<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>

No conflicts of interest declared.

Using our agar-block set-up (Figure 1) Serpula lacrymans grew in direct contact with the gypsum surfaces and into wood blocks (Figure 2), leading to more than 60% weight loss in the control brown-rotted pine blocks (Figure 3). This easily satisfies the ASTM standard goal of &gt;50% decay, and the average coefficient of variation (CV) in decay at was 0.055 at week 16. This data is published in Schilling7. Again, other fungi will require longer incubation in agar-block than in soil-block. For reference, we have successfully run similar designs in several past experiments with a variety of other fungal species, often using scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS) and inductively coupled plasma spectroscopy (ICP-OES) to verify connectivity with exogenous substrates and translocation of calcium and other elements into wood8,9.
 In addition to low variability, there are two other results that our agar-block design reveals. First, there was a treatment effect (Figure 3), where our calcium additions, whether to the agar as CaCl2 or as pure gypsum, inhibited decay by this fungus. This is a useful result because others have theorized calcium enhances decay based on studies with real-world materials like mortar and plaster. Instead, we see decay rates rebound with the addition of iron to the gypsum, suggesting iron, not calcium, is key. The second useful result, however, is not quantified but is instead the color of the mycelia (Figure 4). There are significant and obvious melanization differences in external hyphae among treatments, and researchers have previously noted 'rust' melanization when encountering this fungus on building materials (eg. Lowet al. 2000). In our more recent studies, we found that these effects are lost using a high-iron medium (Figure 5). Collectively, it suggests that this fungus utilizes iron, not calcium, in these materials and that earlier studies, with 'rusty' mycelia reported, were observing iron effect, not calcium effect.
 Overall, these results and a lack of treatment effects using high-iron agar are a very strong demonstration of the control that the agar-block design can provide the researcher, especially in light of the alternative soil-block approach.

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<td>Petri dishes</td>
<td>&nbsp;</td>
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<td>4014</td>
<td>25 x 150 mm</td>
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<td>Agar, Type A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4550</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Millinckrodt</td>
<td>3436-12</td>
<td>&nbsp;</td>
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<td>Potassium phosphate, KH2PO4</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3246-01</td>
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<td>Magnesium sulfate 7-hydrate, MgSO4&bull;7H2O</td>
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<td>230391</td>
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<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
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<td>G8270</td>
<td>Dextrose</td>
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<td>Boric acid, H3BO4</td>
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<td>Mallinckrodt</td>
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<td>Copper(II) sulfate 5-hydrate, CuSO4&bull;5H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>209198</td>
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<td>Ammonium heptamolybdate 4-hydrate, (NH4)6Mo7O24&bull;4H2O</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431346</td>
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EtOH the surface</td>
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<td>BD Biosciences</td>
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<td>Gutter Guard</td>
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Hardware store</td>
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<td>Acros Organics</td>
<td>385355000</td>
<td>&nbsp;</td>
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<td>#4 cork borer</td>
<td>&nbsp;</td>
<td>Boekel</td>
<td>1601</td>
<td>&nbsp;</td>
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<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney</td>
<td>PM-996</td>
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agar-block microcosms for controlled plant tissue decomposition by aerobic fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

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

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

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

Nanyang Technological University

Research

JoVE Journal

Biology

15.0K Views.  University of Minnesota. This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

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.

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

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

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.

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.

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.

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.

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.

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).

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.

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