<meta charset="utf8"></head><body>棘 阿米巴 物种运动的长期跟踪和定量

<ol>
	<li>Randag, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rising+incidence+of+Acanthamoeba+keratitis:+A+7-year+nationwide+survey+and+clinical+assessment+of+risk+factors+and+functional+outcomes.">The rising incidence of Acanthamoeba keratitis: A 7-year nationwide survey and clinical assessment of risk factors and functional outcomes.</a> PLoS One. 14 (9), e0222092 (2019).</li><li>Szentmary, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis+-+Clinical+signs,+differential+diagnosis+and+treatment.">Acanthamoeba keratitis - Clinical signs, differential diagnosis and treatment.</a> J Curr Ophthalmol. 31 (1), 16-23 (2019).</li><li>Carnt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis:+confirmation+of+the+UK+outbreak+and+a+prospective+case-control+study+identifying+contributing+risk+factors.">Acanthamoeba keratitis: confirmation of the UK outbreak and a prospective case-control study identifying contributing risk factors.</a> Br J Ophthalmol. 102 (12), 1621-1628 (2018).</li><li>Verani, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+outbreak+of+Acanthamoeba+keratitis+associated+with+use+of+a+contact+lens+solution,+United+States.">National outbreak of Acanthamoeba keratitis associated with use of a contact lens solution, United States.</a> Emerg Infect Dis. 15 (8), 1236-1242 (2009).</li><li>Tu, E. Y., Joslin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+outbreaks+of+atypical+contact+lens-related+keratitis:+what+have+we+learned.">Recent outbreaks of atypical contact lens-related keratitis: what have we learned.</a> Am J Ophthalmol. 150 (5), 602-608 (2010).</li><li>International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+14729:2001/A1.+Ophthalmic+optics-Contact+lens+care+products-Microbiological+requirements+and+test+methods+for+products+and+regimens+for+hygienic+management+of+contact+lenses.">ISO 14729:2001/A1. Ophthalmic optics-Contact lens care products-Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses.</a> International Organization for Standardization. , (2010).</li><li>. ASC Z80, Parent Committee Meeting Available from: <a target="_blank" href="https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf">https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf</a> (2023)</li><li>Rayamajhee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba,+an+environmental+phagocyte+enhancing+survival+and+transmission+of+human+pathogens.">Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.</a> Trends Parasitol. 38 (11), 975-990 (2022).</li><li>Reverey, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superdiffusion+dominates+intracellular+particle+motion+in+the+supercrowded+cytoplasm+of+pathogenic+Acanthamoeba+castellanii.">Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii.</a> Sci Rep. 5, 11690 (2015).</li><li>Thapa, S., Lukat, N., Selhuber-Unkel, C., Cherstvy, A. G., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+superdiffusion+of+polydisperse+vacuoles+in+highly+motile+amoeboid+cells.">Transient superdiffusion of polydisperse vacuoles in highly motile amoeboid cells.</a> J Chem Phys. 150 (14), 144901 (2019).</li><li>Rudell, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+migration+in+an+electric+field.">Acanthamoeba migration in an electric field.</a> Invest Ophthalmol Vis Sci. 54 (6), 4225-4233 (2013).</li><li>Fukaya, S., Aoki, K., Kobayashi, M., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+the+motility+of+giant+virus-infected+amoebae+using+phase-contrast+microscopic+images.">Kinetic analysis of the motility of giant virus-infected amoebae using phase-contrast microscopic images.</a> Front Microbiol. 10, 3014 (2019).</li><li>Fukaya, S., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+Acanthamoeba+castellanii+infected+with+giant+viruses+quantitatively+revealed+process+of+morphological+and+behavioral+changes+in+host+cells.">Kinetic analysis of Acanthamoeba castellanii infected with giant viruses quantitatively revealed process of morphological and behavioral changes in host cells.</a> Microbiol Spectr. 9 (1), 0036821 (2021).</li><li>Cheng, H. J., Hsu, C. H., Hung, C. L., Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+for+cell+and+particle+tracking+on+microscopy+images+using+algorithms+and+deep+learning+technologies.">A review for cell and particle tracking on microscopy images using algorithms and deep learning technologies.</a> Biomed J. 45 (3), 465-471 (2022).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li><li>Svensson, C. M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untangling+cell+tracks:+Quantifying+cell+migration+by+time+lapse+image+data+analysis.">Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.</a> Cytometry A. 93 (3), 357-370 (2018).</li><li>Piltti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+time-lapse+imaging+and+single-cell+tracking+of+in+vitro+cultured+neural+stem+cells+-+Tools+for+analyzing+dynamics+of+cell+cycle,+migration,+and+lineage+selection.">Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells - Tools for analyzing dynamics of cell cycle, migration, and lineage selection.</a> Methods. 133, 81-90 (2018).</li><li>Campolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+real-time+motility+analysis+of+Acanthamoeba+reveals+sustained+movement+in+absence+of+nutrients.">Continuous real-time motility analysis of Acanthamoeba reveals sustained movement in absence of nutrients.</a> Pathogens. 10 (8), 995 (2021).</li><li>Campolo, A., Patterson, B., Lara, E., Shannon, P., Crary, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+recovery+of+Acanthamoeba+motility+among+surviving+organisms+after+contact+lens+care+disinfection.">Complete recovery of Acanthamoeba motility among surviving organisms after contact lens care disinfection.</a> Microorganisms. 11 (2), 299 (2023).</li><li>Crary, M. J., Walters, R., Shannon, P., Gabriel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+affecting+the+recovery+of+Acanthamoeba+trophozoites.">Variables affecting the recovery of Acanthamoeba trophozoites.</a> Pathogens. 10 (2), 221 (2021).</li><li>. imagej Available from: <a target="_blank" href="https://imagej.net/plugins/trackmate/tutorials/getting-started">https://imagej.net/plugins/trackmate/tutorials/getting-started</a> (2024)</li><li>Cherstvy, A. G., Nagel, O., Beta, C., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Gaussianity,+population+heterogeneity,+and+transient+superdiffusion+in+the+spreading+dynamics+of+amoeboid+cells.">Non-Gaussianity, population heterogeneity, and transient superdiffusion in the spreading dynamics of amoeboid cells.</a> Phys Chem Chem Phys. 20 (35), 23034-23054 (2018).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#188; Ringer&#8217;s solution</td>
			<td>ThermoFisher Scientific</td>
			<td>BR0052G</td>
			<td>Oxoid Ringers Solution Tablets. Follow directions to make one-quarter strength instead of full strength Ringers.&#160;</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette</td>
			<td>Eppendorf Research</td>
			<td>3123000039</td>
			<td>2 &#181;L-20 &#181;L single channel</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette tips</td>
			<td>Neptune Scientific, San Diego, CA, USA</td>
			<td>BT10.N</td>
			<td>10 &#181;L Universal Barrier Tip</td>
		</tr>
		<tr>
			<td>48 well plate</td>
			<td>Millipore Sigma,&#160;</td>
			<td>CLS3548</td>
			<td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td>
		</tr>
		<tr>
			<td>50 mL conical tubes (1 for each sample, 1 for each pass 2 sample)</td>
			<td>Fisher Scientific</td>
			<td>Falcon 352098</td>
			<td>Falcon 50 mL High Clarity Conical Centrifuge Tubes, polypropylene</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Millipore Sigma, Burlington, MA, USA</td>
			<td>CLS3596</td>
			<td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td>
		</tr>
		<tr>
			<td>Acanthamoeba&#160;</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td>ATCC 30461</td>
			<td>This protocol has been verified for ATCC 50655, 30872, 50702, 30010, 30461, 50370, 50703, 30137, PRA-115, and PRA-411</td>
		</tr>
		<tr>
			<td>Axenic culture media (AC6)</td>
			<td>Made in house</td>
			<td>n/a</td>
			<td>Containing 20 g biosate peptone, 5 g glucose, 0.3 g KH2PO4, 10 &#181;g vitamin B12, and 1 glass5 mg&#160;l-methionine per liter of distilled deionized water. Adjust pH to 6.6-6.95 with 1 M NaOH and autoclave at 121 &#176;C for 20 min, store at room temperature for up to 3 months.&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge and appropriate rotor</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>Sorvall ST 40R</td>
			<td>Any equivalent centrifuge and rotor are acceptable so long as it can spin 50 mL conical tubes at 500 x g for 5 min</td>
		</tr>
		<tr>
			<td>Chemotaxis and Migration Tool</td>
			<td></td>
			<td></td>
			<td>Free software based on ImageJ, available at https://ibidi.com/chemotaxis-analysis/171-chemotaxis-and-migration-tool.html</td>
		</tr>
		<tr>
			<td>Disposable hemocytometer</td>
			<td>Bulldog Bio, Portsmouth, NH, USA</td>
			<td>DHC-N01</td>
			<td>Neubauer Improved 2-Chip Disposable Hemocytometer, Individually packaged, Nonpyrogenic</td>
		</tr>
		<tr>
			<td>ImageJ software with Trackmate plugin (this protocol written with Trackmate version 6.0.2.)</td>
			<td></td>
			<td></td>
			<td>Free software developed by the National Institutes of Health, available at imagej.net. Trackmate plugin available at https://imagej.net/plugins/trackmate/</td>
		</tr>
		<tr>
			<td>Microscope, preferably with automated moveable stage</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>A Nikon Eclipse Ti-U Microscope was used in this study and the automated moveable stage was utilized to be able to record images in multiple wells at a time.&#160;</td>
		</tr>
		<tr>
			<td>NIS-Elements software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>BrandTech 26331</td>
			<td>BrandTech accu-jet pro Pipet Controller</td>
		</tr>
		<tr>
			<td>Serological pipette tips</td>
			<td>VWR</td>
			<td>5 mL: 76201-710 
			10 mL: 170356 
			25 mL: 89130-900 
			50 mL: 75816-088</td>
			<td>VWR Serological Pipette, Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Sterile aluminum transmission flow cell</td>
			<td>Biosurface Technologies Corporation, Bozeman, MT, USA</td>
			<td>FC 81-AL</td>
			<td>Anodized aluminum single channel transmission flow cell with 96-well plate footprint for use with an inverted microscope</td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>734-2316</td>
			<td>VWR Tissue Culture Flask, 182.5 cm, Surface treated, Plug seal cap, Sterile</td>
		</tr>
	</tbody>
</table>

quantification of acanthamoeba spp. motility

<meta charset="utf8"></head><body>作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

棘阿米巴属的定量运动性

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential ...

quantification-of-acanthamoeba-spp-motility

Research

JoVE Journal

Biology

Quantification of Acanthamoeba spp. Motility

2.5K 次观看.  Alcon Research, LLC. 我们的研究深入研究了棘阿米巴的自然行为，因为当人类和棘阿米巴不可避免地相遇时，它在自然界中是如此。从这个基础上，我们可以推断出棘阿米巴如何在各种环境和表面（无论是隐形眼镜还是角膜）中相互作用的见解，使我们能够制定有针对性的策略来预防棘阿米巴角膜炎和改善隐形眼镜卫生习惯。从历史上看，棘阿米巴领域在采用遗传学和?...

视频: 作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

科研

生物学

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制:Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

测量果蝇的作物运动性和食物通度

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...