이것은 생산 및 실험 작업은 여기에 친절 리나 아귀레, 길버토 코르도바, 윌리엄 메라, Ximena Bonilla, 그리고 다리오 Viveros 지원 레이 날도 Pareja의 헌신적이고 열정적인 도움을 반영 제시. 우리는 또한 우리가 비디오와 원고를 향상 도움이 덧글에 대한 존 마일즈 세 익명 리뷰어 감사합니다.

1. 식물 <ol><li> 성숙한 식물의 단일 줄기 잘라는 실험 단위로 사용됩니다. 잘라는 심는 재료의 균일을 위해 10cm로 손질합니다. </li><li> 식물 병원균에 의해 오염을 방지하기 위해, 잘라 2 분 3 % 나트륨 차아 염소 산염의 용액에 씻은 후 수돗물로 씻어서 thoughroughly 있습니다. </li><li> 각각의 절단은 CA에 심어져 있습니다. 원통형 PVC 튜브에 무균 토양 36g는 (6.5 cm 높이의 직경 외부 5.3 ㎝) 스티로폼 컵에있는 낮은 끝에 봉인 및 PVC 부싱로 덮인. 절단은 부시의 중심 오프닝에 배치 거품 링에 의해 장소에서 개최됩니다. </li><li> 적절한 토양 수분을 유지하기 위해 필요에 따라 식물은 수정된하고 물결 무늬가있는 있습니다. </li><li> 식물은 뿌리 - 목부 수유의 spittlebug의 nymphs을위한 사이트를 먹이 역할을 외면 뿌리의 개발을 허용, 곤충에 의해 도전되기 전에 한 달 동안 성장을합니다. </li><li> 뿌리 잘라와 PVC 튜브는 부분적으로 루트 아키텍처 고유의 차이에 대한 조정, 더 이상 외면 뿌리의 성장을 촉진하기 전에 침입 8 D에 대한 반전됩니다. 이 단계는 우리가 24헥타르 일을 제공 빛의 인공​​ 소스를 필요로합니다. 이 역전의 치료 실험에서 제거 후 herbivore 저항과 혼란함을 주죠 herbivory에서 탈출을 피하기 위해 각각의 공장은 거의 또는 전혀 외면 뿌리로 복제합니다. 이 제거는 그들의 유전자형의 철폐로 이어지는 결코 드문 있어야합니다. </li></ol> 2. 곤충 <ol><li> 어른 spittlebugs는 현장에서 수집한 종을 발견하고, 그들에 대한 피드에 감염될 brachiariagrasses의 단풍을 제공 산란 연습장에 소개합니다. 여자들은 토양에 알을은 새장의 바닥에 놓인 얕은 이동식 트레이에 포함되어 있다고 믿는다. </li><li> 며칠 후, 흙은 물에 중단되고 계란을 수집 sieves 일련의 (42, 60 및 150 메쉬) 통과. 30 % 생리 식염수에 sieved 자료를 중지하면 미숙 계란 및 잔류 유기물 뒤에두고, 성숙한 난자가 부동하게됩니다. </li><li> 곤충 병원체의 오염을 방지하기 위해, 계란은 5 분 0.5 % 나트륨 차아 염소 산염의 용액에 소독 후 증류수로 씻어서 있습니다. </li><li> 각각의 시험 공장 (실험 장치) 6 성숙 난자로 토양 표면에 감염됩니다. 부화에 가까운만이 성숙한 난자가 infestations 위해 선택됩니다. 곤충의 복부에 대응하는 자신의 사후 부분에 핑크색 표시,,, 그리고 완벽하게 확장 operculum 그들은 두 개의 빨간 곤충의 눈을에 대응하는 자신의 앞쪽에 부분 점의 존재에 의해 확인할 수 있습니다. </li><li> 성공적인 부화은 어떤 안깐 계란은 우리의 식민지에서 neonate spittlebug으로 대체되는 시점에서 4 D 나중에 확인됩니다. </li><li> 테스트 식물은 유전자형 최대 6 복제와 함께 무작위로 전체 블록 설계에 정렬됩니다. </li></ol> 3. 평가 <ol><li> 실험은 CA를 평가합니다. 침입 후 30 D (정확한 시간은 공부 벌레 종류에 따라 다릅니다.) </li><li> 피해 1 눈에 띄는 손상과 죽은 식물 (그림 1) 5에 해당에 해당하는 1-5 시각적 척도에 점수입니다. 식물의 피해를 채점하는 것은 내성을 예측할 수 있습니다. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="그림 1" /> Brachiaria 종의 spittlebug herbivory에 내성을 평가하기 위해 사용 그림 1. 피해 규모. 1 = 없음 감지 손상, 2 = 0~25% 괴사성 단풍, 3 = 25-50% 괴사성 단풍, 4 = 50~75% 괴사성 단풍, 5 = 75-100% 괴사성 단풍. <ol start="3"><li> 곤충 생존들은 최종 instar 또는 성인 무대 (그림 2) 중 하나를 도달하는 곤충을 계산하여 평가합니다. 곤충 생존을위한 채점하는 것은 항생의 예측을 제공합니다. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="그림 2" /> 첫 instar (왼쪽)에서 성인 (오른쪽)에 그림 2. Spittlebug 개발. 평가 날짜 적어도 최종 instar을 (화살표)에 도달만이 벌레가로 계산됩니다 &quot;생존자.&quot; 4. 대표 결과 spittlebug Aeneolamia reducta에 대한 저항을위한 대표적인 분석 심사 Brachiaria의 하이브리드의 결과는 그림 3에 제공됩니다. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="그림 3" /> 그림 3. spittlebug Aeneolamia reducta에 저항 Brachiaria의 하이브리드를 검사의 결과. herbivory 자신의 응답이 음모의 절반 아랫부분에 일인 경우 우리는 식물이 저항하는 것이 좋습니다. 오른쪽에서 왼쪽 및 관용 위에서 아래로 증가 항생 증가. 화살표 우리 내성 검사에서 가리 킵니다.

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관심 없음 충돌 선언하지 않습니다.

herbivore 저항 메커니즘 간의 차별하는 것은 작물 번식 노력 3 밝게하실 수 있습니다. 강력한 항생에 따라 저항이 어떤 상황에서보다 적극적인 해충 biotypes에 대한 선택 압력을 만들 수 있습니다. 한편, 관용에 따라 저항은 오차가 결국 압도 때까지 해충 인구가 증가 허용할 수 있습니다. 튼튼한 저항 번식, 따라서 어떤 저항 메커니즘은 특정 작물 / 해충 시스템에 대한 대부분의 안정성을 부여 것입니다 [케네디 외 볼의 신중하게 고려해야합니다. (1987) 주제] 3 종합 토론. 이 비디오 데모는 brachiariagrasses 6-9에서 spittlebug 저항하는 항생과 관용의 상대적인 기여도를 평가하기위한 몇 가지 이전의 연구에 빌드. 선택 시험 또는 현장 실험과 대조적으로, 노 - 선택 시험 따라서 herbivore 행동 (예, 호스트 환경 설정, 집합)에서 발생하는 차이에 대한 제어를 제공하는 모든 식물이 같은 herbivore 압력을 받게 보장합니다. 이러한 이유로, 그러나, - 선택 시험은 antixenosis에 의해 저항을 평가하기위한 적합하지 않습니다. 인위적으로 곤충 식민지를 양육하는 것은 저항 심사 assays 10 테스트 곤충을 얻기 위해 선호하는 방법입니다. 식민지은 연중 11 시간을 실시하는 실험 알려진 시대의 spittlebugs의 신뢰할 수와 유니폼 소스를 제공합니다. 그것은, 그러나, 정기적으로 식민지 관련 현장 인구 10 유전 이탈하지 않도록하기 위해 식민지에 야생 개인 달이다해야 할 수도 있습니다. 이러한 인위적 - reared herbivores의 적합성을 평가하고, 시간이 지남에 따라 자신의 공격성에 가능한 변화를 모니터링하기 위해서는 모든 검사는 알려진 저항 수준의 적절한 검사를 포함하는 것이 중요합니다. 여러 개의 매개 변수는 신중하게 적절한 저항 심사 분석을 설계 정의해야합니다. 우리가 테스트를 설계하는 생각이 몇 가지 주요 매개 변수는 호스트 식물의 나이, 종 및 spittlebugs의 발달 단계, 침입 수준 및 식물 곤충 담당자 8,9,12 기간을 포함합니다. 실험 및 프로토콜 세련의 여러 반올림은 적절하게 현장에서 herbivore 저항을 예측 신속하고 비용 효율, 신뢰성, 분석에 도달하는 데 필요한 수 있습니다.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

antixenosis, 항생 및 관용 1 : 식물 세 확장 메커니즘을 통해 herbivore 손상에 저항하실 수 있습니다. Antixenosis은 herbivore 다른 식물에게 2를 선택할 수있을 때 발전소를 피할 수있다 정도입니다. 항생은 식물이 1 먹이 herbivore의 적합성에 영향을 미치는 정도입니다. 공차가 공장이 herbivore의 성장과 재생산 1 손상시키지 않고 견딜 또는 herbivore으로 인한 손상을 복구할 수있는 정도입니다. 농업 환경에서 herbivore 저항의 내구성은 농작물 번식 노력 셋 중에 선호하는 저항 메커니즘에 큰 정도에 따라 달라집니다. 우리는 Brachiaria 종에 spittlebug 저항하는 항생과 관용의 상대적인 기여도를 추정하기위한 노 선택 실험을 보여줍니다. 속 Brachiaria의 아프리카 목초의 여러 종의 Neotropics에 귀중한 마초와 파스투레 식물, 그러나 그들이 심각 spittlebugs 여러 토착 종 (Hemiptera : Cercopidae)에 의해 도전을하실 수 있습니다. spittlebugs들의 저항을 평가 4, 식물 vegetatively - 전달됩니다 줄기 잘라 의해 및 spittlebugs는 피드 수있는 피상적인 뿌리의 성장을 허용, 약 한 달 동안 성장을 수있었습니다. 그 시점에서, 각 시험 공장은 개별적으로 부화 근처 여섯 spittlebug 달걀과 도전이다. Infestations는 공장 손상과 곤충의 생존을 평가하기 전에 한 달 동안 진행하기 위해 사용할 수 있습니다. 곤충의 생존을 득점하는 것은 항생의 추정치를 제공하면서 설비의 손상을 득점하는 것은 내성을 예측할 수 있습니다. 이 프로토콜은 상업 brachiariagrases 5 spittlebug 저항을 강화하기 위해 식물 번식 목표를 촉진하고 있습니다.

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이 동영상은 herbivory에 호스트 식물 저항의 메커니즘을 설명 및 저항을 spittlebug하기 위해 항생과 관용의 상대적인 기여도를 추정 노 선택 테스트를 보여줍니다<em&gt; Brachiaria</em&gt; 종.

특성화 Herbivore 저항 메커니즘 : Spittlebugs에 Brachiaria 종. 예로

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

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

13.7K 조회수.  CIAT. 이 동영상은 herbivory에 호스트 식물 저항의 메커니즘을 설명 및 저항을 spittlebug하기 위해 항생과 관용의 상대적인 기여도를 추정 노 선택 테스트를 보여줍니다 Brachiaria 종.

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

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

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생물학

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Herbivore 유도 블루베리 휘발성 물질과 내부 식물 신호

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

삼투 물 투수 계수 (P 측정<sub&gt; F</sub&gt;) 구형 세포 : 예를 들어 식물 절연 원형질체

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

이미징 FITC-dextran 규제 Exocytosis 위한 기자로 서

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM 마이크로코일 성능 교정 및 사용 에 설명 된 메디카고 트런카툴라 뿌리에 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

바이러스 감염시 m6A 및 m5C 에피레미테롬 탐험: HIV를 가진 예

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

원자력 현미경을 사용한 살아있는 식물 장기의 일차 세포벽의 기계적 특성 특성 분석

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Rickettsia spp.를 Tick 세포주에서 형광 단백질 발현 셔틀 벡터로 형질전환하기 위한 전기천공 방법

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay(FluoZin-3 분석을 통한 포유류 세포의 아연 수송 메커니즘 조사)

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

저자 스포트라이트: ZnT1 기능을 통한 세포 아연 조절 탐구 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

In Vitro Zinc Transport Assay를 사용한 포유류 아연 수송체 특성 분석

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...