The authors are grateful to Joachim Theussl (Center of Medical Research, Medical University of Graz) for the excellent technical assistance in the video production and to Monica Farrell (Research Management, Medical University of Graz) as voice talent.

Protocol and video shooting were approved by the institutional review board of the Medical University of Graz (Votum 27-126ex14/15). Written consent for this video publication was obtained from the child and her parent.
1. Instruments and Materials
<ol>
	<li>Cut calibrated (ISO 015/02) and sterile paper points, usually used in endodontic therapy, at the first ring mark in order to standardize their length (Figure 1).</li>
	<li>Apply UV light irradiation at 260 nm for 30 min in order to avoid DNA and RNA contamination of the paper points (Figure 2).</li>
	<li>Autoclave the tubes for storage at 120 &#176;C and 1.2 bar for 20 min and UV-irradiate them as well or employ ready-to-use gamma irradiated tubes.</li>
</ol>
2. Preparation of Subjects
Note: Written informed consent was obtained from the child and her parent prior to enrollment.
<ol>
	<li>Apply cocoa butter to the lips.</li>
	<li>Mount the cheek and tongue retractor for full access to both dental arches.</li>
	<li>Stain the dental plaque with plaque disclosure.</li>
	<li>Remove the dry field apparatus.</li>
	<li>Rinse the mouth with water until the water is colorless.</li>
	<li>Brush teeth thoroughly with an electric tooth brush at 45 &#176; angulation. Apply water only but no toothpaste, as this would alter the oral biofilm.</li>
	<li>Mount the dry field apparatus again, including the tongue guard to keep the mouth open and dry.</li>
	<li>Clean and dry the index teeth with sterile cotton swabs to avoid absorption of supragingival fluid during paper point sampling.</li>
</ol>
3. Biofilm Sampling
<ol>
	<li>Paper point sampling of the subgingival sulcus
	<ol>
		<li>Grasp the paper points with sterile dental tweezers.</li>
		<li>Insert paper points tangentially up to a defined length of 4 mm. Take special care not to traumatize the junctional epithelium (Figure 3).</li>
		<li>Remove paper points after 20 s.</li>
		<li>Collect paper points directly into prepared tubes: place the paper point tip directly in a sterile and DNA-free vial, and cut it at the third mark to a standardized length of 4 mm (Figure 4).</li>
		<li>If indicated by the study protocols, insert two paper points parallel and simultaneously at the same site (Figure 5A).</li>
		<li>Alternatively, collect paper points from several sites if the study protocol requires &#34;pooled samples&#34; (Figure 5B).</li>
		<li>Remove the dry field apparatus for mucosal and saliva sampling.</li>
	</ol>
	</li>
	<li>Mucosal paper point sampling
	<ol>
		<li>Apply paper points to the vestibular fold of the upper cheek (Figure 6).</li>
		<li>Close the cheek and massage it briefly.</li>
		<li>Open the cheek and remove the paper points after 20 s.</li>
		<li>Cut paper points at the third mark and place them in a sterile tube as indicated for subgingival sampling.</li>
	</ol>
	</li>
	<li>Saliva sampling
	<ol>
		<li>Let the child spit unstimulated saliva into a sterilized collection vessel (Figure 7).</li>
	</ol>
	</li>
</ol>
4. Transfer and Storage
<ol>
	<li>Collect paper points as single, pooled, or parallel samples depending on the study design (Figure 8).</li>
	<li>Apply a color-coded storage system to facilitate further sample management (Figure 9).</li>
	<li>Finally, store the samples at &#8722;80 &#176;C pending microbiome analysis.</li>
</ol>

<ol>
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The authors report no conflicts of interest related to this scientific video production.

Bacteria are found at all sites within the oral cavity24,25,26. Many studies have focused on the use of saliva as a sampling medium to map oral colonization as it can easily be sampled through spitting27,28,29,30,31,32. However, salivary biofilm does not reflect subgingival biofilm composition. Thus, the use of other sampling methods is crucial for creating the whole picture, and will evoke new discussions in dental research.Several articles compare the use of curettes and paper points for subgingival sampling of periodontally diseased subjects8,33,34. Yet no research group is known to have focused on the application of standardized sampling devices for different age groups, especially for children19.
In this video, oral biofilm sampling of three oral habitats in healthy children is presented.
Modifications and Troubleshooting:
The aim of the manuscript focuses on the clinical protocol for subgingival biofilm sampling of healthy children. The method of choice refers to sound subgingival sulci where limited space makes sampling especially challenging. In this video the method was applied to early permanent dentition. It can also be applied to mixed or mature dentition, and to children and/or adults. Our sampling method allows for modifications such as single or pooled samples. The latter might become a crucial factor for molecular analyses due to the small amount of DNA collectable from the healthy subgingival sulcus. The choice of the index teeth may be modified on demand; in particular, another tooth may be sampled as an alternative when the epithelium is traumatized and bleeding, thus making the paper point unusable. Parallel sampling using two paper points simultaneously at one site renders sample replicates in one step, in addition to shortening the chair time for children.
With slight modifications diseased periodontal pockets can be sampled. Here paper points will need to be inserted to the full length of the pocket, which can reach up to 10 mm. The length and conus of paper points as well as the depth of insertion must be adapted to the intraoral habitat of interest, but should always be standardized by material and dimensions. Samples can also be taken from the mucosa using paper points. Identical protocols allow comparisons of different oral habitats and various dental materials. Patient preparation and sample storage can be performed as described in this video. For metatranscriptome research, RNA could be sampled with the same method. Thus, after sampling, the paper points should be inserted directly in RNA stabilization solution.
Limitations of the Technique:
Regardless of modifications, the clinical sampling method we describe is limited to the sound subgingival sulcus. Other sampling sites, as for example periodontally diseased pockets, were not part of our study design. As sampling to the full depth of such pockets is needed, paper points might not be large enough to collect samples sufficiently. If the experimental goal is to compare data sampled from sound subgingival sulci and periodontally diseased pockets, it is important to be aware of the inherent bias that is associated with different clinical sampling methods. It therefore is not advisable to compare such data.
A limitation of the method presented here is sampling without the subsequent use of propidium monoazide. Adding this agent directly after sampling allows for specific analysis of only the living cells in the biofilm analyzed. The sampling method described here reflects numbers of living and dead cells. For research of severe periodontitis, paper points will probably need to be replaced by curettes, as biofilm formation in deep pockets is strong. Paper point sampling might not reflect the entire microbial spectrum in these cases, as their surface might be saturated too quickly.
Significance with Respect to Existing Methods:
The proposed manuscript is the first of its kind to standardize subgingival sampling in the healthy sulcus with paper points. Other reports have referred to the use of metal curettes35,36 or paper points 37,38,39, but did not describe the processes that take place prior to sampling, such as plaque control, tooth cleaning, tooth isolation, and drying, as well as the processes that result from inadequate specifications on the sampling technique and time lines.
In two previous articles, we show that the use of paper points is a reproducible method for subgingival biofilm sampling in children22,40. Due to their design, curettes are too large for the shallow sulcus with limited space and too sharp for the tender junctional epithelium. It is crucial to access the subgingival sulcus without traumatizing the epithelium. In this way, bias arising from bleeding is avoided. Thus, the use of the slim and tender paper points is preferred for this area. Paper points are functional to monitor changes in biofilm composition during orthodontic treatment21,41. They are slim and flexible enough to fit between the elements of fixed orthodontic appliances. This is a major advantage to other sampling methods like curettes. Atraumatic sampling is simplified and sampling of small amounts of DNA is possible.
Future Applications:
In this video we demonstrated the method on non-diseased, early, permanent dentition. It also can be applied to mixed or mature dentition. With some adaptations it is possible to sample periodontally diseased sites like teeth or implants and other niches due to dental materials by following the same protocol. Caries research could benefit from this standardized protocol, in particular studies on root caries. The most important lecture from our sampling video is standardization of the protocols to make data comparable, no matter what and how samples are measured. Future video demonstrations could enhance the field of clinical sampling in general.
Oral biofilm obtained as shown in the video is used in clinics and for scientific investigations. This in vivo approach represents a good addition to in vitro molecular techniques such as fluorescence in situ hybridization and confocal laser scanning microscopy15. NGS methods, as the pyrosequencing shown here, applied to the collected biofilm, allow the analysis of the complete microbiota. Calculating the relative abundances of certain bacterial species can be used to support treatments or to compare different patients or different treatments. Together with a standardized sequencing protocol and sequence analysis workflow that we have previously described40, this sampling method allows sequence analysis down to the genus level. Further &#34;meta&#34;-studies such as metatranscriptomic or metabolomic&#160;studies are possible based on the sampling protocol presented in this video.
Critical Steps:
Avoiding contamination is a critical step: sterile instruments have to be applied in a non-sterile in vivo environment. The transfer from the mouth to the bench has to be performed quickly and securely by an experienced examiner to keep chair time for study participants as short as possible. The most critical step in the protocol is the atraumatic insertion of the paper point into the subgingival sulcus. Disruption of the junctional epithelium and bleeding absolutely have to be avoided. Further care has to be taken in order to not contaminate paper points and storage vials with exogenous DNA or RNA. The storage itself then also is a critical step. Samples need to be frozen directly, at best at &#8722;80 &#176;C. This avoids changes in bacterial composition post sampling, which would falsify sequencing results.

Human oral biofilm comprises a broad community consisting mostly of commensals and beneficial microorganisms1,2,3. Species found here colonize all niches that the oral cavity offers4,5,6. Biofilm composition in these niches varies as widely as the habitats. Saliva for example displays different bacterial profiles than plaque samples. In plaque samples of healthy adults, the relative abundance of Actinobacteria is over 20%, while less than 7% is found in saliva. Bacteroidetes and Firmicutes in contrast appear in much higher numbers in saliva7. Thus, it is important to sample at different locations in the mouth in order to get the whole picture. Additionally, various factors such as geographic and ethnic differences, age, sex, and many other factors make it difficult to identify general rules for biofilm development and disease onset8,9. For many years the investigation of periopathogenic and cariogenic biofilm has been a central concern8,10,11,12,13.
In recent years, research on healthy subjects has gained importance not only for a broader understanding of disease but also for the implementation of preventive measures14. New technologies and molecular analyses further elucidate oral biofilm formation and function15,16, and enable the complete profiling of microbial diversity17,18. This is expected to also lead to a new understanding of microbial changes during orthodontic therapy19. An impact on the development of orthodontic biomaterials is foreseeable. The enhanced perspective will shed new light on complications of orthodontic therapy associated with biofilm, such as enamel demineralization and periodontal diseases12,20,21. To permit worldwide data comparisons, it is crucial to standardize all steps in data generation. Minor changes in laboratory procedures can strongly influence the results. In addition, the use of varying computational platforms in data processing can lead to non-comparable datasets. Lastly, the choice of statistical tests and corrections has an influence on the results. However, sampling bias can already occur long before any lab work or bio-computing commences. Non-standardized sampling methods lead to an inherently biased study. In view of low to moderate levels of standardization in the relevant studies, little evidence exists on the relationship between orthodontics and microbiomes19. Therefore, the development of standardized methods would facilitate qualified comparison of data in the field.
This article presents standardized oral biofilm sampling procedures. The protocol is intended to contribute towards generating globally comparable collections of microbial sequence data. A step-by-step protocol for oral biofilm sampling in healthy children is presented. As the method of choice, paper points are inserted atraumatically into the sound subgingival sulcus. To facilitate habitat comparisons, cheek mucosa is sampled according to the same protocol. This article additionally demonstrates parallel and pooled sampling. Furthermore, saliva sampling is shown. A simple color-coded transport and storage system is also presented, facilitating specimen management for further processing. The choice of downstream operations regarding storage medium, metagenomic analyses, and bioinformatics depends on the clinical questions raised by different fields in oral research. In this manuscript, DNA sampling for NGS has been chosen as an example of a possible application for orthodontic research.

<table border="0" cellpadding="0" cellspacing="0" width="1545">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Paper Points Taper .02, sterilised</td>
			<td style="width:247px;">VDW Dental</td>
			<td style="width:119px;">550 029 015</td>
			<td style="width:947px;">http://www.vdw-dental.com/en/products/root-canal-drying/paper-points.html?seminarNo=24&#38;cHash= 
			1107d79ab05653b454fd90a954dc92e2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC Cocoa Butter, Tube of 10 g</td>
			<td style="width:247px;">GC EUROPE N.V.</td>
			<td style="width:119px;">000387</td>
			<td>http://www.gceurope.com/products/detail.php?id=22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Rondells blue</td>
			<td style="width:247px;">DIRECTA AB</td>
			<td>Rondell Tablets</td>
			<td>http://www.directadental.com/exego.aspx?p_id=767</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Great Lakes Nola Dry Field System</td>
			<td style="width:247px;">Great Lakes Ortho</td>
			<td>300-401</td>
			<td>http://www.greatlakesortho.com/commerce/detail/?nPID=1626</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">tooth brush</td>
			<td style="width:247px;">Oral-B</td>
			<td style="width:119px;"></td>
			<td>http://www.oralb-blendamed.de/de-DE/pro</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">micro-Ident plus11</td>
			<td style="width:247px;">Hain Lifescience GmbH</td>
			<td>23312</td>
			<td>http://www.hain-lifescience.de/en/products/microbiology/dental-diagnostics/micro-ident-und-micro-identplus.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">ParoCheck</td>
			<td style="width:247px;">Greiner Bio One International GmbH</td>
			<td>460020</td>
			<td>https://shop.gbo.com/en/row/articles/catalogue/article/0150_00020/12705/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carpegen Perio Diagnostik</td>
			<td style="width:247px;">Carpegen GmbH</td>
			<td style="width:119px;"></td>
			<td>http://www.carpegen.de/en/products-and-services/carpegen-perio-diagnostics.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">tubes Reaktionsgef&#228;&#223;e 1,5 ml</td>
			<td>Lactan</td>
			<td>CH77.1&#160; to CH81.1</td>
			<td>www.lactan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">cotton swabs</td>
			<td>Henry Schein</td>
			<td style="width:119px;">9003182</td>
			<td>www.henryschein.at</td>
		</tr>
	</tbody>
</table>

oral biofilm sampling for microbiome analysis in healthy children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

In the present publication, DNA sampling for NGS is demonstrated as an example of a possible application for orthodontic research. Bacterial DNA is extracted directly from the paper point samples. This DNA can then be used in studies of the microbial composition for clinical or research questions (as summarized in Figure 10).
Modern molecular analysis methods such as the NGS method 454-pyrosequencing give an overview of the complete microbiota of a sample. The DNA of thousands of bacteria is identified simultaneously at different phylogenetic levels over the 16s rRNA sequence differences. Bioinformatic platforms need to be generated as a means of structuring into clusters the information retrieved from the large data sets. Statistical analysis can then be applied to microbiota datasets.
The overall relative abundance of certain bacterial species can be analyzed as can shifts in the microbiota due to changing habitat conditions. Bar charts (Figure 11) and heatmaps (Figure 12) display the subgingival bacterial composition at phylum level or at order level. Various individuals and treatments can be compared by descriptive and inferential statistics. Figure 13 presents a histogram comparison of two modes of subgingival paper point sampling at different taxonomic levels. Table 1 shows the shift in biofilm composition during an in vitro study comparing two time points (T1 and T3). Statistically significant changes were calculated with Wilcoxon signed-rank test and following Bonferroni correction from the 454-pyrosequencing data.
Finally, Principal Coordinate Analysis (PCoA) enables direct 3D comparison at Operational Taxonomic Unit (OTU) level as identified in different samples. The PCoA plots in Figure 14 display intra- and inter-individual differences of the subgingival microbiome in a case series of five children. Figure 15 shows the results of an orthodontic case-control study: pink to red dots are the cases, light to dark blue dots are the controls, all sampled repeatedly over a period of four months. A clear clustering of the cases after the orthodontic intervention (red dots) represents a shift in the microbiome. Blue dots, representing the control group, are evenly distributed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56320/56320fig1.jpg" /> 
Figure 1: Prepare paper points of standardized length. Sterile calibrated paper points are cut at the first ring mark to standardize their length. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56320/56320fig2.jpg" /> 
Figure 2: Sterilize and free paper points from DNA. Standardized paper points are sterilized and UV irradiated in a clean bench. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56320/56320fig3.jpg" /> 
Figure 3: Subgingival paper point insertion. Sterile calibrated paper points are inserted tangentially into the subgingival sulcus until the 4 mm ring mark for 20 s.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56320/56320fig4.jpg" /> 
Figure 4: Cutting paper point into tube. Directly after sampling, paper points are cut at the third ring mark (4 mm) into a sterile and DNA-free 1.5 mL vial. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56320/56320fig5.jpg" /> 
Figure 5: Pooled and parallel sampling. Two paper points are inserted simultaneously into the subgingival sulcus of one tooth (A) or several paper points into the subgingival sulci of several teeth (B). <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56320/56320fig6.jpg" /> 
Figure 6: Mucosal sampling. Sterile calibrated paper points are laid into the mucosa of the vestibular fold. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56320/56320fig7.jpg" /> 
Figure 7: Saliva collection. Unstimulated saliva is spit into a sterile beaker. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56320/56320fig8.jpg" /> 
Figure 8: Sampling scheme. Samples can be collected as single (left side, one tooth each), pooled (several to all stripes in one vial), or parallel (blue stripes, two paper points at one tooth) samples. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56320/56320fig9.jpg" /> 
Figure 9: Color-coded storage. A referring color code for sheets and vials facilitates further sample handling. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56320/56320fig10.jpg" /> 
Figure 10: Subgingival biofilm analysis. Schematic presentation of clinical biofilm sampling (A), DNA extraction (B), and different NGS technologies (C). <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56320/56320fig11.jpg" /> 
Figure 11: Bar chart. Showing relative abundance of bacteria at phylum level analyzed using 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56320/56320fig12.jpg" /> 
Figure 12: Heatmap. Showing relative abundances of bacterial orders analyzed using 454-pyrosequencing. Dark red represents a high relative abundance (&#62; 10%). Dark blue represents a very low to no relative abundance of the respective bacterial order. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56320/56320fig13.jpg" /> 
Figure 13: Histogram. Comparison of two modes of paper point sampling (modes A and B) at different taxonomic levels. Data generated with 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56320/56320fig14.jpg" /> 
Figure 14: 2D PCoA plot. A case series (n=5) showing intra-individual and inter-individual differences as a result of two subgingival sampling methods (1 color is 1 subject).Data generated with 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56320/56320fig15.jpg" /> 
Figure 15: Snapshot of a 3D PCoA plot animation. Showing subgingival microbiome shifts in an orthodontic case control study (n = 16; each group, 3 points in time; cases: pink to red, controls: light to dark blue). Data generated with 454-pyrosequencing. Unpublished data from working group Santigli.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Taxon: Bacteriae</td>
			<td colspan="3">Time point 1 [%]</td>
			<td colspan="3">Time point 3 [%]</td>
			<td colspan="2">Wilcoxon signed-rank test</td>
		</tr>
		<tr>
			<td colspan="2">Phylum/Genus</td>
			<td>25th</td>
			<td>Median</td>
			<td>75th</td>
			<td>25th</td>
			<td>Median</td>
			<td>75th</td>
			<td>p-value</td>
			<td>p-value adjusted#</td>
		</tr>
		<tr>
			<td colspan="2">Other</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Other</td>
			<td>0.70</td>
			<td>1.07</td>
			<td>1.28</td>
			<td>1.32</td>
			<td>1.63</td>
			<td>1.93</td>
			<td>.000</td>
			<td>.005</td>
		</tr>
		<tr>
			<td colspan="2">Actinobacteria</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Actinomyces</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.13</td>
			<td>0.06</td>
			<td>0.30</td>
			<td>0.97</td>
			<td>.001</td>
			<td>.021</td>
		</tr>
		<tr>
			<td colspan="2">Rothia</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.05</td>
			<td>0.00</td>
			<td>0.13</td>
			<td>0.32</td>
			<td>.001</td>
			<td>.009</td>
		</tr>
		<tr>
			<td colspan="2">Bacteroidetes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Prevotella</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.22</td>
			<td>0.15</td>
			<td>1.02</td>
			<td>2.44</td>
			<td>.000</td>
			<td>.006</td>
		</tr>
		<tr>
			<td colspan="2">Firmicutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Other (Bacilli)</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.06</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.10</td>
			<td>.706</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Staphylococcus</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.12</td>
			<td>0.00</td>
			<td>0.07</td>
			<td>0.17</td>
			<td>.548</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">(Gemellaceae)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.07</td>
			<td>0.12</td>
			<td>0.77</td>
			<td>1.24</td>
			<td>.005</td>
			<td>.091</td>
		</tr>
		<tr>
			<td colspan="2">(Lactobacillales)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.00</td>
			<td>0.12</td>
			<td>1.50</td>
			<td>.004</td>
			<td>.073</td>
		</tr>
		<tr>
			<td colspan="2">Granulicatella</td>
			<td>0.09</td>
			<td>0.23</td>
			<td>0.37</td>
			<td>0.64</td>
			<td>1.59</td>
			<td>2.28</td>
			<td>.000</td>
			<td>.003</td>
		</tr>
		<tr>
			<td colspan="2">Lactobacillus</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.18</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>.700</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Other (Streptococcaceae)</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.13</td>
			<td>0.00</td>
			<td>0.10</td>
			<td>0.19</td>
			<td>.768</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">(Streptococcaceae)</td>
			<td>0.04</td>
			<td>0.10</td>
			<td>0.23</td>
			<td>0.12</td>
			<td>0.37</td>
			<td>0.69</td>
			<td>.005</td>
			<td>.083</td>
		</tr>
		<tr>
			<td colspan="2">Streptococcus</td>
			<td>53.42</td>
			<td>61.96</td>
			<td>88.38</td>
			<td>48.44</td>
			<td>60.83</td>
			<td>73.97</td>
			<td>.055</td>
			<td>.930</td>
		</tr>
		<tr>
			<td colspan="2">Other (Lachnospiraceae)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.11</td>
			<td>.003</td>
			<td>.058</td>
		</tr>
		<tr>
			<td colspan="2">Dialister</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>.240</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Veillonella</td>
			<td>3.43</td>
			<td>27.15</td>
			<td>42.78</td>
			<td>6.69</td>
			<td>13.38</td>
			<td>27.05</td>
			<td>.157</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Proteobacteria</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Haemophilus</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.03</td>
			<td>0.31</td>
			<td>0.84</td>
			<td>2.38</td>
			<td>.000</td>
			<td>.008</td>
		</tr>
		<tr>
			<td colspan="10">#p-value Wilcoxon signed-rank test adjusted according Bonferroni correction</td>
		</tr>
		<tr>
			<td colspan="10">A p&#60;0.0026 was considered significant</td>
		</tr>
	</tbody>
</table>
Table 1: Statistical analysis. Differences in the oral microbiome of healthy children at two points in time. Data generated with 454-pyrosequencing. Image modified from Klug et al.23

Watch this Scientific Journal Video about Oral Biofilm Sampling for Microbiome Analysis in Healthy Children at JoVE.com

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

oral-biofilm-sampling-for-microbiome-analysis-in-healthy-children

Research

JoVE Journal

Medicine

17.0K Views.  Medical University of Graz. Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Video: Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

Virtual Prism Adaptation Therapy: Protocol for Validation in Healthy Adults

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is imperative for the successful management of hemispatial neglect. However, the clinical use of various interventions is limited in real clinical practice. Prism adaptation therapy is one of the most evidence-based rehabilitation modalities to treat hemispatial neglect. To overcome any possible shortcoming that may occur with prism therapy, we developed a new system using immersive virtual reality and depth-sensing camera to create a virtual prism adaptation therapy (VPAT). To validate the VPAT system, we designed an experimental protocol investigating the behavioral errors and changes in cortical activation via the VPAT system. Cortical activation was measured by functional near infrared spectroscopy (fNIRS). The experiment consisted of four phases. All four included clicking, pointing or rest applied to right-handed healthy people. Clicking versus pointing was used for investigating the cortical region related with the gross motor task, and pointing with VPAT versus pointing without VPAT was used for investigating the cortical region associated with visuospatial perception. The preliminary results from four healthy participants showed that pointing errors by the VPAT system was similar to the conventional prism adaptation therapy. Further analysis with more participants and fNIRS data, as well as a study in patients with stroke may be required.

This experimental protocol demonstrates the use of virtual prism adaptation therapy (VPAT) in healthy adults and the association between VPAT and functional near infrared spectroscopy to determine the effect of VPAT on cortical activation. Results suggest that VPAT may be feasible and could induce similar behavioral adaptation as conventional prism adaptation therapy.

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is ...

Lower Limb Biomechanical Analysis of Healthy Participants

Biomechanical analysis techniques are useful in the study of human movement. The aim of this study was to introduce a technique for the lower limb biomechanical assessment in healthy participants using commercially available systems. Separate protocols were introduced for the gait analysis and muscle strength testing systems. To ensure maximum accuracy for gait assessment, attention should be given to the marker placements and self-paced treadmill acclimatization time. Similarly, participant positioning, a practice trial, and verbal encouragement are three critical stages in muscle strength testing. The current evidence suggests that the methodology outlined in this article may be effective for the assessment of lower limb biomechanics.

This article introduces a comprehensive experimental methodology on two of the latest technologies available to measure the lower limb biomechanics of individuals.

Biomechanical analysis techniques are useful in the study of human movement. The aim of this study was to introduce a technique for the lower limb ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation

Magnetic resonance imaging (MRI) is an important tool for the clinical assessment of cardiovascular morphology and heart function. It is also the recognized standard-of-care for blood flow quantification based on phase contrast MRI. While such measurement of blood flow has been possible in adults for decades, methods to extend this capability to fetal blood flow have only recently been developed.
Fetal blood flow quantification in major vessels is important for monitoring fetal pathologies such as congenital heart disease (CHD) and fetal growth restriction (FGR). CHD causes alterations in the cardiac structure and vasculature that change the course of blood in the fetus. In FGR, the path of blood flow is altered through the dilation of shunts such that the oxygenated blood supply to the brain is increased. Blood flow quantification enables assessment of the severity of the fetal pathology, which in turn allows for suitable&#160;in utero&#160;patient management and planning for postnatal care.
The primary challenges of applying phase contrast MRI to the human fetus include small blood vessel size, high fetal heart rate, potential MRI data corruption due to maternal respiration, unpredictable fetal movements, and lack of conventional cardiac gating methods to synchronize data acquisition. Here, we describe recent technical developments from our lab that have enabled the quantification of fetal blood flow using phase contrast MRI, including advances in accelerated imaging, motion compensation, and cardiac gating.

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.