This material is based upon research supported (or supported in part by) the Technology Commercialization Office, Salt Lake City, UT, Office of Research and Development, Rehabilitation R&amp;D Service, DVA SLC Health Care System, Salt Lake City, UT, Department of Defense PRMRP Grant (No. PR054520), the Albert &amp; Margaret Hofmann Chair and the Department of Orthopaedics, University of Utah School of Medicine, Salt Lake City, UT; Technical support for the simulations was provided by the Center for Integrative Biomedical Computing of Scientific Computing and Imaging Institute and was made possible in part by software from the NIH/NCRR Center for Integrative Biomedical Computing, P41-RR12553-07.
Additional gratitude is extended to Gwenevere Shaw for assistance with manuscript preparation and Dustin Williams for the image of biofilm.

Part 1: Using Computed Tomography (CT) Scans for Amputee Reconstruction
<ol><li>Retrospective CT scans were collected from the University of Utah and Department of Veteran Affairs hospitals after obtaining IRB and HIPAA approval.</li>
<li>CT scans were selected because they allow clear distinction between tissue types based on x-ray absorbency.</li>
<li>CTs were manually inspected and included in the study based on the absence of metal implants to prevent image artifact.</li>
</ol>
Part 2: Model Generation with Seg3D
<ol>
<li>Files were downloaded as Dicom images and loaded into Seg3D (version 1.11.0, software.sci.utah.edu) as a new volume.</li>
<li>A median filter was used to smooth the imported volumes prior to determining geometrically defined tissue structures.</li>
<li>The tissue boundaries of the bone, bone marrow, organs and adipose tissue were generated by thresholding the CT files interactively (Figure 1). 
<img src="/files/ftp_upload/1237/1237fig1.jpg" alt="Figure 1" /> 
Figure 1: A sagittal cross section of an amputee residual limb thresholded and separated into specific tissue types.</li>
<li>The musculature was obtained by manually setting seed points inside the thresholded muscle tissue and using a confidence connected filter to find all the tissue connected to the seed points. This step eliminated erroneous tissues which may have been grouped together with the muscle based on similar absorbency from CTs.</li>
<li>The skin, which was impossible to discern reliably from the CT images, was generated by dilating the outermost tissue 2 millimeters based on average skin thickness to produce a layer of homogeneous thickness that surrounded the full model1.</li>
<li>Segmentations were manually inspected, corrected to ensure accuracy and combined in a hierarchy into a single label map required for finite element analysis (Figure 1). 
<img src="/files/ftp_upload/1237/1237fig2.jpg" alt="Figure 2" /> 
Figure 2: Representative hierarchical model of a bilateral amputee created with Seg3D.</li>
</ol>
Part 3: Preparation for Finite Element Analysis
<ol>
<li>A 10 cm implant was designed in Matlab to serve as the implanted orthopedic device and cathode for electrical stimulation and imported into SCIRun (version 4.0, software.sci.utah.edu).</li>
</ol>
Part 4: Electrode Placement &amp; Design
<ol>
<li>SCIRun was utilized for electrode design because it supports interactive electrode placement and simulation.</li>
<li>A network was created and modules organized with specific functions to generate the mesh (Figure 3). &nbsp;Modules were important for defining boundary conditions, tissue conductivities, mesh refinements, generating Matlab histograms, recording field data, etc (Table 1). 
<img src="/files/ftp_upload/1237/1237fig3.jpg" alt="Figure 3" /> 
Figure 3: Representative network image from a pilot study using a two band external electrode configuration. 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Table 1 </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td colspan="2">Conductivities Assigned to Segmented Tissues </td>
 </tr>
 <tr>
 <td>Tissue Type</td>
 <td>Conductivities [S/m]</td>
 </tr>
 <tr>
 <td>Organ</td>
 <td>0.22</td>
 </tr>
 <tr>
 <td>Skin</td>
 <td>0.26</td>
 </tr>
 <tr>
 <td>Adipose</td>
 <td>0.09</td>
 </tr>
 <tr>
 <td>Muscle</td>
 <td>0.25</td>
 </tr>
 <tr>
 <td>Cortical Bone</td>
 <td>0.02</td>
 </tr>
 <tr>
 <td>Bone Marrow</td>
 <td>0.07</td>
 </tr>
</tbody>
</table>
</li>
<li>The configurations for the electrodes consisted of a one patch electrode, two patch electrodes, one continuous band and two continuous bands.</li>
<li>External electrode bands were applied to the residual limb of the models generated from patient CT scans and were 1.6 cm in thickness.</li>
<li>Electrode patches were placed as a strip covering approximately half the diameter of the residual limb and were 3 cm in thickness.</li>
<li>The inner cortical implant which represented the osseointegrated implant was set to endosteal diameter to allow for perfect implant fit and fill2.</li>
</ol>
Part 5: Finite Element Analysis
<ol>
<li>The simulations were generated assuming that the electric metrics could be calculated using a quasi-static approach with no time dependency.</li>
<li>The model was computed by solving Laplace&#x2019;s equation for each tissue type generated from the Seg3D segmentations.</li>
<li>The boundary conditions were formed by the electrodes that injected currents and the guideline that current remained within the body.</li>
<li>Since the electrodes and the implant had a much larger conductivity than the surrounding tissues, it was assumed that the implant (cathode) was at a constant potential, likewise the surface electrodes were modeled with a constant potential difference from the percutaneous implant.</li>
<li>To evaluate the efficacy of electrode configuration and sizing, patient specific models were developed and the electrical potential around the implant interface was used to determine localized field strengths.</li>
<li>The model was generated using a hexahedral mesh that consisted of approximately 1.8 million elements which were treated as piecewise homogenous, ohmic and isotropic.</li>
<li>The optimal model for this experiment was selected with a relative difference &lt;5% in voltage gradients confirmed with a mesh sensitivity study to insure model accuracy (Table 2). 
<table border="1" cellspacing="0" cellpadding="0" width="305">
<tbody>
 <tr>
 <td>Table 2 </td>
 <td>&nbsp; </td>
 <td>&nbsp; </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td colspan="3">Mesh Sensitivity Study for Amputee Model</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mesh</td>
 <td>Elements</td>
 <td>Nodes</td>
 <td>Relative Difference</td>
 </tr>
 <tr>
 <td>100 100 50</td>
 <td>149089</td>
 <td>161131</td>
 <td>0.0995</td>
 </tr>
 <tr>
 <td>125 125 75</td>
 <td>350180</td>
 <td>371472</td>
 <td>0.0802</td>
 </tr>
 <tr>
 <td>150 150 100</td>
 <td>673032</td>
 <td>706082</td>
 <td>0.0545</td>
 </tr>
 <tr>
 <td>175 175 125</td>
 <td>1146778</td>
 <td>1194044</td>
 <td>0.0527</td>
 </tr>
 <tr>
 <td>200 200 150</td>
 <td>1796690</td>
 <td>1860772</td>
 <td>0.0439</td>
 </tr>
 <tr>
 <td>250 250 200</td>
 <td>3745038</td>
 <td>3850202</td>
 <td>0.0364</td>
 </tr>
 <tr>
 <td>275 275 225</td>
 <td>5097243</td>
 <td>5226587</td>
 <td>0.0301</td>
 </tr>
 <tr>
 <td>300 300 250 </td>
 <td>6742588</td>
 <td>6898729</td>
 <td>0.0000</td>
 </tr>
 </tbody>
</table>
</li>
<li>Using an iterative solver, the electric metrics in the finite element models were computed for the electrode configurations.</li>
</ol>

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The technology disclosed in the article, specifically utilizing an orthopedic implant as a functional cathode to increase skeletal attachment, reduce rehabilitation and prevent bacterial adhesion is a novel idea invented by the first author in the journal submission. An invention disclosure form has been submitted to the University of Utah and a United States provisional patent has been filed protecting all technology disclosed.

Understanding the Electrical Stimulation Paradigm
Enhancements in medical care and evacuation strategies on the field of combat have lead to an increased number of warriors surviving disastrous war related injuries. While the improved survival rate is a medical advancement, servicemen and women are returning from combat with amputations that require intensive follow-up care, extensive rehabilitation and expensive prosthetic services from the Veteran Affairs Health Care System3. Congressional reports detail that over 1,000 war-related amputations have occurred as a result of Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) conflicts4.
In the case of OEF and OIF veterans, approximately 15% of returning warriors have lost multiple limbs and a significant number of returning servicemen and women have short residual limbs where socket technology is not an option or has been rejected by the patient. The reported discontinued use of upper extremity prosthesis even exceeds 50% because the fixation devices are cumbersome and difficult to use comfortably5. Lower extremity prostheses are equally problematic and the common problems associated with soft tissue sockets include the inability to walk on challenging terrain6, limited residual limb length7, patient discomfort5, concern with non-physiological loading8, irritation from heterotopic ossification9 and risk of debilitating diseases10. However, osseointegration technology is a novel surgical technique that may reduce pain11, skin irritation12, enhance osseoperception13, improve mobility6, decrease pressure sores associated with sockets6, reduce energy for ambulation7,14 and better serve veteran and warriors with limited residual limb length15.
Despite the numerous physical and psychological advantages of osseointegration, the associated surgical procedures require more advanced infection prevention treatment streategies16, require long rehabilitation programs and include restrictive weight bearing protocols which may last up to 1.5 years postoperative17. Because the viability of the host bone and length of the residual limb is important for muscle attachment and functionality, developing new devices to improve osseointegration is key for returning servicemen and women. Therefore, development of an osseointegrated intelligent implant design (OIID) system which uses controlled electrical stimulation may reduce the length of rehabilitation and increase skeletal attachment for veteran and warrior amputees. However, since no current device is commercially available and directed for use with percutaneous osseointegrated implants, the motivation of the program is to confirm safety and efficacy with finite element analysis.
Understanding the role of electrical stimulation in bone remodeling, specifically the deposition of osteoids and mineralization, has remained speculative. However, electrical activity observed in bone may be the result of mechanical loading18,19 and therefore an electrical stimulus may be an effective mechanism for inducing bone repair19. The logic behind the hypothesis is explained in a fracture healing model. When long bones are loaded, the side in tension becomes electropositive and the compression side electronegative20,21, however, once a bone is broken, the site will remain electronegative with respect to the surrounding environment until healing has commenced and homeostasis resumed21. Simulating the natural healing cascade with an electrical signal has been believed to assist with calcium deposition22, slight alterations in oxygen content and pH23, recruitment of growth factors22 and assists with osteoblast migration and secretion of additional extracellular matrix24.
The premise that electrical stimulation alone could govern complete bone repair has been redefined and the current new hypothesis proposes that complete unions are formed by mechanical loads and an electrical stimulation co-stimulus19. The electrical impulses observed in vivo are associated with piezoelectric deformation of collagen or the large electro-kinetic currents produced by ionic constituents flowing past mineral portions of the bone matrix25. In fact, spontaneous potentials have been reported in bone as large as 6 millivolts and correlates with an increase in the mineral apposition rate of bone26.
Early work by Brighton and Friedenberg18,21,27,28 used the concept of electrical stimulation for bone regeneration in the 1960s and 1970s and demonstrated that direct current could be used to repair non-unions in a shorter period of time when compared to traditional healing methods. Additional models have investigated bone formation with restrictive weight bearing and demonstrated a thirty-one percent increase in osteogenic activity between controls and electrically stimulated limbs25.
While researchers in the field of electrical stimulation have paved the way for understanding the mechanism for osteoblast matrix deposition with electrical stimulation, inadequate understanding has limited the expansion of this technology. While there are many cases of successful healing of non-unions and fracture healing models, examples of patient discomfort and failed attempts are replete in the literature as well29. The problem with electrical stimulation occurs from scientists and clinicians controlling the wrong electric metrics and concentrating solely on current magnitudes. Previous researchers have looked to current as the &#x201C;magic bullet&#x201D; to fixing the approximate 500,000 non-unions which occur annually30. However, repeatability between models has been limited from joule heating complications31 and not determining current densities32. In fact, all manufactured biomedical devices must be limited to a current density less than 2 mA/cm2 as outlined by the International Electrotechnical Commission to prevent localized tissue necrosis and patient discomfort33.
Aside from assisting with skeletal fixation, controlled electrical stimulation may also prevent bacterial adhesion on orthopedic implants and reduce the risk for osteomyelitis and biofilm formation34-37. Biofilm formation on orthopedic devices lead to patient complications and significant distress for those who depend on these devices38. Emphasis is placed on the necessity to have completely sterilized instrumentation and implants prior to surgery39 , however it is often difficult to diagnose bacteria adhesion as evident from many negative cultured cases which indeed are infected40. This problem is often coupled with the fact that biofilms are slow growing in nature40, cannot be growth accurately in vitro39, depend on the type of bacteria cells, cleanliness of the surface and the immune system of the affected person39. Investigation of European transfemoral amputees with oseeointegration technology reveal the most frequent problem is infection (frequent superficial infections, 1/3 periprosthetic infections)41. While there have been vast improvements in surgical preparation, eradicating bacteria is one of the fundamental factors for improving osseointegration since biofilms are between 500-5000 thousand times more difficult to eradicate due to their non-platonic form34,35,39. Therefore, utilizing electrical stimulation as a modality for removing harmful bacterial colonies and increasing skeletal fixation are important factors for ensuring the protection of patient health and OIID efficacy.
The advantages of using veteran and warrior amputees are that the relative youth and otherwise good health of these individuals make them an ideal population for aggressive rehabilitation and a percutaneous post will serve as an ambulatory aid and may be developed as an exposed cathode for electrical stimulation. The presence of an osseointegrated implant does not require additional surgical procedures to insert electrical components, allows the device to be controlled externally and prevents further risk of infection42. Therefore, by understanding the method of current injection into the residual limb of veteran and warrior amputees, an electric field on the magnitude of 1-10 V/cm may be established, controlled and measured at the implant interface. It is hypothesized that this will allow safe levels of electricity to be delivered, capable of inducing osteoblast migration and improving skeletal attachment. An electric field of this degree will increase the quantity and quality of bone at the implant interface, and improve the prospects for accelerated rehabilitation and skeletal fixation for an amputee. Use of electrical stimulation has not been investigated as a modality to accelerate osseointegration in an intramedullary prosthetic implant and presents numerous opportunities for translational research to improve patient care.
Experimental Results
The necessity for patient specific models with a percutaneous electrical stimulation device was supported in the study. The simulations developed for the proposed biomedical device may have the capabilities of expediting skeletal attachment by increasing osteoblast migration and preventing bacterial adhesion27,34,36,39. Computation modeling has effectively shown that 1-10 V/cm electric fields and current densities below 2 mA/cm2 may be generated using the implant as a functional cathode and is most homogenously distributed using a two band external electrode. The OIID system may be the first step to resolving the classic problem associated with electrical stimulation; the inability to define current pathways in the human body43. Therefore, establishing tools for enhancing skeletal attachment may assist with reducing the length of rehabilitation required for an osseointegrated procedure.
Utilizing electrical stimulation for older amputees is also a critical aspect which must be explored as well. Bone mass is maximum a decade after skeletal growth ceases but decreases significantly by the eighth and ninth decade44. As long bones change with age, the endosteal diameter tends to increase more rapidly than the periosteal diameter which may lead to implant loosening45. This problem coupled with the reduction of strain on bones by weaker muscles may contribute to debilitating diseases such as osteoporosis and osteopenia45 and require additional treatment options for patients with osseointegrated implants. However, controlled electrical stimulation and mechanical loading may act as a synergistic catalyst of bone ongrowth and maintain host bone bed integrity with elderly patients using an OIID system.

bioelectric analyses of an osseointegrated intelligent implant design system for amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

Watch this Scientific Journal Video about Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees at JoVE.com

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

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JoVE Journal

Biology

13.9K vues.  Department of Veteran Affairs. Il est nécessaire de développer des alternatives attache une prothèse à cause de la perte attribuée à un membre vasculaires occlusives et les traumatismes. Le but de ce travail est d'introduire une osséointégrés système intelligent design de l'implant pour augmenter la fixation du squelette et de réduire les taux d'infection périprothétique pour les patients nécessitant des technologies ostéo-intégrés.

Vidéo: Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 &#176;C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...