<ol>
	<li>ASTM D1413-07. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+test+method+for+testing+wood+preservatives+by+laboratory+soil-block+cultures.+.">Standard test method for testing wood preservatives by laboratory soil-block cultures. .</a> Annual Book of ASTM Standards. , 185-192 (2007).</li><li>Bravery, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A miniaturized wood block for the rapid evaluation of wood preservative fungicides. , (1978).</li><li>Low, G. A., Young, M. E., Martin, P., Palfreyman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+relationship+between+the+dry+rot+fungus+Serpula+lacrymans+and+selected+forms+of+masonry.">Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry.</a> Int. Biodeterior. Biodegrad. 46, 141-150 (2000).</li><li>Nicolas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+I+(One/1)+-+Degradation+and+Protection+of+Wood+(Syracuse+Wood+Science+Series+#5).">Volume I (One/1) - Degradation and Protection of Wood (Syracuse Wood Science Series #5).</a> Wood Deterioration and Its Prevention by Preservative Treatments. , (1973).</li><li>Schilling, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+calcium-based+materials+and+iron+impurities+on+wood+degradation+by+the+brown+rot+fungus+Serpula+lacrymans.">Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus Serpula lacrymans.</a> Holzforschung. 64, 93-99 (2010).</li></ol>

אין ניגודי אינטרסים הכריז.

באמצעות אגר לחסום שלנו הגדרת (איור 1) Serpula lacrymans גדל במגע ישיר עם משטחי גבס לתוך גושי עץ (איור 2), מובילה לאובדן יותר מ 60% במשקל של חום נרקב לשלוט בלוקים אורן (איור 3 ). זה בקלות עונה על המטרה תקן ASTM של ריקבון&gt; 50%, ואת הממוצע של מקדם השונות (C V) ב ריקבון בבית היה 0.055 ב?...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>4014</td>
<td>25 x 150 mm</td>
</tr>
<tr>
<td>Agar, Type A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4550</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium nitrate, NH4NO3</td>
<td>&nbsp;</td>
<td>Millinckrodt</td>
<td>3436-12</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate, KH2PO4</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3246-01</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate 7-hydrate, MgSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>230391</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>Dextrose</td>
</tr>
<tr>
<td>Boric acid, H3BO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>2549-04</td>
<td>&nbsp;</td>
<tr>
<td>Zinc sulfate 7-hydrate, ZnSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>8880-12</td>
<td>&nbsp;</td>
<tr>
<td>Manganous chloride 4-hydrate, MnCl2&bull;4H2O</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>2540-04</td>
<td>&nbsp;</td>
<tr>
<td>Copper(II) sulfate 5-hydrate, CuSO4&bull;5H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>209198</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium heptamolybdate 4-hydrate, (NH4)6Mo7O24&bull;4H2O</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431346</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride dihydrate, CaCl2&bull;2H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>4160-12</td>
<td>&nbsp;</td>
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<td>Sodium chloride, NaCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>7581-12</td>
<td>&nbsp;</td>
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<td>Ferrous sulfate 7-hydrate, FeSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>5056-12</td>
<td>&nbsp;</td>
<tr>
<td>Pipet-aid</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>4-000-110</td>
<td>Cordless 
EtOH the surface</td>
</tr>
<tr>
<td>10 ml sterile polystyrene pipette</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>Gutter Guard</td>
<td>&nbsp;</td>
<td>Thermwell Products Co.</td>
<td>VX620</td>
<td>Pre-scrubbed with soap 
Hardware store</td>
</tr>
<tr>
<td>Calcium sulfate hemihydrate, CaSO4&bull;0.5H2O</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>385355000</td>
<td>&nbsp;</td>
<tr>
<td>#4 cork borer</td>
<td>&nbsp;</td>
<td>Boekel</td>
<td>1601</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney</td>
<td>PM-996</td>
<td>&nbsp;</td>		</tbody>
		</table>
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agar-block microcosms for controlled plant tissue decomposition by aerobic fungi

שתי השיטות העיקריות לחקר ביודגרדציה פטרייתי של רקמות הצמח lignocellulosic פותחו עבור בדיקות משמר עץ (אדמה לחסום; אגר בלוק). זה טוב כי קיבל אדמה לחסום מיקרוקוסמוס להניב שיעורי עששת גבוה יותר, פחות בעיות רטיבות, השתנות נמוך יותר בקרב מחקרים, ועל סף גבוה יותר של רעילות חומר משמר. אדמה היא לחסום בדיקות ובכך הטכניקה מנוצל יותר כבר טופל על ידי האגודה האמריקאית לבדיקות וחומרים (ASTM) (שיטה D 1413-1407). העיצוב אדמה לחסום יש חסרונות, לעומת זאת, שימוש במקורות אדמה מקומית משתנה וגם להגביל את השליטה של ​​חומרים מזינים חיצוני (אקסוגני) לרקמות מתפורר. חסרונות אלה התפתחו כמו בעיה ביישום שיטה זו, מטרות אחרות מחקר פופולרי יותר ויותר. אלה מטרות המודרני כוללים lignocellulosics משפיל למחקר Bioenergy, בדיקות bioremediation של שיתוף מטבוליזם רעלים, הערכת המנגנונים חמצוני, ועוד אלמנטים translocated מעקב לאורך רשתות hyphal. קרקע, בלוקים לא להשאיל לשלוט מספיק ביישומים אלה. גישה מעודן אגר לחסום הכרחי. כאן, אנו משתמשים ריקבון חום עץ משפיל פטרייה Serpula lacrymans להשפיל עץ אגר לחסום מיקרוקוסמוס, באמצעות עמוק צלחות פטרי עם אגר סידן נמוכה. אנחנו בודקים את התפקיד של אקסוגניים גבס על ריקבון בסדרה פעמי, כדי להדגים את השירות ואת השתנות צפוי. רחובות אחד לוח לקרוע (חתך אורך) מותנים, שקל, autoclaved, והציג בסביבה נקייה מחיידקים, על גבי רשת פלסטיק. חיסונים פטרייתיים הם על הפנים כל בלוק, עם גבס אקסוגניים נוסף על ממשקים. היבולים הם aseptic עד הקציר הרסני הסופי. מיקרוקוסמוס אלה נועדו לחסום להימנע ממגע עם אגר או קירות פטרי צלחת. עיבוי ממוזער במהלך צלחת שופך במהלך הדגירה. לבסוף, inoculum / גבס / עץ המרווח ממוזער אך מבלי לאפשר קשר. אלה ההיבטים הטכניים של עיצוב פחות אגר לחסום גם הם הגורמים השכיחים ביותר של כישלון המקור המפתח של השתנות בין המחקרים. פרסום וידאו ולכן הוא שימושי במקרה הזה, ואנחנו מראים השתנות נמוך, תוצאות באיכות גבוהה.

Watch this Scientific Journal Video about Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi at JoVE.com

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

וידאו זה מדגים גישה בסביבה מבוקרת ללמוד השפלה של רקמות הצמח lignocellulosic ידי פטריות אירובית. היכולת לשלוט במקורות מזין לחות היא יתרון המפתח של אגר, לחסום מיקרוקוסמוס, אך הגישה לרוב מניב הצלחה מעורבת. אנו כתובת החסרונות קריטי תשואה לשחזור, השתנות נמוך התוצאות.

אגר-Block מיקרוקוסמוס של פירוק רקמות הצמח מבוקרת על ידי פטריות אירובית

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

agar-block-microcosms-for-controlled-plant-tissue-decomposition

Research

JoVE Journal

Biology

15.0K Views.  University of Minnesota. וידאו זה מדגים גישה בסביבה מבוקרת ללמוד השפלה של רקמות הצמח lignocellulosic ידי פטריות אירובית. היכולת לשלוט במקורות מזין לחות היא יתרון המפתח של אגר, לחסום מיקרוקוסמוס, אך הגישה לרוב מניב הצלחה מעורבת. אנו כתובת החסרונות קריטי תשואה לשחזור, השתנות נמוך התוצאות.

Video: Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

בדיקת חסמים פיזיולוגיים להעברת נגיפי ב כנימות שימוש Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virus-Induced להשתקת גנים (VIGS) ב ניקוטיאנה benthamiana עגבניות

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

זיהוי של פנוטיפים עיכוב גדילה מושרה על ידי הבעת effectors סוג חיידקית השלישי שמרים

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

קישור סיכוני טריפה, מתח פיסיולוגי אוכלי עשב ופירוק מיקרוביאלית המלטת צמח

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

במעקב 4 ממדי Vivo של תאי גזע ואבות היווצרות דם במבוגרי עכבר calvarial מח עצם

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

CRISPR וקטור בינוי אפיון תפוקה גבוהה של שינויי DNA על ידי דור של שורשי עגבניות שעירים

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

-Cut ההיברידי: שיטת חתך משופרת עבור דוגמאות רקמות צמח סרבן

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

הפעלת Autophagy ידי פעילות גופנית אירובית בעכברים

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.