Optimization of sequence and chiral content enhances therapeutic potential of tilapia piscidin peptides.

Because antimicrobial peptides (AMPs) often exhibit broad-spectrum bactericidal potency, we sought to develop peptide-based antimicrobials for potential clinical use against drug-resistant pathogens. To accomplish this goal, we first optimized the amino acid sequence of a broad-spectrum AMP known as Tilapia Piscidin 4 (TP4). Then, we used the optimized sequence to create a pair of heterochiral variants (TP4-α and TP4-ß) with different percentages of D-enantiomers, as poly-L peptides often exhibit poor pharmacokinetic profiles. The conformations of the peptide pair exhibited inverted chirality according to CD and NMR spectroscopic analyses. Both heterochiral peptides displayed enhanced stability and low hemolysis activities. Irrespective of their different d-enantiomer contents, both heterochiral peptides exhibited bactericidal activities in the presence of human serum or physiological enzymes. However, the peptide with higher d-amino acid content (TP4-ß) caused better bacterial clearance when tested in mice infected with NDM-1 K. pneumoniae. In addition, we observed a relatively higher hydrogen bonding affinity in a simulation of the interaction between TP4-ß and a model bacterial membrane. In sum, our results demonstrate that the current design strategy may be applicable for development of new molecules with enhanced stability and in vivo antimicrobial activity.

Sequential rearrangement and stereochemical reorganization to design an antimicrobial peptide with enhanced stability.

Antimicrobial peptides (AMPs) are natural molecules that function within the innate immune system to counteract pathogenic invasion and minimize the detrimental consequences of infection. However, utilizing these molecules for medical applications has been challenging. In this study, we selected a model AMP with poor stability, Tilapia Piscidin 4 (TP4), and modified its sequence and chirality (TP4-γ) to improve its potential for clinical application. The strategy of chirality inversion was inspired by the cereulide peptide, which has a DDLL enantiomer pattern and exhibits exceptional stability. Sequential substitution of key residues and selective chirality inversion yielded a less toxic peptide with enhanced stability and notable antimicrobial activity. In addition to its superior stability profile and antimicrobial activity, TP4-γ treatment reduced the level of LPS-induced nitric oxide (NO) release in a macrophage cell line. This reduction in NO release may reflect anti-inflammatory properties, as NO is widely known to promote inflammatory processes. Hence, our heterochiral peptide construct shows a more suitable pharmacokinetic profile than its parental compound, and further studies are warranted to develop the molecule for potential clinical application.

Rational Design of Stapled Antimicrobial Peptides to Enhance Stability and In Vivo Potency against Polymicrobial Sepsis.

In this work, we sought to develop a TP4-based stapled peptide that can be used to counter polymicrobial sepsis. First, we segregated the TP4 sequence into hydrophobic and cationic/hydrophilic zones and substituted the preferred residue, lysine, as the sole cationic amino acid. These modifications minimized the intensity of cationic or hydrophobic characteristics within small segments. Then, we incorporated single or multiple staples into the peptide chain, bracketing the cationic/hydrophilic segments to improve pharmacological suitability. Using this approach, we were able to develop an AMP with low toxicity and notable in vivo efficacy. IMPORTANCE In our in vitro studies, one dual stapled peptide out of the series of candidates (TP4-3: FIIXKKSXGLFKKKAGAXKKKXIKK) showed significant activity, low toxicity, and high stability (in 50% human serum). When tested in cecal ligation and puncture (CLP) mouse models of polymicrobial sepsis, TP4-3 improved survival (87.5% on day 7). Furthermore, TP4-3 enhanced the activity of meropenem against polymicrobial sepsis (100% survival on day 7) compared to meropenem alone (37.5% survival on day 7). Molecules such as TP4-3 may be well suited for a wide variety of clinical applications.

Voluntary exercise improves voiding function and bladder hyperalgesia in an animal model of stress-induced visceral hypersensitivity: A multidisciplinary approach to the study of urologic chronic pelvic pain syndrome research network study.

OBJECTIVE: The underlying mechanism of interstitial cystitis/bladder pain syndrome (IC/BPS) is not well understood and evaluation of current therapeutic interventions has not identified any generally effective treatments. Physical activity has shown beneficial effects on individuals suffering from chronic pain. Anxiety-prone rats exposed to water avoidance stress (WAS) develop urinary frequency and lower bladder sensory thresholds with high face and construct validity for the study of IC/BPS. The aim of this study was to evaluate the role of chronic voluntary exercise on urinary frequency, voiding function, and hyperalgesia in animals exposed to WAS. MATERIALS AND METHODS: Twenty-six female Wistar-Kyoto rats were exposed to WAS and thereafter randomized to either voluntary exercise for 3 weeks or sedentary groups. Voiding parameters were assessed at baseline, post-WAS, and weekly for 3 weeks. Before euthanasia, the animals underwent cystometrogram (CMG), external urinary sphincter electromyography, and assessment of visceromotor response (VMR) to isotonic bladder distension (IBD). RESULTS: WAS exposure resulted in adverse changes in voiding parameters. Compared with sedentary animals, animals in the voluntary exercise group had improved voiding parameters during metabolic cage and CMG testing, as well as improved bladder sensory thresholds as determined by VMR during IBD. CONCLUSION: Voluntary exercise in an animal model of chronic stress leads to improvement in voiding function and visceral bladder hyperalgesia.

Comparison between Polybutylcyanoacrylate Nanoparticles with Either Surface-Adsorbed or Encapsulated Brain-Derived Neurotrophic Factor on the Neural Differentiation of iPSCs.

The brain-derived neurotrophic factor (BDNF) is vital in the neural differentiation of neural stem/progenitor cells, and together may have therapeutic potential for neural regeneration. In this study, a multiplexed polybutylcyanoacrylate nanoparticle (PBCA NP) delivery platform was constructed, incorporating either surface-adsorbed or encapsulated BDNF for the induction of neural differentiation in induced pleuripotent stem cells (iPSCs), where tween 80 (T80) and superparamagnetic iron oxide (SPIO) were added for central nervous system (CNS) targeting and magnetic resonance (MR) image tracking, respectively. Both methods by which the BDNF was carried resulted in loading efficiencies greater than 95%. The nanoparticle-mediated delivery of BDNF resulted in neural differentiation of iPSCs detected on immunofluorescence staining as early as 7 days, with enhanced differentiation efficiency by 1.3-fold compared to the control on flow cytometry; the delivery system of surface-adsorbed BDNF gave rise to cells that had the best neural development than the encapsulated formulation. T80-coating disrupted the in vitro blood⁻brain barrier model with a corresponding 1.5- to two-fold increase in permeability. SPIO-loaded PBCA NPs exhibited a concentration-dependent, rapid decay in signal intensity on the phantom MR experiment. This study demonstrates the versatility of the PBCA NP, and the surface-adsorption of BDNF is the preferred method of delivery for the differentiation of iPSCs.

Spinal cord stimulation ameliorates detrusor over-activity and visceromotor pain responses in rats with cystitis.

AIM: Interstitial cystitis/painful bladder syndrome/(IC/PBS) results in recurring pain in the bladder and surrounding pelvic region caused by abnormal excitability of micturition reflexes. Spinal cord stimulation (SCS) is currently clinically used for the attenuation of neuropathic and visceral pain. The present study examined whether SCS at upper lumbar segments modulates detrusor overactivity and visceral hyperalgesia associated with cystitis in a rat model of cyclophosphamide (CYP)-induced cystitis. METHODS: Cystitis was induced by intraperitoneal injection of CYP (200 mg/kg) in six adult female Sprague Dawley rats 48 h prior to urodynamic recordings. Another six rats served as-controls with saline injection. Cystometry and the external urethral sphincter (EUS) electromyography during bladder infusion were evaluated under urethane anesthesia. The visceromotor reflexes (VMR) obtained from the external abdominal oblique muscle were quantified during bladder infusion and isotonic bladder distension (IBD), respectively. After baseline recordings were taken, SCS was applied on the dorsal surface of L3 for 25 min. Urodynamic recordings and VMR during bladder infusion and IBD were repeated 2 h after SCS. RESULTS: CYP resulted in detrusor overactivity, stronger EUS tonic contractions, and increased VMR. SCS significantly reduced non-voiding contractions, prolonged EUS relaxation, and delayed VMR appearance during bladder infusion as well as significantly decreased VMR during IBD in cystitis rats. CONCLUSION: SCS improved bladder function and EUS relaxation during bladder infusion and significantly attenuated visceral nociceptive-related VMR during IBD in cystitis rats. SCS may have therapeutic potential for patients with hyperalgesia and IC/PBS.

Investigations of urethral sphincter activity in mice with bladder hyperalgesia before and after drug administration of gabapentin.

PURPOSE: This study investigated the effect of gabapentin on lower urinary tract dysfunction focusing on urethral activities and cystitis-induced hyperalgesia in a mouse model of painful bladder syndrome/interstitial cystitis (PBS/IC). The electromyography (EMG) of external urethral sphincter (EUS) was difficult to obtain, but contained useful information to examine the drug effect in mice. METHODS: Female C57BL/6J mice were intraperitoneally (ip) administration with either saline or 200 mg/kg of cyclophosphamide (CYP) 48 h before experimental evaluation. Cystitis mice were treated with administration of gabapentin (25 or 50 mg/kg, ip). Cystometry and EUS EMG were obtained and analyzed during continuous bladder infusion. The visceral pain-related visceromotor reflex (VMR) was recorded in response to isotonic bladder distension. RESULTS: Cystitis mice showed shorter inter-contraction intervals and increased occurrence of non-voiding contractions during bladder infusion, with increased VMR during isotonic bladder distension, indicating cystitis-induced bladder hyperalgesia. Gabapentin (50 mg/kg) suppressed effects of CYP on cystometry, but not on EUS EMG activity, during bladder infusion. The effect on urodynamic recordings lasted 4 h. VMR was significantly reduced by gabapentin. CONCLUSIONS: The present study showed that CYP-induced cystitis in mice is a model of visceral hyperalgesia affecting detrusor contractions, not urethral activations. The technique of using EUS EMG to evaluate the drug effects on urethral activities is novel and useful for future investigations. Gabapentin can be as a potential treatment for detrusor overactivity and PBS/IC.

Mapping and neuromodulation of lower urinary tract function using spinal cord stimulation in female rats.

Spinal cord epidural stimulation (SCS) represents a form of neuromodulation for the management of spasticity and pain. This technology has recently emerged as a new approach for potentially augmenting locomotion and voiding function in humans and rodents after spinal cord injury. However, the effect of SCS on micturition has not been studied extensively. Here, SCS was first applied as a direct stimulus onto individual segmental levels of the lumbar spinal cord in rats to map evoked external urethral sphincter (EUS) electromyography activity and SCS-induced voiding contractions. SCS of L2-3 inhibited EUS tonic activity, and SCS on L3 (L3/SCS) inhibited EUS tonic activity and elicited EUS bursting. In contrast, SCS of L1 and L4-6 evoked EUS tonic contractions, which resembled the urethral guarding reflex during bladder storage. Next, the effects of a bilateral pelvic nerve crush (PNC) injury on urodynamic function were examined at 14 days post-operatively. The PNC injury resulted in decreased voiding efficiency and maximum intravesical pressure, whereas the post-voiding residual volume was increased, suggestive of an underactive bladder. Finally, L3/SCS was performed to induce a voiding contraction and enable voiding in rats with a PNC injury. Voiding efficiency was significantly increased, and the residual volume was decreased by L3/SCS in rats after the PNC injury. We conclude that L3/SCS may be used to induce micturition reflexes in a partially filled bladder, reduce urethral resistance, and augment bladder emptying after PNC injury.

Improved thrombolytic effect with focused ultrasound and neuroprotective agent against acute carotid artery thrombosis in rat.

Combination therapy with focused ultrasound (FUS) and a neuroprotective agent, BNG-1, was examined in an acute carotid thrombotic occlusion model using LED irradiation in rat to improve the thrombolytic effect of rt-PA. Seven treatment groups included (A) intravenous bolus injection of 0.45 mg/kg rt-PA, (B) intravenous bolus injection of 0.9 mg/kg, (C) sonothrombolysis with FUS alone, (D) oral administration of 2 g/kg BNG-1 for 7 days alone, (E) A + D, (F) A + C, and (G) A + C + D. Four comparison groups were made including (H) 0.45 mg/kg rt-PA 20% bolus +80% IV fusion + FUS, (I) 0.9 mg/kg rt-PA with 10% bolus + 90% intravenous fusion, (J) B + C, (K) B + D. At 7 days after carotid occlusion, small-animal carotid ultrasound and 7 T MR angiography showed the recanalization rate of ≤50% stenosis was 50% in group B and 83% in group I, but 0% in groups A and C and 17% in group D. Combination therapy improved recanalization rate to 50-63% in groups E and F, to 67-83% in groups J and K, and to 100% in groups G and H. Our study demonstrated combination therapy with different remedies can be a feasible strategy to improve the thrombolytic effect of rt-PA.

Synthesis of composite magnetic nanoparticles Fe3O4 with alendronate for osteoporosis treatment.

Osteoporosis is a result of imbalance between bone formation by osteoblasts and resorption by osteoclasts (OCs). In the present study, we investigated the potential of limiting the aggravation of osteoporosis by reducing the activity of OCs through thermolysis. The proposed method is to synthesize bisphosphonate (Bis)-conjugated iron (II, III) oxide (Fe3O4) nanoparticles and incorporate them into OCs. The cells should be subsequently exposed to radiofrequency (RF) to induce thermolysis. In this study, particles of Fe3O4 were first synthesized by chemical co-precipitation and then coated with dextran (Dex). The Dex/Fe3O4 particles were then conjugated with Bis to form Bis/Dex/Fe3O4. Transmission electron microscopy revealed that the average diameter of the Bis/Dex/Fe3O4 particles was ~20 nm. All three kinds of nanoparticles were found to have cubic inverse spinel structure of Fe3O4 by the X-ray diffraction analysis. Fourier transform infrared spectroscopy confirmed that the Dex/Fe3O4 and Bis/Dex/Fe3O4 nanoparticles possessed their respective Dex and Bis functional groups, while a superconducting quantum interference device magnetometer measured the magnetic moment to be 24.5 emu. In addition, the Bis/Dex/Fe3O4 nanoparticles were fully dispersed in double-distilled water. Osteoblasts and OCs were individually cultured with the nanoparticles, and an MTT assay revealed that they were non-cytotoxic. An RF system (42 kHz and 450 A) was used to raise the temperature of the nanoparticles for 20 minutes, and the thermal effect was found to be sufficient to destroy OCs. Furthermore, in vivo studies verified that nanoparticles were indeed magnetic resonance imaging contrast agents and that they accumulated after being injected into the body of rats. In conclusion, we developed a water-dispersible magnetic nanoparticle that had RF-induced thermogenic properties, and the results indicated that the Bis/Dex/Fe3O4 nanoparticle had the potential for controlling osteoporosis.

A rat model of thrombosis in common carotid artery induced by implantable wireless light-emitting diode device.

This work has developed a novel approach to form common carotid artery (CCA) thrombus in rats with a wireless implantable light-emitting diode (LED) device. The device mainly consists of an external controller and an internal LED assembly. The controller was responsible for wirelessly transmitting electrical power. The internal LED assembly served as an implant to receive the power and irradiate light on CCA. The thrombus formation was identified with animal sonography, 7 T magnetic resonance imaging, and histopathologic examination. The present study showed that a LED assembly implanted on the outer surface of CCA could induce acute occlusion with single irradiation with 6 mW/cm(2) LED for 4 h. If intermittent irradiation with 4.3-4.5 mW/cm(2) LED for 2 h was shut off for 30 min, then irradiation for another 2 h was applied; the thrombus was observed to grow gradually and was totally occluded at 7 days. Compared with the contralateral CCA without LED irradiation, the arterial endothelium in the LED-irradiated artery was discontinued. Our study has shown that, by adjusting the duration of irradiation and the power intensity of LED, it is possible to produce acute occlusion and progressive thrombosis, which can be used as an animal model for antithrombotic drug development.

Preparation and characterization of biocompatible and thermoresponsive micelles based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) grafted on polysuccinimide for drug delivery.

Biocompatible and temperature-sensitive amphiphilic polymeric micelles comprised of poly(succinimide)-g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PSI-g-poly(NIPAAm-co-DMAAm)) were synthesized to use as new drug carriers. The PSI-co-poly(PNIPAAm-co-DMAAm) polymers were prepared by nucleophilic opening of poly(succinimide) using amino-terminated poly(NIPAAm-co-DMAAm). The lower critical solution temperature of the copolymer was 40.6℃ higher than normal human body temperature. The blank polymeric micelles were observed to have a regular spherical shape, and the particle sizes were approximately 85 nm. This copolymer exhibited no significant cytotoxicity and hemolysis indicated that the micelles had good biocompatibility. In addition, these polymeric micelles encapsulated the anti-inflammatory drug, hesperetin, in the inner core with a drug loading content of approximately 20%. The release profiles of hesperetin showed a significant temperature-sensitive switching behavior. The hesperetin release response was dramatically lower at a temperature below the lower critical solution temperature as compared with a temperature above the lower critical solution temperature. The lipopolysaccharide-induced nitric oxide production inhibition experiments demonstrated that hesperetin-encapsulated micelles showed a significant reduction. In this study, the biocompatible temperature-sensitive micelles based on PSI-g-poly(NIPAAm-co-DMAAm) have great potential to act as a suitable carrier for drug delivery.

Laser-induced carotid artery injury model in the rat for therapeutic agent screening.

The aims of this study were to establish a rat model of carotid artery injury and to evaluate its suitability for evaluating therapeutic agents active against endothelial proliferation. Wistar-Kyoto rats were injected intravenously with the photochemically reactive dyes rose bengal or Evans blue, and the carotid artery was then focally irradiated with laser light of the appropriate wavelength. Histological sections of the carotid artery were analyzed to determine the appropriate parameters for this model. Ferulic acid was used to assess the suitability of this model for drug screening. No animal died as a result of the photochemical treatment. Endothelial proliferation in the carotid artery was observed in rats injected with rose Bengal and exposed to green laser light. Ferulic acid (400 mg/kg per day) significantly (p<0.05) reduced endothelial proliferation in the carotid artery 28 days after injury in dye-treated animals compared with vehicle-treated animals. This simple experimental rat model is suitable for studying factors inhibiting endothelial thickening after vessel damage and for developing therapeutic strategies active against endothelial proliferation.