We wish to thank Miss Shin-Fun Wu for her help in the experiments. This study was supported by a grant from the Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) and IRB 100-4470A3, 104-2462A3, Taiwan.

The testing performed in the laboratory was carried out in accordance with the requirements of the Clinical Laboratory Improvement Amendments (CLIA) of 1988, the regulations approved by the Institutional Review Board of Chang Gung Memorial Hospital and University (License no. 100-4470A3 and 104-2462A3). Informed consent was obtained from all patients.
1. Blood Specimen Collection
<ol>
	<li>Collect whole blood into commercially available EDTA-treated tubes. Mix gently and store blood sample at 4 &#176;C until processing.</li>
</ol>
2. DNA Extraction from Peripheral Blood
Use a DNA Extraction Kit for the genomic DNA extraction (see Table of Materials).
<ol>
	<li>Add 10 mL of Solution 1 to the 4.0 mL blood sample. Incubate the sample on ice for 10 min.</li>
	<li>Centrifuge the sample at 2,000 x g for 5 min at 4 &#176;C. Remove and discard supernatant carefully. Resuspend the pellet in 3 mL of Solution 1.</li>
	<li>Centrifuge the sample at 2,000 x g for 5 min at 4 &#176;C again and discard supernatant. Resuspend the pellet in 3 mL of Solution 2.</li>
	<li>Add 10 &#181;L of pronase stock solution (225 mg/mL) to get a final concentration of 100 &#181;g/mL and mix well. Incubate at 37 &#176;C overnight.</li>
	<li>Chill the tube on ice for 10 min. Add 0.8 mL of Solution 3 to the sample and invert 3 - 5 times. Place sample on ice for 5 min.</li>
	<li>Centrifuge the sample at 3,000 x g for 15 min at 4 &#176;C. Carefully transfer the supernatant to a 15 mL sterile conical centrifuge tube.</li>
	<li>Add 6 &#181;L of RNase stock solution (10 mg/mL) to get a final concentration of 20 &#181;g/mL. Incubate at 37 &#176;C for 15 min.</li>
	<li>Add 2.5 mL of isopropyl alcohol and mix thoroughly by gently inverting several times to precipitate DNA (strands of white, flocculent material will form). Using a large-bore pipet, transfer the DNA precipitant to a new 1.5 mL centrifuge tube.</li>
	<li>Centrifuge at 12,000 x g for 3 min at 4 &#176;C. Discard supernatant carefully. Wash DNA pellet by adding 500 &#181;L of 70% ethanol.</li>
	<li>Centrifuge at 12,000 x g for 1 min and discard supernatant. Air dry DNA pellet at room temperature. Add 100 &#181;L of ddH2O and incubate at 65 &#176;C for 15 min to resuspend DNA.</li>
</ol>
3. Genomic DNA Quantification
Use a spectrophotometer (see Table of Materials) to detect the quantity and quality of the genomic DNA.
<ol>
	<li>Log into the computer attached to the spectrophotometer machine. Open the software.</li>
	<li>Initialize the spectrophotometer machine:
	<ol>
		<li>Click &#34;Nucleic Acid&#34; on the spectrophotometer software.</li>
		<li>Clean the pedestal with a disposable paper wipe and purified water.</li>
	</ol>
	</li>
	<li>Blank the spectrophotometer:
	<ol>
		<li>Load 2 &#181;L of purified water on the pedestal.</li>
		<li>Lower the upper arm of the spectrophotometer and click &#34;Blank&#34; to calibrate it.</li>
		<li>When it is done, lift the upper arm and dry the pedestal with a wipe.</li>
	</ol>
	</li>
	<li>Measure the sample:
	<ol>
		<li>Click &#34;DNA-50&#34; on Sample Type.</li>
		<li>Load 2 &#181;L of DNA sample on the pedestal.</li>
		<li>Lower the upper arm and click &#34;Measure&#34; to measure the A260/A280 ratio and the concentration of the sample.</li>
		<li>Clean the pedestal in between each run with a wipe and purified water.</li>
	</ol>
	</li>
	<li>Click &#34;Print Screen&#34; to collect the data.</li>
</ol>
4. Genetic analyses of mutations
Amplify the target DNA with the PCR4. Perform PCR in a 25 &#181;L reaction mixture using a DNA Polymerase kit (see Table of Materials). Table 1 shows the TTR gene intronic primers.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>gene</td>
			<td>Primer sequence</td>
		</tr>
		<tr>
			<td>Exon1</td>
			<td>F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217; 
			R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon2</td>
			<td>F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217; 
			R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon3</td>
			<td>F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217; 
			R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon4</td>
			<td>F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217; 
			R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1. TTR gene intronic primers. (NCBI Reference Sequence: NC_000018.10)
<ol>
	<li>Add the reagents in Table 2 to a 0.2 mL PCR tube.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>10x Buffer</td>
			<td>2.5</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1.5 mM</td>
		</tr>
		<tr>
			<td>360 GC Enhancer</td>
			<td>1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>360 DNA Polymerase</td>
			<td>0.2</td>
			<td>2 U/reaction</td>
		</tr>
		<tr>
			<td>dNTP Mix (25 mM each)</td>
			<td>2</td>
			<td>2 mM each</td>
		</tr>
		<tr>
			<td>Primer F (10 &#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M</td>
		</tr>
		<tr>
			<td>Primer R (10 &#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M</td>
		</tr>
		<tr>
			<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR-grade water</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td>25</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2. PCR reaction conditions.
<ol start="2">
	<li>Mix gently and briefly centrifuge on a benchtop centrifuge to collect all components to the bottom of the tube. Place tube in a thermocycler.</li>
	<li>Perform PCR for 30 cycles. Each cycle consists of a denaturation step for 30 s at 94 &#176;C, primer annealing for 30 s at 58 &#176;C, and polymerase extension for 45 s at 72 &#176;C (see Table 3).</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature (&#176;C)</td>
			<td>Time</td>
			<td>cycle</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>95</td>
			<td>5 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DNA denaturation step</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Primer annealing step</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Polymerase extension step</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Post elongation step</td>
			<td>72</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>End of PCR cycling</td>
			<td>4</td>
			<td>Indefinite</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3. Cycling conditions for amplifying PCR products.
<ol start="4">
	<li>Validate the PCR product by visualizing with gel electrophoresis:
	<ol>
		<li>Prepare a 1.2% agarose gel:
		<ol>
			<li>Mix 1.2 g of agarose powder with 100 mL of 0.5x TBE in a microwavable flask. Microwave for 2 min until the agarose is completely dissolved. Cool down the agarose mixture to 55 &#176;C.</li>
			<li>Add 2 &#181;L of a ethidium bromide solution (10 mg/mL) to the agarose mixture and mix gently. Pour the agarose mixture into a gel tray to about 5 - 7 mm, then add the comb(s) in place. Allow it to solidify (at least 30 min) and carefully remove comb(s).</li>
		</ol>
		</li>
		<li>Load samples and run an agarose gel:
		<ol>
			<li>Place gel and tray into the electrophoresis cell. Fill the electrophoresis cell with 0.5x TBE until the gel is covered.</li>
			<li>Carefully load 2 &#181;L of a 100 bp DNA ladder (see Table of Materials) into the one lane of the gel as molecular size marker. Load dye/sample mixture (5 &#181;L of PCR product mix with 1 &#181;L 6x&#160;loading dye) into the additional lanes.</li>
			<li>Turn on the power supply. Run the gel for 25 min at 100 V.</li>
			<li>Remove the gel from the electrophoresis cell. Visualize the DNA fragments under UV light in an imaging system.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. DNA sequencing
<ol>
	<li>Purify PCR products using a commercial kit and sequence commercially or with an automatic sequencer (see Table of Materials).</li>
	<li>Submit nucleotide sequences to the NCBI BLAST server to compare the nucleotide query to the nucleotide databases10. After the results are displayed, perform sequence alignments and identify the expected mutation.</li>
</ol>

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The authors have nothing to disclose.

There are two critical steps within the protocol. First, in order to have sufficient number of white blood cells, a hemodiluted specimen should be avoided11. Second, the use of appropriate PCR primers is fundamental to obtain reliable results12. We used the Primer-BLAST web tool to design the primers4,13; a minimum of 40 base pairs on each side of the four TTR exons should be covered. We also run BLAST on NCBI to ch...

Transthyretin (TTR) amyloidosis (ATTR) is the most common form of hereditary systemic amyloidosis1, and can be caused by an autosomal dominantly inherited mutation in the transthyretin (TTR) gene2. TTR mutations destabilize the tetrameric protein structure and lead to its dissociation into monomers that reassembles into amyloid fibrils2. More than 100 amyloidogenic TTR mutations have been reported worldwide1. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutation and have advantages including avoiding the handling of radioactively labeled probes in comparison with southern blot3. PCR is a&#160;fast, easy, cheap, and reliable technique that has been applied to numerous fields in modern sciences4.
The early diagnosis of this progressive and fatal disease is challenging given its phenotypic heterogeneity. With increasing attention and broader recognition on the early manifestations of ATTR as well as the emerging treatments5, appropriate diagnostic studies including TTR genetic test are therefore critically fundamental to improve prognosis. Furthermore, different mutations are associated with different penetrance of the trait, age of onset, patterns of progression, disease severity, median survival, efficacy of liver transplantation, or TTR stabilizers2,6, and variable degrees of neurological and cardiological involvement, which have great implications for genetic counseling7,8,9. Besides, a highly accurate genetic test is the only tool that differentiates the two distinct types of ATTR: hereditary (mutant) and wild type (non-mutant form, senile systemic amyloidosis, SSA)7. It is imperative to confirm the types of ATTR, because the therapies vary widely2. Therefore, there is an increasing necessity to describe the stepwise protocol of the TTR genetic test.
The molecular approach to detect the mutation will be illustrated using Ala97Ser, the most common endemic mutation in Taiwan, as an example. Modifications in the DNA extraction step reduce the amount of the three solutions used and yields a sufficient amount of DNA. In this protocol, all four TTR exons were analyzed, while regions including 5&#39; upstream, 3&#39; downstream, promoters, introns, and untranslated regions (UTR) were not sequenced.

<table border="0" cellpadding="0" cellspacing="0" style="width:951px;" width="951">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">EDTA-treated tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:347px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA Extraction Kit</td>
			<td style="width:199px;">Stratagen</td>
			<td style="width:347px;">200600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">NanoDrop ND2000 spectrophotometer&#160;</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:347px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Delicate Task Wipers</td>
			<td style="width:199px;">Kimberly-Clark</td>
			<td style="width:347px;">Kimtech Science Kimwipes</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">AmpliTaq Gold 360 DNA Polymerase kit</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">4398823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">TTR gene intronic primers&#160;</td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">thermocycler</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">electrophoresis cell&#160;</td>
			<td style="width:199px;">ADVANCE</td>
			<td style="width:347px;">Mupid-2plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA ladder</td>
			<td style="width:199px;">Protech</td>
			<td style="width:347px;">PT-M1-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">dye</td>
			<td style="width:199px;">BioLabs</td>
			<td style="width:347px;">B7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">AlphaImager EC</td>
			<td style="width:199px;">Alpha Innotech</td>
			<td style="width:347px;">AlphaImager EC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">automatic sequencer&#160;</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">3730xl DNA Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin amyloidosis (ATTR). ATTR is a rare but fatal disease with heterogeneous phenotypes; therefore, the diagnosis is sometimes delayed. With increasing attention and broader recognition on early manifestations of ATTR as well as emerging treatments, appropriate diagnostic studies, including the transthyretin (TTR) genetic test, to confirm the types and variants of ATTR are therefore fundamental to improve the prognosis. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutations much more quickly and safer than conventional methods such as southern blot. Herein, we demonstrate genetic confirmation of the ATTR Ala97Ser mutation, the most common endemic mutation in Taiwan. The protocol comprises four main steps: collecting whole blood specimen, DNA extraction, genetic analysis of all four TTR exons with PCR, and DNA sequencing.

Agarose gel electrophoresis of two patients and one healthy individual revealed bands of the expected sizes, including a 454 bp PCR product for exon 4 of the TTR gene (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg" /> 
Figure 1. Gel electrophoresis depicting PCR amplified TTR gene. Normal: a healthy indiv...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

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JoVE Journal

Medicine

7.6K Views.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

Video: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...