This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Teaching Reform Research Project of China Pharmaceutical University (2019XJYB18).

1. Bacterial strains
NOTE: For this experiment, nine standard strains were used, including Salmonella paratyphi A, Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, Proteus vuigaris, and Klebsiella pneumoniae (Table 1). Salmonella paratyphi A, Fusobacterium nucleatum, Pseudomonas aeruginosa, and Proteus vuigaris can produce H2S, as outlined in previous literature1.
<ol>
	<li>Preparation of bacterial culture
	<ol>
		<li>Transfer one bacterial colony of Salmonella paratyphi A, Salmonella paratyphi B, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, and Klebsiella pneumoniae from a Luria-Bertani (LB) agar plate to 100 mL of LB medium and culture at 37 &#176;C for 12-16 h until the bacterial concentration is about 1 x 109 cells/mL (as indicated by OD600 = 1).</li>
		<li>Transfer one bacterial colony of Fusobacterium nucleatum and Proteus vuigaris from trypticase soy broth (TSB) agar plates to 100 mL of TSB medium and culture at 37 &#176;C anaerobically for 24 h until the bacterial concentration is about 1 x 109 cells/mL (as indicated by OD600 = 1).</li>
	</ol>
	</li>
</ol>
2. H2S detection assay
<ol>
	<li>Hydrogen sulfide production test
	<ol>
		<li>Mix 100 &#181;L of bacterial culture with 100 &#181;L of newly prepared bismuth solution (pH 8.0; 10 mM bismuth (III) chloride, 0.4 M triethanolamine-HCl, 20 mM pyridoxal 5-phosphate monohydrate, 20 mM EDTA, and 40 mM L-cysteine) in the 96-well microtiter plates and culture for 20 min at 37 &#176;C. For each bacterial strain, perform the analysis in triplicate.</li>
		<li>After 20 min, check for color change. If the color of the solution changes from light yellow to black, this indicates that the bacteria is able to produce H2S. Repeat this measurement 3x.</li>
	</ol>
	</li>
	<li>Sensitivity of the method
	<ol>
		<li>Determine the sensitivity of the method using different concentrations of sodium hydrosulfide (NaHS): 2 mM, 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.2 mM, 0.1 mM, and 0 mM, mixed with BS solution 14.</li>
		<li>Determine the presence of HS&#8722;/S2&#8722; by observing the formation of a black BS precipitate. Score the color of the wells using a visual scale from no color production (-) to darkest black color production (++++++).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Thompson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+group+of+hydrogen+sulphide+producing+bacteria.">The group of hydrogen sulphide producing bacteria.</a> Journal of Medical Research. 42 (184), 383-389 (1921).</li><li>Ono, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+hydropersulfide+inactivates+&#946;-lactam+antibiotics+with+formation+of+ring-opened+carbothioic+s-acids+in+bacteria.">Cysteine hydropersulfide inactivates &#946;-lactam antibiotics with formation of ring-opened carbothioic s-acids in bacteria.</a> ACS Chemical Biology. 16 (4), 731-739 (2021).</li><li>Mironov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+H2S-mediated+protection+against+oxidative+stress+in+Escherichia+coli.">Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (23), 6022-6027 (2017).</li><li>Shen, Y., Shen, Z., Luo, S., Guo, W., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardioprotective+effects+of+hydrogen+sulfide+in+heart+diseases:+From+molecular+mechanisms+to+therapeutic+potential.">The cardioprotective effects of hydrogen sulfide in heart diseases: From molecular mechanisms to therapeutic potential.</a> Oxidative Medicine and Cellular Longevity. 2015, 925167 (2015).</li><li>Salloum, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide+and+cardioprotection-mechanistic+insights+and+clinical+translatability.">Hydrogen sulfide and cardioprotection-mechanistic insights and clinical translatability.</a> Pharmacology &amp; Therapeutics. 152, 11-17 (2015).</li><li>Wallace, J. L., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide-based+therapeutics:+Exploiting+a+unique+but+ubiquitous+gasotransmitter.">Hydrogen sulfide-based therapeutics: Exploiting a unique but ubiquitous gasotransmitter.</a> Nature Reviews. Drug Discovery. 14 (5), 329-345 (2015).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hydrogen+sulfide+in+ischemia-reperfusion+injury.">Role of hydrogen sulfide in ischemia-reperfusion injury.</a> Oxidative Medicine and Cellular Longevity. , 186908 (2015).</li><li>Truong, D. H., Eghbal, M. A., Hindmarsh, W., Roth, S. H., O'Brien, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+hydrogen+sulfide+toxicity.">Molecular mechanisms of hydrogen sulfide toxicity.</a> Drug Metabolism Reviews. 38 (4), 733-744 (2006).</li><li>Shatalin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+bacterial+H2S+biogenesis+targeting+antibiotic+resistance+and+tolerance.">Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.</a> Science. 372 (6547), 1169-1175 (2021).</li><li>Fr&#225;vega, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Typhimurium+exhibits+fluoroquinolone+resistance+mediated+by+the+accumulation+of+the+antioxidant+molecule+H2S+in+a+CysK-dependent+manner.">Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.</a> The Journal of Antimicrobial Chemotherapy. 71 (12), 3409-3415 (2016).</li><li>Luhachack, L., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gasotransmitters:+An+innate+defense+against+antibiotics.">Bacterial gasotransmitters: An innate defense against antibiotics.</a> Current Opinion in Microbiology. 21, 13-17 (2014).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide+production+from+cysteine+and+homocysteine+by+periodontal+and+oral+bacteria.">Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.</a> Journal of Periodontology. 80 (11), 1845-1851 (2009).</li><li>Basic, A., Blomqvist, S., Carl&#233;n, A., Dahl&#233;n, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+bacterial+hydrogen+sulfide+production+in+vitro.">Estimation of bacterial hydrogen sulfide production in vitro.</a> Journal of Oral Microbiology. 7, 28166 (2015).</li><li>Rosolina, S. M., Carpenter, T. S., Xue, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bismuth-based,+disposable+sensor+for+the+detection+of+hydrogen+sulfide+gas.">Bismuth-based, disposable sensor for the detection of hydrogen sulfide gas.</a> Analytical Chemistry. 88 (3), 1553-1558 (2016).</li><li>Barton, L. L., Fauque, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry,+physiology+and+biotechnology+of+sulfate-reducing+bacteria.">Biochemistry, physiology and biotechnology of sulfate-reducing bacteria.</a> Advances in Applied Microbiology. 68, 41-98 (2009).</li><li>Shatalin, K., Shatalina, E., Mironov, A., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2S:+A+universal+defense+against+antibiotics+in+bacteria.">H2S: A universal defense against antibiotics in bacteria.</a> Science. 334 (6058), 986-990 (2011).</li><li>Schnabel, B., Caplin, J. L., Cooper, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+H2S+test+to+screen+for+the+detection+of+sulphur-+and+sulphate-reducing+bacteria+of+faecal+origin+in+water.">Modification of the H2S test to screen for the detection of sulphur- and sulphate-reducing bacteria of faecal origin in water.</a> Water Supply. 21 (1), 59-79 (2021).</li><li>Netzer, R., Ribi&#269;i&#263;, D., Aas, M., Cav&#233;, L., Dhawan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+of+priority+bacteria+in+aquaculture+using+digital+PCR.">Absolute quantification of priority bacteria in aquaculture using digital PCR.</a> Journal of Microbiological Methods. 183, 106171 (2021).</li></ol>

The authors declare no conflicts of interest.

The hydrogen sulfide production test is one of the conventional phenotypic tests for the identification and differentiation of bacterial strains. Many bacterial species can produce hydrogen sulfide in their natural environment, such as aquatic water. These bacterial species include Salmonella sp., Citrobacter sp., Proteus sp., Pseudomonas&#160;sp., some strains of Klebsiella sp., Escherichia coli, and some species of anaerobic Clostridia15...

Hydrogen sulfide producing bacteria can utilize sulfur-containing amino acids and proteins to produce hydrogen sulfide (H2S). The production of H2S occurs usually in gram-negative Enterobacteriaceae family bacteria and also in&#160;members of Citrobacter spp., Proteus spp., Edwardsiella spp., and Shewanella spp.1. These bacteria have the ability to reduce sulphate into hydrogen sulfide (H2S) in order to obtain energy. Hydrogen sulfide has been implicated in the development of bacterial drug resistance. H2S protects bacteria from the toxicity of reactive oxygen species (ROS), thus antagonizing the antibacterial effect of antibiotics2,3. H2S also has an important physiological effect in maintaining homeostasis. At supraphysiological levels, H2S has been shown to be profoundly toxic to the body. In the human body, H2S has another role as a gas signaling molecule that is involved in a variety of physiological and pathological processes. H2S can regulate the systolic function of the heart and plays an important physiological role in relaxing blood vessels, inhibiting vascular remodeling, and protecting the myocardium4,5. H2S also plays an important role in regulating the nervous system and digestive tract6,7. It has been found that, when exposed to bactericidal antibiotics, bacteria produce lethal reactive oxygen species (ROS) leading to cell death8,9,10,11.
As a common biochemical test in microbiological laboratory courses, the hydrogen sulfide test is an important experiment in the identification of bacteria, especially bacteria of the family Enterobacteriaceae. At present, the hydrogen sulfide test is usually performed on a large number of sulfur-containing amino acids and lead acetate medium inoculated with the bacteria to be tested. After a period of incubation (2-3 days), the results are judged by observing whether the culture medium or lead acetate paper strip is blackened because of lead acetate production11. However, these traditional methods are not only tedious and time-consuming but also prone to inhibition of bacterial growth due to the toxic effect of heavy metal salts in sulfur-containing medium, which often leads to negative results. A bismuth-based method has been established for the detection of H2S12,13. H2S can react with bismuth, forming black bismuth sulfide precipitation. In order to conduct a reform for this biochemical test, a simple and quick method with no side effects on bacterial growth needs to be established. Here, we set up a simple method for the detection of hydrogen sulfide producing bacteria grown in an in vitro environment using bismuth sulfide as a substrate in a 96-well microtiter plate format.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bismuth (III)chloride</td>
			<td>Shanghai Macklin Biochemical Co., Ltd</td>
			<td>7787-60-2</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Nanjing Chemical Reagent Co., Ltd</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterococcus faecalis&#160;</td>
			<td>ATCC&#160;</td>
			<td>19433</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium&#160;nucleatum&#160;</td>
			<td>ATCC&#160;</td>
			<td>25586</td>
			<td></td>
		</tr>
		<tr>
			<td>Klebsiella pneumoniae&#160;</td>
			<td>ATCC&#160;</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Amresco</td>
			<td>52-90-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteus&#160;vuigaris&#160;</td>
			<td>CMCC&#160;</td>
			<td>49027</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella paratyphi A</td>
			<td>CMCC</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella paratyphi B</td>
			<td>CMCC</td>
			<td>50094</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus&#160;aureus&#160;</td>
			<td>ATCC&#160;</td>
			<td>25923</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine-HCl</td>
			<td>Shanghai Aladdin Biochemical Technology Co., Ltd.</td>
			<td>637-39-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a sensitive visual method for the detection of hydrogen sulfide producing bacteria

Hydrogen sulfide (H2S) is a toxic gas produced by bacteria in the proteolysis of sulfur-containing amino acids and proteins that plays an important role in human health. The H2S production test is one of the important bacterial biochemical identification tests. The traditional methods are not only tedious and time-consuming but also prone to inhibition of bacterial growth due to the toxic effect of heavy metal salts in sulfur-containing medium, which often leads to negative results. Here, we established a simple and sensitive method to detect H2S in bacteria. This method is a modified version of bismuth sulfide (BS) precipitation that uses 96-well transparent microtiter plates. Bacterial culture was combined with bismuth solution containing L-cysteine and cultivated for 20 min, at the end of which a black precipitate was observed.The visual detection limit for H2S was 0.2 mM. Based on the visual color change, the simple, high-throughput, and rapid detection of H2S producing bacteria can be achieved. In summary, this method can be used to identify H2S production in bacteria.

Detection of hydrogen sulfide producing bacteria 
The performance of the H2S test was investigated using pure cultures of selected bacterial strains, as listed in Table 1. The results indicated that Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Pseudomonas aeruginosa,&#160;and Proteus vuigaris can produce H2S with black BS precipitate, while Salmonella paratyphi A, Staphylococcus <e...

Watch this Scientific Journal Video about A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria at JoVE.com

A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria

Here, we present a protocol to detect hydrogen sulfide producing bacteria with a modified protocol used for bismuth sulfide (BS) precipitation. The key advantages of this method are that it is easy to evaluate and does not require specialized equipment.

Hydrogen sulfide (H2S) is a toxic gas produced by bacteria in the proteolysis of sulfur-containing amino acids and proteins that plays an important role ...

a-sensitive-visual-method-for-detection-hydrogen-sulfide-producing

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Biology

3.4K Views.  China Pharmaceutical University. Here, we present a protocol to detect hydrogen sulfide producing bacteria with a modified protocol used for bismuth sulfide (BS) precipitation. The key advantages of this method are that it is easy to evaluate and does not require specialized equipment.

Video: A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...