We express our sincere appreciation for the financial support received from the Connotation Construction Project of the 14th Five-Year Plan of the University of Tibetan Medicine (2022ZYYGH12), the 2022 Open Subjects of the Key Laboratory of Tibetan Medicine and Basic Education of the Ministry of Education at the University of Tibetan Medicine (ZYYJC-22-04), the Key Research and Development Program of Ningxia (2023BEG02012), and the Xinglin Scholar Research Promotion Project of Chengdu University of Traditional Chinese Medicine (XKTD2022013).

The compound solution, protein solution, and dye were introduced into a PBS solution. Subsequently, the samples underwent gradual heating using a digital heating shaking dry bath, and the thermal stability of the proteins was evaluated by measuring the change in fluorescence intensity within the complex system. The detailed steps are outlined below, and Figure 1 illustrates an overview of the protocol.
1. Solution preparation
<ol>
	<li>Prepare a...

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

<ol>
	<li>Cairang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+standardization+of+clinical+application+of+Tibetan+medicine+&amp;#34;five+roots&amp;#34.">Study on the standardization of clinical application of Tibetan medicine &amp;#34;five roots&amp;#34.</a> Guid J Trad Chin Med Pharm. 28 (11), 133136 (2022).</li><li>Li, L., 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=Effects+of+Xigui+extract+on+immunosuppressive+mice+induced+by+cyclophosphamide.">Effects of Xigui extract on immunosuppressive mice induced by cyclophosphamide.</a> Chin J Hosp Pharm. 37 (03), 244-247 (2017).</li><li>Lu, H., 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=Effects+of+Vicatia+thibertica+de+boiss+on+immunologic+and+hematopoietic+function+of+mice.">Effects of Vicatia thibertica de boiss on immunologic and hematopoietic function of mice.</a> J Dali Univ. 2 (02), 6-10 (2017).</li><li>Dong, S., Zhang, X., Hu, Y., Gong, X., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+constituents,+quality+control+and+pharmacology+research+progress+of+Xigui.">Chemical constituents, quality control and pharmacology research progress of Xigui.</a> Chin J Ethnomed Ethnopharm. 27 (13), 40-42 (2018).</li><li>Lu, Z., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+total+flavone+extract+from+Shuiqin+(Oenanthe+Javanica)+on+immune+function+of+immunosuppression+mice.">Effects of total flavone extract from Shuiqin (Oenanthe Javanica) on immune function of immunosuppression mice.</a> Chin J Trad Med Sci Technol. 23 (04), 423-425 (2016).</li><li>Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+agonist+antibodies+in+cancer+immunotherapy.">CD40 agonist antibodies in cancer immunotherapy.</a> Annu Rev Med. 71, 47-58 (2020).</li><li>Grewal, I. S., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+and+CD154+in+cell-mediated+immunity.">CD40 and CD154 in cell-mediated immunity.</a> Annu Rev Immunol. 16, 111-135 (1998).</li><li>Long, K. B., 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=IFN&#947;+and+CCL2+cooperate+to+redirect+tumor-infiltrating+monocytes+to+degrade+fibrosis+and+enhance+chemotherapy+efficacy+in+pancreatic+carcinoma.">IFN&#947; and CCL2 cooperate to redirect tumor-infiltrating monocytes to degrade fibrosis and enhance chemotherapy efficacy in pancreatic carcinoma.</a> Cancer Discov. 6 (4), 400-413 (2016).</li><li>He, Y., 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=The+roles+of+regulatory+B+cells+in+cancer.">The roles of regulatory B cells in cancer.</a> J Immunol Res. 2014, 215471 (2014).</li><li>Eliopoulos, A. G., 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=CD40+induces+apoptosis+in+carcinoma+cells+through+activation+of+cytotoxic+ligands+of+the+tumor+necrosis+factor+superfamily.">CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.</a> Mol Cell Biol. 20 (15), 5503-5515 (2000).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2 (9), 2212-2221 (2007).</li><li>Rosa, N., 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=Meltdown:+A+Tool+to+help+in+the+interpretation+of+thermal+melt+curves+acquired+by+differential+scanning+fluorimetry.">Meltdown: A Tool to help in the interpretation of thermal melt curves acquired by differential scanning fluorimetry.</a> J Biomol Screen. 20 (7), 898-905 (2015).</li><li>Hellman, L. M., 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=Differential+scanning+fluorimetry+based+assessments+of+the+thermal+and+kinetic+stability+of+peptide-MHC+complexes.">Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.</a> J Immunol Methods. 432, 95-101 (2016).</li><li>Pantoliano, M. W., 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=High-density+miniaturized+thermal+shift+assays+as+a+general+strategy+for+drug+discovery.">High-density miniaturized thermal shift assays as a general strategy for drug discovery.</a> J Biomol Screen. 6 (6), 429-440 (2001).</li><li>Gorny, H., 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=Combining+nano-differential+scanning+fluorimetry+and+microscale+thermophoresis+to+investigate+VDAC1+interaction+with+small+molecules.">Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules.</a> J EnzymeInhib Med Chem. 38 (1), 2121821 (2023).</li><li>Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+denaturation+methods+in+the+study+of+protein+folding.">Thermal denaturation methods in the study of protein folding.</a> Methods Enzymol. 259, 144-168 (1995).</li><li>Phiri, M. J., Mofokeng, J. P., Phiri, M. M., Mngomezulu, M., Tywabi-Ngeva, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical,+thermal+and+morphological+properties+of+polybutylene+succinate-waste+pineapple+leaf+fibres+composites.">Chemical, thermal and morphological properties of polybutylene succinate-waste pineapple leaf fibres composites.</a> Heliyon. 9 (11), 21238 (2023).</li><li>Zhou, C., Yu, M., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparation+of+three+measuring+methods+for+thermodynamic+stability+of+protein.">Comparation of three measuring methods for thermodynamic stability of protein.</a> Anal Test Technol Instruments. 27 (04), 252-259 (2021).</li><li>Hou, Y., 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=Salidroside+intensifies+mitochondrial+function+of+CoCl2-damaged+HT22+cells+by+stimulating+PI3K-AKT-MAPK+signaling+pathway.">Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.</a> Phytomedicine. 109, 154568 (2023).</li><li>Wang, X., 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=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiola+crenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> J Ethnopharmacol. 293, 115278 (2022).</li><li>Magnez, R., Bailly, C., Thuru, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+as+a+tool+to+study+protein+interactions+and+their+implication+in+human+diseases.">Microscale thermophoresis as a tool to study protein interactions and their implication in human diseases.</a> Int J Mol Sci. 23 (14), 7672 (2022).</li><li>Kamat, V., Rafique, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+binding+kinetic+assay+on+the+bio-layer+interferometry+(BLI)+biosensor+to+characterize+antibody-antigen+interactions.">Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions.</a> Anal Biochem. 536, 16-31 (2017).</li><li>Freyer, M. W., Lewis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry:+experimental+design,+data+analysis,+and+probing+macromolecule/ligand+binding+and+kinetic+interactions.">Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions.</a> Methods Cell Biol. 84, 79-113 (2008).</li><li>Sharma, R., 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=Atosiban+and+Rutin+exhibit+anti-mycobacterial+activity+-+An+integrated+computational+and+biophysical+insight+toward+drug+repurposing+strategy+against+Mycobacterium+tuberculosis+targeting+its+essential+enzyme+HemD.">Atosiban and Rutin exhibit anti-mycobacterial activity - An integrated computational and biophysical insight toward drug repurposing strategy against Mycobacterium tuberculosis targeting its essential enzyme HemD.</a> Int J Biol Macromol. 253, 127208 (2023).</li><li>Gr&#228;dler, U., 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=Biophysical+and+structural+characterization+of+the+impacts+of+MET+phosphorylation+on+tepotinib+binding.">Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.</a> J Biol Chem. 299 (11), 105328 (2023).</li><li>Xu, Y., 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=Differential+scanning+fluorimetry+and+its+application+in+the+study+of+protein.">Differential scanning fluorimetry and its application in the study of protein.</a> Chemistry of Life. 38 (03), 351-357 (2018).</li></ol>

DSF, also known as the thermal shift assay or thermal fluorescence assay, is a technique employed to detect the process of thermal denaturation of proteins in samples by monitoring changes in the fluorescence signal of the test sample or dye during a slow, programmed temperature increase. Initially established by Pantoliano14, DSF serves as a high-throughput method. The main procedure involves elevating the temperature on a computer-controlled heated plate, emitting excitation light using a long-w...

Vicatia thibetica H. Boissieu, a plant in the umbelliferous family, is commonly used in Tibetan medicine and represents one of the essential components of the five basic ingredients (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew., and Gymnadenia conopsea (L.) R. Br.)1. Mainly distributed in southwest China, such as northwest Yunnan, western Sichuan, Tibet, and other areas, its dried root serves as a local substitute for Angelica1. The root, known for its fragrance, is often utilized as a stew seasoning, and the leaves, referred to as Tibetan celery, contribute to delectable dishes. Thus, Vicatia thibetica holds significance not only as a unique medicinal plant but also as a food source in Tibet.
Both domestic and international research indicates that Vicatia thibetica possesses blood-replenishing and qi (power or energy)-invigorating properties, beneficial for regulating menstruation. It is employed to address symptoms of irregular menstrual dysmenorrhea caused by palpitation and blood deficiency, exhibiting pharmacological effects such as antioxidant regulation of body immunity2. The alcohol extract of Vicatia thibetica has demonstrated the ability to restore body mass and organ index in immunocompromised mice induced by cyclophosphamide. Additionally, it increases the activity of superoxide dismutase in serum and reduces the content of malondialdehyde, suggesting an improvement in antioxidant capacity and a balancing effect on lipid peroxidation2,3. Simultaneously, it enhances the number of blood cells and hemoglobin, which not only objectively reflects the body&#39;s hematopoietic function but also plays a crucial role in the immune system2,3.
Umbelliferone, characterized by acicular crystals and a bitter taste, possesses a small molecular weight, is volatile, and can be distilled with steam. It sublimates easily, has low solubility in water, and high solubility in organic matter. As one of the main chemical components of Vicatia thibetica, umbelliferone exhibits immunomodulatory effects on cellular immunity, humoral immunity, and non-specific immunity in hydrocortisone-induced immunosuppression mouse models4,5.
The cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed in immune cells6. Its homologous ligand, CD154, also known as CD40L, is a type II transmembrane protein expressed by activated T lymphocytes. CD40 activation has the capacity to up-regulate the expression of co-stimulatory molecules on the surface of dendritic cells (DC) and monocytes. This process promotes the antigen presentation function of major histocompatibility complex (MHC) molecules and further activates CD8+ T cells7.
Macrophages play a crucial role in the formation and regulation of the tumor microenvironment, and CD40 activation can enhance the remodeling of the tumor microenvironment by macrophages8. CD40 signal activation significantly influences the proliferation and activation of B cells. B cells, when activated by CD40, can function as effective antigen-presenting cells. They present antigens, generate effector T cell activity, and thereby contribute to anti-tumor effects9. Moreover, CD40 activation in tumor cells can induce apoptosis and inhibit tumor growth10. CD40 primarily transduces signals by controlling the activity of non-receptor tyrosine protein kinases, including Lyn, Fyn, Syk, and others. Additionally, it has the ability to stimulate Bcl-xL, Cdk4, and Cdk6 proteins, activate Rel/NF-kB transcription factors, and phosphorylate CG-2 and PI3K10.
The Differential Scanning Fluorescence (DSF) method is widely employed to assess the impact of various environmental conditions, such as buffer composition, temperature, and small molecule ligands, on the thermal stability of protein structures. The commonly utilized dye for DSF is an orange, environmentally sensitive, hydrophobic dye. Under normal conditions, the protein structure is folded, concealing its hydrophobic segment internally. As the temperature increases, the protein&#39;s hydrophobic region becomes more exposed, resulting in a gradual breakdown of the natural protein structure. The dye selectively binds to this exposed protein portion, amplifying its fluorescence. Tm values are then calculated by monitoring changes in the fluorescence signal detection11. To a certain extent, variations in Tm values can gauge shifts in protein stability due to mutations, changes in buffers, or ligand binding. Furthermore, it can indicate structural alterations during the protein folding process12. This approach offers precise data, a broad temperature range, high sensitivity, and minimal protein sample loss13.
In this study, fluorescence determination was conducted using a fluorescent microplate reader instead of a fluorescence quantitative polymerase chain reaction (PCR) apparatus. This modification enables DSF detection in laboratories lacking a fluorescent quantitative PCR instrument, making the method less complex and reducing the steps required for instrument setup, thereby simplifying the experimental process. However, there are certain drawbacks to this approach. While the complexity is reduced, the procedure becomes more cumbersome. Manual fluorescence detection at different temperatures is necessary, and automatic and continuous collection of fluorescence from the system cannot be achieved. Thus, this study utilized the DSF technique to explore the interaction between CD40 protein and umbelliferone, providing novel insights into the molecular mechanisms of Tibetan medicine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Our protocol provides a significant advantage by allowing use without specialized equipment. Unlike traditional fluorescence quantitative PCR methods, it simplifies the experimental process, requiring fewer steps and reducing overall complexity, making it safer and easier to conduct. This experiment requires the operator to be very familiar with the experimental procedure and the fluorescent microplate reader, and not to waste too much time during the solution heating and the fluorescence detection.We will focus on the molecular mechanism by which umbelliferone binds to CD14 protein to produce immune effects.

The orange dye consistently exhibited stable fluorescence excitation at Ex = 470 nm and Em = 570 nm, both at room temperature and elevated temperatures. An optimal dilution ratio of 1:500 was determined (Figure 2A,B). Detection of the Tm value proved challenging when the concentration of CD40 protein was below 15 &#181;g/mL (Figure 3A,B). However, at a concentration of 15 &#181;g/mL, a stable Tm value of 51.82 &#176;C was detec...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

detection-cd40-protein-umbelliferone-interaction-via-differential

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Medicine

340 vues.  University of Tibetan Medicine. Notre protocole offre un avantage significatif en permettant une utilisation sans équipement spécialisé. Contrairement aux méthodes traditionnelles de PCR quantitative par fluorescence, elle simplifie le processus expérimental, nécessitant moins d’étapes et réduisant la complexité globale, ce qui le rend plus sûr et plus facile à réaliser. Cette expérience nécessite que l’opérateur soit très familier avec le procédé expérimental et le lecteur de microplaques fluorescent...

Vidéo: Pleins feux sur l’auteur : Rationaliser les méthodes de PCR pour une accessibilité et une efficacité accrues

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data...

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enhancing density maps by removing the majority of particles in single particle cryogenic electron microscopy final stacks

Author Spotlight: Optimizing Cryo-EM Analysis with CryoSieve for Enhanced Particle Selection Efficiency

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world...

Enhancing Cryo-EM Map Quality with CryoSieve Particle Selection

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subjective refraction test using a smartphone for vision screening

Author Spotlight: Empowering Vision Care Accessibility &#8212; A Mobile App for Refractive Error Screening in Underserved Communities

This paper presents the protocols and clinical validation data for using a smartphone app to subjectively measure refractive...

Smartphone App for Measuring Refractive Errors and Inter-Pupillary Distance

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hairy root transformation and regeneration in arabidopsis thaliana and brassica napus

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

&lt;em&gt;Agrobacterium&lt;/em&gt;-Mediated Hairy Root Induction and Regeneration in Plants

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digital droplet pcr method for the quantification of aav transduction efficiency in murine retina

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

methods for electroporation and transformation confirmation in limosilactobacillus reuteri dsm20016

Author Spotlight: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016  

Here, we present protocols for working with Limosilactobacillus reuteri DSM20016, detailing growth, plasmid transformation, colony PCR, fluorescent reporter protein measurement, and limited plasmid mini-prep, as well as common issues and troubleshooting. These protocols allow the measurement of reporter proteins in DSM20016, or confirmation via colony PCR if no reporter is involved.

Measurement of Reporter Proteins in &lt;em&gt;Limosilactobacillus reuteri&lt;/em&gt; DSM20016

automatic surgery in transcatheter aortic valve replacement using augmented reality

Author Spotlight: Revolutionizing Remote Surgery with Augmented Reality and Robotics for Enhanced Precision and Accessibility

This article presents the design and implementation of an automatic surgery module based on augmented reality (AR)- based 3D reconstruction. The system enables remote surgery by allowing surgeons to inspect reconstructed features and replicate surgical hand movements as if they were performing the surgery in close proximity.

Optimized 3D Reconstruction and Motion Mapping for Remote AR-Assisted Surgery

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improved hysteroscopic resection of endometrial polyps using 6-fr micro-scissors and forceps

Author Spotlight: A Reproductive Hysteroscopy Approach for Complete Endometrial Polyp Removal and Enhanced Endometrial Receptivity

The novel technique presented here employs a combination of 6-Fr micro-scissors and forceps for hysteroscopic treatment of endometrial polyps, demonstrating encouraging outcomes for infertile patients afflicted with this...

Hysteroscopic Technique with Micro-Scissors and Forceps for Polyp Removal

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new features in visual dynamics 3.0

Author Spotlight: Streamlining Visual Dynamics to Simplify Molecular Dynamics Simulations Using Gromacs

Visual Dynamics is an open-source tool that accelerates implementations and learning in molecular dynamics simulation using Gromacs. The presented protocol will guide you through the steps to perform a protein-ligand simulation prepared in ACPYPE with ease and general steps to other simulation...

Visual Dynamics: Simplifying Molecular Dynamics Simulations

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microscopic varicocelectomy under local anesthesia as the treatment of varicocele

Author Spotlight: Advancing Varicocele Treatment Through Enhanced Surgical Techniques

Varicocele accounts for 35%-40% of infertility cases among men of reproductive age. The international standard treatment method is microscopic varicocelectomy, typically performed under general anesthesia. This study proposes and implements microscopic varicocelectomy under local anesthesia, which provides more humane care for patients and reduces the economic...

Microsurgical Varicocele Removal Under Local Anesthesia

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quantification of adeno-associated viral genomes in purified vector samples by digital droplet polymerase chain reaction

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome...

Standardized Protocol for Viral Genome Quantification Using Digital Droplet PCR

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a neural implant design toolbox for nonhuman primates

Author Spotlight: Streamlined Brain and Skull Modeling for Enhanced Neurosurgical Planning in NHP Research

This paper outlines automated processes for nonhuman primate neurosurgical planning based on magnetic resonance imaging (MRI) scans. These techniques use procedural steps in programming and design platforms to support customized implant design for NHPs. The validity of each component can then be confirmed using three-dimensional (3D) printed life-size anatomical...

Automated 3D Brain and Skull Modeling from MRI for NHP Neurosurgical Planning

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an automated culture system for maintaining and differentiating human-induced pluripotent stem cells

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of...

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

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techniques for rapidly sampling six crucial organs in adult xenopus

Author Spotlight: Standardizing Tissue Sampling in Proteomics and Immunochemistry Research

This article presents a guide for sampling six significant and diverse organs in adult Xenopus that can be rapidly and easily accessed: the heart ventricle, liver lobe, pancreas, fat bodies, paired kidneys, and...

Standardized Dissection and Sampling Protocol for Adult &lt;em&gt;Xenopus&lt;/em&gt; Organs

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drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and sirna screening applications

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA...

an efficient and reproducible method for producing composite plants by agrobacterium rhizogenes-based hairy root transformation

Author Spotlight: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite...

One-Step Hairy Root Transformation Method for Advancing Root-Related Biology

a method for manipulating blood glucose and measuring resulting changes in cognitive accessibility of target stimuli

A Method for Manipulating Blood Glucose and Measuring Resulting Changes in Cognitive Accessibility of Target Stimuli

The authors introduce a method for manipulating blood glucose and measuring resulting changes in cognitive accessibility of target words using a lexical decision...

creating a box-cavity defect model in the cortical bone of rat femora

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

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monochrome multiplex quantitative pcr telomere length measurement 

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based...

Accurate Telomere Length Assessment in Human Population Studies

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acupuncture treatment in a mouse model of chronic hypoxia-induced cognitive dysfunction

Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.