Quercetin Improves the Apoptotic Index and Oxidative Stress in Post-Thaw Dog Sperm

This study was conducted to investigate if quercetin (QRN) may ameliorate apoptosis and oxidative stress in post-thaw dog sperm. Herein, we evaluated the post-thaw apoptosis and oxidative stress after treatment with QRN (control, 25, 50, and 100 µM) in freezing of dog semen. The oxidative stress index was significantly affected (p<0.05) between the various concentrations of QRN and the control (17.56 ± 1.02, 7.54 ± 0.48, 5.66 ±0.80, and 10.41 ± 0.69), respectively. The apoptosis index was 9.1 ± 1.34, 6.66 ± 0.58, 6.77 ± 0.66, and 5.38 ± 0.86 in the control, and 25, 50, and 100 µM QRN treatment groups, respectively (p< 0.05). The effects of ameliorated cryo-induced damage by QRN on post-thaw sperm quality were also observed through improved structural and functional tests. Sperm treated with 50 µM QRN showed significantly higher motility (51.8 ± 2.1% vs. 43.1 ± 1.4%, P < 0.05), survival rates (46.9 ± 0.7% vs. 43.9 ± 0.4%, P < 0.05), and mucus penetration than control group, respectively. Results demonstrate that supplementing freezing buffer with 50 µM QRN reduced oxidative damage and improved the quality of post-thaw dog sperm.

Alkaline pH increases swimming speed and facilitates mucus penetration for Vibrio cholerae

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two V. cholerae strains (El Tor C6706 and Classical O395) in mucus using single cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae is able to penetrate mucus using flagellar motility and that alkaline pH increases swimming speed, and consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive haemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Overall, our results support that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection. IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single cell tracking that V. cholerae is able to penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome, will offer new strategies to fight cholera.

Oral nanotherapeutics with enhanced mucus penetration and ROS-responsive drug release capacities for delivery of curcumin to colitis tissues

The therapeutic efficacies of oral nanotherapeutics for ulcerative colitis (UC) are seriously hindered by the lack of mucus-penetrating capacity and uncontrolled drug release. To overcome these limitations, the surface of...

Acidic pH reduces Vibrio cholerae motility in mucus by weakening flagellar motor torque

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between the epithelial tissues and luminal microbes. Enteropathogens, such as Vibrio cholerae, must compromise or circumvent the mucus barrier to establish a successful infection. We investigated how motile V. cholerae is able to penetrate mucus using single cell tracking in unprocessed porcine intestinal mucus. We found that changes in pH within the range of what has been measured in the human small intestine indirectly affect V. cholerae flagellar motor torque, and consequently, mucus penetration. Microrheological measurements indicate that the viscoelasticity of mucus does not change substantially within the physiological pH range and that commercially available mucins do not form gels when rehydrated. Finally, we found that besides the reduction in motor torque, El Tor and Classical biotypes have different responses to acidic pH. For El Tor, acidic pH promotes surface attachment that is mediated by activation of the mannose-sensitive haemagglutinin (MshA) pilus without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Overall, our results support that the high torque of V. cholerae flagellar motor is critical for mucus penetration and that the pH gradient in the small intestine is likely an important factor in determining the preferred site of infection.Author summaryThe diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, V. cholerae’s interactions with intestinal mucus has not been extensively investigated. In this report, we demonstrate using single cell tracking that V. cholerae is able to penetrate native intestinal mucus using flagellar motility. In addition, we found that a strong motor torque is required for mucus penetration and, that torque is weakened in acidic environments even though the motor is powered by a sodium potential. This finding has important implications for understanding the dynamics of infection because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with V. cholerae’s flagellar motility, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome, will offer new strategies to fight cholera.

Microfluidics as tool to prepare size-tunable PLGA nanoparticles with high curcumin encapsulation for efficient mucus penetration

Great challenges still remain to develop drug carriers able to penetrate biological barriers (such as the dense mucus in cystic fibrosis) and for the treatment of bacteria residing in biofilms, embedded in mucus. Drug carrier systems such as nanoparticles (NPs) require proper surface chemistry and small size to ensure their permeability through the hydrogel-like systems. We have employed a microfluidic system to fabricate poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with a muco-penetrating stabilizer (Pluronic), with a tunable hydrodynamic diameter ranging from 40 nm to 160 nm. The size dependence was evaluated by varying different parameters during preparation, namely polymer concentration, stabilizer concentration, solvent nature, the width of the focus mixing channel, flow rate ratio and total flow rate. Furthermore, the influence of the length of the focus mixing channel on the size was evaluated in order to better understand the nucleation–growth mechanism. Surprisingly, the channel length was revealed to have no effect on particle size for the chosen settings. In addition, curcumin was loaded (EE% of ≈68%) very efficiently into the nanoparticles. Finally, the permeability of muco-penetrating PLGA NPs through pulmonary human mucus was assessed; small NPs with a diameter of less than 100 nm showed fast permeation, underlining the potential of microfluidics for such pharmaceutical applications.

M13 phage display to identify a permeating peptide against hyperconcentrated mucin

ABSTRACTMucus is an impregnable barrier for drug delivery across the epithelia for treatment of mucosal-associated diseases. While current carriers are promising for mucus penetration, their surface chemistries do not possess chemical complexity to probe and identify optimal physicochemical properties desired for mucus penetration. As initial study, we use M13 phage display presenting random peptides to select peptides that can facilitate permeation through hyperconcentrated mucin. Here, a net-neutral charge, hydrophilic peptide was identified to facilitate transport of phage and fluorophore conjugates through mucin barrier compared to controls. This initial finding warrants further study to understand how composition and spatial distribution of physicochemical properties of peptides can be optimized to improve transport across the mucus barrier.