선택의 여지가 실험도 : <ol><li> 조심스럽게 플라스틱 컵에 대한 관심의 Arabidopsis 공장을 묶으십시오. 이 경우 : 콜 - 0 (WT)와 JA의 생산에 장애인 돌연변이. 당신이 나뭇잎에 손상을 제한 있는지 확인하십시오. 그걸 위해서는이 페인트의 뒷면을 사용합니다. </li><li> 양배추에있는 식민지에서 갓 부화 유충을 위해 좋은 페인트를 사용하십시오. </li><li> Arabidopsis 식물에 유충을 조심스럽게 넣습니다. </li><li> 일 4, 7, 10 중량 유충. 팁 : 식물 성장 (1 유충 1 공장의 전체 생명주기를 수행 수) 증가 짧은 하루 조건 (8 시간 조명) 아래 식물을 성장. </li></ol> 선택 실험 : <ol><li> 두 식물은 각각의 유전자형 중 하나를 선택합니다. 베스트는 트레이 효과 같은 다른 요인 등을 제한 같은 냄비에 식물을 성장하는 것입니다 </li><li> 나비 케이지에있는 식물을 놓으십시오. </li><li> 비옥한 여성을 선택합니다. 난 보통 적극적으로 양육 식민지에서 알을 누워있는 여자를 잡으. </li><li> 24 시간을위한 두 식물의 선택은 새장에있는 여성을 풀어주고있다. </li></ol> 팁 1 : 학습의 영향을 제거하기 위해, 이후의 선택 시험에 대한 여자를 재사용하지 않습니다. 팁 2 : 하나는 비슷한 성장 표현형을 가지고 식물을 비교할 수 있습니다. 또는, 당신은 Erlenmeyer 플라스크 위에 각 유전자형에서 나뭇잎을 탑재하고 여성들의 매력을 비교해 볼 수있을 텐데. 방문하십시오 <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.032607.092825">식물 생물학의 연간 리뷰</a> 곤충 herbivory이 프로토콜과 식물 면역에 대한 자세한 내용은.

choice and no-choice assays for testing the resistance of a. thaliana to chewing insects

작은 흰 배추 나비의 유충은 농업 설정에서 해충입니다. 이 애벌레의 종류가 같은 양배추 많은 작물을 포함 양배추 가족 식물에서 피드, 곤충의 브로콜리, Val Thorens 호텔 콩나물 등 리어링은 온실의 양배추 식물에 이루어집니다. 최소한 두 케이지는 피에리스 rapae의 양육을 위해 필요합니다. 애벌레와 어른, 나비를 포함하는 다른 하나는. 식물 호르몬이 곤충 해충에 대한 저항의 유해 식물 화학 물질의 역할을 조사하기 위해 두 실험을 보여줍니다. 첫째, jasmonic 산성 (JA - 자주 곤충에 대한 저항에 표시된 식물 호르몬)의 역할 결정 씹는 곤충 피에리스 rapae에 저항 인치 캐타필러 성장 JA 생산에 장애인 야생 타입과 돌연변이 식물에 비교할 수 있습니다. 애벌레가 합성 또는 JA에서 결함있는 단일 공장에서 존속하도록 강요되기 때문에이 실험은, &quot;아무도 선택&quot;으로 간주되지 않습니다. 둘째, 우리는 산란 (달걀 부설) 신호로 사용됩니다 glucosinolates의 역할을 조사 실험을 보여줍니다. 여기서 우리는 여성의 나비가 두 유전자형의 식물에 자신의 알을 낳기 위해 선택할 수있는 &quot;선택&quot;실험에 glucosinolate 생산​​ 장애인 WT 및 돌연변이 Arabidopsis을 사용합니다. 이 비디오는 두 assays뿐만 아니라 대표적인 결과에 대한 실험 설정을 보여줍니다.

Watch this Scientific Journal Video about Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects at JoVE.com

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

씹는 곤충 herbivores 식물의 저항은 여러 가지 방법으로 테스트할 수 있습니다. 여기, 우리는 선택과 해충 종 피에리스 rapae에 대한 저항을 식별하는 모델 식물 Arabidopsis thaliana의와 노 선택 실험을 셋업하는 방법을 보여줍니다.

씹는 곤충에 A.의 thaliana의 저항을 테스트하기 위해 선택과 노 - 선택 Assays

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

choice-no-choice-assays-for-testing-resistance-thaliana-to-chewing

Research

JoVE Journal

Biology

17.1K 조회수.  Cornell University. 씹는 곤충 herbivores 식물의 저항은 여러 가지 방법으로 테스트할 수 있습니다. 여기, 우리는 선택과 해충 종 피에리스 rapae에 대한 저항을 식별하는 모델 식물 Arabidopsis thaliana의와 노 선택 실험을 셋업하는 방법을 보여줍니다.

비디오: Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

의 간질 발작의 돌연변이에 대한 Neurocircuit의 Assays Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

약리 및 기능성 유전자 Assays은 Planarian의 재생을 조작하는<em자포니카> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

계면 세포 핵의 기계적 성질을 알아내기 위해 Biophysical Assays : 기판 스트레인 신청 및 Microneedle 조작

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

말라리아 모기의 개발, 다산과 생존자에 성적인 의약품의 시험 효과에 대한 독성 시험 법

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

인 - 더 - 필드 응용 프로그램에 대한 설계 및 앱 타머 - 금 나노 입자 기반 비색 분석법 개발

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Whitefly에서 Endosymbionts의 추출을위한 방법<em&gt; 베미 시아 타바시</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

모기에 소설 Insecticidal 화학 물질의 독성을 테스트 하기 위한 프로토콜

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

모델 식물 애기장대 의 Thigmomorphogenesis의 연구를 위한 노동 절약 및 반복 가능한 터치 포스 신호 돌연변이 스크린 프로토콜

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Finite Element Approach for Locating the Center of Resistance of Maxillary Teeth

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of teeth range from traditional radiographic and physical measurements to in vitro analysis on models or cadaver specimens. Techniques involving finite element analysis of high-dose micro-CT scans of models and single teeth have shown a lot of promise, but little has been done with newer, low-dose, and low resolution cone beam computed tomography (CBCT) images. Also, the CRES for only a few select teeth (i.e., maxillary central incisor, canine, and first molar) have been described; the rest have been largely ignored. There is also a need to describe the methodology of determining the CRES in detail, so that it becomes easy to replicate and build upon.
This study used routine CBCT patient images for developing tools and a workflow to obtain finite element models for locating the CRES of maxillary teeth. The CBCT volume images were manipulated to extract three-dimensional (3D) biological structures relevant in determining the CRES of the maxillary teeth by segmentation. The segmented objects were cleaned and converted into a virtual mesh made up tetrahedral (tet4) triangles having a maximum edge length of 1 mm with 3matic software. The models were further converted into a solid volumetric mesh of tetrahedrons with a maximum edge length of 1 mm for use in finite element analysis. The engineering software, Abaqus, was used to preprocess the models to create an assembly and set material properties, interaction conditions, boundary conditions, and load applications. The loads, when analyzed, simulated the stresses and strains on the system, aiding in locating the CRES. This study is the first step in accurate prediction of tooth movement.

This study outlines the necessary tools for utilizing low-dose three-dimensional cone beam-based patient images of the maxilla and maxillary teeth to obtain finite element models. These patient models are then used to accurately locate the CRES of all the maxillary teeth.

상악 치아의 저항 중심을 찾기위한 유한 요소 접근 법

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of ...