Name: the use of chemostats in microbial systems biology
Uploaded: 2013-10-14T20:45:00
Description: Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

Questo lavoro è stato sostenuto da start up fondi dalla New York University. Ringraziamo Maitreya Dunham e Matt Brauer che ha inizialmente sviluppato l&#39;uso di bioreattori Sixfors come chemostati.

Il principio di coltura continuo utilizzando un chemostat può essere realizzato in una varietà di implementazioni. In tutti chemostati è essenziale per avere 1) metodi per mantenere la sterilità di tutti i componenti, 2) una cultura ben miscelato, 3) aerazione adeguata del recipiente di coltura e 4) in modo affidabile Inoltre media e rimozione cultura. Qui, descriviamo l&#39;uso di un bioreattore Sixfors (Infors Inc) come chemostat utilizzando metodi che possono essere facilmente adattato alle configurazioni alterna...

<ol>
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Chemostati permettono la coltivazione di microbi in condizioni di steady-state di crescita controllata. Le cellule crescono continuamente ad una velocità costante risultante in un ambiente esterno invariante. Questo è in contrasto con i metodi di coltura batch in cui l&#39;ambiente esterno è in continua evoluzione e il tasso di crescita delle cellule è determinata dalla complessa interazione di ambiente e genotipo. Pertanto, un vantaggio importante di coltura microbi in chemostati oltre colture in lotti è la capaci...

La crescita delle cellule è regolata da complesse reti di interazione dei fattori genetici e ambientali 1,2. La regolazione multifattoriale della crescita cellulare richiede un approccio a livello di sistema allo studio. Tuttavia, lo studio rigoroso della crescita cellulare regolamentato è contestata dalla difficoltà di controllare sperimentalmente la velocità con cui le cellule crescono. Inoltre, anche nelle condizioni più semplici esperimenti extracellulari sono frequentemente dinamico e complesso come cellule alterano continuamente il loro ambiente mentre proliferano. Una soluzione a questi problemi è fornita dal chemostat: un metodo di coltura di cellule che consente il controllo sperimentale dei tassi di crescita delle cellule in ambienti definiti, invarianti e controllati. Il metodo di coltura continuo utilizzando un chemostat è stato descritto in modo indipendente da Monod 3 e Novick &amp; Szilard 4 nel 1950. Come originariamente concepito, le cellule sono coltivate in un volume fisso di supporto che è constantemente diluita con l&#39;aggiunta di nuovi media e la rimozione simultanea di vecchi media e cellule (Figura 1). Accoppiate equazioni differenziali ordinarie (Figura 2) descrivono il tasso di variazione della densità delle cellule (x) e la concentrazione di una sostanza nutritiva (s) crescita limitativo nel recipiente chemostat. È importante sottolineare che questo sistema di equazioni prevede un unico (diverso da zero) stabile allo stato stazionario (Figura 3) con l&#39;implicazione notevole che allo stato stazionario, il tasso di crescita specifico delle cellule (cioè il costante tasso di crescita esponenziale), è pari al tasso alla quale la coltura è diluito (D). Variando la velocità di diluizione è possibile stabilire popolazioni steady-state di cellule a tassi di crescita diversi e in diverse condizioni di scarsità di nutrienti. Il controllo sperimentale del tasso di crescita utilizzando chemostati è stato fondamentale per lo sviluppo di una comprensione di come i cambiamenti fisiologia cellularecon tassi di crescita 5,6. Tuttavia, questo ex pilastro dei metodi microbiologici divenne sempre più oscuro durante l&#39;esplosione nella ricerca di biologia molecolare nel corso del tardo ventesimo secolo. Oggi, rinnovato interesse nel controllo della crescita in entrambi i microbi e organismi multicellulari e l&#39;avvento di metodi genoma scala per analisi a livello di sistema ha rinnovato motivazione per l&#39;uso di chemostati. Qui, descriviamo tre applicazioni che capitalizzano il controllo preciso dei tassi di crescita delle cellule e l&#39;ambiente esterno che sono unicamente possibile con chemostati. In primo luogo, si descrive l&#39;uso di chemostati per studiare come l&#39;abbondanza di migliaia di biomolecole - come trascrizioni e metaboliti - sono coordinatamente regolato con il tasso di crescita. In secondo luogo, si descrive come chemostati possono essere utilizzati per ottenere stime precise delle differenze di tasso di crescita tra i diversi genotipi in ambienti nutrienti limitata con esperimenti di competizione. In terzo luogo, si descrive come chemostati puòessere usato per studiare l&#39;evoluzione adattativa di cellule che crescono in ambienti poveri di nutrienti costanti. Questi esempi esemplificano i modi in cui i chemostati stanno consentendo indagini sistemi a livello di regolazione della crescita delle cellule, geni da interazioni ambientali e l&#39;evoluzione adattativa.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Infors-HT Sixfors Chemostat</td>
			<td>Appropriate Technical Resources, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Bottle 9.5 L</td>
			<td>Fisher Scientific</td>
			<td>02-887-1</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Pinchcock</td>
			<td>Fisher Scientific</td>
			<td>05-867</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Stopper, Size 12, Green Neoprene</td>
			<td>Cole-Palmer</td>
			<td>EW-62991-42</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Straight Connector</td>
			<td>Cole-Palmer</td>
			<td>EW-30703-02</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>General purpose ties 4 in</td>
			<td>Fisher Scientific</td>
			<td>NC9557052</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone Rubber</td>
			<td>Small Parts</td>
			<td>B000FMWTDE</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone, 3/8 in OD</td>
			<td>Fisher Scientific</td>
			<td>02-587-1Q</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone, 7/32 in OD</td>
			<td>Fisher Scientific</td>
			<td>02-587-1E</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Stainless Steel, 3/16 in OD</td>
			<td>McMaster-Carr</td>
			<td>6100K164</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Stainless Steel, 3/8 in OD</td>
			<td>McMaster-Carr</td>
			<td>6100K161</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Hook Connectors</td>
			<td>Fisher Scientific</td>
			<td>14-66-18Q</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Ratchet Clamp</td>
			<td>Cole-Palmer</td>
			<td>EW-06403-11</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Luer, Female</td>
			<td>Cole-Palmer</td>
			<td>EW-45512-34</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Luer, Male</td>
			<td>Cole-Palmer</td>
			<td>EW-45513-04</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Millipore Aervent MTGR05010 62 mm Filter, 0.2 &mu;m</td>
			<td>Fisher Scientific</td>
			<td>MTGR05010</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>PTFE Acrodisc CR 13 mm filters, 0.2 &mu;m</td>
			<td>Fisher Scientific</td>
			<td>NC9131037</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Direct-Reading Flowtube for Air</td>
			<td>Cole-Palmer</td>
			<td>EW-32047-77</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Direct-Reading Flowtube for Nitrogen</td>
			<td>Cole-Palmer</td>
			<td>EW-32048-63</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Gas Proportioner Multitube Frames</td>
			<td>Cole-Palmer</td>
			<td>EW-03218-50</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Regulator, Two-Stage Analytical</td>
			<td>Airgas</td>
			<td>Y12-N145D580</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Hose Adaptor, Stainless Steel</td>
			<td>Airgas</td>
			<td>Y99-26450</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Hose Male Adaptor</td>
			<td>Airgas</td>
			<td>WES544</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Norprene Tubing</td>
			<td>US Plastics</td>
			<td>57280</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Tripod Base</td>
			<td>Cole-Palmer</td>
			<td>EW-03218-58</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Valve Cartridges</td>
			<td>Cole-Palmer</td>
			<td>EW-03217-92</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Carboy 10 L</td>
			<td>Fisher Scientific</td>
			<td>02-963-2A</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>Steritop Sterile Vacuum Bottle-Top Filters, 1,000 ml, PES membrane; for 45 mm neck size</td>
			<td>Fisher Scientific</td>
			<td>SCGP-T10-RE</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>Media Bottle 100 ml, 45 mm neck size</td>
			<td>Fisher Scientific</td>
			<td>FB-800-100</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>calcium chloride&middot;2H2O</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>magnesium sulfate&middot;7H2O</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>AC424205000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>AC423400010</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>copper sulfate&middot;5H2O</td>
			<td>Sigma Aldrich</td>
			<td>209198</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium iodide</td>
			<td>Sigma Aldrich</td>
			<td>60400</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>ferric chloride&middot;6H2O</td>
			<td>Fisher Scientific</td>
			<td>I88-100</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>manganese sulfate&middot;H2O</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>sodium molybdate&middot;2H2O</td>
			<td>Sigma Aldrich</td>
			<td>M7634</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>zinc sulfate&middot;7H2O</td>
			<td>Fisher Scientific</td>
			<td>Z68-500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>biotin</td>
			<td>Fisher Scientific</td>
			<td>BP232-1</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>calcium pantothenate</td>
			<td>Fisher Scientific</td>
			<td>AC24330-1000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>folic acid</td>
			<td>Sigma Aldrich</td>
			<td>F7876</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>inositol (aka myo-inositol)</td>
			<td>Fisher Scientific</td>
			<td>AC12226-1000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>niacin (aka nicotinic acid)</td>
			<td>Sigma Aldrich</td>
			<td>N4126</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>p-aminobenzoic acid</td>
			<td>Fisher Scientific</td>
			<td>AC14621-2500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>pyridoxine HCl</td>
			<td>Sigma Aldrich</td>
			<td>P9755</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>riboflavin</td>
			<td>Sigma Aldrich</td>
			<td>R4500-25G</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>thiamine HCl</td>
			<td>Fisher Scientific</td>
			<td>BP892-100</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Sigma Aldrich</td>
			<td>L8000-100G</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma Aldrich</td>
			<td>U0750</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>DF0155-08-5</td>
			<td>Media Reagents</td>
		</tr>
	</tbody>
</table>

the use of chemostats in microbial systems biology

Le cellule regolano il loro tasso di crescita in risposta a segnali dal mondo esterno. Con la crescita della cellula, diversi processi cellulari devono essere coordinati tra cui la sintesi macromolecolare, metabolismo e infine, l&#39;impegno per il ciclo di divisione cellulare. Il chemostato, un metodo per controllare sperimentalmente tasso di crescita cellulare, fornisce un potente mezzo di sistematicamente studiare come impatti tasso di crescita processi cellulari - tra l&#39;espressione genica e del metabolismo - e le reti di regolazione che controllano il tasso di crescita delle cellule. Quando mantenuta per centinaia di generazioni chemostati può essere usato per studiare l&#39;evoluzione adattativa di microbi in condizioni ambientali che limitano la crescita cellulare. Descriviamo il principio di culture chemostato, dimostriamo loro funzionamento e fornire esempi delle loro varie applicazioni. Dopo un periodo di inattività dopo la loro introduzione a metà del XX secolo, la convergenza delle metodologie genoma scala con una rinnovata nelteresse nella regolazione della crescita cellulare e la base molecolare della evoluzione adattativa è stimolare una rinascita nell&#39;uso di chemostati nella ricerca biologica.

Uno dei principali vantaggi di chemostati è la capacità di controllare la velocità di crescita delle cellule sperimentalmente variando il grado di diluizione. Nel lievito erba, Saccharomyces cerevisiae, la morfologia di una cellula è informativo della fase del ciclo di divisione cellulare. Popolazioni con tassi di crescita contengono una percentuale maggiore di divisione attivamente cellule come determinato misurando la frazione di cellule unbudded (Figura 5A). Analisi di espressione mRNA globale di...

Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

The Use of Chemostats in Microbial Systems Biology

Il tasso di crescita delle cellule è un processo regolato e un determinante fondamentale della fisiologia cellulare. Coltura continua utilizzando chemostati consente il controllo estrinseco di crescita cellulare mediante limitazione nutritiva facilitare lo studio delle reti molecolari che controllano la crescita cellulare e come tali reti evolvono per ottimizzare la crescita cellulare.

L&#39;uso di chemostati in Biologia microbica Sistemi

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

the-use-of-chemostats-in-microbial-systems-biology

Research

JoVE Journal

Biology

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Indagare la comunità microbica nel hindgut Termite - Intervista

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Ricerca

Biologia

Microbial Communities in Nature and Laboratory - Interview

Comunità microbica in natura e laboratorio - Intervista

Biology of Microbial Communities  - Interview

Biologia delle comunità microbiche - Intervista

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Utilizzando Micro-Electro-Mechanical Systems (MEMS) per sviluppare strumenti diagnostici

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

L&#39;uso del Biofeedback in Clinica Realtà Virtuale: Il Progetto INTREPID

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Finestra su un micromondo: Sistemi Microfluidic semplice per studiare il trasporto microbica in mezzi porosi

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

Determinare il contributo dei sistemi di energia durante l&#39;allenamento

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

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.

Collegamento rischio di predazione, stress Herbivore fisiologica e decomposizione microbica dei Rifiuti vegetali

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

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...