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Wound infection and excessive blood loss are the two major challenges associated with trauma injuries that account for 10% of annual deaths in the United States. Nitric oxide (NO) is a gasotransmitter cell signaling molecule that plays a crucial role in the natural wound healing process due to its antibacterial, anti-inflammatory, cell proliferation, and tissue remodeling abilities. Tranexamic acid (TXA), a prothrombotic agent, has been used topically and systemically to control blood loss in reported cases of epistaxis and combat-related trauma injuries. Its properties could be incorporated in wound dressings to induce immediate clot formation, which is a key factor in controlling excessive blood loss. This study introduces a novel, instant clot-forming NO-releasing dressing, and fabricated using a strategic bi-layer configuration. The layer adjacent to the wound was designed with TXA suspended on a resinous bed of propolis, which is a natural bioadhesive possessing antibacterial and anti-inflammatory properties. The base layer, located furthest away from the wound, has an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), embedded in a polymeric bed of Carbosil®, a copolymer of polycarbonate urethane and silicone. Propolis was integrated with a uniform layer of TXA in variable concentrations: 2.5, 5.0, and 7.5 vol % of propolis. This design of the TXA-SNAP-propolis (T-SP) wound dressing allows TXA to form a more stable clot by preventing the lysis of fibrin. The lactate dehydrogenase-based platelet adhesion assay showed an increase in fibrin activation with 7.5% T-SP as compared with control within the first 15 min of its application. A scanning electron microscope (SEM) confirmed the presence of a dense fibrin network stabilizing the clot for fabricated dressing. The antibacterial activity of NO and propolis resulted in a 98.9 ± 1% and 99.4 ± 1% reduction in the colony-forming unit of Staphylococcus aureus and multidrug-resistant Acinetobacter baumannii, respectively, which puts forward the fabricated dressing as an emergency first aid for traumatic injuries, preventing excessive blood loss and soil-borne infections., (© 2024 Wiley Periodicals LLC.)

Recent clinical trials of drugs enhancing cyclic guanosine monophosphate (cGMP) signaling for cardiovascular diseases have renewed interest in cGMP biology within the kidney. However, the role of cGMP signaling in glomerular endothelial cells (GECs) and podocytes remains largely unexplored. Using acute kidney slices from mice expressing the FRET-based cGMP biosensor cGi500 in endothelial cells or podocytes enabled real-time visualization of cGMP. Stimulation with atrial natriuretic peptide (ANP) or SNAP (NO donor) and various phosphodiesterase (PDE) inhibitors elevated intracellular cGMP in both cell types. GECs showed a transient cGMP response upon particulate or soluble guanylyl cyclase activation, while the cGMP response in podocytes reached a plateau following ANP administration. Co-stimulation (ANP + SNAP) led to an additive response in GECs. The administration of PDE inhibitors revealed a broader basal PDE activity in GECs dominated by PDE2a. In podocytes, basal PDE activity was mainly restricted to PDE3 and PDE5 activity. Our data demonstrate the existence of both guanylyl cyclase pathways in GECs and podocytes with cell-specific differences in cGMP synthesis and degradation, potentially suggesting new therapeutic options for kidney diseases., (© 2024. The Author(s).)

The l -arginine ( l -Arg)/nitric oxide/cyclic GMP/potassium channel (K ATP ) pathway and opioid receptors are known to play critical roles in pain perception and the antinociceptive effects of various compounds. While there is evidence suggesting that the analgesic effects of rutin may involve nitric oxide modulation, the direct link between rutin and the l -Arg/nitric oxide/cyclic GMP/K ATP pathway in the context of pain modulation requires further investigation. The antinociceptive effect of rutin was studied in male NMRI mice using the formalin test. To investigate the role of the l -Arg/nitric oxide/cyclic GMP/K ATP pathway and opioid receptors, the mice were pretreated intraperitoneally with different substances. These substances included l -Arg (a precursor of nitric oxide), S-nitroso- N -acetylpenicillamine (SNAP, a nitric oxide donor), N(gamma)-nitro- l -arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase), sildenafil (an inhibitor of phosphodiesterase enzyme), glibenclamide (a K ATP channel blocker), and naloxone (an opioid receptor antagonist). All pretreatments were administered 20 min before the administration of the most effective dose of rutin. Based on our investigation, it was found that rutin exhibited a dose-dependent antinociceptive effect. The administration of SNAP enhanced the analgesic effects of rutin during both the initial and secondary phases. Moreover, L-NAME, naloxone, and glibenclamide reduced the analgesic effects of rutin in both the primary and secondary phases. In conclusion, rutin holds significant value as a flavonoid with analgesic properties, and its analgesic effect is directly mediated through the nitric oxide/cyclic GMP/K ATP channel pathway., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Photothermal therapy (PTT) shows promise in cancer treatments due to its good spatiotemporal selectivity and minimal invasiveness. However, PTT has some problems such as excessive heat damage to normal tissues, tumor thermo-resistance caused by heat shock proteins (HSPs), and limited efficacy of monotherapy. Here, we construct a patch named "partitioned microneedles" (PMN-SNAP/CuS), which separates the "catalyst" bovine serum albumin-based copper sulfide nanoparticles (CuS@BSA NPs) and the "reactant" S -nitroso- N -acetylpenicillamine (SNAP) into different regions of microneedles, for enhancing mild PTT (mPTT) of melanoma. PMN-SNAP/CuS showed an excellent photothermal effect, Fenton-like catalytic activity, and nitric oxide (NO) generation ability. The combination of NO and reactive oxygen species (ROS) produced by PMN-SNAP/CuS effectively blocked the synthesis of HSPs at the source and enhanced the efficacy of mPTT. Both in vitro and in vivo results proved that PMN-SNAP/CuS significantly enhanced the inhibition of melanoma under 808 nm laser irradiation. In conclusion, our partitioned microneedle strategy based on the combination of enhanced mPTT and gas therapy (GT) provides a promising approach to enhance the therapeutic effect on melanoma.

Healthcare associated infections (HCAI) represent a significant burden worldwide contributing to morbidity and mortality and result in substantial economic consequences equating to billions annually. Although the impacts of HCAI have been felt for many years, the coronavirus pandemic has had a profound effect, escalating rates of HCAI, even with extensive preventative measures such as vaccination, personal protective equipment, and deep cleaning regimes. Therefore, there is an urgent need for new solutions to mitigate this serious health emergency. In this paper, the fabrication of nitric oxide (NO) releasing dual action polymer coatings for use in healthcare applications is described. The coatings are doped with the NO donor S -nitroso- N -acetylpenicillamine (SNAP) and release high payloads of NO in a sustained manner for in excess of 50 hours. These coatings are extensively characterized in multiple biologically relevant solutions and the antibacterial/antiviral efficacy is studied. For the first time, we assess antibacterial activity in a time course study (1, 2, 4 and 24 h) in both nutrient rich and nutrient poor conditions. Coatings exhibit excellent activity against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA), with up to complete reduction observed over 24 hours. Additionally, when tested against SARS-CoV-2, the coatings significantly reduced active virus in as little as 10 minutes. These promising results suggest that these coatings could be a valuable addition to existing preventative measures in the fight against HCAIs.

The acidic byproducts of bacteria in plaque around orthodontic brackets contribute to white spot lesion (WSL) formation. Nitric oxide (NO) has antibacterial properties, hindering biofilm formation and inhibiting the growth of oral microbes. Materials that mimic NO release could prevent oral bacteria-related pathologies. This study aims to integrate S-nitroso-acetylpenicillamine (SNAP), a promising NO donor, into orthodontic elastomeric ligatures, apply an additional polymer coating, and evaluate the NO-release kinetics and antimicrobial activity against Streptococus mutans . SNAP was added to clear elastomeric chains (8 loops, 23 mm long) at three concentrations (50, 75, 100 mg/mL, and a control). Chains were then coated, via electrospinning, with additional polymer (Elastollan ® ) to aid in extending the NO release. NO flux was measured daily for 30 days. Samples with 75 mg/mL SNAP + Elastollan ® were tested against S. mutans for inhibition of biofilm formation on and around the chain. SNAP was successfully integrated into ligatures at each concentration. Only the 75 mg/mL SNAP chains maintained their elasticity. After polymer coating, samples exhibited a significant burst of NO on the first day, exceeding the machine's reading capacity, which gradually decreased over 29 days. Ligatures also inhibited S. mutans growth and biofilm formation. Future research will assess their mechanical properties and cytotoxicity. This study presents a novel strategy to address white spot lesion (WSL) formation and bacterial-related pathologies by utilizing nitric oxide-releasing materials. Manufactured chains with antimicrobial properties provide a promising solution for orthodontic challenges, showing significant potential for academic-industrial collaboration and commercial viability.

Bacteria-associated infections and thrombus formation are the two major complications plaguing the application of blood-contacting medical devices. Therefore, functionalized surfaces and drug delivery for passive and active antifouling strategies have been employed. Herein, we report the novel integration of bio-inspired superhydrophobicity with nitric oxide release to obtain a functional polymeric material with anti-thrombogenic and antimicrobial characteristics. The nitric oxide release acts as an antimicrobial agent and platelet inhibitor, while the superhydrophobic components prevent non-specific biofouling. Widely used medical-grade silicone rubber (SR) substrates that are known to be susceptible to biofilm and thrombus formation were dip-coated with fluorinated silicon dioxide (SiO 2 ) and silver (Ag) nanoparticles (NPs) using an adhesive polymer as a binder. Thereafter, the resulting superhydrophobic (SH) SR substrates were impregnated with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) to obtain a superhydrophobic, Ag-bound, NO-releasing (SH-SiAgNO) surface. The SH-SiAgNO surfaces had the lowest amount of viable adhered E. coli (> 99.9 % reduction), S. aureus (> 99.8 % reduction), and platelets (> 96.1 % reduction) as compared to controls while demonstrating no cytotoxic effects on fibroblast cells. Thus, this innovative approach is the first to combine SNAP with an antifouling SH polymer surface that possesses the immense potential to minimize medical device-associated complications without using conventional systemic anticoagulation and antibiotic treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacterial biofilms play a central role in the development and progression of periodontitis, a chronic inflammatory condition that affects the oral cavity. One solution to current treatment constraints is using nitric oxide (NO)─with inherent antimicrobial properties. In this study, an antimicrobial coating is developed from the NO donor S -nitroso- N -acetylpenicillamine (SNAP) embedded within polyethylene glycol (PEG) to prevent periodontitis. The SNAP-PEG coating design enabled a controlled NO release, achieving tunable NO levels for more than 24 h. Testing the SNAP-PEG composite on dental floss showed its effectiveness as a uniform and bioactive coating. The coating exhibited antibacterial properties against Streptococcus mutans and Escherichia coli , with inhibition zones measuring up to 7.50 ± 0.28 and 14.80 ± 0.46 mm 2 , respectively. Furthermore, SNAP-PEG coating materials were found to be stable when stored at room temperature, with 93.65% of SNAP remaining after 28 d. The coatings were biocompatible against HGF and hFOB 1.19 cells through a 24 h controlled release study. This study presents a facile method to utilize controlled NO release with dental antimicrobial coatings comprising SNAP-PEG. This coating can be easily applied to various substrates, providing a user-friendly approach for targeted self-care in managing gingival infections associated with periodontitis.

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Hitesh Handa is a founder of Nytricx INC, which is involved in exploring possibilities of using nitric oxide-releasing materials for medical applications., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Urinary tract infections (UTIs) are some of the most common infections seen in humans, affecting over half of the female population. Though easily and quickly treatable, if gone untreated for too long, UTIs can lead to narrowing of the urethra as well as bladder and kidney infections. Due to the disease potential, it is crucial to mitigate the development of UTIs throughout healthcare. Unfortunately, sexual activity and the use of condoms have been identified as common risk factors for the development of sexually acquired UTIs. Therefore, this study outlines a potential alteration to existing condom technology to decrease the risk of developing sexually acquired UTIs using S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor. Herein, varying concentrations of SNAP are integrated into commercialized condoms through a facile solvent swelling method. Physical characterization studies showed that 72%-100% of the ultimate tensile strength was maintained with lower SNAP concentrations, validating the modified condom's mechanical integrity. Additionally, the evaluation of room-temperature storage stability via NO release analysis outlined a lack of special storage conditions needed compared to commercial products. Moreover, these samples exhibited >90% relative cell viability and >96% bacterial killing, proving biocompatibility and antimicrobial properties. SNAP-Latex maintains the desired condom durability while demonstrating excellent potential as an effective new contraceptive technology to mitigate the occurrence of sexually acquired UTIs., (© 2024 Wiley Periodicals LLC.)

S-Nitroso-N-acetylpenicillamine (SNAP) is among the most common nitric oxide (NO)-donor molecules and its solid-state photolytic decomposition has potential for inhaled nitric oxide (iNO) therapy. The photochemical NO release kinetics and mechanism were investigated by exposing solid-state SNAP to a narrow-band LED as a function of nominal wavelength and intensity of incident light. The photolytic efficiency, decomposition products, and the photolytic pathways of the SNAP were examined. The maximum light penetration depth through the solid layer of SNAP was determined by an optical microscope and found to be within 100-200 μm, depending on the wavelength of light. The photolysis of solid-state SNAP to generate NO along with the stable thiyl (RS·) radical was confirmed using Electron Spin Resonance (ESR) spectroscopy. The fate of the RS· radical in the solid phase was studied both in the presence and absence of O 2 using NMR, IR, ESR, and UPLC-MS. The changes in the morphology of SNAP due to its photolysis were examined using PXRD and SEM. The stable thiyl radical formed from the photolysis of solid SNAP was found to be reactive with another adjacent thiyl radical to form a disulfide (RSSR) or with oxygen to form various sulfonyl and sulfonyl peroxyl radicals {RS(O) x O·, x = 0 to 7}. However, the thiyl radical did not recombine with NO to reform the SNAP. From the PXRD data, it was found that the SNAP loses its crystallinity by generating the NO after photolysis. The initial release of NO during photolysis was increased with increased intensity of light, whereas the maximum light penetration depth was unaffected by light intensity. The knowledge gained about the photochemical reactions of SNAP may provide important insight in designing portable photoinduced NO-releasing devices for iNO therapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The emergence of antibiotic-resistant bacteria poses a pressing threat to global health and is a leading cause of healthcare-related morbidity and mortality. Herein, we report the fabrication of medical-grade polymers incorporated with a dual-action S-nitroso-N-acetylpenicillamine-functionalized ampicillin (SNAPicillin) conjugated molecule through a solvent evaporation process. The resulting SNAPicillin-incorporated polymer materials act as broad-spectrum antibacterial surfaces that improve the administration efficacy of conventional antibiotics through the targeted release of both nitric oxide and ampicillin. The polymer surfaces were characterized by scanning electron microscopy and static contact angle measurements. The nitric oxide (NO) release profile and diffusion of SNAPicillin from polymers were quantified using a chemiluminescence-based nitric oxide analyzer (NOA) and ultraviolet-visible (UV-vis) spectroscopy. As a result, the films had up to 2.96 × 10 -7 mol cm -2 of total NO released within 24 hr. In addition, >79 % of the SNAPicillin reservoir was preserved in the polymers after 24 hr of incubation in the physiological environment, indicating their longer-term NO release ability and therapeutic window for antibacterial effects. The SNAPicillin-incorporated polymers reduced the viability of adhered bacteria in culture, with >95 % reduction found against clinically relevant strains of Staphylococcus aureus (S. aureus). Furthermore, SNAPicillin-modified surfaces did not elicit a cytotoxic effect toward 3T3 mouse fibroblast cells, supporting the material's biocompatibility in vitro. These results indicate that the complementary effects of NO-release and ampicillin in SNAPicillin-eluting polymers can enhance the properties of commonly infected medical device surfaces for antibacterial purposes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Currently, the controlled release of nitric oxide (NO) plays a crucial role in various biomedical applications. However, injectable NO-releasing materials remain an underexplored research field to date. In this study, via the incorporation of S-nitroso-N-acetyl-penicillamine (SNAP) as an NO donor, a family of NO-releasing injectable hydrogels was synthesized through the in situ cross-linking between sodium alginate and calcium ion induced by D-(+)-gluconate δ-lactone as an initiator. Initially, the organic functional groups and the corresponding morphologies of the resulting injectable hydrogels were characterized by IR and SEM spectroscopies, respectively. The NO release times of hydrogels with different SNAP loading amounts could reach up to 36-47 h. Due to the release of NO, the highest antibacterial rates of these injectable hydrogels against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were up to 95 %, respectively. Furthermore, the matrix of these hydrogels demonstrated great water absorption ability, swelling behavior, and degradation performance. Finally, we expect that these NO-releasing injectable hydrogels could have great potential applications various biomedical material fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

This report discusses potential therapies for treating human coronaviruses (HCoVs) and their economic impact. Specifically, we explore therapeutics that can support the body's immune response, including immunoglobulin (Ig)A, IgG and T-cell responses, to inhibit the viral replication cycle and improve respiratory function. We hypothesize that carbon quantum dots conjugated with S-nitroso-N-acetylpenicillamine (SNAP) could be a synergistic alternative cure for treating respiratory injuries caused by HCoV infections. To achieve this, we propose developing aerosol sprays containing SNAP moieties that release nitric oxide and are conjugated onto promising nanostructured materials. These sprays could combat HCoVs by inhibiting viral replication and improving respiratory function. Furthermore, they could potentially provide other benefits, such as providing novel possibilities for nasal vaccines in the future., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We have previously reported that L-arginine, a nitric oxide synthase substrate, inhibits the basolateral 10-pS Cl- channel through the cGMP/PKG signaling pathway in the thick ascending limb (TAL). As a NO releasing agent, the effect of S-nitroso-N-acetyl-penicillamine (SNAP) on the channel activity was examined in thick ascending limb of C57BL/6 mice in the present study. SNAP inhibited the basolateral 10-pS Cl- channel in a dose-dependent manner with an IC50 value of 6.6 μM. The inhibitory effect of SNAP was abolished not only by NO scavenger (carboxy-PTIO) but also by blockers of soluble guanylate cyclase (ODQ or LY-83583), indicating that the cGMP-dependent signaling pathway is involved. Moreover, the inhibitory effect of SNAP on the channel was strongly attenuated by a protein kinase G (PKG)-specific inhibitor, KT-5823, but not by the PDE2 inhibitor, BAY-60-7550. We concluded that SNAP inhibited the basolateral 10-pS Cl- channels in the TAL through a cGMP/PKG signaling pathway. As the 10-pS Cl- channel is important for regulation of NaCl absorption along the nephron, these data suggest that SNAP might be served as a regulator to prevent high-salt absorption related diseases, such as hypertension., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biofilm formation on biomaterial interfaces and the development of antibiotic-resistant bacteria have decreased the effectiveness of traditional antibiotic treatment of infections. In this project, ampicillin, a commonly used antibiotic, was conjugated with S -nitroso- N -acetylpenicillamine (SNAP), an S -nitrosothiol compound (RSNO) used for controlled nitric oxide (NO) release. This novel multifunctional molecule is the first of its kind to provide combined antibiotic and NO treatment of infectious pathogens. Characterization of the molecule included NMR, FTIR, and mass spectrometry. NO release behavior was also measured and compared to pure, unmodified SNAP. When evaluating the antimicrobial efficacy, the synthesized SNAPicillin molecule showed the lowest MIC value against Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-resistant Staphylococcus aureus compared to ampicillin and SNAP alone. SNAPicillin also displayed enhanced biofilm dispersal and killing of both bacterial strains when treating a 48 h biofilm preformed on a polymer surface. The antibacterial results combined with the biocompatibility of the molecule show great promise for infection prevention and treatment of polymeric interfaces to reduce medical device-related infections.

Small diameter vascular grafts (SDVGs) are associated with a high failure rate due to poor endothelialization. The incorporation of a nitric oxide (NO) releasing system improves biocompatibility by using the NO effect to promote endothelial cell (EC) migration and proliferation while preventing bacterial infection. To circumvent the instability of NO donors and to prolong NO releasing, S-nitroso-N-acetyl-D-penicillamine (SNAP) as a NO donor was loaded in multi-walled carbon nanotubes (MWCNTs). Successful loading was confirmed with a maximum SNAP amount of ~ 5% (w/w) by TEM, CHNS analysis and FTIR spectra. SDVGs were 3D printed from polycaprolactone (PCL) and coated with a 1:1 ratio of polyethylene glycol and PCL dopped with different concentrations of SNAP-loaded matrix and combinations of MWCNTs-OH. Coating with 10% (w/w) SNAP-matrix-10% (w/w) SNAP-MWCNT-OH showed a diminished burst release and 18 days of NO release in the range of 0.5-4 × 10 -10  mol cm -2  min -1 similar to the NO release from healthy endothelium. NO-releasing SDVGs were cytocompatible, significantly enhanced EC proliferation and migration and diminished bacterial viability. The newly developed SNAP-loaded MWCNT-OH has a great potential to develop NO releasing biomaterials with a prolonged, controlled NO release promoting in-situ endothelialization and tissue integration in vivo, even as an approach towards personalized medicine., (© 2023. The Author(s).)

The controlled release of nitric oxide (NO) is significantly crucial in the NO-related biomedical field. In the current work, the controlled release of NO from alginate microspheres was achieved through the direct impregnation of S-nitroso-N-acetyl-penicillamine (SNAP) in the gelation of sodium alginate with calcium ions. The loading rate of SNAP in alginate microspheres was obtained in a range of 0.69 %‑27.5 %. Specifically, the longest NO release time reached up to ∼93 h. Furthermore, the structure, thermal properties, and morphology were fully characterized. During the antibacterial studies, the NO-releasing spheres can produce a great bactericidal effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The alginate microspheres impregnated with 315 mg SNAP (sphere size: 2.88 mm) can effectively reduce the number of bacteria by 7 orders of magnitude with an inhibition rate up to 100 %. Therefore, we anticipated that these NO-releasing alginate microspheres would have great potential for biomedical-related applications., Competing Interests: Declaration of competing interest No competing financial interests or personal relationships were needed to be declared in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Physical incorporation of nitric oxide (NO) releasing materials in biomedical grade polymer matrices to fabricate antimicrobial coatings and devices is an economically viable process. However, achieving long-term NO release with a minimum or no leaching of the NO donor from the polymer matrix is still a challenging task. Herein, ( N -acetyl- S -nitrosopenicillaminyl)- S -nitrosopenicillamine (SNAP-SNAP), a penicillamine dipeptide NO-releasing molecule, is incorporated into a commercially available biomedical grade silicone rubber (SR) to fabricate a NO-releasing coating (SNAP-SNAP/SR). The storage stabilities of the SNAP-SNAP powder and SNAP-SNAP/SR coating were analyzed at different temperatures. The SNAP-SNAP/SR coatings with varying wt % of SNAP-SNAP showed a tunable and sustained NO release for up to 6 weeks. Further, S -nitroso- N -acetylpenicillamine (SNAP), a well-explored NO-releasing molecule, was incorporated into a biomedical grade silicone polymer to fabricate a NO-releasing coating (SNAP/SR) and a comparative analysis of the NO release and S -nitrosothiol (RSNO) leaching behavior of 10 wt % SNAP-SNAP/SR and 10 wt % SNAP/SR was studied. Interestingly, the 10 wt % SNAP-SNAP/SR coatings exhibited ∼36% higher NO release and 4 times less leaching of NO donors than the 10 wt % SNAP/SR coatings. Further, the 10 wt % SNAP-SNAP/SR coatings exhibited promising antibacterial properties against Staphylococcus aureus and Escherichia coli due to the persistent release of NO. The 10 wt % SNAP-SNAP/SR coatings were also found to be biocompatible against NIH 3T3 mouse fibroblast cells. These results corroborate the sustained stability and NO-releasing properties of the SNAP-SNAP in a silicone polymer matrix and demonstrate the potential for the SNAP-SNAP/SR polymer in the fabrication of long-term indwelling biomedical devices and implants to enhance biocompatibility and resist device-related infections.

The presence of bacteria and biofilm on medical device surfaces has been linked to serious infections, increased health care costs, and failure of medical devices. Therefore, antimicrobial biointerfaces and medical devices that can thwart microbial attachment and biofilm formation are urgently needed. Both nitric oxide (NO) and chlorhexidine diacetate (CHXD) possess broad-spectrum antibacterial properties. In the past, individual polymer release systems of CHXD and NO donor S-nitroso-N-acetylpenicillamine (SNAP) incorporated polymer platforms have attracted considerable attention for biomedical/therapeutic applications. However, the combination of the two surfaces has not yet been explored. Herein, the synergy of NO and CHXD was evaluated to create an antimicrobial medical-grade silicone rubber. The 10 wt% SNAP films were fabricated using solvent casting with a topcoat of CHXD (1, 3, and 5 wt%) to generate a dual-active antibacterial interface. Chemiluminescence studies confirmed the NO release from SNAP-CHXD films at physiologically relevant levels (0.5-4 × 10 -10  mol min -1  cm -2 ) for at least 3 weeks and CHXD release for at least 7 days. Further characterization of the films via SEM-EDS confirmed uniform distribution of SNAP and presence of CHXD within the polymer films without substantial morphological changes, as confirmed by contact angle hysteresis. Moreover, the dual-active SNAP-CHXD films were able to significantly reduce Escherichia coli and Staphylococcus aureus bacteria (>3-log reduction) compared to controls with no explicit toxicity towards mouse fibroblast cells. The synergy between the two potent antimicrobial agents will help combat bacterial contamination on biointerfaces and enhance the longevity of medical devices., (© 2022 Wiley Periodicals LLC.)

Therapeutic agents can be linked to nanoparticles to fortify their selectivity and targeted delivery while impeding systemic toxicity and efficacy loss. Titanium dioxide nanoparticles (TiNPs) owe their rise in biomedical sciences to their versatile applicability, although the lack of inherent antibacterial properties limits its application and necessitates the addition of bactericidal agents along with TiNPs. Structural modifications can improve TiNP's antibacterial impact. The antibacterial efficacy of nitric oxide (NO) against a broad spectrum of bacterial strains is well established. For the first time, S -nitroso- N -acetylpenicillamine (SNAP), an NO donor molecule, was covalently immobilized on TiNPs to form the NO-releasing TiNP-SNAP nanoparticles. The TiNPs were silanized with 3-aminopropyl triethoxysilane, and N -acetyl-d-penicillamine was grafted to them via an amide bond. The nitrosation was carried out by t -butyl nitrite to conjugate the NO-rich SNAP moiety to the surface. The total NO immobilization was measured to be 127.55 ± 4.68 nmol mg -1 using the gold standard chemiluminescence NO analyzer. The NO payload can be released from the TiNP-SNAP under physiological conditions for up to 20 h. The TiNP-SNAP exhibited a concentration-dependent antimicrobial efficiency. At 5 mg mL -1 , more than 99.99 and 99.70% reduction in viable Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, respectively, were observed. No significant cytotoxicity was observed against 3T3 mouse fibroblast cells at all the test concentrations determined by the CCK-8 assay. TiNP-SNAP is a promising and versatile nanoparticle that can significantly impact the usage of TiNPs in a wide variety of applications, such as biomaterial coatings, tissue engineering scaffolds, or wound dressings.

Although numerous prevention and intervention techniques have been developed to counteract catheter-associated urinary tract infections (CAUTIs), urinary catheters remain one of the most common sources of hospital-acquired infections. Nitric oxide (NO), a gaseous free radical responsible for regulating many physiological functions in the body, has gained immense popularity due to its potent, broad-spectrum antimicrobial activity, which is capable of combating medical device-associated infections. In this work, a straightforward solvent-swelling method was used to load the NO donor S -nitroso- N -acetyl-penicillamine (SNAP) into commercial latex catheters (SNAP-UCs) for the first time. The effects of swelling catheters with different concentrations of SNAP solutions (25-125 mg/mL SNAP in tetrahydrofuran (THF)) were studied by measuring the NO release kinetics, SNAP loading, and SNAP leaching. SNAP-UCs impregnated with a 50 mg/mL SNAP-THF solution were found to maximize the amount of SNAP loaded into the latex (0.115 ± 0.009 mg SNAP/mg catheter) and showed physiological levels of NO release (>2 × 10 -10 mol min -1 cm -2 ) over 7 days and minimal SNAP leaching (<2%). SNAP-UCs showed impressive in vitro contact-based and diffusible antimicrobial efficacy against three CAUTI-associated pathogens, reducing the viability of adhered and planktonic Escherichia coli , Proteus mirabilis , and Staphylococcus aureus by ∼98.0 to 99.1% (adhered) and 86.3-96.3% (planktonic) compared to control latex catheters. In vitro cytotoxicity against 3T3 mouse fibroblasts using a CCK-8 assay showed that SNAP-UCs were noncytotoxic (>90% viability). In summary, SNAP-UCs show stable, noncytotoxic NO release characteristics capable of potent, broad-spectrum antimicrobial activity, demonstrating great potential for reducing the devastating effects associated with CAUTIs.

The host immune system plays a pivotal role in the containment of Mycobacterium tuberculosis (Mtb) infection, and host-directed therapy (HDT) is emerging as an effective strategy to treat tuberculosis (TB), especially drug-resistant TB. Previous studies revealed that expression of sirtuin 7 (SIRT7), a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, was downregulated in macrophages after Mycobacterial infection. Inhibition of SIRT7 with the pan-sirtuin family inhibitor nicotinamide (NAM), or by silencing SIRT7 expression, promoted intracellular growth of Mtb and restricted the generation of nitric oxide (NO). Addition of the exogenous NO donor SNAP abrogated the increased bacterial burden in NAM-treated or SIRT7-silenced macrophages. Furthermore, SIRT7-silenced macrophages displayed a lower frequency of early apoptotic cells after Mycobacterial infection, and this could be reversed by providing exogenous NO. Overall, this study clarified a SIRT7-mediated protective mechanism against Mycobacterial infection through regulation of NO production and apoptosis. SIRT7 therefore has potential to be exploited as a novel effective target for HDT of TB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Liu, Zhou, Ou, Xiao, Li, Wang, Wang, Liu and Zhang.)

Attachment of a nitric oxide (NO) donor to an electrospun polymer has the potential to improve its proliferative and antimicrobial capabilities. This study presents the novel, covalent attachment of S -nitroso- N -acetylpenicillamine (SNAP) to polyacrylonitrile (PAN) fibers. By attaching the NO donor to the polymer, rather than blending it, leaching is reduced to maintain a NO flux within the physiologically relevant range for a longer duration, while limiting any cytotoxic effects. The synthesized fibers were characterized using a variety of techniques such as scanning electron microscopy, 1 H NMR, and drop shape analysis. Due to the antimicrobial activity of NO, the SNAP-PAN fibers demonstrated a 2-log reduction of S. aureus adhesion. Furthermore, the extended zone of inhibition of S. aureus by SNAP-PAN demonstrates the ability of NO to impact the environment surrounding the material, in addition to the environment in direct contact with it. The combination of NO release, hydrophilicity of PAN, and the fibrous network led to increased fibroblast proliferation and attachment, potentially expanding the fibers as an improved cell scaffolding platform. The results from this study demonstrate a novel preparation and design of NO-releasing fibers to provide multiple benefits for a variety of biomedical applications.

Controlled release of drugs from medical implants is an effective approach to reducing foreign body reactions and infections. We report here on a one-step 3D printing strategy to create drug-eluting polymer devices with a drug-loaded bulk and a drug-free coating. The spontaneously formed drug-free coating dramatically reduces the surface roughness of the implantable devices and serves as a protective layer to suppress the burst release of drugs. A high viscosity liquid silicone that can be extruded based on its shear-thinning property and quickly vulcanize upon exposure to ambient moisture is used as the ink for 3D printing. S -Nitrosothiol type nitric oxide (NO) donors in their crystalline forms are selected as model drugs because of the potent antimicrobial, antithrombotic, and anti-inflammatory properties of NO. Direct ink writing of the homogenized polymer-drug mixtures generates rough and ill-defined device surfaces because of the exposed S -nitrosothiol microparticles. When a low-viscosity silicone (polydimethylsiloxane) is added into the ink, this silicone diffuses outward upon deposition to form a drug-free outermost layer without compromising the integrity of the printed structures. S -Nitrosoglutathione (GSNO) or S -nitroso- N -acetylpenicillamine (SNAP) embedded in the printed silicone matrix releases NO under physiological conditions from days to about one month. The microsized drug crystals are well-preserved in the ink preparation and printing processes, which is one reason for the sustained NO release. Biofilm and cytotoxicity experiments confirmed the antibacterial property and safety of the printed NO-releasing devices. This additive manufacturing platform does not require dissolution of drugs and involves no thermal or UV processes and, therefore, offers unique opportunities to produce drug-eluting silicone devices in a customized manner.

As a promising flavonoid, naringenin has shown potential anti-inflammatory and antioxidant properties mainly in inflammatory pain models by oral administration. Therefore, we investigated the antinociceptive activity of this compound by intraperitoneally (i.p.) administration, as well as, associated mechanism of action considering the involvement of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/potassium channel (KATP) pathway and opioid receptors. The antinociceptive effect of naringenin was evaluated in male NMRI mice using formalin test at early and late phases. To assess the involvement of L-arginine/NO/cGMP/KATP pathway and opioid receptors, mice were pretreated i.p. with L-arginine (NO precursor), S-nitroso-N-acetylpenicillamine (SNAP, NO donor), N(gamma)-nitro-L-arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase), sildenafil (inhibitor of phosphodiesterase enzyme), glibenclamide (KATP channel blocker) and naloxone (an opioid receptor antagonist), respectively 20 min before administration of the most effective dose of naringenin. Naringenin showed a dose-dependent antinociceptive effect at both early and late phases of the formalin test. The dose of 100 mg/kg of naringenin was identified as the most effective dose and selected for further experiments. Our mechanistic evaluations showed that L-arginine, SNAP and sildenafil could enhance the antinociceptive effects of naringenin, revealing the critical role of NO and cGMP during its antinociceptive effect. On the other hand, glibenclamide and naloxone could mitigate the antinociceptive potential of naringenin at both phases of formalin test, which confirmed the associated role of KATP channels and opioid receptors. In conclusion, naringenin could be a promising antinociceptive agent acting through opioid receptors and L-arginine/NO/cGMP/KATP channel pathway., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) play important roles in the development of insulin resistance. In glomerular podocytes, crosstalk between these two enzymes may be altered under hyperglycemic conditions. SIRT1 protein levels and activity and AMPK phosphorylation decrease under hyperglycemic conditions, with concomitant inhibition of the effect of insulin on glucose uptake into these cells. Nitric oxide (NO)-dependent regulatory signaling pathways have been shown to be downregulated under diabetic conditions. The present study examined the involvement of the NO synthase (NOS)/NO pathway in the regulation of SIRT1-AMPK signaling and glucose uptake in podocytes. We examined the effects of NOS/NO pathway alterations on SIRT1/AMPK signaling and glucose uptake using pharmacological tools and a small-interfering transfection approach. We also examined the ability of the NOS/NO pathway to protect podocytes against high glucose-induced alterations of SIRT1/AMPK signaling and insulin-dependent glucose uptake. Inhibition of the NOS/NO pathway reduced SIRT1 protein levels and activity, leading to a decrease in AMPK phosphorylation and blockade of the effect of insulin on glucose uptake. Treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented high glucose-induced decreases in SIRT1 and AMPK activity and increased GLUT4 protein expression, thereby improving glucose uptake in podocytes. These findings suggest that inhibition of the NOS/NO pathway may result in alterations of the effects of insulin on glucose uptake in podocytes. In turn, the enhancement of NOS/NO pathway activity may prevent these deleterious effects of high glucose concentrations, thus bidirectionally stimulating the SIRT1-AMPK reciprocal activation loop., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Despite technological advancement, nosocomial infections are prevalent due to the rise of antibiotic resistance. A combinatorial approach with multimechanistic antibacterial activity is desired for an effective antibacterial medical device surface strategy. In this study, an antimicrobial peptide, nisin, is immobilized onto biomimetic nitric oxide (NO)-releasing medical-grade silicone rubber (SR) via mussel-inspired polydopamine (PDA) as a bonding agent to reduce the risk of infection. Immobilization of nisin on NO-releasing SR (SR-SNAP-Nisin) and the surface characteristics were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy and contact angle measurements. The NO release profile (7 days) and diffusion of SNAP from SR-SNAP-Nisin were quantified using chemiluminescence-based nitric oxide analyzers and UV-vis spectroscopy, respectively. Nisin quantification showed a greater affinity of nisin immobilization toward SNAP-doped SR. Matrix-assisted laser desorption/ionization mass spectrometry analysis on surface nisin leaching for 120 h under physiological conditions demonstrated the stability of nisin immobilization on PDA coatings. SR-SNAP-Nisin shows versatile in vitro anti-infection efficacy against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in the planktonic and adhered states. Furthermore, the combination of NO and nisin has a superior ability to impair biofilm formation on polymer surfaces. SR-SNAP-Nisin leachates did not elicit cytotoxicity toward mouse fibroblast cells and human umbilical vein endothelial cells, indicating the biocompatibility of the material in vitro . The preventative and therapeutic potential of SR-SNAP-Nisin dictated by two bioactive agents may offer a promising antibacterial surface strategy.

Rises in local neural activity trigger local increases of cerebral blood flow, which is essential to match local energy demands. However, the specific location of microvascular flow control is incompletely understood. Here, we used two-photon microscopy to observe brain microvasculature in vivo. Small spatial movement of a three-dimensional (3D) vasculature makes it challenging to precisely measure vessel diameter at a single x-y plane. To overcome this problem, we carried out four-dimensional (x-y-z-t) imaging of brain microvessels during exposure to vasoactive molecules in order to constrain the impact of brain movements on the recordings. We demonstrate that rises in synaptic activity, acetylcholine, nitric oxide, cyclic guanosine monophosphate, ATP-sensitive potassium channels, and endothelin-1 exert far greater effects on brain precapillary sphincters and first-order capillaries than on penetrating arterioles or downstream capillaries, but with similar kinetics. The high level of responsiveness at precapillary sphincters and first-order capillaries was matched by a higher level of α-smooth muscle actin in pericytes as compared to penetrating arterioles and downstream capillaries. Mathematical modeling based on 3D vasculature reconstruction showed that precapillary sphincters predominantly regulate capillary blood flow and pressure as compared to penetrating arterioles and downstream capillaries. Our results confirm a key role for precapillary sphincters and pericytes on first-order capillaries as sensors and effectors of endothelium- or brain-derived vascular signals., Competing Interests: The authors declare no competing interest.

Mitochondria play essential roles in eukaryotic cells for glucose metabolism to produce ATP. In Schizosaccharomyces pombe, transcription factor Rst2 can be activated upon glucose deprivation. However, the link between Rst2 and mitochondrial function remains elusive. Here, we monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system, and found that inhibition of mitochondrial complex III/IV caused cells to produce reactive oxygen species (ROS) and nitric oxide (NO), which in turn activated Rst2. Furthermore, Rst2-GFP was observed to translocate from cytoplasm to nucleus upon mitochondrial complex III/IV inhibitors treatment, and deletion of genes associated with complex III/IV resulted in delayed process of Rst2-GFP nuclear exportation under glucose-rich condition. In particular, we found that Rst2 was phosphorylated following the treatment of complex III/IV inhibitors or SNAP. Altogether, our findings suggest that mitochondrial complex III/IV participates in the activation of Rst2 through ROS and NO generation in Schizosaccharomyces pombe., (© 2021 John Wiley & Sons Ltd.)

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

The current use of implantable and indwelling medical is limited due to potential microbial colonization leading to severe ailments. The aim of this work is to develop bioactive polymers that can be customized based on patient needs and help prevent bacterial infection. Potential benefits of additive manufacturing technology are integrated with the antimicrobial properties of nitric oxide (NO) to develop NO-releasing biocompatible polymer interfaces for addressing bacterial infections. Using filament-based additive manufacturing and polycarbonateurethane-silicone (PCU-Sil) a range of films possessing unique porosities (Disk-60, Disk-40, solid, capped) were fabricated. The films were impregnated with S-nitroso-N-acetyl-penicillamine (SNAP) using a solvent-swelling process. The Disk-60 porous films had the greatest amount of SNAP (19.59 wt%) as measured by UV-vis spectroscopy. Scanning electron microscopy and energy-dispersive X-ray spectroscopy confirmed an even distribution of SNAP throughout the polymer. The films exhibited structure-based tunable NO-release at physiological levels ranging from 7-14 days for solid and porous films, as measured by chemiluminescence. The antibacterial efficacy of the films was studied against Staphylococcus aureus using 24 h in vitro bacterial adhesion assay. The results demonstrated a >99% reduction of viable bacteria on the surface of all the NO-releasing films compared to unmodified PCU-Sil controls. The combination of 3D-printing technology with NO-releasing properties represents a promising technique to develop customized medical devices (such as 3D-scaffolds, catheters, etc.) with distinct NO-release levels that can provide antimicrobial properties and enhanced biocompatibility.

Social recognition memory (SRM) forms the basis of social relationships of animals. It is essential for social interaction and adaptive behavior, reproduction and species survival. Evidence demonstrates that social deficits of psychiatric disorders such as autism and schizophrenia are caused by alterations in SRM processing by the hippocampus and amygdala. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptors PAC1, VPAC1 and VPAC2 are highly expressed in these regions. PACAP is a pleiotropic neuropeptide that modulates synaptic function and plasticity and is thought to be involved in social behavior. PACAP signaling also stimulates the nitric oxide (NO) production and targets outcomes to synapses. In the present work, we investigate the effect of the infusion of PACAP-38 (endogenous neuropeptide and potent stimulator of adenylyl cyclase), PACAP 6-38 (PAC1/VPAC2 receptors antagonist) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) in the CA1 region of the hippocampus and in the basolateral amygdala (BLA) on the consolidation of SRM. For this, male Wistar rats with cannulae implanted in CA1 or in BLA were subjected to a social discrimination paradigm, which is based on the natural ability of rodents to investigate unfamiliar conspecifics more than familiar one. In the sample phase (acquisition), animals were exposed to a juvenile conspecific for 1 h. Immediately, 60 or 150 min after, animals received one of different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. Animals that received infusions of PACAP 6-38 (40 pg/side) into CA1 immediately after the sample phase or into BLA immediately or 60 min after the sample phase were unable to recognize the familiar juvenile during the retention test. This impairment was abolished by the coinfusion of PACAP 6-38 plus SNAP (5 μg/side). These results show that the blockade of PACAP/PAC1/VPAC2 signaling in the CA1 and BLA during a restricted post-acquisition time window impairs the consolidation of SRM and that the SNAP is able to abolish this deficit. Findings like this could potentially be used in the future to influence studies of psychiatric disorders involving social behavior., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS -/- ) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS -/- microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dysfunction of the pancreatic β cells leads to several chronic disorders including diabetes mellitus. Several mediators and mechanisms are known to be involved in the regulation of β cell secretory function. In this study, we propose that cytokine-induced nitric oxide (NO) production interacts with cholinergic mechanisms to modulate insulin secretion from pancreatic β cells. Using a rat insulinoma cell line INS-1, we demonstrated that β cell viability decreases significantly in the presence of SNAP (NO donor) in a concentration- and time-dependent manner. Cell viability was also found to be decreased in the presence of a combined treatment of SNAP with SMN (muscarinic receptor antagonist). We then investigated the impact of these findings on insulin secretion and found a significant reduction in glucose uptake by INS-1 cells in the presence of SNAP and SMN as compared with control. Nitric oxide synthase 3 gene expression was found to be significantly reduced in response to combined treatment with SNAP and SMN suggesting an interaction between the cholinergic and nitrergic systems. The analysis of gene and protein expression further pin-pointed the involvement of M 3 muscarinic receptors in the cholinergic pathway. Upon treatment with cytokines, reduced cell viability was observed in the presence of TNF-α and IFN-γ. A significant reduction in insulin secretion was also noted after treatment with TNF-α and IFN-γ and IL1-β. The findings of the present study have shown for the first time that the inhibition of the excitatory effects of cholinergic pathways on glucose-induced insulin secretion may cause β cell injury and dysfunction of insulin secretion in response to cytokine-induced NO production.

The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations' health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nitric oxide (NO) regulates a variety of epidermal functions, including epidermal proliferation, differentiation and cutaneous wound healing. However, whether nitric oxide (NO) and its synthetic enzymes regulate epidermal permeability barrier homeostasis is not clear. In the present study, we employed inducible nitric oxide synthase (iNOS) KO mice to explore the role of iNOS in epidermal permeability barrier homeostasis. Our results showed that iNOS mice displayed a comparable levels of basal transepidermal water loss rates, stratum corneum hydration and skin surface pH to their wild-type mice, but epidermal permeability barrier recovery was significantly delayed both 2 and 4 hours after acute barrier disruption by tape stripping. In parallel, expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes were lower in iNOS KO mice versus wild-type controls. Topical applications of two structurally unrelated NO donors to iNOS KO mice improved permeability barrier recovery kinetics and upregulated expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes. Together, these results indicate that iNOS and its product regulate epidermal permeability barrier homeostasis in mice., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: In this study, we examined the effect of acute hyponatremia associated with vasopressin (AVP) on the responses of the isolated rat's MCAs and PAs to acidosis, nitric oxide donor (SNAP) and to endothelium-dependent vasodilator ATP., Methods: The studies were performed on isolated, perfused and pressurized MCAs and PAs in control conditions and during AVP-associated hyponatremia. Hyponatremia was induced in vitro by lowering Na + concentration from 144 to 121 mmol/L in intra- and extravascular fluid in the presence of AVP., Results: Parenchymal arterioles showed greater response to an increase in H + and K + ions concentration and to ATP in comparison with MCAs in control normonatremic conditions. Both PAs and MCAs constricted in response to acute hyponatremia associated with AVP. Interestingly, disordered regulation of vascular tone was observed in PAs but not in MCAs. The abnormalities in the regulation comprised a significant reduction of PA response to acidosis and the absence of the response to the administration of SNAP or ATP., Conclusions: Arginine vasopressin-associated hyponatremia leads to constriction and dysregulation of PAs which may impair neurovascular coupling., (© 2020 John Wiley & Sons Ltd.)

Introduction: Stem cell-derived cardiac myocytes are potential sources for testing cardiocytoprotective molecules against ischemia/reperfusion injury in vitro., Materials and Methods: Here we performed a systematic analysis of two different induced pluripotent stem cell lines (iPSC 3.4 and 4.1) and an embryonic stem cell (ESC) line-derived cardiac myocytes at two different developmental stages. Cell viability in simulated ischemia/reperfusion (SI/R)-induced injury and a known cardiocytoprotective NO-donor, S-nitroso-n-acetylpenicillamine (SNAP) was tested., Results: After analysis of full embryoid bodies (EBs) and cardiac marker (VCAM and cardiac troponin I) positive cells of three lines at 6 conditions (32 different conditions altogether), we found significant SI/R injury-induced cell death in both full EBs and VCAM+ cardiac cells at later stage of their differentiation. Moreover, full EBs of the iPS 4.1 cell line after oxidative stress induction by SNAP was protected at day-8 samples., Conclusion: We have shown that 4.1 iPS-derived cardiomyocyte line could serve as a testing platform for cardiocytoprotection., Competing Interests: Declaration of Competing Interest Author Kobolák, Julianna; Bock, István; Fehér, Anita; Dinnyes, Andras was employed by the company Biotalentum Ltd. Author Anikó Görbe, Péter Ferdinandy and Judit Pipis was employed by the company Pharmahungary Group Ltd. The remaining authors declare that the research was conducted in the absence of any commercial for financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A previous study demonstrated that glutathione (GSH) produces specific antidepressant-like effect in the forced swimming test (FST), a predictive test of antidepressant activity. The present study investigated the involvement of multiple cellular targets implicated in the antidepressant-like effect of GSH in the FST. The antidepressant-like effect of GSH (300 nmol/site, icv) lasted up to 3 h when mice were submitted to FST. The central administration of oxidized GSH (GSSG, 3-300 nmol/site) did not alter the behavior of mice submitted to the FST. Furthermore, the combined treatment of sub-effective doses of GSH (100 nmol/site, icv) with a sub-effective dose of classical antidepressants (fluoxetine 10 mg/kg, and imipramine 5 mg/kg, ip) presented synergistic effect by decreasing the immobility time in the FST. The antidepressant-like effect of GSH was abolished by prazosin (1 mg/kg, ip, α 1 -adrenoceptor antagonist), baclofen (1 mg/kg, ip, GABAB receptor agonist), bicuculline (1 mg/kg, ip, GABAA receptor antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/site, icv, NO donor), but not by yohimbine (1 mg/kg, ip, α 2 -adrenoceptor antagonist). The NMDA receptor antagonists, MK-801(0.001 mg/kg, ip) or GMP (0.5 mg/kg, ip), potentiated the effect of a sub-effective dose of GSH in the FST. These results suggest that the antidepressant-like effect induced by GSH is connected to the activation of α1 adrenergic and GABAA receptors, as well as the inhibition of GABAB and NMDA receptors and NO biosyntesis. We speculate that redox-mediated signaling on the extracelular portion of cell membrane receptors would be a common mechanism of action of GSH., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chemoresistance is a main obstacle in ovarian cancer therapy and new treatment strategies and further information regarding the mechanism of the medication cisplatin are urgently needed. Nitric oxide has a critical role in modulating the activity of chemotherapeutic drugs. Our previous work showed that connexin32 contributed to cisplatin resistance. However, whether nitric oxide is involved in connexin32-mediated cisplatin resistance remains unknown. In this study, using A2780 and A2780 cisplatin-resistant cells, we found that S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, attenuated cisplatin toxicity by decreasing gap junctions in A2780 cells. Enhancement of gap junctions using retinoic acid reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin toxicity. In A2780 cisplatin-resistant cells, however, S-nitroso-N-acetyl-penicillamine enhanced cisplatin toxicity by decreasing connexin32 expression. Downregulation of connexin32 expression by small interfering RNA exacerbated the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity and upregulation of connexin32 expression by pcDNA transfection reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity. Our study suggests for the first time that combining cisplatin with nitric oxide in clinical therapies for ovarian cancer should be avoided before cisplatin resistance emerges. The present study provides a productive area of further study for increasing the efficacy of cisplatin by combining cisplatin with the specific inhibitors or enhancers of nitric oxide in clinical treatment., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

The chances of ventilator-associated pneumonia (VAP) increases 6-20 folds when an endotracheal tube (ETT) is placed in a patient. VAP is one of the most common hospital-acquired infections and comprises 86% of the nosocomial pneumonia cases. This study introduces the idea of nitric oxide-releasing ETTs (NORel-ETTs) fabricated by the incorporation of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) into commercially available ETTs via solvent swelling. The impregnation of SNAP provides NO release over a 7-day period without altering the mechanical properties of the ETT. The NORel-ETTs successfully reduced the bacterial infection from a commonly found pathogen in VAP, Pseudomonas aeruginosa, by 92.72 ± 0.97% when compared with the control ETTs. Overall, this study presents the incorporation of the active release of a bactericidal agent in ETTs as an efficient strategy to prevent the risk of VAP., (© 2020 Wiley Periodicals, Inc.)

The management of thrombosis and bacterial infection is critical to ensure the functionality of medical devices. While administration of anticoagulants is the current antithrombotic clinical practice, a variety of complications, such as uncontrolled hemorrhages or heparin-induced thrombocytopenia, can occur. Additionally, infection rates remain a costly and deadly complication associated with use of these medical devices. It has been hypothesized that if a synthetic surface could mimic the biochemical mechanisms of the endothelium of blood vessels, thrombosis could be reduced, anticoagulant use could be avoided, and infection could be prevented. Herein, the interfacial biochemical effects of the endothelium were mimicked by altering the surface of medical grade silicone rubber (SR). Surface modification was accomplished via heparin surface immobilization (Hep) and the inclusion of a nitric oxide (NO) donor into the SR polymeric matrix to achieve synergistic effects (Hep-NO-SR). An in vitro bacteria adhesion study revealed that Hep-NO-SR exhibited a 99.46 ± 0.17% reduction in viable bacteria adhesion compared to SR. An in vitro platelet study revealed Hep-NO-SR reduced platelet adhesion by 84.12 ± 6.19% compared to SR, while not generating a cytotoxic response against fibroblast cells. In a 4 h extracorporeal circuit model without systemic anticoagulation, all Hep-NO-SR samples were able to maintain baseline platelet count and device patency; whereas 66% of SR samples clotted within the first 2 h of study. Results indicate that Hep-NO-SR creates a more hemocompatible and antibacterial surface by mimicking two key biochemical functions of the native endothelium.

The release of the neuropeptide CGRP from the trigeminal ganglion neurons (TGNs) plays a central role in migraine. Whereas CGRP can activate NO release from ganglionic glial cells, NO in turn enhances CGRP release. However, it remains unclear how NO promotes CGRP release. Here, we report that the NO donor SNAP triggered CGRP release from cultured primary TGNs. This event was associated with GSK-3β activation and Akt inactivation. Immunofluorescent staining revealed that GSK-3β primarily located in neurons. Furthermore, GSK-3β inhibition resulted in a marked reduction in expression of CGRP as well as other migraine-related factors, including substance P, cholecystokinin, and prostaglandin E2. Last, exposure to SNAP also activated NF-κB, while NF-κB inhibition prevented the induction of CGRP by SNAP. Interestingly, this event was blocked by GSK-3β inhibition, in association with inhibition of NF-κB/p65 expression and nuclear translocation. Together, these findings argue that NO could stimulate TGNs to release of CGRP as well as other migraine-related factors, likely by activating GSK-3β, providing a novel mechanism underlying a potential feed-forward loop between NO and CGRP in migraine. They also raise a possibility that GSK-3β might act to trigger migraine through activation of NF-κB, suggesting a link between neuroinflammation and migraine.

Ethanol (EtOH) exposure causes alterations of motor coordination, balance, behavior, speech, and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission. The cerebellar cortical molecular layer interneuron-Purkinje cell (MLI-PC) synapses are critical for various information integration and transmission, which are sensitive to acute and chronic EtOH exposure. The aim of this study is to investigate the effect of chronic ethanol exposure on the facial stimulation-evoked MLI-PC synaptic transmission in urethane-anesthetized mice, by electrophysiological recording and pharmacological methods. Under current-clamp recording conditions, air-puff stimulation of ipsilateral whisker pad evoked MLI-PC synaptic transmission, which expressed an inhibitory component (P1) followed by a pause of simple spike (SS) firing in cerebellar PCs. Chronic ethanol exposure did not change the latency of the facial stimulation-evoked responses in cerebellar PCs, but induced significant enhancement of the stimulation-evoked MLI-PC synaptic transmission, which expressed increases in amplitude of P1 and pause of SS firing. The amplitude of P1 and pause of SS in ethanol exposure group were significant higher than that in control group. Cerebellar surface application of nitric oxide synthesis (NOS) inhibitor, L-NNA (5 mM) significantly decreased the amplitude of P1 and the pause of SS firing in EtOH exposure group, but did no effect on control group. In contrast, cerebellar surface application of NO donor, SNAP (100 μM) significantly increased the amplitude of P1 and the pause of SS firing in control group, but not in EtOH exposure group. These results indicated that chronic EtOH exposure significantly facilitated the sensory-evoked MLI-PC synaptic transmission via NO signaling pathway in mouse cerebellar cortex., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia, neutrophil-mediated inflammation, and lung edema formation. Whereas lung damage might be alleviated by nitric oxide (NO), goal of this study was to evaluate if intratracheal NO donor S-nitroso-N-acetylpenicillamine (SNAP) can positively influence the lung functions in experimental model of ARDS. New Zealand rabbits with respiratory failure induced by saline lavage (30 ml/kg, 9+/-3 times) were divided into: ARDS group without therapy, ARDS group treated with SNAP (7 mg/kg i.t.), and healthy Control group. During 5 h of ventilation, respiratory parameters (blood gases, ventilatory pressures) were estimated. After anesthetics overdosing, left lung was saline-lavaged and cell count, cell viability and protein content in bronchoalveolar lavage fluid (BALF) were measured. Right lung tissue was used for estimation of wet/dry weight ratio, concentration of NO metabolites, and histomorphological investigation. Repetitive lung lavage induced lung injury, worsened gas exchange, and damaged alveolar-capillary membrane. Administration of SNAP reduced cell count in BALF, lung edema formation, NO metabolites, and histopathological signs of injury, and improved respiratory parameters. Treatment with intratracheal SNAP alleviated lung injury and edema and improved lung functions in a saline-lavaged model of ARDS suggesting a potential of NO donors also for patients with ARDS.

Glioblastoma multiforme (GBM) is the most frequently occurring and gravest primary tumor of the CNS in adults. The development of chemoresistance to temozolomide (TMZ), the first-line chemotherapy for GBM, is an important factor contributing to poor treatment outcomes. Down-regulation of O -6-methylguanine-DNA methyltransferase (MGMT) expression in GBM cells is an attractive strategy for overcoming TMZ resistance and improving outcomes. This study revealed that the nitric oxide (NO) donor S -nitroso- N -acetylpenicillamine (SNAP) exerts antitumorigenic effects on TMZ-sensitive and TMZ-resistant (TMZ-R) glioma cells. Pretreatment with SNAP not only induced apoptosis, mitochondrial dysfunction, and hypoxia-inducing factor 1, but also resensitized TMZ-R GBM cells to TMZ through down-regulation of MGMT expression. SNAP acted principally through post-translational modification of p53, phosphorylated N-myc downstream regulated gene 1, and MGMT protein stability in TMZ-R GBM cells. Additionally, when applied together, SNAP and TMZ enhanced the inhibition of tumor growth in vitro and in vivo . This study sheds new light on a potential strategy to overcome TMZ resistance in GBM and thus possesses the potential for prolonging survival of patients with GBM.-Tsai, C.-K., Huang, L.-C., Wu, Y.-P., Kan, I.-Y., Hueng, D.-Y. SNAP reverses temozolomide resistance in human glioblastoma multiforme cells through down-regulation of MGMT.

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4 + T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2-36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4 + T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4 + T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4 + T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: Determining how obesity affects function in human myometrial arteries, to help understand why childbirth has poor outcomes in obese women., Methods: Myometrial arteries were studied from 84 biopsies. Contraction (vasopressin and U-46619) and relaxation (carbachol, bradykinin, SNAP) was assessed using wire myography. eNOS activity was assessed using L-NAME. Cholesterol was reduced using methyl-β-cyclodextrin to determine whether it altered responses. Differences in endothelial cell intracellular Ca 2+ signalling were assessed using confocal microscopy., Results: The effects of BMI on relaxation were agonist specific and very marked; all vessels, irrespective of BMI, relaxed to bradykinin but 0% of vessels (0/13) from obese women relaxed to carbachol, compared to 59% (10/17) from normal weight women. Cholesterol-lowering drugs did not restore carbachol responses (n = 6). All vessels, irrespective of BMI, relaxed when NO was directly released by SNAP (n = 19). Inhibition of eNOS with L-NAME had a significant effect in normal but not overweight/obese vessels. Compared to bradykinin, a lower proportion of endothelial cells responded to carbachol and the amplitude of the calcium response was significantly less, in all vessels. Furthermore, a significantly lower proportion of endothelial cells responded to carbachol in the overweight/obese group compared to control. In contrast to relaxation, the effect of contractile agonists was unchanged with increasing BMI., Conclusions: The ability of human myometrial arteries to relax is significantly impaired with obesity, and our data suggest this is due to a deficit in endothelial calcium signalling. This inability to recover following compression during contractions, might contribute to poor labours in obese women., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)