liquid phase in thinlayer chromatography (TLC) is the solvent used to prepare the thin layer of adsorbent. However, as developing solvent molecules move through the sorbent, polar solvent molecules may bind to the immobilized support and become the sorbent. Preparation of the Sorbent The support medium may be a sheet of cellulose or a glass or plastic plate covered with a thin coating of silica gel, alumina, or cellulose. Large sheets of cellulose chromatography paper are available in different porosities. These may be cut to the appropriate size and used without further treatment. The paper should never be handled with bare fingers. Although thin-layer plates can easily be prepared, it is much more convenient to purchase ready-made plates. These are available in a variety of sizes, materials, and thicknesses of stationary support. They are relatively inexpensive and have a more uniform support thickness than handmade plates. Figure 5.2 outlines the application procedure. The sample to be analyzed is usually dissolved in a volatile solvent. A very small drop of solution is spotted onto the plate with a disposable microcapillary pipet and allowed to dry; then the spotting process is repeated by superimposing more drops on the original spot. The exact amount of sample applied is critical. There must be enough sample so the developed spots can be detected, but overloading will lead to “tailing” and lack of resolution. Finding the proper sample size is a matter of trial and error. It is usually recommended that two or three spots of different concentrations be applied for each sample tested. Spots should be applied along a very faint line drawn with a pencil and ruler. TLC plates should not be heavily scratched or marked. Identifying marks may be made on the top of the chromatogram, where solvent does not reach.

A wide selection of solvent systems is available in the biochemical literature. If a new solvent system must be developed, a preliminary analysis must be done on the sample with a series of solvents. Solvents can be rapidly screened by developing several small chromatograms in small sealed bottles containing the solvents. For the actual analysis, the sample should be run on a larger plate with appropriate standards in a development chamber (Figure 5.3). The chamber must be airtight and saturated with solvent vapors. Filter paper on two sides of the chamber, as shown in Figure 5.3, enhances vaporization of the solvent. Paper chromatograms may be developed in either of two types of arrangements—ascending or descending solvent flow. Descending solvent flow leads to faster development because of assistance by gravity, and it can offer better resolution for compounds with small values because the solvent can be allowed to run off the paper. Values cannot be determined under these conditions, but it is useful for qualitative separations. Two-dimensional chromatography is used for especially difficult separations. The chromatogram is developed in one direction by a solvent system; air dried, turned 90 and developed in a second solvent system.


Detection and Measurement of Components
Unless the components in the sample are colored, their location on a chromatogram will not be obvious after solvent development. Several methods can be used to locate the spots, including fluorescence, radioactivity, and treatment with chemicals that develop colors. Substances that are highly conjugated may be detected by fluorescence under a UV lamp. Chromatograms may be treated with different types of reagents to develop a color. Universal reagents produce a colored spot with any organic compound. When a solvent-developed plate is sprayed with concentrated H_2SO_4 and heated at 100 for a few minutes, all organic substances appear as black spots. Amore convenient universal reagent is I_2 the solvent-developed chromatogram is placed in an enclosed chamber containing a few crystals of I_2 the I_2 vapor reacts with most organic substances on the plate to produce brown spots. The spots are more intense with unsaturated compounds. Specific reagents react with a particular class of compound. For example, rhodamine B is often used for visualization of lipids, ninhydrin for amino acids, and aniline phthalate for carbohydrates. The position of each component of a mixture is quantified by calculating the distance traveled by the component relative to the distance traveled by the solvent. This is called relative mobility and symbolized by In Figure 5.2D, the R_f values for components B and C are calculated. The R_f for a substance is a constant for a certain set of experimental conditions. However, it varies with solvent, type of stationary support (paper, alumina, silica gel), temperature, humidity, and other environmental factors. Values are always reported along with solvent and temperature.

Applications of Planar Chromatography
Thin-layer chromatography is now more widely used than paper chromatography. In addition to its greater resolving power, TLC is faster and plates are available with several sorbents (cellulose, alumina, silica gel). Partition chromatography as described in this section may be applied to two major types of problems: (1) identification of unknown samples, and (2) isolation of the components of a mixture. The first application is, by far, the more widely used. Paper chromatography and TLC require only a minute sample size, the analysis is fast and inexpensive, and detection is straightforward. Unknown samples are applied to a plate along with appropriate standards, and the chromatogram is developed as a single experiment. In this way, any changes in experimental conditions (temperature, humidity, etc.) affect standards and unknowns to the same extent. It is then possible to compare the values directly. Purified substances can be isolated from developed chromatograms; however, only tiny amounts are present. In paper chromatography, the spot may be cut out with scissors and the piece of paper extracted with an appropriate solvent. Isolation of a substance from a TLC plate is accomplished by scraping the solid support from the region of the spot with a knife edge or razor blade and extracting the sorbent with a solvent. “Preparative” thin-layer plates with a thick coating of sorbent (up to 2 mm) are especially useful because they have higher sample capacity.

SDS-PAGE
Sample preparation
Samples may be any material containing proteins, for example prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure. Cells may also be broken open by one of the above mechanical methods.
In the case of tissues or cells, a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis.
The sample to be analyzed is mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Heating the samples to at least 60°C further promotes protein denaturation, helping SDS to bind.
A tracking dye may be added to the protein solution. This typically has a higher electrophoretic mobility than the proteins to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.

Preparing acrylamide gels
The gels typically consist of acrylamide, bisacrylamide, SDS, and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel after it is poured, as butanol removes bubbles and makes the surface smooth. A source of free radicals and a stabilizer such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction results in a gel because of the added bisacrylamide, generally about 1 part in 35 relative to acrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of acrylamide to bisacrylamide can be varied for special purposes. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight proteins, while much higher percentages are needed to resolve smaller proteins. Determining how much of the various solutions to mix together to make gels of particular acrylamide concentration is possible.
Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Electrophoresis
Various buffer systems are used in SDS-PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
An electric field is applied across the gel, causing the negatively charged proteins to migrate across the gel towards the positive (+) electrode (anode). Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance). After a set amount of time (usually a few hours, though this depends on the voltage applied across the gel; protein migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages) the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Proteins may therefore be separated roughly according to size (and thus molecular weight); however certain glycoproteins behave anomalously on SDS gels.
Polymerase chain reaction
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
•	Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.
•	Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
•	Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.
•	Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
•	Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
•	Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
Northern blot
A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.
A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction, thus preventing RNA degradation by high temperatures. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by microarrays or RT-PCR.
Gels
The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure. The gels can be stained with ethidium bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. Polyacrylamide gel electrophoeresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. An RNA ladder is often run alongside the samples on an electrophoresis gel to observe the size of fragments obtained but in total RNA samples the ribosomal subunits can act as size markers. Since the large ribosomal subunit is 28S (approximately 5kb) and the small ribosomal subunit is 18S (approximately 2kb) two prominent bands will appear on the gel, the larger at close to twice the intensity of the smaller.
Probes
Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence. RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise. Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot. The probes need to be labelled either with radioactive isotopes (32P) or with chemiluminescence in which alkaline phosphatase or horseradish peroxidase break down chemiluminescent substrates producing a detectable emission of light.  The chemiluminescent labelling can occur in two ways: either the probe is attached to the enzyme, or the probe is labelled with a ligand (e.g. biotin) for which the antibody (e.g. avidin or streptavidin) is attached to the enzyme.  X-ray film can detect both the radioactive and chemiluminescent signals and many researchers prefer the chemiluminescent signals because they are faster, more sensitive, and reduce the health hazards that go along with radioactive labels. The same membrane can be probed up to five times without a significant loss of the target RNA.
Blood culture
A minimum of 10 ml of blood is taken through venipuncture and injected into two or more "blood bottles" with specific media for aerobic and anaerobic organisms. A common media used for anaerobes is thioglycollate broth. The blood is collected using aseptic technique. This requires that both the tops of the culture bottles and the venipuncture site of the patient are cleaned prior to collection with swabs 70% isopropyl alcohol.
To maximise the diagnostic yield of blood cultures multiple sets of cultures (each set consisting of aerobic & anaerobic vials filled with 3–10 mL) may be ordered by medical staff. A common protocol used in US hospitals includes the following:
•	Set 1 = L. antecubital fossa at 0 minutes
•	Set 2 = R. antecubital fossa at 30 minutes
•	Set 3 = L. or R. antecubital fossa at 90 minutes
Ordering multiple sets of cultures increases the probability of discovering a pathogenic organism in the blood and reduces the probability of having a positive culture due to skin contaminants.
After inoculating the culture vials, advisably with new needles and not the ones used for venepuncture, they are sent to the clinical pathology microbiology department. Here the bottles are entered into a blood culture machine, which incubate the specimens at body temperature. The blood culture instrument reports positive blood cultures (cultures with bacteria present, thus indicating the patient is "bacteremic"). Most cultures are monitored for 5 days after which negative vials are removed.
If a vial is positive, a microbiologist will perform a Gram Stain on the blood for a rapid, general ID of the bacteria, which they will report to the attending physician of the bacteremic patient. The blood is also subcultured or "subbed" onto agar plates to isolate the pathogenic organism for culture and suceptibility testing, which takes up to 3 days. This culture & sensitivity (C&S) process identifies the species of bacteria. Antibiotic sensitivities are then assessed on the bacterial isolate to inform clinicians on appropriate antibiotics for treatment. Some guidelines for infective endocarditis recommend taking up to 6 sets of blood for culture (around 60 ml).
Results:[translators note: the graphs have since been confiscated by employer so I am unable to include them, sorry about that I will try and summarize the data as best as I can]
Elevated levels of following hormones:
Testosterone 2000
Estradiol 25 ng/dL
Aldosterone 37 ng/dL
Elevated levels of the following excitatory neurotransmitters:
norepinephrine
epinephrine
histamine
Red blood cells:
Hemoglobin 200 g/L
Hematocrit .85
White blood cells:
White cell count- 25*10^9/L
High concentration of 7 unknown organic compounds (speculation in the Discussion)
 4 unknown types of RNA were found in the sample indicating the presences of an unknown species of viruses
4 unidentified chromosomes (new configurations: named the k configuration by the lab staff)
Discussion:
	The results of the Biochemical analysis have proven to be quite enlightening about the unusual cellular behavior observed under the microscope. It has also raised many more questions about the circumstances under which the patient died [translators note: again there is a note at the top of the page that says “best not to ask.”] Aside from the circumstances under which the patient passed, there were a number of unusual characteristics that were detected via Thin-Layer Chromatography in particular extremely elevated levels of excitatory neurotransmitters, well beyond what would be consider fatal in ordinary humans. Our staff geneticist has hypothesized that one of the results of the new chromosomes may be a more robust nervous system (she has also expressed an interest in examining the liver and kidneys,) though she also notes that the patient was likely suffering from a form of leukemia as indicated by the high white blood cell count. Our staff endocrinologist believes that the elevated levels of the excitatory neurotransmitter epinephrine in conjunction with elevated levels of testosterone are evidence of sophisticated efforts to enhance the patient’s strength and endurance. It should be noted though that this hypothesis cannot be confirmed without examining the body or a sample of muscle tissue.
	One of the most interesting things revealed by the Biochemical analysis is the presence of unidentified organic compounds. 7 unique compounds were identified and all of them were confirmed to be alien to the human body as we understand it. While we could not identify the compounds themselves we have been able to identify compounds that were similar in structure. Again it should be noted that while the following compounds are similar to the unidentified ones in structure and composition they may not have the same effects. This is made especially relevant by the altered state of the patients DNA.
Compounds of similar composition and structure:
MDMA (3,4-methylenedioxy-N-methylamphetamine)
Psilocin (4-HO-DMT)
Phencyclidine ((1-phenylcyclohexyl)piperidine)
Lophophine (MMDPEA, 3-methoxy-4,5-methylenedioxyphenethylamine)
Benzoylmethylecgonine
Ephedrine
Bufotenin (5-HO-DMT, N,N-dimethylserotonin)
	We are unsure whether these compounds were introduced into the patients system or whether they were produced internally as part of some new organ system. We are however certain that these compounds are somehow related to the 4 extra chromosomes that were revealed by the PCR. Our staff geneticist was especially excited about this as not only do these chromosomes contain genes that have never been seen before but they were also in a new configuration, a single line with two branches near the middle (she has taken to calling this configuration a K chromosome.) She believes that these new chromosomes were introduced in order to activate a previously dormant segment of the patients “junk” DNA. It’s difficult to know exactly what the effects of these new genes would be without being able to observe a live patient, though it should be noted that the living cells in the blood sample were able to metabolize the unidentified compounds in part thanks to these new chromosomes. On a more disturbing note these same genes also allowed the living cells to alter their internal structures in response to their environment as confirmed by the Northern blot.
	The blood culture gave the most interesting results of all of the tests, as it was the only test that confirmed our suspicions about genetic manipulation. After the blood had been cultured and another Northern Blot performed, 4 unknown types of RNA were found in the sample. This indicated the presence of previously unidentified viruses which we were able to reconstruct via the smart sequencing software from the Orochi Corporation. We believe that these viruses were used to alter massive swaths of the patients DNA in a very short amount of time. The effects of this kind of massive alteration on a human would be hard to predict but given the rapid pace of the alteration, it is safe to say that they would be unpleasant at the very least. Our virologist was extraordinarily helpful in understanding the nature of these viruses; he believes that these viruses are themselves the product of genetic manipulation which is why we have been unable to identify them. However again thanks to the smart sequencing software our virologist was able to identify the “ancestors” of the unknown viruses which are as follows
Ancestor viruses:
The Rabies virus
The Coronavirus
The Human immunodeficiency virus
The Monkeypox virus
	The virus that resembles the Coronavirus also seems to be related to the virus that was isolated in another sample last month, though that particular version was not quite as virulent. This Coronavirus is particularly virulent as it was particularly difficult to deactivate and contain. It not only was able to infect any cell we introduced it to but, it was also able to turn its host cell into a kind of anti white blood cell, devouring any healthy cells that it came into contact with. It was also able to change its method of transmission depending on its environment. Meaning if it came into contact with water, it would spread via ingested water, if you removed the water it becomes airborne, and if it infected a subject it would spread via bodily fluids (blood, saliva, sexual fluids etc.)All of the mice we exposed to the virus expired in a matter of hours and all of them followed the same cycle. At first their body temperature would steadily rise, and then they began to have seizures. Next black mucus would begin to excrete from their eyes and their hair follicles and then they would die. It should also be noted that the staff needed to use an experimental plasma furnace to destroy the mice as the normal furnace was not hot enough to destroy the bodies. The blood samples were destroyed par the clients request and all materials that have come in to contact with the samples have been replaced as well (we have billed the listed account as instructed).Thus concludes our analysis of the blood sample. We strongly encourage the client to bring more samples as we believe there is much more we can learn about the anomalies encountered during this analysis, provided that we are able to examine the samples in their proper context.