This research was financially supported by the earmarked fund for Jiangsu Natural Science Fund (BK20170185), Project from Jiangsu Fisheries Administrator (Y2017-30), National Natural Science Foundation of China (NFSC31801575), China Agricultures Research System (CARS-45-26), national first-class discipline program of Food Science and Technology (JUFSTR20180201), and Yi Tong-Jiangsu Postdoctoral Program.

1. Sample preparation
<ol>
	<li>Thaw frozen sturgeon meat under flowing water below 20 &#176;C. Then, clean, skin off and cut the fish into cubes (2 cm &#215; 2 cm &#215; 1.5 cm). 
	NOTE: Raw material could be fresh or frozen sturgeon meat.</li>
	<li>Mix fish cubes with 6-12% (w/v) salt solution at a rate of 1:1 (fish cube mass to solution volume), at 10 &#176;C for 1-3 h.</li>
	<li>Put the marinated fish cubes onto a stainless-steel gridding plate and dry fish cubes at 40-60 &#176;C for about 6-10 h by hot air, or dry the fish cubes in vacuum, until the water content decreases to 50-60%.</li>
	<li>Mix dried fish cubes with the flavor solution inoculating 0.4-1.6% (w/w, starter culture mass to fish cube mass) S. cerevisiae at a rate of 1:1 (fish cube mass to solution volume). Seal the mixture in a container and ferment at 25-35 &#176;C for 6-10 h. 
	NOTE: The fermentation must be performed under anaerobic environment.
	<ol>
		<li>Prepare the flavor solution to include 25% spices liquid, 40% yellow wine, 7% white wine, 25% cane sugar, 2% monosodium glutamate, and 1% vinegar in mass fraction.</li>
		<li>Prepare the spice liquid is as follows. Mix spices including 1.2 units of scallions, 1 unit of ginger, 0.6 units of star anise, 0.6 units of fennel, 0.3 units of green tea and 0.3 units of pepper with 40 units of water and boil for 0.5 h. Collect the filtrate. Mix the residue with another 40 units of water and boil for 0.5 h again. Combine filtrates of the two boils and make up to 100 units using boiled water. 
		NOTE: It is a must to pause the protocol here until the spice liquid has cooled to the room temperature before the addition of starter culture. This pause could avoid inactivation of yeast by heat.</li>
	</ol>
	</li>
	<li>Seal fermented fish cubes in vacuum package with marinating and fermenting solutions at a rate of 11-13:1 (fish cube mass to solution volume). The vacuum strength, sealing time and cooling time are 0.085 MPa, 3.6 s and 5.5 s, respectively. 
	NOTE: The vacuum package material is polyethylene terephthalate/cast polypropylene (PET/CPP). The package weight is 50-80 g.</li>
	<li>Sterilize packed fish cubes before storage and sale. The sterilization F-value should be more than 4.5 min. The sterilization temperature and time are 115-121 &#176;C and 10-20 min, respectively. Maintain pressure constant at 0.12 MPa for cooling after sterilization. 
	NOTE: When sterilization F-value is 4.5 min and sterilization temperature is set as 121 &#176;C, the sterilization time is 11.4 min in the equipment conditions of this article. Revise the sterilization time when the type of equipment used is different.</li>
</ol>
2. Estimation of shelf life of fermented sturgeon meat product
NOTE: The estimation of shelf life of fermented sturgeon meat product uses accelerated shelf life testing (ASLT) method with Arrhenius model according to the method of Wahyuni et al. with some modifications18.
<ol>
	<li>Store products at 20 &#176;C, 30 &#176;C, 40 &#176;C.</li>
	<li>Predict the shelf life by the Arrhenius model using rancidity rates. Measure rancidity rates by testing acid values (AV) under different temperatures.</li>
	<li>Measure AV on the 0th, 14th, 28th, 42nd, 56th, 70th days.</li>
</ol>
3. Chemical analysis
<ol>
	<li>Determination of salt content 
	NOTE: We measure salt content according to the method proposed by Zeng et al.19.
	<ol>
		<li>Accurately weigh 1 g of the sample and add 10 mL of 0.1 M AgNO3 + 10 mL of HNO3.</li>
		<li>Heat the mixture gently on an induction-cooker for 10 min.</li>
		<li>Cool the mixture with running water, and add distilled water (50 mL) and ferric alum indicator (5 mL).</li>
		<li>Titrate the mixture with standard 0.1 M KSCN until the solution turns permanent brownish-red. 
		NOTE: Calculate and express the results as % NaCl.</li>
	</ol>
	</li>
	<li>Determination of moisture content
	<ol>
		<li>Determine moisture content according to the method of Zeng et al.19.</li>
	</ol>
	</li>
	<li>Determination of pH value 
	NOTE: We measure pH value according to the method of Zeng et al.6.
	<ol>
		<li>Homogenize 10 g of the sample with 90 mL of deionized water.</li>
		<li>Measure the pH value with a digital pH meter.</li>
	</ol>
	</li>
</ol>
4. Headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS) analysis
NOTE: We measure flavor according to the method of Gao et al. with some modification17.
<ol>
	<li>Extraction of volatile flavors
	<ol>
		<li>Weigh 2 g of the sample accurately, and then put it into the sample vial.</li>
		<li>Add 2.5 mL of saturated sodium chloride solution and 0.5 M 2,4,6-trimethylpyridine into the sample vial with a rotor, and then close the vial cap.</li>
		<li>Put the sample vial on the magnetic stirrer and stir for 10 min to mix the sample homogenously.</li>
		<li>Insert the fiber into the headspace of the sample vial.</li>
		<li>Adsorb at 60 &#176;C for 30 min, and then desorb at 250 &#176;C for 3 min in the injection port.</li>
	</ol>
	</li>
	<li>Analysis of flavor by GC-MS
	<ol>
		<li>Refer to the Table of Materials for details of the instrument used.</li>
		<li>Use Helium (purity &#62; 99.995%) as the carrier gas, and set the flow rate to 0.9 mL/min.</li>
		<li>Set the column temperature to 40 &#176;C for 3 min initially, and then increase to 90 &#176;C at a rate of 5 &#176;C/min. Subsequently, increase to 230 &#176;C at a rate of 10 &#176;C/min.</li>
		<li>Run MS in electron ionization (EI)+ mode, and set electron energy as 70 eV. Set scan range from 30 m/z to 500 m/z. Set emission current to 80 &#956;A. Use interface and source temperatures of 250 and 200 &#176;C, respectively.</li>
		<li>Identify the volatile flavor compounds by NIST2005 and Willey 7 standard libraries. Semi-quantify the retention index (RI) of volatile compounds using an internal standard (2,4,6-trimethylpyridine).</li>
		<li>Calculate the concentration of volatile flavor compounds by comparing the peak area of each flavor compound with that of the internal standard. Express the results as &#956;g/kg.</li>
	</ol>
	</li>
</ol>
5. Texture profile analysis
NOTE: Analyze texture profile by following a previous study20.
<ol>
	<li>Perform texture profile analysis (TPA) with a texture analyzer equipped with a cylindrical probe (P/36R).</li>
	<li>Apply two consecutive cycles. Deformation degree of TPA is 50% and the trigger force is 5 x g.</li>
	<li>The speed of cylindrical probe for pre-, in- and pro-test, are 2, 1 and 5 mm/s, respectively. Calculate the texture parameters by its inbuilt software.</li>
</ol>
6. Color measurement
NOTE: Measure color according to the method of Czerner et al. with some modifications21.
<ol>
	<li>Measure samples by using colorimeter. Record the lightness value (L*), greenness/redness value (a*) and blueness/yellowness value (b*) of the samples.</li>
	<li>Measure five replicates for each sample.</li>
</ol>
7. Sensory evaluation
<ol>
	<li>Conduct sensory analysis of samples by at least 20 trained panelists (23 panelists as an example, 12 man and 11 women, ages 20-40) using a previously method with some modifications20.</li>
	<li>Score the samples from 0 to 10 for taste, flavor, color and texture. Score 0 represents &#34;dislike extremely&#34;&#160;and score 10 represents for &#34;like extremely&#34;. 
	NOTE: Scoring criteria are shown in Table 1. The overall score is composed of a sum of different contributors (30% taste score + 30% flavor score + 20% color score + 20% texture score). An overall score of 6 is viewed as the borderline of acceptable quality.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Quality parameter</td>
			<td>Description</td>
			<td>Score</td>
		</tr>
		<tr>
			<td rowspan="4">Taste</td>
			<td>Acceptable sweet and salty; harmonious taste; wine taste; not astringent</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Too heavy or light (sweet or salty); harmonious taste; wine taste; not astringent</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Too heavy or light (sweet or salty); light wine- and after- taste; astringent</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Too light wine- and after- taste; obviously astringent</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Flavor</td>
			<td>Mellow wine flavor; rich fermentation aroma; no weird smell</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Mellow wine flavor; light fermentation aroma; no weird smell</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Light wine flavor; light fermentation aroma; light weird smell</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>No wine aroma; light fermentation aroma; obviously weird smell</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Appearance</td>
			<td>Russet color; glossy appearance; bright surface</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Russet color; bright surface</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Yellow color; nonuniform</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Pale color; rough surface</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td rowspan="4">Texture</td>
			<td>Acceptable chewiness</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>6-8</td>
		</tr>
		<tr>
			<td>Light springiness</td>
			<td>3-6</td>
		</tr>
		<tr>
			<td>Soft texture; coarse mouth-feel</td>
			<td>0-3</td>
		</tr>
	</tbody>
</table>
Table 1: Scoring criteria for sensory quality of fermented wine-aroma sturgeon meat product.

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The authors have nothing to disclose.

In this study, a new technique for the production of high-quality fermented fish product with wine aroma and tests for sensory properties are provided. The key processes of this technique are marinating, drying, fermentation and sterilization. In the marinating process, the salt concentration, the temperature and the time all affect the textural properties of the fish. The hardness and chewiness of the product increase gradually with the increase of salt concentration (0-12%, w/v) and the prolongation of marinating time ...

Current commercial marinated fish product in China has the problem of heavy salty taste, insufficient wine aroma, poor elasticity and pale color, which decreases the acceptability to consumers. Therefore, a new technique for a high-quality fish meat product with wine aroma needs to be created and optimized.
In recent years, application of modern fermentation techniques in meat and fish has attracted attention from more and more researchers1,2,3,4. By inoculation of starter cultures into meat and fish, food safety has been enhanced, the processing time has been shortened; and the product sensory properties have been modified. Saithong et al.5 isolated lactobacillus bacteria (LAB) from natural plaa-som and used this LAB as a starter culture, which induced high acidity and suppressed pathogenic bacteria. Zeng et al.6 reported that inoculation with the autochthonous starter cultures reduced fermentation time and improved the sensory properties of samples. Casaburi et al.7 claimed that the use of microbial starter cultures influence the development of aroma in fermented meats. In these starter cultures, S. cerevisiae could produce wine aroma by alcoholic fermentation and could also give the product other improved organoleptic qualities. Therefore, S. cerevisiae is a suitable starter culture for wine-aroma products8,9,10 and wine-aroma fish product could be made by S. cerevisiae.
In the process of making wine-aroma fish product, the texture of meat and fish could be affected by salt content, water content, pH, protein denaturation, etc. Therefore, marinating, drying, fermentation and sterilization could all influence the texture characteristics. The formation of flavor and taste is complicated and is mainly affected by marinating and fermentation, because it is highly related to hydrolysis of carbohydrates, proteins and lipids, and mild lipid oxidation11,12. It could also be affected by addition of spices13. For development of color, Maillard reaction occurs which is involved in the process of fermentation and sterilization10.
This article could provide technical support for the industrialization of fermented fish product with wine aroma, which is of great significance to the development of the fish processing industry. This technique could improve taste of product by increased proteolysis (more free amino acids and TCA-soluble peptides), modify flavor mainly by alcohols (ethanol, 1-octen-3-ol, 2-methyl-1-propanol and 3-methyl-1-butanol), esters (ethyl acetate) and aldehydes (nonanal, 3-methylbutanal and benzaldehyde), increase mouthfeel by higher hardness, springiness, gumminess and chewiness, and give more attractive russet color by and a bright surface14. It also gives consumers convenience because the product can be stored at room temperature. As described by other previous studies15,16,17, fermentation with S. cerevisiae has also been proved to significantly improve organoleptic qualities in other meat or fish products.
It is worth noting that the introduced protocol could also be applied in other species of fish, such as grass carp, silver carp, black carp, bighead carp, cod, salmon, etc. For high quality of fish products, fish without processing should be used, such as fresh fish, fish in ice or frozen fish stored for less than 1 year. Besides, since mild lipid oxidation could enhance flavor while extensive lipid oxidation brings unpleasant flavor, fish with less fat is preferred or skimmed fish is recommended.

<table>
	<tbody>
		<tr>
			<td>2,4,6-trimethylpyridine</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Purity 98%</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorimeter</td>
			<td>Hunterlab</td>
			<td>UltraScan Pro1166</td>
			<td></td>
		</tr>
		<tr>
			<td>DB-WAX column</td>
			<td>Agilent</td>
			<td>30 m &#215; 0.25 mm &#215; 0.25&#956;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pH meter</td>
			<td>Mettler toledo Instrument (Shanghai) Co., Ltd.</td>
			<td>DELTA-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>DHG-9070A</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen sturgeon</td>
			<td>Huada Marine Industry Group Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph-mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSQ Quantum XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidities incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>LHS-250HC-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae</td>
			<td>Angel Yeast Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Spices</td>
			<td>Auchan Supermarket</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization pot</td>
			<td>Longqiang Machinery Technology Co., Ltd.</td>
			<td>RHS-03-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Supelco</td>
			<td>Sigma</td>
			<td>65&#956;m, PDMS/DVB</td>
			<td></td>
		</tr>
		<tr>
			<td>Texture analyzer</td>
			<td>Stable Micro Systems, Ltd.</td>
			<td>TA-XT2i</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum package machine</td>
			<td>Quanzhou Yiminxin Electromechanical Co., Ltd.</td>
			<td>YMX-958-10L</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of high-quality fermented fish product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The suitable salt concentration, marinating time and temperature make the product&#8217;s textural quality better. The best marinating conditions were as follows: the salt concentration of 8% in the marinating solution; marinating time of 2 h; and marinating temperature of 10 &#176;C. See Figure 1.
In drying process, the final moisture content and drying temperature could influence the texture and sensory quality. The best drying conditions were as follows: moistu...

Watch this Scientific Journal Video about Preparation of High-Quality Fermented Fish Product at JoVE.com

Preparation of High-Quality Fermented Fish Product

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

preparation-of-high-quality-fermented-fish-product

Research

JoVE Journal

Biochemistry

10.1K Views.  Jiangnan University. The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

Video: Preparation of High-Quality Fermented Fish Product

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...