Name: inactivation of pathogens via visible-light photolysis of riboflavin-5&#8242;-phosphate
Uploaded: 2022-04-06T13:40:00
Description: Watch this Scientific Journal Video about Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5â€²-Phosphate at JoVE.com

The authors are grateful to Dr. Tak-Wah Wong and Mr. Zong-Jhe Hsieh for their support with experiments.

1. Photolysis system setup
<ol>
	<li>Mount six light-emitting diodes (LED) (DC 12 V) on the inside of an opaque plastic cup (8 cm x 7 cm) as shown in Figure 1 to establish a photolysis system31.</li>
	<li>Add reactants (see below) into the glass test tubes (13 mm in diameter and 100 mm in height) and secure the tubes at the top of the cup as shown in Figure 1. Place the experimental setup in a room with a steady tempera...

A photosensitizer increases the photochemical reaction of chemical compounds to generate ROS. Pathogenic microorganisms can be inactivated by light irradiation in the presence of photosensitizers. This study determines the aPDI of S. aureus due to ROS generated by violet light irradiation of an exogenous photosensitizer, FMN.
As shown in Figure 3, for FMN, the absorbance at 444 nm is reduced significantly after 5 min of irradiation using violet or blue li...

Riboflavin-5&#8242;-phosphate (FMN) is formed by phosphorylation at the riboflavin 5&#8242;-position of the ribityl side-chain and is required by all flavoproteins for numerous cellular processes to generate energy. It also plays the role of vitamin for some functions in the human body1. FMN is approximately 200 times more soluble in water than riboflavin2.
The antibacterial photodynamic inactivation (aPDI) of bacteria is an efficient way to control resistance to bacteria3,4 because it does not depend on the mode of bacterial resistance. Clinically, aPDI is used to treat soft tissue infections in order to decrease infection of nosocomial skin due to multi-resistant bacteria5,6,7,8,9. aPDI also produces cell death by generating reactive oxygen species (ROS). ROS, such as superoxide radicals (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />), singlet oxygen, hydroxyl radicals (&#8226;OH), and peroxyl radicals, are free radicals or molecules that contain reactive oxygen10,11,12 and are normally reactive13. Similar to DNA damage that is caused by ROS, membrane peroxidation and destruction of endothelial cells are also adverse biochemical reactions that are attributed to ROS in cells12.
The use of aPDI for pathogenic bacteria&#160;involves a visible or UV light source to inactivate microorganisms in the presence of chemical compounds, such as methylthioninium chloride14, PEI-ce6 conjugate15, porphyrin16, titanium dioxide17, toluidine blue O18, and zinc oxide nanoparticles19. Toluidine blue O and methylene blue are phenothiazinium dyes and methylene blue has toxic properties. Zinc oxide nanoparticles and UV irradiation are linked to adverse health and environmental effects. As such, the exploitation of a reliable, secure, and simple photosensitizer via photolysis under visible irradiation deserves further study.
The micronutrient, riboflavin or FMN, is not toxic and is indeed used for food manufacturing or feeding20. Both FMN and riboflavin are highly sensitive to light irradiation2. Under UV1,2,21,22,23 and blue light irradiation10,24, these two compounds achieve an excited state. The activated riboflavin or FMN that is produced by photolysis is promoted to its triplet state and ROS are generated simultaneously2,25. Kumar et al. reported that riboflavin activated by UV light selectively causes increased injury to the guanine moiety of DNA in pathogenic microorganisms26. Under irradiation by UV light, photodynamically activated riboflavin is demonstrated to promote the generation of 8-OH-dG, which is a biomarker for oxidative stress, in double-stranded DNA27. It is reported that S. aureus and E. coli are deactivated by ROS during riboflavin or FMN photolysis10,24,28. A previous study by the authors showed that the photolytic reactions involving riboflavin and FMN reduce crystal violet, a triarylmethane dye and an antibacterial agent that generates <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />, and eliminate most of the antimicrobial capability of crystal violet28. When flavin adenine dinucleotide or FMN is irradiated by blue light, the resulting ROS produce apoptosis in HeLa cells for their poisoning in vitro29. Using photochemical treatment in the presence of riboflavin, Cui et al. inactivated lymphocytes by inhibiting their proliferation and cytokine production30.
The photolysis of riboflavin is used for the inactivation of blood pathogen by UV10,24, but blood components can be impaired under UV light irradiation30. It is also reported that platelets exposed to UV progressively enhance the performance of the activation markers P-selectin and LIMP-CD63 on their membranes. The cytotoxicity of UV and high-intensity irradiation needs to be investigated and a photosensitizer that is uncomplicated and safe during an FMN photoreaction involving visible light would be of great use.
Light of shorter wavelengths has more energy and is much more likely to cause severe damage to cells. However, in the presence of a suitable photosensitizer, irradiation with low-intensity violet light can inhibit pathogenic microorganisms. The photosensitization and the generation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> by FMN when irradiated with violet light is thus important to study, in order to determine the pathway by which ROS from FMN photolysis increases the inactivation of bacteria.
Antimicrobial control is a common issue and the development of new antibiotics frequently takes decades. After irradiation with violet light, photo&#173;inactivation that is intermediated by FMN can annihilate environmental pathogenic bacteria. This study presents an effective antimicrobial protocol in vitro using violet light for driving FMN photolysis and thus generating <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> for aPDI. The microbial viability of S. aureus is used to determine the feasibility of FMN-induced aPDI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blue, green and red LED lights</td>
			<td>Vita LED Technologies Co., Tainan, Taiwan</td>
			<td></td>
			<td>DC 12 V 5050</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>190186</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared thermometer</td>
			<td>Raytek Co. Santa Cruz, CA</td>
			<td>MT4</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Difco Co., NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>1.05707</td>
			<td></td>
		</tr>
		<tr>
			<td>NBT</td>
			<td>Bio Basic, Inc. Markham, Ontario, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>China tech Co., New Taipei City, Taiwan</td>
			<td>YP30-3-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin 5&#8242;-phosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>R7774</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>New England BioLabs, Inc. Ipswich, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solar power meter</td>
			<td>Tenmars Electronics Co., Taipei, Taiwan</td>
			<td>TM-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus subsp. aureus</td>
			<td>Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan</td>
			<td>10451</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis optical spectrometer</td>
			<td>Ocean Optics, Dunedin, FL</td>
			<td>USB4000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Hitachi High-Tech Science Corporation,Tokyo, Japan</td>
			<td>U-2900</td>
			<td></td>
		</tr>
		<tr>
			<td>Violet LED</td>
			<td>Long-hui Electronic Co., LTD, Dongguan, China</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inactivation of pathogens via visible-light photolysis of riboflavin-5&#8242;-phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Effect of light wavelength on FMN 
The absorbance spectra of 0.1 mM FMN that is irradiated using blue, green, red, and violet LEDs are shown in Figure 3. There are two peaks for FMN (372 nm and 444 nm) for the dark control. Green and red light have no effect because changes in the spectra are insignificant. On the other hand, the respective absorbance of FMN at 444 nm is reduced by about 19% and 34%, respectively,&#160;after blue and violet light irradiation at 10 W/m<s...

Watch this Scientific Journal Video about Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5â€²-Phosphate at JoVE.com

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

This protocol uses visible violet light irradiation to induce flavin mononucleotide photolysis. This produces a large amount of free radicals, which inhibit pathogenic bacteria such as Staphylococcus aureus and E.coli. We have tested flavin mononucleotide as an appropriate photosensitizer.In this protocol, we use visible violet light which is safe and doesn't require expensive equipment. This method can be used for the therapy of injured skin or infected subcutaneous tissues by inserting an optical fiber for illumination. It can also be applied to the sanitation practice in food industry.Demonstrating the procedure will be Tang-Yu Chen, a graduate student from my laboratory To begin, mount six light-emitting diodes of 12 volts each on the inside of an opaque plastic cup. Take a glass test tube with a diameter of 13 millimeters and a height of 100 millimeters and add the reactants to the tube. Secure the tubes at the top of the cup and place this setup at a constant temperature of 25 three degrees Celsius.Monitor and record the temperature of test units throughout the photolytic reactions by an infrared thermometer. Next, prepare a 0.1-millimolar FMN solution in 100-millimolar potassium phosphate buffer at pH 7.8. Expose three milliliters of FMN samples each to blue, green, red, or violet light for five minutes.In parallel, keep three milliliters of FMN solution in the dark as control. Place the sample into a UV-visible spectrophotometer and record the absorbance of irradiated samples in the 250-to 750-nanometer range. To prepare for the detection of superoxide radicals, first add 109.3 milligrams of L-methionine to 73.3 milliliters of 100-millimolar phosphate buffer at pH 7.8.Into the solution, add 10 milligrams of NBT powder and 1.5 milliliters of 0.117-millimolar FMN. Expose one milliliter of the reaction solution to blue or violet LED light for one to five minutes. Record the absorbance of 516 nanometers to detect formazan, which is produced by the photochemical reaction of NBT.Inoculate a colony of Staphylococcus aureus into 10 milliliters of lysogeny broth in a 15-milliliter screw-capped test tube. Grow the culture at 37 degrees Celsius for 16 hours in a shaker. Transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube.Dilute the culture to an optical density of 0.5 at 600 nanometers by adding sterilized water. Then, transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube. Centrifuge at 14, 000 G for 10 minutes and decant the supernatant.Add one milliliter of FMN buffered solution to the cell pellet and vortex. For irradiated control, add one milliliter of phosphate buffer. Transfer one milliliter of bacterial cell solution containing 30-micromolar FMN and phosphate buffer into glass tubes.Place them for a radiation under violet light at 10 watts per meter square for 30 minutes. Then, transfer one milliliter of bacterial cell solution containing 30-60-and 120-micromolar FMN and phosphate buffer in glass tubes. Irradiate with blue light for 120 minutes at 20 watts per meter square.Also, irradiate a tube containing one milliliter of 120-micromolar FMN for 60 minutes. After irradiation, add 0.2 milliliters of the reaction solution onto a luria agar plate. Spread the solution using an L-shaped glass rod and incubate overnight at 37 degrees Celsius.On the next day, calculate the viable plate count and inactivation rate of Staphylococcus aureus. Prepare the staphylococcus aureus samples as described earlier to obtain a cell pellet. To 75 milliliters of phosphate buffer add 0.1093 grams of L-methionine, 0.1 gram of NBT, and 25 milliliters of FMN solution.Add one milliliter of each reactant solution with varying FMN concentrations to the cell pellets and vortex. Irradiate the solutions under violet light at 10 watts per meter square for 10 minutes. Centrifuge the mixture at 14, 000 G for 10 minutes and decant the supernatant.Resuspend the pellet in one milliliter of dimethyl sulfoxide to extract the reduced NBT. Record the absorbance at 560 nanometers. Irradiation of FMN with green and red light showed no effect on the peaks at 372 and 444 nanometers compared to dark control, whereas the reduction in absorbance of FMN at 444 nanometers was observed under blue and violet light, indicating increased photolysis.Detection of superoxide radicals formed during FMN photolysis under blue or violet light was done using NBT reduction. The photochemical effect of FMN was dependent on the irradiation time. FMN photolysis using blue or violet light irradiation resulted in significant inactivation of Staphylococcus aureus.Dose-dependent inactivation was observed for blue light irradiation, with 97%inactivation achieved for 120-micromolar FMN. Under violet light, an inactivation rate of 96%was achieved for staphylococcus aureus, with 30-micromolar FMN showing higher efficacy for bacterial inactivation. NBT reduction showed that the photolytic effect of FMN under violet light in Staphylococcus aureus was proportional to FMN concentration.In untreated conditions, a lower abundance of superoxide radicals was indicated by low absorbance at 560 nanometers, whereas higher absorbance values in FMN treatment showed abundant production of superoxide radicals, resulting in increased photochemical effect. It is critical to have a good LED light source. The spectral width of the violet light regime must be narrow to avoid any overlap with UV absorption, as UV radiation may be hazardous.Following the procedure, other chemical compounds such as tetracycline, doxycycline, catechin, and can be tested for photolysis similar to FMN.

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Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine branches, particularly cell therapy, are novel approaches to improve and restore the urethral sphincter function. Even though injection of active functional cells is routinely performed in clinical settings by needle and syringe, these approaches have significant disadvantages and limitations. In this context, needle-free waterjet (WJ) technology is a feasible and innovative method that can inject viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we used WJ to deliver porcine adipose tissue-derived stromal cells (pADSCs) into cadaveric urethral tissue and subsequently investigated the effect of WJ delivery on cell yield and viability. We also assessed the biomechanical features (i.e., elasticity) by atomic force microscopy (AFM) measurements. We showed that WJ delivered pADSCs were significantly reduced in their cellular elasticity. The viability was significantly lower compared to controls but is still above 80%.

We present a method of cell injection via needle free waterjet technology coupled with a sequela of post-delivery investigations in terms of cellular viability, proliferation, and elasticity measurements.

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...