The authors would like to acknowledge Marcos Di Falco for his expertise and advice with the preparation of the samples for nano-LC MS/MS as well as Dr. Zoran Minic from the University of Regina for the LC MS/MS analysis. This work was supported by NSERC (DG402214-2011) and CRC (950-221184) funding. D.C. was supported by Concordia Institute for Water, Energy, and Sustainable Systems and FQRNT.

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The authors have nothing to disclose.

الحفاظ على عينة هو المفتاح لدراسات metaproteomic وأظهرت الأعمال السابقة أن الحل استقرار RNA هو عازلة تخزين مفيد لتخزين الخلايا قبل البروتين استخراج 28. من الناحية المثالية، سوف يتم الحفاظ على عينات في الموقع لنفي التحولات في تعبير البروتين أثناء التعامل مع 33،34....

الكائنات الحية الدقيقة في كل مكان وتلعب أدوارا أساسية في دورات الأرض البيولوجية الكيميائية 1. حاليا، هناك العديد من المناهج الجزيئية المتاحة لتحديد خصائص بنية المجتمع الميكروبي والوظيفة. الأكثر شيوعا هو تحليل 16S الريباسي الجينات تسلسل PCR تضخيم من الحمض النووي البيئية 2-4. عيوب تحليل الجينات 16S الريباسي هو أنه لا يقدم سوى معلومات عن هوية النشوء والتطور وبنية المجتمع، مع القليل من المعلومات عن وظيفة التمثيل الغذائي. في المقابل، تقترب مثل metagenomics، metatranscriptomics وmetaproteomics تقديم معلومات عن بنية المجتمع والتمثيل الغذائي. توفر Metagenomics، أو تحليل المحتوى الجيني لفيف من الكائنات الحية، معلومات عن هيكل والإمكانات الوظيفية للمجتمع 5-8. وعلى الرغم من قوة، وهذه الإمكانات الوظيفية قد لا تتوافق مع الأيضيةأنشطة الكائنات الحية. ويمثل النمط الوراثي للكائن التي كتبها جيناته، كل منها يمكن نسخها إلى RNA وكذلك ترجمت إلى البروتين، مما أدى إلى النمط الظاهري. وهكذا، للمساعدة في فهم النشاط الوظيفي الميكروبي في بيئة وتحليل ما بعد الجينوم يجب أن يتم تنفيذ 9. Metatranscriptomics، أو تحليل النصوص RNA هو مفيد لأنه يكشف الجينات التي تم نسخها في أي بيئة معينة. ومع ذلك، مستويات مرنا لا تتطابق دائما مستويات البروتين يناظرها بسبب تنظيم متعدية، RNA نصف الحياة، وحقيقة أن نسخ متعددة من البروتين يمكن أن تتولد عن كل مرنا 10. لهذه الأسباب metaproteomics ومن المسلم به الآن كأداة هامة لعلم الأحياء الدقيقة البيئي. يحلل metaproteomic شيوعا استخدام نهج البروتين طلقات نارية حيث يتم تنقية كاملة القريب من البروتينات في عينة المعقدة وتحليلها في وقت واحد، وعادة من خلال ENالهضم zymatic إلى ببتيدات والتحليل على مطياف الكتلة. يستخدم لاحق جنبا إلى جنب مطياف الكتلة (MS / MS) &quot;البصمات الببتيد&quot; لتحديد تسلسل الببتيد والمحتملين البروتين من أصل بواسطة قاعدة بيانات البروتين البحث (لاستعراض رؤية 11). لقد حان عمل البروتين شوطا طويلا في السنوات ال 25 الماضية بفضل الزيادة في توافر البيانات الجينومية وزيادة حساسية ودقة الطيف الجماعية والسماح لتحديد البروتين الإنتاجية العالية وتقدير 11،12. منذ البروتينات هي المنتج النهائي في التعبير الجيني، يمكن للبيانات metaproteomic تساعد في تحديد الكائنات التي تنشط في أي بيئة معينة وما البروتينات فإنهم يعبرون. هذا هو المفيد عند محاولة تحديد كيف يمكن لمجموعة معينة من المتغيرات البيئية سوف تؤثر على النمط الظاهري للكائن الحي أو المجتمع. في وقت مبكر، واستخدمت دراسات metaproteomic يستند إلى MS-MS / في المحيط لتحديد بروتينات معينة في المستهدفةالأنساب الميكروبية، مع أول دراسة تركز على ضوء مدفوعة مضخة البروتون proteorhodopsin في البكتيريا البحرية SAR11 13. وفي الآونة الأخيرة، توضيح التحليلات metaproteomic المقارنة التفاضلية أنماط التعبير البروتين بين المجتمعات المعقدة. ومن الأمثلة على ذلك تحديد التحولات الزمنية في عملية التمثيل الغذائي في المناطق الساحلية شمال غرب المحيط الأطلسي 14 أو شبه الجزيرة القطبية الجنوبية 5. وقد وصفت دراسات أخرى الاختلافات في أنماط التعبير البروتين عبر النطاقات المكانية، على سبيل المثال، على طول قطاع من الجغرافي من تلفيف المحيط منخفضة في المغذيات إلى نظام الموجات المتقلبة الساحلي مثمرة للغاية (15). للحصول على مزيد من الآراء metaproteomics نوصي شنايدر وآخرون (2010) 9 ويليامز وآخرون (2014) 16. كما تم توظيف البروتينات المستهدفة في السنوات الأخيرة لتحديد التعبير من المسارات الأيضية محددة في البيئة 17،18. ذره هي ثلاث مراحل رئيسية في التحليل metaproteomic. المرحلة الأولى هي إعداد نموذج، والذي يتضمن جمع العينات، تحلل الخلية وتركيز البروتين. جمع العينات في علم الأحياء المجهرية البحرية غالبا ما ينطوي على ترشيح مياه البحر من خلال ما قبل التصفية لإزالة الخلايا حقيقية النواة أكبر والجزيئات والبكتيريا المرتبطة الجسيمات، تليها الترشيح للقبض على الخلايا الميكروبية تعيش حرة، عادة مع استخدام خرطوشة 0.22 ميكرومتر وحدة تصفية 19،20. وincased هذه المرشحات في اسطوانة بلاستيكية وسيكون لتحلل الخلايا والبروتين بروتوكول الاستخراج التي يمكن القيام بها داخل وحدة مرشح أن يكون أداة قيمة. مرة واحدة يتم الحصول على الكتلة الحيوية، ويجب أن يكون هي lysed الخلايا للسماح لاستخراج البروتين. يمكن استخدام العديد من الطرق، بما في ذلك غوانيدين، حمض الهيدروكلوريك تحلل 21 والصوديوم دوديسيل كبريتات (SDS) وسائل تحلل المستندة. على الرغم من المنظفات مثل SDS هي فعالة جدا في تعطيل الأغشية والانحلال العديد من أنواع البروتين، concentratiإضافات منخفضة كما يمكن أن تتداخل مع 0.1٪ المصب الهضم من البروتين وتحليل MS 22. قلق كبير من الآثار السلبية للSDS على كفاءة الهضم التربسين، حل السلطة من العكسي اللوني المرحلة السائلة والأيونات قمع أو تراكم داخل المصدر أيون 23. المرحلة الثانية هي تجزئة وتحليل، حيث يتعرضون للبروتينات الهضم الأنزيمي تليها تحليل LC MS / MS، مما أدى إلى نمط تجزئة صباحا / ض التي يمكن استخدامها للتأكد من تسلسل الحمض الأميني الأساسي للالببتيد زيتية الأولي. لا يمكن أن يؤديها أساليب الهضم المختلفة تبعا لأنواع المنظفات المستخدمة، وكذلك المصب سير العمل مطياف الكتلة. في بروتوكول لدينا، ويستخدم 1-D PAGE الكهربائي تليها إزالة SDS من الجل من أجل إزالة أي تلوث المنظفات. تحليل البروتينات التي يصعب إذابة، مثل بروتينات الغشاء، يتطلب استخدام concen عاليةtrations من SDS أو المنظفات الأخرى. وهذا يؤدي إلى مشاكل التوافق مع SDS هلام الكهربائي. إذا كان الهدف من الدراسة يتطلب الإذابة هذه بجد لإذابة البروتينات، ونظام أنبوب هلام يمكن استخدامها 22،24. يتضمن طريقة أنبوب هلام البروتينات داخل المصفوفة هلام دون استخدام الكهربائي. بعد ذلك يتم إزالة أي المنظفات المستخدمة لإذابة قبل هضم البروتين. المرحلة الثالثة هي تحليل بيوينفورمتيك. في هذه المرحلة يتم البحث في البيانات الببتيد MS / MS ضد قاعدة بيانات للتسلسل النوكليوتيدات ترجمتها لتحديد الببتيدات والبروتينات موجودة في العينة. تحديد الببتيدات يعتمد على قاعدة بيانات يتم البحث عنها ضد. يتم البحث بيانات metaproteomic البحرية عادة ضد قواعد البيانات تتألف من الجينوم المرجعية والبيانات metagenomic مثل مجموعة البيانات العالمية المحيط أخذ العينات 25، فضلا عن خلية تضخيم الجينوم واحدة من لى غير المزروعةneages 26،27. ويمكن أيضا زيادة تحديد البروتين من خلال إدراج تسلسل metagenomic من نفس العينة كما يتضح من البيانات metaproteomic وقد اشتق 5. نحن هنا نقدم بروتوكول لتوليد الببتيدات مناسبة لتحليل يستند إلى MS-MS / الكتلة الحيوية الميكروبية التي تم جمعها من طريق الترشيح وتخزينها في محلول استقرار RNA. بروتوكول الموصوفة هنا يسمح للDNA والبروتين لتكون معزولة من نفس العينة حتى يتسنى لجميع الخطوات المؤدية إلى البروتين والأمطار DNA متطابقة. من منظور عملي، مطلوب أقل الترشيح منذ مطلوب مرشح واحد فقط لكل من البروتين واستخراج الحمض النووي. ونود أيضا أن نعترف أنه تم إنشاء هذا البروتوكول من خلال الجمع بين والتكيف وتعديل بروتوكولين المنشورة سابقا. يتم تكييفها الخطوات تحلل الخلية من سايتو وآخرون (2011) 28 ويتم تكييف التربسين في جل هضم مكون من ShevcheNKO وآخرون. (2007) 29.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sterivex -GP 0.22 &#956;m filter unit</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Bio Basic</td>
			<td>77-86-1 or TB0196-500G</td>
			<td>Protein Extraction/ SDS PAGE gel</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-1G</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio Basic</td>
			<td>15-21-3</td>
			<td>Protein Extraction/ SDS PAGE gel</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bio Basic</td>
			<td>6381-92-6</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>10K Amicon Filter</td>
			<td>Millipore</td>
			<td>UFC801024</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337-4L</td>
			<td>Protein Precipitation</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>67-64-1</td>
			<td>Protein Precipitation</td>
		</tr>
		<tr>
			<td>MPC Protein Precipitation reagent</td>
			<td>Epicenter</td>
			<td>mmP03750</td>
			<td>DNA Precipitation</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>67-63-0</td>
			<td>DNA Precipitation</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td>DNA Quantification</td>
		</tr>
		<tr>
			<td>Qubit Protein Assay kit</td>
			<td>Life Technologies</td>
			<td>Q33211</td>
			<td>Protein Quantification</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Bio Basic</td>
			<td>57-50-1</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio Rad</td>
			<td>161-0800</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Bio Rad</td>
			<td>161-0700</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>Bio Rad</td>
			<td>161-0158</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio Rad</td>
			<td>161-0718</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>Bio Basic</td>
			<td>60-24-2</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Laemmli Sample Buffer</td>
			<td>Bio Rad</td>
			<td>161-0737</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Kaleidoscope Ladder</td>
			<td>Bio Rad</td>
			<td>161-0375EDU</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>VWR</td>
			<td>CABDH6044-4</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>NH4HCO3</td>
			<td>Bio Basic</td>
			<td>1066-33-7</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-1G</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F0507-500ML</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>HPLC grade H2O</td>
			<td>Sigma-Aldrich</td>
			<td>270733-4L</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Bio Basic</td>
			<td>144-48-9</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 1.5ml</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>2mL ROBO vial 9mm</td>
			<td>Candian Life Science</td>
			<td>VT009/C395SB</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>PP BM insert, No spring</td>
			<td>Candian Life Science</td>
			<td>4025P-631</td>
			<td>In-gel Trypsin digest</td>
		</tr>
	</tbody>
</table>

an aquatic microbial metaproteomics workflow: from cells to tryptic peptides suitable for tandem mass spectrometry-based analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

كما مظاهرة، أجرينا البروتوكول على عينتين مياه البحر التي تم جمعها من سطح والكلوروفيل أقصى المحيط الساحلي في شمال كندا. بينما في عرض البحر، صدر 6-7 L من مياه البحر من خلال 3 ميكرون GF / D prefilter، ثم تم جمع الخلايا الميكروبية على 0.22 ميكرون وحدة تصفية خرطوشة بعد بروتوكول والش <e...

Watch this Scientific Journal Video about An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis at JoVE.com

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

والمائية الميكروبية Metaproteomics سير العمل: من الخلايا إلى زيتية الببتيدات مناسبة لتحليل يستند الطيف، جنبا إلى جنب الشامل

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

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8.7K Views.  Concordia University. This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Video: An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

توصيف وتطبيق العينات سلبية لمراقبة المبيدات في المياه

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Alternative Method of Removing Otoliths from Sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

طريقة بديلة لإزالة Otoliths من سمك الحفش

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

استخراج وتحليل الميكروبية الفوسفوليبيدية الأحماض الدهنية في التربة

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

البحوث الأساسية في الطب البلازما-نهج إنتاجية من السوائل للخلايا

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

توصيف الأغشية الحيوية المائية مع التدفق الخلوي

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

عزل وتحليل المجتمعات الميكروبية في التربة، رهيزوسفيري، وجذور في تجارب الأعشاب المعمرة

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

بروتوكول تجريبي لفحص ملفات تعريف الاستجابة السلوكية في الأسماك اليرقات: تطبيق على الكافيين منشط للأعصاب

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

تحديد كل-والأنواع الكيميائية أميد السلفونات المتعددة مع سير عمل مجتمعة المستهدفة وغير المستهدفة من الفحص الكتلي عالية الدقة

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

البكتيريا التي تحمل علامة الفلورسنت كجهاز تتبع للكشف عن مسارات جديدة لتدفق الكربون العضوي في النظم الإيكولوجية المائية

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency response analysis (cFRA) on proton-exchange membrane (PEM) fuel cells. During cFRA experiments, the modulated concentration feed was sent to the cathode of the cell at different frequencies. The electric response, which can be cell potential or current depending on the control applied on the cell, was registered in order to formulate a frequency response transfer function. Unlike traditional electrochemical impedance spectroscopy (EIS), the novel cFRA methodology makes it possible to separate the contribution of different mass transport phenomena from the kinetic charge transfer processes in the frequency response spectra of the cell. Moreover, cFRA is able to differentiate between varying humidification states of the cathode. In this protocol, the focus is on the detailed description of the procedure to perform cFRA experiments. The most critical steps of the measurements and future improvements to the technique are discussed.

We present a protocol for concentration alternating frequency response analysis of fuel cells, a promising new method of studying fuel cell dynamics.

دليل للتركيز بالتناوب تحليل استجابه التردد من خلايا الوقود

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency ...