iGEM 2021 — Team HKBU

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# Team:HKBU/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:HKBU/Engineering

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# Engineering

## Sclerostin: A Complicated Spaghetti Strand

We have [understood our aim:](https://2021.igem.org/Team%3AHKBU/Description) to create a sclerostin inhibitor that will both eliminate osteogenesis imperfecta (OI) *and* allow sclerostin to continue perform its functions to reduce the risk of contracting cardiovascular diseases via inflammation in the arteries. As we've seen with the example of romosozumab, we cannot take a one-size-fits-all solution and just inhibit the entire molecule without question. We must dive deeper.

First, if sclerostin has multiple functions, which parts of the protein is responsible for what function? If we can determine which component is responsible for binding to LRP-5/6 and which for atheroma/aneurysm prevention, then we would only need to target the problematic component to hit two birds with one stone.

Typically, in most proteins, there are four types of structures present:

| Type of structure | General description |
| --- | --- |
| **Primary[1]** | * Refers to the sequence of amino acids that make up the protein (in humans, 20 of such standard L-α-amino acids are used for protein synthesis) * Side chains present on amino acids give rise to different chemical, physical, and structural properties to proteins |
| **Secondary[2]** | * refers to the  *distinct and characteristic*  local structural conformations in the protein * Dependent on hydrogen bonding, and the alignment of hydrogen bonds * Main types:   1. α-helix: right-hand coiled strand, hydrogen bonding is typically intra-strand   2. β-sheet: pairs of strands lie side by side to form a flat sheet, hydrogen bonding is typically inter-strand   3. ω-loops[3]: a loop of six or more amino acid residues and any amino acid sequence; residues that make up the beginning and end of the loop are close together in space with no intervening lengths of regular secondary structural motifs |
| **Tertiary[4]** | * Refers to the overall 3-D arrangement of the protein molecule * After the primary structure is set, proteins will bend and twist to achieve the maximum stability *or* lowest energy state |
| **Quaternary\*[5]** *(optional)* | * Refers to how the constituent polypeptide chains in a protein (known as protein subunits) interact with each other and arrange themselves to form a larger aggregate protein complex * Stabilised by hydrogen bonding, disulphide bridges, and salt bridges |

A polypeptide strand will fold and bend under the right conditions (e.g. surrounding pH, temperature, presence of water and hydrophobic surfaces, etc.) into a protein and gain its tertiary structure.
During this process, an arbitrary number of protein loops may form between the N- and C-termini at the ends of the strand as the strand condenses in size.
Each protein loop may provide unique binding sites and thus the ability to express different properties to the protein, and each loop may be activated or disabled individually without disrupting the exhibition of other protein loops in the same protein molecule. Note that protein loops are not necessary due to the presence of ω-loops.

Sclerostin has three loops present in its secondary structure[6], illustrated below in Fig E.1:

![3D NMR structure of sclerostin.](https://static.igem.org/mediawiki/2021/6/63/T--HKBU--sclerostin_3d.png)

Fig E.1: 3D NMR structure of sclerostin[6].

## Different Functions of Sclerostin

Remember the [antibody](/Team%3AHKBU/Description#3) from before? According to Fig. E.2 below, romosozumab binds to human sclerostin via loop 2 and loop 3. Thus, it can be deduced that the loop responsible for causing OI in affected patients and the loop responsible for protection against cardiovascular diseases is loop 2 and loop 3. To assign the properties to the loops, further experiments must be performed.

![A graph showing the affinity of romosozumab to the different loops in sclerostin.](https://static.igem.org/mediawiki/2021/3/3a/T--HKBU--loop2_3_romosozumab_binding.png)

Fig E.2: A graph showing the affinity of romosozumab to the different loops in sclerostin[6]

To distinguish which loops are responsible for which function of sclerostin, we have created two main types of variants of the sclerostin molecule, one deficient in loop 3 only, and one deficient in both loop 2 and 3, and compared their affinities by administering them to OI mice with Apolipoprotein E deficiency (ApoE-/-).

![](https://static.igem.org/mediawiki/2021/1/17/T--HKBU--scl-loops.png)

Fig E.3: Diagram of sclerostin variants and their beneficial suppression effects on the cardiovascular system.

As seen from Fig E.3, the loop 2- and loop 3-deficient sclerostin had lost its suppressive effects on expression of inflammatory cytokines and chemokine in vitro, whereas the loop 3-deficient sclerostin had maintained such functions.

![](https://static.igem.org/mediawiki/2021/b/b6/T--HKBU--loop2_loop3_data.png)

Fig E.4: Demonstration of the cardiovascular effects of sclerostin and its variants.

Not only that, according to Figs E.4, the loop 3-deficient variant sclerostin and the full-length sclerostin both had similar effects in the suppression of activity in inflammatory cytokines and chemokine, and thus the progression of aortic aneurysms and atherosclerosis in mice affected with OI as well. It can thus be concluded that sclerostin's loop 2 is responsible for offering protection against cardiovascular diseases, while loop 3 is responsible for disrupting the Wnt signalling pathway, and thus causing OI.

Great! Now we have our bullseye: loop 3 in a sclerostin molecule. Now all we need is something to inhibit its effects.

## What are aptamers?

**Aptamers**  are a class of functional oligonucleotides which can bind to a wide variety of specific targets, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells, with high affinities and specificities.

They are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches, and can exist in many different types.
A useful distinction between aptamers and normal DNA/RNA strands is that while they can all encode genetic information, aptamers are developed specifically to achieve a high affinity with a specific molecule, receptor or binding site.
In a way, they are similar to the many variants of glue (e.g. epoxy, spray adhesives, white craft glue, cyanoacrylate adhesives, etc.) that are well adapted to particular situations.

Their extremely high affinities and other favourable properties, such as ease of administration, lowered costs, reduction of immunosuppressive effects upon and after injection, and many more over other forms of specific treatments (e.g. antibody treatment) make them optimal candidates for this task.
For the full list of benefits, please refer to [here.](https://2021.igem.org/Team%3AHKBU/Contribution#2)

## SELEX

To synthesise the desired aptamer for this project, our team has utilised the SELEX (**S**equential **E**volution of **L**igands by **EX**ponential enrichment) process[7] and modified the aptamer such that the product aptamer will be most suitable for this undertaking.

![General overview of the SELEX process used in this project.](https://static.igem.org/mediawiki/2021/b/b1/T--HKBU--selex.png)

Fig E.5: General overview of the SELEX process used in this project.

Below is a general overview of the process:

1. A random single-stranded DNA (ssDNA) library of random oligonucleotides is prepared.
2. Filter through the library with positive selection by removing all ssDNAs that have a low affinity to normal human sclerostin.
3. Elute the DNA strands that are now bound to the sclerostin molecule sample via a solvent.
4. Filter the remaining strands again with negative selection by removing all strands that have a high affinity to loop 3-deficient human sclerostin.
5. Elute the unbound DNA strands from the loop 3-deficient sclerostin and its bound strands in the mixture with a solvent.
6. Administer PCR amplification on the remaining strands.

![General overview of the SELEX process used in this project.](https://static.igem.org/mediawiki/2021/f/f8/T--HKBU--aptamer_data1.png)

Fig E.6: Affinity data for aptamers in the positive and negative selection rounds in SELEX cycles, and the specificity and affinity data of the Ostamer to sclerostin loop 3.

Comparing the affinities of aptamers in SELEX, the unselected library has the lowest light absorbance, signalling that lots of aptamer sequences did not bind to the positive target well. After the 10th round, the affinity to human sclerostin was mediocre, as shown by the low absorbance rate. After 20 rounds, the affinity to human sclerostin was high.

After extracting the different sequences, we found that one sequence binds to loop 3 of sclerostin while having extremely low affinities for the other loops of sclerostin, and the affinity increased as the concentration of the aptamer increased. This means that this aptamer sequence satisfies the criteria of only binding to loop 3 of sclerostin.

Wonderful! We now have our little aptamer molecule that targets sclerostin's loop 3.

## Additional Chemical Modifications

As aptamers are short oligonucleotide sequences, they are cleared quickly in the body via interactions with biomolecules and the kidneys.
As a result, the average time of oligonucleotide decay in blood ranges from several minutes to several tens of minutes depending on the oligonucleotide concentration and conformational structure.[8]

Such a short time range is unacceptable for most therapeutic applications, and thus several methods for protecting aptamers against degradation by nucleases have been developed.

For our project, we will use chemical modifications to extend the half-life of the aptamer, similar to the method used by another FDA-approved aptamer-based drug, Macugen[8].

![](https://static.igem.org/mediawiki/2021/d/da/T--HKBU--apt_mod_1.png)
![](https://static.igem.org/mediawiki/2021/3/3d/T--HKBU--apt_mod_2.png)

Fig E.7, 8: Chemical modifications added to the ostamer.

## References

[1] Sanger F (1952). "The arrangement of amino acids in proteins". In M.L. Anson; Kenneth Bailey; John T. Edsall (eds.). Advances in Protein Chemistry. 7. pp. 1–67.
[2] Linderstrøm-Lang KU (1952). Lane Medical Lectures: Proteins and Enzymes. Stanford University Press. p. 115.
[3] Leszczynski, JF; Rose, GD (1986). "Loops in globular proteins: a novel category of secondary structure". Science. 234 (4778): 849–855.
[4] Branden C. and Tooze J. "Introduction to Protein Structure" Garland Publishing, New York. 1990 and 1991.
[5] Berg JM, Tymoczko JL, Stryer L (2002). "Section 3.5: Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures". Biochemistry (5. ed., 4. print. ed.). New York, NY [u.a.]: W. H. Freeman.
[6] Veverka V et al. (2009). Characterization of the structural features and interactions of Sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. The Journal of biological chemistry. 284. 10890-900.
[7] Mallikaratchy P (2017). "Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens". Molecules. 22 (2): 215.
[8] Lakhin, A. V., Tarantul, V. Z., & Gening, L. V. (2013). Aptamers: problems, solutions and prospects. Acta naturae, 5(4), 34–43.

# HKBU iGEM 2021

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# Team:HKBU/Experiments

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# Experimental Protocols

## Studying Bone Anabolism in Mice with Osteogenesis Imperfecta (OI)

![](https://static.igem.org/mediawiki/2021/3/3a/T--HKBU--expt2.png)

Fig Ex.1: Experimental design to evaluate bone anabolism in OI mice. [unpublished]

## Studying Cardiovascular Risks in Mice with OI and Apolipoprotein E Deficiency (ApoE-/-)

![](https://static.igem.org/mediawiki/2021/4/4e/T--HKBU--expt3.png)

Fig Ex.2: Experimental design to evaluate cardiovascular risks in OI-ApoE-/- mice. [unpublished]

## Studying Bone Anabolism in Mice with OI with human sclerostin (hSOST)

![](https://static.igem.org/mediawiki/2021/9/91/T--HKBU--expt1.png)

Fig Ex.3: Experimental design to evaluate bone anabolism in OI mice with human sclerostin. [unpublished]

We have devised a number of experimental protocols for our investigation, presented as follows.

## Micro-CT analysis

Analysis of the trabecular bone at the left distal femoral metaphysis, the left proximal tibia metaphysis and the fourth lumbar vertebrae, as well as the cortical bone at the left femoral mid-shaft is performed with micro-CT[1].

Images of femur, tibia, and vertebrae will be reconstructed and calibrated at the isotropic voxel size of 12.5 and 17.5 μm, respectively[2]. Every measurement uses the same filtering and segmentation values. Using the Scanco evaluation software, regions of interest will be defined for both trabecular and cortical parameters.

**For trabecular bones**, a central region will be selected equivalent to 70% of the vertebral body height and extended from proximally to the end of the distal growth plate toward the vertebral body.
We drew freehand the trabeculae regions of interest on 100 sequential slices to ensure it will be within the endosteal envelope.
Trabecular bone parameters, including trabecular volume per total volume (Tb.BV/TV), trabecular volumetric bone mineral density (Tb.vBMD), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular structure model index (Tb.SMI) and trabecular connectivity density (Tb.conn.D) will be calculated.

**For cortical bones**, 100 slices will be measured at the exact center and at the distal 50% of femur length using the automated thresholding algorithm.
 Trabeculae in contact with cortical bone will be manually removed from the regions of interest.
Cortical bone parameters, including cortical volumetric bone mineral density (Ct.vBMD) and cortical thickness (Ct.Th) will be calculated[a].

## Bone histomorphometric analysis

After sacrificing, the left distal femoral metaphysis, the left proximal tibial metaphysis and the fifth lumbar vertebrae, as well as the left femoral mid-shaft will be fixed in 4% Paraformaldehyde for 48 h, dehydrated in an increased 10%, 20%, 30% concentrations of sucrose (dilution in 1x PBS) for 24 h in each concentration and embedded without decalcification in an optimal cutting temperature compound[3].

After embedding, the proximal region of the samples will be sectioned longitudinally, and the histomorphometric analyses of trabecular bone will be performed at the above four sites. The frozen tissue specimens will be obtained at a thickness of 5 μm with CryoStar NX50[4].

The sites will be consistent with the selected sites of Micro-CT. Fluorescence micrographs for the bone sections will be captured by a Q500MC fluorescence microscope[5]. The parameters of bone dynamic histomorphometric analysis for trabecular bone and cortical bone includes bone formation rate (BFR/BS) and mineral apposition rate (MAR).

The analysis will be performed using a professional histomorphometric analysis system[6] and the parameters will be calculated and expressed according to the ASBMR standardized nomenclature for bone histomorphometry[b].

## Mechanical tests

The fifth lumbar vertebra (Lv5) and the right femora will be used to describe the bone mechanical properties through the compression test and the three-point test by using a universal testing machine[7] with a 2.5 kN load cell.

**For compression tests**, Lv5s will be isolated from vertebral columns and constructed into a cylinder with two parallel planes (5-7 cm) before the test. The Lv5s will be positioned horizontally to the base. Load are applied constantly with displacement rate of 1 mm/min. After failure, the load vs. displacement curves will be recorded and the failure force (N) and ultimate strength (MPa) will be calculated for statistical analysis.

**For three-point tests**, femora will be loaded in the anterior-posterior direction with the span set as 5 mm. Load will be applied with a constant displacement rate of 1 mm/min at the femur mid-shaft. After failure, the load vs. displacement curves will be recorded and the failure force (N), stiffness and fracture energy (J) will be calculated for statistical analysis.

## Inflammatory cytokine and chemokine analysis

The concentration of inflammatory cytokines (IL-6, TNFα) and chemokines (MCP-1) in serum of mice were determined by ELISA kit[8] in triplicate following manufacturer’s instructions[c].

## Aortic aneurysm analysis

Immediately after sacrifice, the aortas were perfused via left ventricle with ice-cold saline, isolated from the fat and connective tissues under Zeiss Stemi 305 Stereomicroscope with AxioCam 208 Color Camera, and then fixed in 4% paraformaldehyde (PFA). The aortas with or without aneurysm formation were defined according to Daugherty's modified classification.

The incidence of AA of each group was determined as follows:

![](https://static.igem.org/mediawiki/2021/b/b2/T--HKBU--equation_1.png)

The maximum outer diameters of thoracic aorta and suprarenal aorta of each mouse were determined by Zeiss software[9; c, d].

## Atherosclerosis analysis

The atherosclerotic plaque was quantified by measuring the surface area of the Oil Red O-positive lesions on en face preparation of aortic arches. In addition, the saline-perfused upper half of the heart including the aortic root was directly embedded in an optimal cutting temperature compound[10], frozen in liquid nitrogen, and cryo-sectioned (10 μm).

The ratio of atherosclerotic plaque area to total cross cryo-section area of aortic root was examined by Oil Red O staining, and quantified by colorimetric analysis using Image J software[c, d].

## Notes and References

[1] Version 6.5, vivaCT40, SCANCO Medical AG, Bassersdorf, Switzerland.
[2] 70 kVp, 114 μA, 200 ms integration time, 260 thresholds, 1200 mg HA/cm3.
[3] Sakura Finetek, Co. Ltd., Tokyo, Japan.
[4] Thermo Fisher Scientific, Waltham, MA, USA.
[5] Leica, Bensheim, Germany.
[6] BIOQUANT OSTEO, Nashville, TN, USA.
[7] H25KS Series, Hounsfield Test Equipment Ltd, Redhill, UK.
[8] eBioscience.
[9] Carl Zeiss Far East Co., Ltd., Germany.
[10] O.C.T., Sakura Finetek, Co. Ltd., Tokyo, Japan.
[a] Zhang, Guo et al. 2012, Liang, Guo et al. 2015.
[b] Li, Liu et al. 2016.
[c] Krishna, Seto et al. 2017.
[d] Zong-Kang Zhang, 2016.

# HKBU iGEM 2021

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# Team:HKBU/Results

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# Results

*For more information on the protocols used in the experiments, please refer to [here.](https://2021.igem.org/Team%3AHKBU/Experiments) Knowledge of abbreviations stated in the experimental protocols will be assumed in this page. Other abbreviations will be defined.*

## Ostamer Affinity Evaluation

We have created our desired aptamer to solve the main focus of our project: to design a sclerostin inhibitor without sacrificing the cardiovascular properties of sclerostin.
Hence, we are to first evaluate the affinity between the Ostamer and human sclerostin to ensure that the effects of the Ostamer will be exerted.

Affinity evaluation of the Ostamer has already been completed [here.](https://2021.igem.org/Team%3AHKBU/Proof_Of_Concept#1)

## Chemical Modifications

As normal aptamers have a very short half-life when present in human serum[1], we have performed additional chemical modifications to O to increase serum stability.

Affinity comparisons between modified ostamer (O') and O has already been completed [here.](https://2021.igem.org/Team%3AHKBU/Proof_Of_Concept#2)

## Ostamer Efficacy Evaluation

Efficacy evaluation of the Ostamer has already been completed [here.](https://2021.igem.org/Team%3AHKBU/Proof_Of_Concept#3)

## Induced Cardiovascular Effects Evaluation

We have determined that the Ostamer is able to promote bone anabolism effectively and reverse the effects brought about by osteogenesis imperfecta (OI). However, we must also determine the cardiovascular risks that this promotion of bone anabolism will bring.

We have used mice affected by OI and Apolipoprotein E deficiency (ApoE-/-).

![](https://static.igem.org/mediawiki/2021/9/9a/T--HKBU--Apc_AA-5.png)

Fig R.1: Graphs comparing the serum levels of inflammatory cytokines and chemokine with different reagents in OI-ApoE-/- mice. [unpublished data]

Angiotensin II (AngII) is a naturally-occuring hormone that acts on the central nervous system to increase vasopressin production, and also acts on venous and arterial smooth muscle to cause vasoconstriction[2]. The presence of this hormone will lead to a higher blood pressure due to vasoconstriction, and this feature is used in medications that treats hypotension resulting from septic shock or other distributive shock[3].
Synthetic AngII (identical to its natural counterpart) is used to allow the cardiovascular effects of the reagents used to be exaggerated, allowing for better comparisons against the conditions present in normal blood.

Examining the effects of O' against the antibody, from these three graphs, it can be determines that cytokine and chemokine activity induced by the introduction of the antibody is at around 2.5 to 3.2 times that amount induced by the introduction of O', which is about the same level as in normal blood.

As control, loop2 mutant sclerostin (loop2m, does not suppress cardiovascular effects, but still binds to the antibody, thereby inhibiting the antibody) was also introduced with and without the sclerostin antibody (in separate trials) and the data (along with data provided by literature[4]) above suggests that the reason behind the increased cytokine and chemokine activity was due to the interactions between the antibody and sclerostin at loop 3, re-confirming that loop 3 of sclerostin is responsible for cardiovascular risk regulation.

![](https://static.igem.org/mediawiki/2021/0/03/T--HKBU--cvr-1.png)

Fig R.2: Graphs and images displaying the progression of aortic aneurysms (AA) in OI-ApoE-/- mice under different infusions. [unpublished data]

As seen from R.5, compared to the baseline (AngII+veh), the introduction of the sclerostin antibody has nearly doubled AA incidence, and that the introduction of O' decreased AA incidence by roughly 15%.

On the left, it can be seen that the size of the aortic aneurysm in the aorta with the introduction of O' is much smaller than that of the baseline. On the right, the maximum diameter of the aneurysms in the aortas when O' was introduced was roughly similar (slightly smaller) to that of the baseline, but that when the antibody was introduced were nearly double the size for the thoracic aorta, and 1.2 times the size for the suprarenal aorta.

This shows that O' slightly decreases the risk of developing aortic aneurysms in mice, while the antibody increases that risk by a huge amount.

![](https://static.igem.org/mediawiki/2021/4/45/T--HKBU--cvr-2.png)

Fig R.3: Graphs and images displaying the progression of atherosclerosis in mice under different infusions. [unpublished data]

Oil Red O stain is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections, and can make fat more visible in various cuts in pathology[5]. The staining techniques used are non-destructive (i.e. does not destroy the exhibit and does not prevent the use of other techniques) and mainly targets fat deposits on the surface of porous exhibits[6]. This will allow atheromas deposited on the aortas to be stained for easy identification.

The graphs on the right show that the amount of positive stain deposited into the atheroma tissue was double the baseline when the sclerostin antibody was introduced, while that when O' was introduced did not change.

The maximum diameter of the aortic arch in mice was slightly smaller when O' was introduced, but had increased in size when the sclerostin antibody was introduced.
The images of aortic arches agree with this, with significantly less Oil Red O stain deposited on the aortic arches for when O' was introduced than that when the sclerostin antibody was introduced.
From the images of cross-sections of the aortas, it can be clearly seen that when O' was introduced, the diameter of the aortic root was much wider than that when the sclerostin antibody was introduced.

This shows that O' slightly decreases the risk of developing atherosclerosis in mice, while the antibody increases that risk by a huge amount.

Combining the results from above, it can be concluded that the induced cardiovascular effects by the addition of the ostamer does not affect the risk of contracting cardiovascular diseases in OI-ApoE-/- mice, and the risk is maintained at a significantly lower level than that induced by the addition of the sclerostin antibody.

## Conclusions

In summary of the results and conclusions above, we have successfully created an aptamer-based sclerostin inhibitor that does not impose severe cardiovascular risk on mice, and our experiments have demonstrated that our ostamer is technically superior to the available sclerostin antibody.

![](https://static.igem.org/mediawiki/2021/6/6e/T--HKBU--fda.png)

Fig R.7: US FDA Orphan Drug Designation of our aptamer-based drug.

We have received US FDA Orphan Drug Designation, and our team is now in the process of applying for clinical trials for human testing to ensure that the bone anabolic and cardiovascular effects described above are sustained in humans.

## References

[1] Thiviyanathan, V., & Gorenstein, D. G. (2012). Aptamers and the next generation of diagnostic reagents. Proteomics. Clinical applications, 6(11-12), 563–573.
[2] Chawla LS, Busse L, Brasha-Mitchell E, Davison D, Honiq J, Alotaibi Z, Seneff MG (October 2014). "Intravenous angiotensin II for the treatment of high-output shock (ATHOS trial): a pilot study". Critical Care. 18 (5): 534.
[3] Kaufman MB (March 2018). "Pharmaceutical Approval Update". P & T. 43 (3): 141–170.
[4] Veverka V et al. (2009). Characterization of the structural features and interactions of Sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. The Journal of biological chemistry. 284. 10890-900.
[5]["Forensic Pathology".](http://library.med.utah.edu/WebPath/FORHTML/FOR002.html)
[6] Beaudoin, A. New technique for revealing latent fingerprints on wet, porous surfaces: Oil Red O. Journal of Forensic Identification, 2004, 54 (4), 413-421.

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