העבודה נתמכה, בין השאר, על ידי המכון הלאומי של המרכז לבריאות של אקסלנס למחקר ביולוגי [מספר מענק 1P20GM109005], הקונסורציום ארקנסו שטח גרנט דרך התעופה הלאומית שטח המינהל [NNX15AK32A מספר מענק], לבין המכון הלאומי לבטיחות תעסוקתית בריאות (NIOSH) [מספר מענק 2T420H008436]. המחברים מבקשים להודות כריסטופר Fettes עבור הגהה ועריכה כתב היד הזה.

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The authors have nothing to disclose.

מחקר ציטוגנטית או ניתוח מיקרוסקופי של מספרים או מבנים של כרומוזומים, בעיקר מתפשט metaphase, מספק מידע חיוני עבור הפרוגנוזה, הערכת סיכונים, וטיפול במחלות שונות. עכשיו זה מבוסס היטב, כי אבנורמליות ציטוגנטית מקושרת עם ההתקדמות והתפתחות של מחלות שונות, לרבות סרטן. נכון להיום,...

הערכה הוא כי חשיפה לחומר חלקיקים (PM) גורמת מעל 3 מיליון מקרי מוות מדי שנה עודפת, בעיקר ממחלות לב-ריאה וסרטן ריאות סרטן 1. ואכן, אחר הצהריים הוכר מסרטנים לבני אדם על ידי הסוכנות הבינלאומית לחקר הסרטן (קבוצה 1), כמו סיכון מוגבר לסרטן ריאות עם העלייה ברמות החשיפה PM הוכח 2. מעניין לציין שכמעט כל תאי סרטן נמל ליקויים מספריים ו / או מבניים כרומוזומליות. פחמן אורגן חלקיקים הנו נגזר, מורכב, תערובת הטרוגנית, אשר תלוי הפצת רכב וגדל על פליטה גם לשינויים פיסיים וכימיים. זה החשבונות של 35-55% ממסת עירוני PM 2.5 (PM 2.5 מיקרומטר בגודל קטן יותר) ויותר מ 60% של PM רקע כפריים קונטיננטלית 2.5 המונית 3, 4. השבר המסיס במים מהווה 30-90% של תרסיס אורגני. מספר רב של תרכובות אורגניותזוהה, כולל פחמימנים אליפטיות, פחמימנים ארומטיים פוליציקליים, ונגזר מחומצן ו הניטריות שלהם, אלדהידים ו כהלי aliphatic, חומצות שומן חופשיות ומלחיהם, חומצות קרבוקסיליות-di, תרכובות רבות תכליתיות, חלבונים, מקרומולקולות דמוי גומין (HULIS) באמצעות שיטות chromatographic בשילוב עם טכניקות ספקטרומטריה 5-8. תרכובות אלה מהוות פחות מ 10-20% של פחמן אורגן חלקיקים, ובכך ביותר של פחמן אורגני היא 9 ידוע. ראיה נסיונית עולה כי cytotoxicity, סטרס חמצוני, ודלקת מעורבים בהתפתחות מצבים פתולוגיים הקשורים PM. הוכח לאחרונה, עם זאת, כי חשיפת PM גם תוצאות מספר שינויים אפיגנטיים, כוללים שינויי מתילציה DNA של אלמנטים חוזרים, בשני ניסיוני במערכות במבחנה בבני אדם 10-12. מעניין במיוחד הן ההשפעות שלPM על DNA לווין - לווינים ראשיים ומשניות - אשר נמצאים באזור הטרוכרומטין ברחבי centromere של הכרומוזומים. זה היה הראה כי תופעות אלה עשויות להיות עקביות מטבעו, כפי שהם יכולים להיות מזוהים לפחות 72 שעות לאחר חשיפת 12. שינויים מתילציה DNA, במיוחד סביב צנטרומר, עלול לגרום להצטברות של תעתיקי mRNA DNA לווין, פשרה שלמות כרומוזומליות במהלך חלוקת התא, ולאחר מכן, לגרום להתפתחות של מגוון מצבים פתולוגיים 13. התאמת הגישה ציטוגנטית לניתוח CAs כמו שינויי נקודת סיום של אפיגנטיים הנגרמים על ידי ראש הממשלה, ובכך, היא בעלת חשיבות גבוהה. כאן אנו מדווחים הגישה לאיסוף PM הסביבה והכנת, בחשיפה במבחנה וניתוח של רשויות אישורים באמצעות מערכת מודל בעכברים מקרופאג RAW264.7. המקרופאגים מהווים את קו ההגנה הראשון נגד הגופים הזרים בשאיפה, ולכן לאקו התאו משמש הקים את המודל הנפוץ ביותר ב טוקסיקולוגיה חלקיקי 11, 12, 14, 15.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Total suspended particulate sampler</td>
			<td style="width:179px;">Tisch Environmental</td>
			<td style="width:119px;">TE-5170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Bruker Avance III NMR spectrometer&#160;</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TopSpin 3.5/pl2 software</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">ACD/NMR Processor Academic Edition</td>
			<td style="width:179px;">ACD/Labs</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAW264.7 murine macrophages</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">High glucose DMEM GlutaMAX media</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10569010</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Fetal Bovine Serum</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15140163</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">25200056</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PBS, pH 7.4</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10010049</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:261px;">KaryoMAX Colcemid Solution in PBS</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15212012</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">KaryoMAX Potassium Chloride Solution</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10575090</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol (HPLC)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">A452N1-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Acetic Acid, Glacial</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1185-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">Decon Contrad 70 Liquid Detergent</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">04-355-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Wright and Wright-Giemsa Stain Solutions</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-200733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Permount Mounting Medium</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Axio Imager 2</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

חשיפה לחומר חלקיקי (PM) הוא החשש העיקרי בעולם הבריאות, אשר עשוי לגרום נזק לרכיבים תאיים שונים, לרבות החומר הגנטי גרעיני. כדי להעריך את ההשפעה של PM על שלמות גנטית גרעינית, סטיות כרומוזומליות מבניות הבקיע מתפשט metaphase של תאי מקרופאג העכבר RAW264.7. PM נאסף מאוויר הסביבה עם סמפלר חלקיקים מרחף הכולל בנפח גבוה. החומר שנאסף solubilized ומסוננים לשמור על מסיסים במים, חלק בסדר. החלקיקים מאופיינים עבור הרכב כימי על ידי ספקטרוסקופיה בתהודה מגנטית גרעינית (NMR). ריכוזים שונים של השעית חלקיק מתווספים על תרבות במבחנה של מקרופאגים עכבר RAW264.7 במשך זמן חשיפה כולל של 72 שעות, יחד עם לתאי קבוצת ביקורת. בסוף החשיפה, התרבות מטופלת עם colcemid לעצור תאי metaphase. תאים אז שנקטפו, שטופלו פתרון hypotonic, קבוע acetomethanol, דropped גבי שקופיות זכוכית ולבסוף מוכתם פתרון Giemsa. שקופיות נבדקות כדי להעריך את סטיות כרומוזומליות מבניות (CA) ב metaphase פורשת בהגדלה 1,000X באמצעות מיקרוסקופ שדה בהיר. 50 עד 100 התפשטות metaphase הם הבקיע עבור כל קבוצת הטיפול. טכניקה זו מותאמת לצורך זיהוי של סטיות כרומוזומליות מבניות (CA), כגון הפסקות כרומטידה מסוג כרומטידה מהסוג חילופי, שברי acentric, כרומוזומים dicentric וטבעת, דקות כפולות, endoreduplication, ו טרנסלוקציות Robertsonian במבחנה לאחר חשיפת PM. זוהי שיטה רבת עוצמה כדי לשייך הסיום ציטוגנטית ומבוססת לשינויים אפיגנטיים.

בזהירות רבה יש לנקוט בבחירת המיקום של סמפלר TSP, כמו גם את הזמן של השנה כאשר מתבצע איסוף. ההרכב הכימי כמו גם את הגודל של חלקיקים עשויים להשפיע על התוצאות באופן משמעותי. החומר שנאסף צריך להיות גלוי נגד המסנן הלבן. התפשט metaphase עכבר רגיל יהיו 40 כרומוזומים a...

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

פרוטוקול זה מתאר שיטות כימות ואפיון של סטיות כרומוזומליות במבחנה מקרופאגים העכבר RAW264.7 לאחר הטיפול עם חומר חלקיקי באוויר הסביבה.

ניתוח של סטיות כרומוזומליות Matter הנגרמת חלקיקי אמביינט שימוש<em&gt; במבחנה</em&gt; מערכת

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

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Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

8.6K Views.  University of Arkansas for Medical Sciences.  פרוטוקול זה מתאר שיטות כימות ואפיון של סטיות כרומוזומליות במבחנה מקרופאגים העכבר RAW264.7 לאחר הטיפול עם חומר חלקיקי באוויר הסביבה. 

Video: Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

במבחנת תמלול מבחנים ויישומם ותרופות דיסקברי

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

איתור של סטיות יציבות אינטר-כרומוזומליות ידי קרינה נפוצה<em&gt; באתרו</em&gt; הכלאה (mFISH) ו ספקטרלית Karyotyping (SKY) בעכברים מוקרנים

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

ניתוח של C-KIT ליגנד האמרגן באמצעות Immunoprecipitation הכרומטין

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

קביעת Location(s) כרומוזומלית האופטימלית עבור רכיב ה-DNA ב- Escherichia coli באמצעות גישה מוזרה בתיווך Transposon

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

פרופיל תזמון שכפול ה-DNA באמצעות דג זברה כמערכת מודל In Vivo

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR בתיווך ארגון מחדש של הכרומטין לולאה

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

מדידת רמות פעילות גופנית ב- דרוזופילה melanogaster באמצעות מערכת כימות בתרגיל סיבוב (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

שימוש בחוץ גופית וצורה בתא לחקור מולקולה קטנה המושרה שינויים מבניים Pre-mRNA

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

קביעת שיעור הפריה הביצית של כנימת עש הטבק בטכניקה Cytogenetic

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

אפיון של בידול במבחנה של Keratinocytes האדם העיקרי על ידי ניתוח RNA-Seq

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...