Therefore, infection control measures are necessary to prevent infection of dental personnel during dental treatments. The use of a preprocedural mouth rinse with chlorhexidine (CHX), cetylpyridinium chloride (CPC), or hydrogen peroxide (H2O2) solution for 30–60 s may reduce the viral load and may protect the personnel in a dental practice. In the present study the virucidal effect of the mouth rinsing solutions ViruProX® with 0.05% CPC and 1.5% H2O2 and BacterX® pro containing 0.1% CHX, 0.05% CPC, and 0.005% sodium fluoride (F-) was investigated in vitro. The mouth rinsing solutions successfully inactivated infectious SARS-CoV-2 particles, the causative agent of coronavirus disease 2019 (COVID-19), within 30 s. To determine the effective components, CHX, CPC, H2O2, and a combination of CHX and CPC, were tested against SARS-CoV-2 in addition. While a combination of CPC and CHX as well as CPC alone led to a significant reduction of infectious viral particles, H2O2 and CHX alone had no virucidal effect against SARS-CoV-2. It can be assumed that preprocedural rinsing of the mouth with ViruProX® or BacterX® pro will reduce the viral load in the oral cavity and could thus lower the transmission of SARS-CoV-2 in dental practice.The  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and has become a pandemic with more than 111 million confirmed cases and nearly 2.5 million deaths worldwide (as of February 2021), making it a significant threat to global health (WHO Coronavirus Disease (COVID-19) Dashboard) [shboard) ,4,5.2O2)), which have an antimicrobial activity or fungicidal effect. They can damage or destroy membranes, and some have been proven effective against several viruses [influenza A virus, parainfluenza virus, herpes simplex virus 1, and hepatitis B virus but not against nonenveloped viruses [2O2. Among others it has been demonstrated to be effective against adenoviruses, rhinovirus, influenza A virus, and coronavirus strain E229 [influenza A virus [Due to close physical contact, as well as aerosol formation during dental treatment, dentists and their staff are at increased risk for infection and may be a source of further transmission ,7. There viruses ,9,10. Th viruses . Mouth r viruses ,13,14. Hain E229 ,16,17. C A virus ,19. It i A virus .coronavirus. Official recommendations for dental practice therefore give little guidance on specific compounds for preprocedural mouth rinses. Recommendations by the World Health Organization (WHO) include the use of 1% H2O2 and 0.2% PI solutions. The Center for Disease Control and Prevention (CDC) lists CHX, essential oils, PI, and CPC to be used in preprocedural mouth rinses [®, which contains CPC and H2O2, and BacterX® pro, containing CHX, CPC, and sodium fluoride, for their virucidal activity against SARS-CoV-2 in vitro. Furthermore, the individual components of these mouth rinsing solutions were tested to find out which has antiviral properties against SARS-CoV-2. According to the manufacturer ViruProX® and BacterX® pro are usually applied pure to rinse the oral cavity for 20–40 s. To evaluate if this procedure is sufficient to successfully inactivate SARS-CoV-2, in the following study infectious SARS-CoV-2 particles were incubated with the two solutions and some of their individual components for 30 s and analyzed for their virucidal efficacy. As the null hypothesis of the study it was assumed that none of the tested solutions would have a virucidal activity against SARS-CoV-2 and therefore will show no significant difference compared to the medium control. This hypothesis should be rejected in the present study.To date, there is little scientific evidence to recommend mouthwashes with antiviral effects against SARS-CoV-2 to reduce viral load in the oral cavity and until now it is not known which mouth rinsing solutions or which components are effective against this novel h rinses ,22. TherSevere acute respiratory syndrome coronavirus 2  was provided by the Westfaelische Wilhelms-University Muenster Institute of Virology (IVM) and passaged three times on Vero E6 cells. Titer: 1.25 × 107 plaque forming units per milliliter (pfu/mL). Vero E6 cells, Cercopithecus aethiops kidney epithelial cells were obtained from the American Type Culture Collection (ATCC). Cells were grown at 37 °C in Iscove Modified Dulbecco Media (IMDM)  supplemented with 10% fetal bovine serum (FBS)  and 1% Penicillin-Streptomycin .® (0.05% CPC and 1.5% H2O2)  and BacterX® pro   (2O2 solution were used in this study (The mouth rinses ViruProXGermany)  and someis study .6 pfu) in 80 µL infection medium (IMDM (Thermo Fisher Scientific), 5% FCS (Capricorn Scientific GmbH), 1% Penicillin-Streptomycin (Sigma Aldrich)) were diluted 1:2 with the mouth rinsing solutions ViruProX® or BacterX® pro or components thereof. In parallel a nonvirucidal medium control of SARS-CoV-2 with infection medium (1 × 106 pfu in 80 µL infection medium + 80 µL infection medium) and a no-virus control (80 µL infection medium + 80 µL test solution) were prepared accordingly. The samples were mixed briefly and incubated at 37 °C for 30 s. Immediately after, 1440 µL infection medium were added leading to a 1:20 dilution of either virus or test solution. Afterwards, the sample was diluted in infection medium up to 1:40,000, which was analyzed in technical duplicates in a plaque assay to allow for separated, single plaques per well and to avoid cytotoxic effects due to the test solutions.Infectious SARS-CoV-2 particles (1 × 106 cells/well) were seeded in 6-well plates and incubated for 24 h at 37 °C, 5% CO2 in a humidified incubator. The next day the cells were washed once with infection medium before inoculation with 500 µL/well of the prediluted virucidal activity test samples for 1 h at 37 °C, 5% CO2 in a humidified incubator. The virucidal activity test samples were defined as the “mouth rinsing solutions” , the “medium control” (SARS-CoV-2 particles + infection medium) and the “no-virus control” (mouth rinse solution + infection medium). The no-virus control was prepared to rule out potential cytopathic effects by the test solutions. After incubation, the inoculum was removed completely, and the cells were overlaid with Avicel-Medium , 10% Minimum Essential Media (MEM) (Thermo Fisher Scientific), 0.01% DEAE-Dextran (Sigma Aldrich), 2.8% NaHCO3 , 1% Penicillin-Streptomycin (Sigma Aldrich), 0.2% BSA , 1% L-Glutamine (Sigma Aldrich)) for 72 h at 37 °C, 5% CO2 in a humidified incubator. The Avicel-Medium was removed, the cells were rinsed twice with PBS (Thermo Fisher Scientific) and fixed with 4% Roti-Histofix (Carl Roth) in PBS  for 30 min at 4 °C before staining with crystal violet solution , 10% ethanol in ddH2O). Plaques were counted to determine the virus titer in the sample compared to the medium control. Statistical analysis was performed using unpaired t-test (p < 0.05). Furthermore, the crystal violet staining was dissolved with methanol and the absorbance was measured at 595 nm, for quantification of the cytopathic effect. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (p < 0.05) using GraphPad Prism version 9.0.1 .Vero E6 cells (1 × 10® and BacterX® pro against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the mouth rinsing solutions were incubated for 30 s with 1 × 106 plaque forming units (pfu) of SARS-CoV-2 strain FI-100. The remaining infectivity was tested by a standard plaque assay using Vero E6 cells. To avoid a cytotoxic effect of ViruProX® and BacterX® pro and their components on Vero E6 cells, the test solution/virus mixture was diluted 1:40,000. This also resulted in an amount of virus particles that was suitable to be counted in the plaque assay. A large number of plaques (>50) was found when Vero E6 cells were incubated with a mixture of SARS-CoV-2 and a medium control (® (® pro (6 pfu below the detection limit (8 × 104 pfu/mL). When mouth rinsing solutions were incubated with Vero E6 cells alone in the same concentration as in the mixture with SARS-CoV-2, no plaques or cell destruction was found, indicating that the mouth rinsing solutions showed no cytotoxic effect  compared to the medium control, while BacterX® pro led to a reduction by ≥8.4 × 106 pfu/mL (≥2.0 log10 fold). The combination of 0.1% CHX with 0.05% CPC (6 pfu/mL and 5.6 × 106 pfu/mL/1.2 log10 fold and 0.7 log10 fold). While 0.1% CHX  (® contains 0.05% cetylpyridinium chloride (CPC) and 1.5% hydrogen peroxide (H2O2) and BacterX® pro contains 0.1% chlorhexidine (CHX), 0.05% CPC, and 0.005% fluoride (F-). BacterX® is also available with ethanol but, as ethanol may have a virucidal effect per se, the version without ethanol was tested here. When testing 0.05% CPC, 0.1% CHX, and 1.5% H2O2 alone, only 0.05% CPC led to a significant plaque reduction compared to the medium control (reduction by 0.7 log10 fold)  than 0.05% CPC alone, although 0.1% CHX alone did not reduce the viral titer significantly  A,B. The 10 fold) D–F. Thusficantly , meaning and CPC . Howeveropies/mL . The virhe mouth . Using 12O2 against SARS-CoV-2. They showed a minimal virucidal effect for 3% H2O2 and 6% H2O2 in a 1:2 dilution with the virus for 30 s, comparable to our setup, which resulted in a reduction of the viral titer by 1.0 to 1.8 log10 fold compared to the virus control [2O2 and 0.015% dequalinium chloride, a compound related to CPC in mouth rinses. The concentrations used were comparable to the present study. Consistent with our results, they observed a strong virucidal effect for dequalinium chloride (reduction by ≥2.6 to ≥3.11 log10 fold) and they also found a small reduction for CHX (reduction by 0.78 to 1.17 log10 fold) and H2O2 (reduction by 0.33 to 0.78 log10 fold) [2O2 compared to our study might be found in the different SARS-CoV-2 isolates used. As Meister et al. [2O2 [n = 2–6) they could show a significant reduction in the viral titer  for CPC 5 min and 6 h post rinse. No significance was reached for the CHX group compared to the water control [Similar studies investigating antiviral properties of mouth rinse solutions have been performed previously. Bidra et al.  investig control . Another10 fold) . An explr et al.  observedal. [2O2 . Further control . The res® and BacterX® pro. Both successfully inactivated 1 × 106 pfu SARS-CoV-2. Furthermore, after testing the individual components, it could be shown that 0.05% CPC was able to strongly reduce infectious SARS-CoV-2 particles within 30 s incubation. Therefore, it can be hypothesized that CPC, which is present in both mouth rinses, is the effective component and we would recommend the use of mouth rinses containing CPC, like ViruProX® and BacterX® pro, in preprocedural use in dental practice to reduce the risk of transmission of SARS-CoV-2 during dental treatments. Another possible indication outside the dental practice could be rinsing and gurgling of the mouth with BacterX® pro in order reduce the total load of SARS-CoV-2 after a possible encounter. BacterX® pro is in contrast to ViruProX® a cosmetic product according to EU regulations and may be used at home, too [® and BacterX® pro would need to be conducted to further support our findings and determine the duration of viral reduction in the oral cavity to ensure they exceed the duration for standard dental procedures. In regard to the results presented here, an update of guidelines recommending preprocedural mouth rinsing with solutions based on H2O2 or CHX might be advisable.The present study demonstrates the virucidal activity against SARS-CoV-2 for both mouth rinses ViruProX


Solanum lycopersicum L. is affected among other pests and diseases, by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), causing important economic losses worldwide. Antimicrobial peptides (AMPs) are amphipathic cationic oligopeptides with which the development of pathogenic microorganisms has been inhibited. Therefore, in this study, we evaluate antimicrobial activity of mesoporous silica nanoparticles (MSN5.4) loaded with human β-defensin-2 (hβD2) and two mutants (TRX-hβD2-M and hβD2-M) against Cmm. hβD2, TRX-hβD2-M and hβD2-M presented a half-maximum inhibitory concentration (IC50) of 3.64, 1.56 and 6.17 μg/mL, respectively. MSNs had average particle sizes of 140 nm (SEM) and a tunable pore diameter of 4.8 up to 5.4 nm (BJH). AMPs were adsorbed more than 99% into MSN and a first release after 24 h was observed. The MSN loaded with the AMPs inhibited the growth of Cmm in solid and liquid media. It was also determined that MSNs protect AMPs from enzymatic degradation when the MSN/AMPs complexes were exposed to a pepsin treatment. An improved AMP performance was registered when it was adsorbed in the mesoporous matrix. The present study could expand the applications of MSNs loaded with AMPs as a biological control and provide new tools for the management of phytopathogenic microorganisms. S. lycopersicum L., of which just in 2019 production exceeded 180 million tons [Cmm is a Gram-positive bacterium distributed worldwide that causes bacterial tomato canker disease, which can cause large economic losses to farmers if not treated properly and in a timely manner [Cmm from non-pathogenic Clavibacter strains [Cmm can survive inside contaminated seeds and plant residues for months or even years [One of the most important crops worldwide is tomato, the fruit of ion tons . Cmm is y manner . The use strains . However strains . The inf strains ,7,8. Depen years .Cmm and other diseases are based mainly on application of chemical pesticides [Cmm, among which the application of inorganic nanomaterials with antimicrobial activity such as nanoparticles (NPs) of Ag, ZnO, Cu, etc., the biological control (competition between microorganisms), the introduction of resistant cultivars and the direct application of biomolecules with antimicrobial activity to cultivars [Actinobacillus actinomycetemcomitans, Candida albicans and Pseudomona aeruginosa [Currently the control of sticides ,11. Howesticides ,12. Otheultivars ,14,15. Dultivars . They arultivars ,17,18,19ruginosa . The exaruginosa ,22,23,24ruginosa . An alteruginosa .Cmm.The matrix of the nanocapsules can be constructed from proteins, polysaccharides and synthetic polymers . Mesopor2PO4 , K2HPO4 , NaCl , pepsin from porcine gastric mucosa (Sigma Aldrich), yeast extract  and casein peptone (DIBICO). For the synthesis of MSN, deionized water with a resistivity of 18.2 MΩ•cm was used. For all other experiments double distilled water was employed.N,N-dimethylhexadecylamine , Pluronic F127 , cetyltrimelammonium bromide , NaOH , tetraethyl orthosilicate , anhydrous glycerol , glucose , imidazole , Tris base , KHEscherichia coli was cloned between the restriction sites NcoI and XhoI within the vector pUC57 (pUC57-hβD2) by the company GenScript. The plasmid was reconstituted within Milli-Q water and transformed into chemocompetent E. coli Mach1 T1 cells. Subsequently, pUC57-hβD2 and the expression vector pCri-4a extracted with the commercial kit ZR Plasmid Miniprep-Classic  were subjected to a double restriction assay with the enzymes NcoI and XhoI. The restricted products hβD2 and pCri-4a were purified from with the Zymoclean Gel DNA Recovery Kit (Zymo Research) from a 1% agarose gel and ligated together with the enzyme T4 DNA Ligase. Chemically competent cells of E. coli Mach1 T1 were transformed with the pCri-4a-hβD2 construction. Recombinant plasmids were sequenced in both directions. The expression vector pCri-4a allows the expression of a translated fusion to a 6-histidine tag, followed by sequence encoding to the thioredoxin-binding protein (TrxA) and contain a tobacco etch virus protease (TEV) cleavage site (The amino acid sequence of hβD2 was obtained from the Protein Data Bank (1FD3). The sequence of the gene that codes for hβD2 with optimized codons for age site A. In ordE. coli Rosetta (DE3) previously transformed with the plasmid pCri-4a-hβD2 or pCri--hβD2-M was seeded in 50 mL lysogeny broth  supplemented with kanamycin sulfate (50 μg/mL), chloramphenicol (20 μg/mL) and glucose (5 g/L). The culture was incubated at 37 °C for 16 h at 180 rpm for 16 h. 4 L of Terrific Broth medium  supplemented with the same antibiotics and sugar mentioned above were inoculated with 1% from the preinoculum. The cultures were kept under a constant temperature (37 °C) and shaking (200 rpm) until the OD600 reached 0.7–0.8. The expression of the fusion proteins was induced with 1 mM isopropyl β-d-galactopyranoside (IPTG). After carrying out the induction, the culture was incubated at 18 °C for 16 h at 150 rpm. The cells were harvested by centrifugation at 5000× g for 30 min at 4 °C and stored at −80 °C until protein extraction.2PO4, 0.5 M NaCl, 20 mM imidazole, pH 7.4) and then lysed by sonication within an ice bath using the Ultrasonic Homogenizer 750 . The cell lysate was centrifuged at 164,391× g for 30 min at 4 °C. The supernatant obtained loaded on a HisTrap HP 5 mL column previously equilibrated with buffer A. Proteins were eluted through a linear gradient (1–100%) with buffer B  for 15 CV, the fusion proteins were resolved in 10% Tricine-SDS-PAGE. To carry out the cleavage of the fusion proteins by TEV protease, the eluted fractions containing the protein of interest were subjected to a buffer exchange from B to C  using a HiPrep 26/10 Desalting column. The desalted fractions were mixed and the TEV enzyme was added in a ratio of 1:100 v/v, the reaction mixture was incubated at 4 °C for16 h. After cleavage process, the samples were re-purified using a HisTrap HP column using the previously mentioned conditions. The proteins that did not bound to the column, (corresponding to the AMPs) were concentrated to 0.5 mL with a centrifugal concentrator with a 3 kDa cut-off. To determine the concentration and yield of total protein in each of the purification processes, a commercial Pierce ™ BCA ™ kit  was used.Cell pellets (approximately 15 g) were resuspended in buffer A  of 0.99.The particle size distribution of the NPs was determined by dynamic light scattering (DLS) with a Zetasizer Nano ZSP . To find out the possible functional groups of the material under study, Fourier transform infrared spectroscopy (FT-IR) was performed using an infrared spectrophotometer Nicolet 6700 . Raman spectroscopy was used to determine the different vibrational modes of the MSN, for this analysis the AFM-Raman NTEGRA SPECTRA II system  was used using a blue solid-state laser (455 nm). The analyses were carried out at room temperature by scanning a range of 200–1400 nm in the Raman shift. The morphology, size and porosity of the MSN were determined by transmission electron microscopy (TEM) and scanning (SEM). The samples for TEM were visualized in a JEM 2010 transmission electron microscope  using an acceleration of 200 KV. The samples were also observed in an AURIGA 3916-FESEM scanning electron microscope  using an acceleration of 2 KV. The nitrogen adsorption-desorption isotherms of MSN were obtained using a Minisorb II instrument  at 77 K. Prior to analysis, the samples were degassed at room temperature for 24 h. Specific surface areas were determined based on adsorption data using the Brunauer-Emmett-Teller (BET) model whilefrom the branches of the desorption isotherms and using the Barret-Joyner-Halenda (BJH) model, the effective volume and size distributions of pores were determined. Finally, the total pore volume was calculated based on the amount of NCmm was cultivated in Mueller-Hinton broth (MHB) at 37 °C for 16 h at 180 rpm. Employing 0.5 mL of the previous culture, 50 mL of fresh MHB were inoculated. The culture was maintained under the conditions mentioned above until the OD600 reached 0.2–0.3. 9.5 mL of MHB agar prewarmed to 37 °C were added to 0.5 mL of the culture. It was homogenized and then poured onto a Petri dish containing MHB agar to form a uniform layer of 2 mm. Once the surface was dry, different volumes  of the defensins under study (50 μM) were added to the surface. The plates were incubated for 24 h at 28 °C, then the formed inhibition halos were measured. CH3COOH (acid acetic)10 mM pH 2.4 and Tris 50 mM pH 8.4 were used both as negative controls. The IC50 was determined based on that reported by Tay and Liu [Cmm. and Liu ,37. Thisg for 15 min at 4 °C at the end of the incubation period. The supernatant was carefully removed and analyzed by the Pierce ™ BCA Protein kit to determine the percentage of total protein concentration and thus indirectly estimate the adsorption of AMPs by MSN5.4. AMPs in PBS at the same concentration were used as controls.A microtube were filled with 5 mg of MSN5.4 (previously sterilized by UV light), 0.5 mg of AMPs and an amount of PBS 67 mM pH 7.0 to complete a final volume of 1 mL. The mixture was kept under constant stirring for 16 h at 4 °C. The samples were centrifuged at 10,000× g for 15 min at 4 °C, the resulting pellet was resuspended in 1 mL of sterile PBS. Supernatants were kept being analyzed by the Pierce™ BCA Protein kit  to determine the total protein concentration that was being released. These experiments were performed by triplicates.About the release of AMPs, the methodology referred to in the previous paragraph was re-performed. The removal of the supernatant was done, and the pellet was dried in sterile conditions for 2 h. After 1 mL of sterilized PBS was added to the pellet and shaken with the aid of a vortex to obtain a homogeneous mixture, the microtubes remained at constant agitation and temperature 28 ± 2 °C throughout the whole analysis period plotted . For each sampling point, microtubes were centrifuged at 10,000× Cmm . The plates were incubated for 24 h at 28 °C, the diameter of the inhibition halos was measured. MSN (5 mg/mL) and AMPs (0.5 mg/mL) were employed as controls. These experiments were performed by triplicates.The antimicrobial activity of the MSN/AMPs complexes was evaluated both in solid and liquid media. For the solid medium, the adsorption process was carried out as described in the previous section. The pellet was resuspended in PBS (1 mL). Afterwards, different volumes of the MSN/AMPs mixture were deposited onto Petri dishes with MHB medium and Cmm preinoculum was first placed on MHB medium and incubated at 28 °C for 24 h at 180 rpm. Cells were inoculated to MHB medium in proportion 1:100 v/v and kept under the same conditions until the OD600 reached 0.5. From the previous culture, 200 µL were taken and added to 40 mL of fresh MHB medium to have a cell concentration of 2.5 × 106 colony forming units per mL (CFU/mL); this was referred as the “diluted culture”. To set the adsorption process of the AMPs by MSN, after removing the supernatant and drying the MSN/AMPs, complexes were resuspended in 3 mL of diluted Cmm culture and incubated at 28 °C for 24 h at 180 rpm. After incubation period, serial dilutions were performed up to 1 × 106 of which 100 µL were taken and boxes were planted with MHB medium. The plates were incubated at 28 °C for 72 h and growth colonies were counted, expressed as CFU/mL. Controls were supplemented with MSN (5 mg/mL) and AMPs (0.5 mg/mL) in PBS [To evaluate the antimicrobial activity of the MSN/AMPs complexes at liquid medium, a ) in PBS . The expg for 10 min at 4 °C. The resulting pellet was washed twice with deionized water and centrifuged at 5000× g for 10 min at 4 °C between each wash. MSN/AMPs complexes were resuspended in 3 mL of diluted Cmm culture and incubated at 28 °C for 24 h at 180 rpm. After the incubation period, serial dilutions were performed up to 1 × 106 of which 100 µL were taken and Petri dishes were poured with 30 mL MHB medium. The plates were incubated at 28 °C for 72 h and the colonies were counted, bacterial growth was expressed as CFU/mL. Controls were prepared with MSN (5 mg/mL) in PBS and AMPs (1 mg/mL) treated with protease (1 mg/mL) in a 5:1 ratio (peptide:enzyme) [After removing the supernatant and drying the MSN/AMP complexes, 200 µL of the pepsin (1 mg/mL) were added and incubated at 37 °C for 16 h under constant shaking. The microtubes were then centrifuged at 5000× :enzyme) . The expp-values, p-values of ≤ 0.0028 were considered statistically significant.Data of measurements of the growth inhibition halos and the CFU/mL were presented as the mean and the bars represent the standard deviation. Data analysis was performed using one-way analysis of variance (ANOVA). The Tukey’s test was used to calculate Two gene constructs were designed, encoding the defensin hβD2 and the mutant (hβD2-M), the open reading frame (ORF) encoding these peptides, cloned in the expression vectors are shown in 2+-IMAC, a band near to 4.6 kDa assigned to hβD2 was observed. Similar results were obtained for TRX-hβD2-M and hβD2-M, the complete purification processes for both peptides are summarized in Recombinant proteins were present in both soluble and insoluble fraction. The TRX-hβD2 fusion protein had a molecular weight of 18 kDa which coincides with the theoretical weight. After cleavage by TEV protease and analyzing the unbound fraction of a second purification step by NiCmm was evaluated. CH3COOH pH 2.4 and Tris 50 mM pH 8.4 did not show antimicrobial activity against the strain under study, this same behavior was observed for TRX-hβD2 under both pH conditions. On the other hand, hβD2, TRX-hβD2-M and hβD2-M exhibited inhibition halos at the evaluated pH , stretching vibrations of the Si-O covalent bond, stretching vibrations in the plane of the Si-O bond of the silanol groups (Si-OH), vibrations symmetric tension of the Si-O-Si bonds and symmetric bending vibrations of the Si-O-Si bonds of the siloxane bonds, respectively [−1 associated with the vibration of oxygen in Si–O–Si and the vibration of the silanol group (Si-O-H), respectively  had the best characteristics with mean pore size, pore volume (BJH model) and specific surface area (BET model) of 5.4 nm, 1.77 cm3/g and 1021.4 m2/g, respectively ; however, after that time its release rate was gradually increased, even exceeding TRX-hβD2-M A. As farCmm was performed in both solid  and liquid medium (quantitative). The bioactivity evaluation of the MSN5.4 loaded with the AMPs against Cmm growth inhibition by 96, 47 and 94% when hβD2, hβD2-M and TRX-hβD2-M were adsorbed on MSN5.4 (loaded with 0.5 mg of AMPs), respectively .To determine whether MSN protected AMPs from enzymatic degradation, a proteolytic degradation study was carried out using the peptidase pepsin. Considering the diversity of proteases in nature, further investigation will be required to evaluate other enzymes likely present in tomato crops. The results of this preliminary study indicate that AMPs without the mesoporous matrix are not susceptible to the catalytic activity of pepsin since hβD2, TRX-hβD2-M and hβD2-M inhibited the growth of ectively D. HoweveIt has been shown that hβD2, as well as other AMPs, can inhibit the growth of multidrug resistant strains ,34. In tCmm both in solid and liquid medium. Based on the bibliographic review that was carried out, the IC50 value presented by hβD2 and hβD2-M (acid buffer) is lower than that reported for inhibiting Cmm using AMPs [hβD2, TRX-hβD2-M and hβD2-M showed antimicrobial activity on ing AMPs ,43,44.50 than hβD2, the reason why hβD2-M exhibits better antimicrobial activity against Cmm compared to hβD2 could be attributed to our replacement of Pro45 by an Arg at the carboxyl terminal end. By changing the content of Arg in the sequence of an AMP, a higher net positive charge was obtained due to a guanidinium functional group present in the replaced amino acid. Thus a promoting of long-range electrostatic attraction towards negatively charged bacterial membranes is done [Cmm. Likewise, it is important to emphasize that hβD2-M presents antimicrobial activity even when it is fused to TrxA (TRX-hβD2-M), until now there are few reports indicating that an AMP presents activity linked to a fusion protein [It should be mentioned that the hβD2-M mutant presented a lower IC is done . The mut protein ,48.2 with an imminent type of HI hysteresis loop which are characteristic of mesopores in the form of independent thin cylindrical channels with a wide size distribution [MSN5.4 and MSN4.8 presented a type IV adsorption-desorption isotherm of Nribution ,49. On tribution ,50.software, the dimensions of the AMPs are less than the pore diameter of the MSN5.4 . The above suggests that the material in the liquid MHB presented some alterations on its surface such as the blocking of its pores by some compound in the medium as a consequence the complex had a lower antimicrobial activity [Cmm, this is because the IC50 concentration was exceeded. It is possible that hβD2-M modifies its charge (more cationic) but since it does not have the matrix present and the most anionic is the wall of the bacteria, its activity is not affected antimicrobial.The MSN/hβD2 and MSN/TRX-hβD2 complexes in liquid medium greatly inhibited the growth of activity ,52. As mStaphylococcus aureus compared to the peptide without the mesoporous matrix. It is very important to mention that our AMPs were not so susceptible to pepsin degradation. Since they still significantly inhibited the growth of Cmm, after exposition to pepsin. However, when they were adsorbed on MSN5.4, their antimicrobial activity was significantly higher. On the other hand, the MSN/TRX-hβD2-M complex presented a lower antimicrobial activity in contrast to the peptide without the matrix, here it is possible that the pepsin has carried out some type of interaction with the material causing the clogging of the pores and being TRX-hβD2-M the largest protein had a lower degree of release and therefore the complex presented a lower antimicrobial activity against Cmm [Cmm when it is infecting S. lycopersicum plants in advanced stages of development and in this way its spread could be reduced.The adsorption and posterior release of multiple AMPs has been carried out in different mesoporous matrices ,32,38, iinst Cmm . AccordiE. coli inhibit the growth of Cmm in both solid and liquid tests. The MSN/AMPs complexes (hβD2 and TRX-hβD2-M) in solution after being treated with a pepsin were shown to have better antimicrobial activity against Cmm compared to AMPs without the mesoporous matrix. Thus, AMPs and complex MSN/AMPs are excellent candidates for the potential management of bacterial tomato canker, as well as other diseases caused by the genus Clavibacter.In this work, we demonstrated that recombinant expressed AMPs hβD2, TRX-hβD2-M and hβD2-M in


To separate these signaling modalities, we have used βArr1 and βArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and βArr1-KO mice, without effects in βArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and βArr1-KO animals. By contrast, in βArr2-KO mice head twitch responses are low with LSD and this psychedelic is without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks the LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and βArr1-KOs, but not in βArr2-KOs. MDL restores LSD-mediated disruption of PPI in WT mice; haloperidol is required for normalization of PPI in βArr1-KOs. Collectively, these results reveal that LSD’s psychedelic drug-like actions appear to require βArr2.Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT LSD alters sensation, perception, thought, mood, sense of time and space, and consciousness of self in humans2. Since LSD-induced states bear many similarities to early acute phases of psychosis2 and because serotonin (5-HT) and LSD both contain an indolamine moiety, Woolley and Shaw3 proposed that aberrant 5-HT levels in brain may produce mental disturbances including psychosis. This suggestion gave rise to the 5-HT hypothesis for schizophrenia and stimulated researchers to study LSD in hopes of gaining a better understanding of the disorder. However, this research was largely curtailed when LSD was classified as a DEA Schedule I drug in the 1960’s. Recent research has revealed that LSD has medicinal value in treating cluster headaches4, anxiety and depressive disorders in life-threatening conditions when combined with psychotherapy5, and it may have potential for studying human consciousness and substance abuse7.Lysergic acid diethylamide (LSD) is a prototypical psychedelic drug and is one of the most potent drugs in this class1. Thus, it is not surprising that LSD has high affinities for all thirteen 5-HT G protein coupled receptors (GPCRs)10. Besides 5-HT receptors, LSD activates other biogenic amine GPCRs8 and this polypharmacology may contribute to LSD’s many actions. One activity in particular regarding LSD is its hallucinogenic actions. This activity is ascribed to 5-HT2A receptor (5-HT2AR) stimulation since in drug discrimination studies, discrimination-derived potency is correlated with hallucinogenic potency in humans11. Because the same psychedelics produce head twitches in mice, this response is used as a proxy for hallucinations in humans12, even though non-psychedelic drugs like 5-hydroxytryptophan induce robust head-twitch responses (HTRs)13. Hallucinogen-induced HTRs in rodents are blocked by the highly selective 5-HT2AR antagonist MDL10090716 and are absent in htr2A knockout (KO) mice18. In addition, human studies have shown the hallucinogenic actions of LSD are blocked with the 5-HT2AR preferring antagonist ketanserin19. Thus, the hallucinogenic effects of LSD appear to be mediated through the 5-HT2AR20.LSD shares structural similarities to 5-HTq protein and to non-visual arrestin mediated signaling24. Recent experiments reveal the 5-HT2AR preferentially activates Gq family members, with moderate activity at Gz, and minimal activities at Gi-, G12/13-, and Gs-family members25. However, the 5-HT2AR binds to both β-arrestin 1 (βArr1) and βArr2 proteins in vitro and is complexed with these βArrs in cortical neurons in vivo24. Note, within the arrestin family the non-visual βArr1 and βArr2 are termed Arr2 and Arr3, respectively. While most GPCR agonists, like 5-HT, activate both G protein and βArr signaling, ligand binding can activate also G protein-dependent signaling while serving to activate or inhibit βArr-mediated signaling. Hence, a given ligand can act as an agonist at one pathway while inhibiting the other pathway or it can possess combinations of these actions. This property is termed functional selectivity or biased signaling28 and ligands have been developed to exploit these signaling features29. Although LSD activates G protein signaling at many GPCRs10, this psychedelic stimulates βArr-mediated responses at most tested biogenic amine GPCRs8. Interestingly, LSD displays βArr-biased signaling at the 5-HT2AR25. Most 5-HT2AR-containing neurons express both βArr1 and βArr224, and global βArr1 and βArr2 knockout (KO) mice have been generated31. Since LSD is βArr biased at the 5-HT2AR, the present investigations were conducted to determine whether LSD produces behavioral effects that were differential among the wild-type (WT) and βArr1-KO, and WT and βArr2-KO mice.The 5-HT2AR is a rhodopsin family member of GPCRs that is coupled to GArrb1 (βArr1 protein) or Arrb2 (βArr2 protein) genes were obtained from 129 libraries, the constructs were injected into ES cells that were microinjected into C57BL/6 blastocysts31. These chimeric mice were backcrossed to C57BL/6J mice. Both the βArr1 and βArr2 mice are on a C57BL/6J genetic background and are maintained as separate strains. However, their behavioral responses are somewhat different between the strains. All experiments have an approximate equal mix of mutant and relevant WT littermates. No sex effects were detected in any experiments.The 36. We examined responses to LSD in the global βArr1-KO and global βArr2-KO mice to determine whether disruption of either gene product could modify the behavioral responses to this hallucinogen and to test whether 5-HT2AR antagonism could block these effects. Locomotor, rearing, and stereotypical activities were monitored at 5-min intervals over the 120 min test in both the βArr1 and βArr2 genotypes . When administered with LSD, both doses of MDL blocked the locomotor-stimulating effects of this psychedelic.When cumulative baseline locomotion was examined in βArr1 mice, activity was not differentiated by genotype or by the pre-assigned treatment condition  (Supplementary Table p = 0.024). Nevertheless, Bonferroni post-hoc analyses only identified a trend between the group treated with LSD and the group given MDL alone (p = 0.062)  . Hence, all three doses of the 5-HT2AR antagonist were efficacious in suppressing the LSD-induced hyperlocomotion to control levels. Although LSD increased locomotor activity in βArr2-KO mice, it was not significantly different from any other treatment group.When baseline motor activities were evaluated in the βArr2 mice, no significant differences were found  . Rearing was higher also in mice given 0.05 mg/kg MDL plus LSD than the controls . When 0.1 or 0.5 mg/kg MDL was given with LSD, both doses reduced the LSD-stimulated rearing activities to control levels . By comparison, LSD was without effect in the βArr2-KO mice.Similar to locomotion, LSD also stimulated rearing activities to a greater extent in WT compared to βArr2-KO mice . Notably, 0.5 mg/kg MDL abrogated the LSD effects (p = 0.003) by bringing levels to those of the controls. Together, these results indicate that LSD stimulates locomotor responses in the WT and βArr1-KO animals. LSD stimulated also locomotor, rearing, and stereotypical activities in WT mice from the βArr2 strain. The 5-HT2AR antagonist blocks these LSD-stimulated activities. By striking comparison, LSD failed to significantly stimulate these same motor responses in the βArr2-KO mice above that of the controls.An assessment of stereotypical activities failed to find any genotype differences between the βArr2 mice   Fig. a,b,d. Whp values < 0.001) . Nonetheless, both 0.1 and 0.5 mg/kg MDL decreased retrograde walking to control levels when administered with LSD .Besides HTRs and grooming, LSD was efficacious in potentiating retrograde walking in the WT and βArr1-KO mice compared to the vehicle and MDL controls (01) Fig. c. Responp values < 0.001) . Notably, both 0.1 and 0.5 mg/kg MDL significantly reduced the LSD-stimulated responses —with the higher MDL dose being the more efficacious in suppressing HTRs to control levels (p < 0.001). In βArr2-KO mice, the LSD and the 0.05 and 0.1 mg/kg MDL plus LSD treatments increased HTRs compared to the vehicle and MDL controls . Only 0.5 mg/kg MDL was sufficient to normalize the LSD-stimulated response to control levels in the βArr2-KO mice (p = 0.019).In contradistinction to βArr1 mice, genotype differences were present for βArr2 animals. HTRs were significantly increased in the LSD and 0.05 mg/kg MDL plus LSD groups of WT relative to βArr2-KO mice (01) Fig. a. In WT p values ≤ 0.016) . While 0.05 mg/kg MDL failed to block the LSD effects, both of the 0.1 and 0.5 mg/kg doses were efficacious in normalizing the responses to that of the controls . In βArr2-KO animals, the duration of grooming to LSD was not significantly different from the vehicle and MDL controls. Nevertheless, grooming was enhanced in mice administered 0.05 mg/kg MDL plus LSD compared to all groups , except those given LSD alone.For grooming, the durations of responding were higher in WT than in the βArr2-KO groups administered LSD alone, 0.05 mg/kg MDL plus LSD, or 0.5 mg/kg MDL with LSD (16) Fig. b. In WT 42, we examined grooming in detail as it has a chained organization of responses in rodents43. Note, since in our video recordings the WT mice in the βArr1 and βArr2 strains responded similarly to the vehicle and MDL controls, as well as to LSD and the 0.5 mg/kg MDL plus LSD treatments for grooming, recordings from only one of the WT strains is presented. Analyses of the video-recordings confirmed that all genotypes engaged in a normal sequence of grooming beginning with the face, progressing down the body, and ending at the feet or tail  . Although 0.05 mg/kg MDL was ineffective in decreasing this LSD-stimulated behavior, both 0.1 and 0.5 mg/kg MDL suppressed this response to that of controls . By contrast, LSD was without any significant effect on retrograde walking in the βArr2-KO animals compared to its vehicle and MDL controls.Aside from abnormalities in the organization of grooming, LSD also induced retrograde walking and stimulated nose-poking behaviors. Incidences of retrograde walking were increased significantly in WT mice in the groups given LSD or 0.05 MDL plus LSD compared to the βArr2-KO groups (p < 0.001) . All doses of the 5-HT2AR antagonist reduced the LSD-stimulated nose poking to the levels of the vehicle and MDL controls. No treatment effects were noted among the βArr2-KO animals.Similar to retrograde walking, nose-poking behavior was increased by LSD in WT relative to βArr2-KO mice (01) Fig. d. In WT In summary, responses to LSD across these LSD-stimulated behaviors were similar between the WT and βArr1-KO mice and the 5-HT2AR antagonist reduced these responses to levels of the vehicle and MDL controls. By contrast, the WT mice responded quite differently from the βArr2-KO animals. HTRs, grooming, retrograde waking, and nose-poking to LSD were significantly higher in WT than in βArr2-KO mice. Notably, LSD disrupted the sequences of grooming in the WT and βArr1-KO mice; βArr2-KO animals were unaffected. Nonetheless, divergent responses to MDL alone or to MDL plus LSD were observed among the genotypes.44. βArr1 mice were pre-treated with the vehicle or with 0.1 or 0.5 mg/kg MDL. Subsequently, they were administered the vehicle or 0.3 mg/kg LSD and tested in PPI. No significant genotype or treatment effects were observed for null activity or in response to the 120 dB startle stimulus  . Both 0.1 and 0.5 mg/kg MDL normalized PPI in WT mice to control levels. In βArr1-KO animals, PPI was still significantly disrupted in mice administered 0.1 mg/kg MDL with LSD relative to controls . Although PPI responses in the 0.5 mg/kg MDL plus LSD group were not significantly different from these controls, they were also not significantly different from the LSD group. Hence, LSD disrupted PPI in both WT and βArr1-KO mice, while MDL restored PPI only in WT animals.LSD disrupts PPI in both rats and humans and the response can be restored with 5-HT2AR antagonists45, we tested whether this antipsychotic drug could normalize the LSD-disrupted PPI in the βArr1-KO mice. Overall treatment effects were found where null activities were higher in the 0.1 mg/kg haloperidol plus LSD group than in mice treated with the vehicle or haloperidol alone    . Here, haloperidol normalized the LSD-disrupted PPI to control levels in both WT and βArr1-KO mice.Since haloperidol can normalize PPI in mouse modelsp values ≤ 0.003) . In WT animals, LSD disrupted PPI compared to the MDL and vehicle controls . PPI remained disrupted in the 0.05 mg/kg MDL plus LSD group relative to the MDL control (p = 0.050). However, PPI was normalized to controls with 0.1 mg/kg MDL. By comparison, LSD was without effect in the βArr2-KO mice. Collectively, these findings show that LSD disrupts PPI in both genotypes of the βArr1 mice. PPI was disrupted also with LSD in the WT animals from the βArr2 strain. The 5-HT2AR antagonist restored PPI in both WT strains, whereby haloperidol was required to normalize it in βArr1-KO mice. By contrast, PPI in βArr2-KO mice was unaffected by LSD.PPI responses in the βArr2 mice were examined also. Overall null activity was decreased in the 0.1 mg/kg MDL plus LSD group compared to the vehicle control and the LSD group 36; the possible reasons for this discrepancy in mice versus rats are unclear. When βArr2 mice were tested, LSD disrupted PPI selectively only in WT mice. Notably, βArr2-KO mice were completely unresponsive to this psychedelic. As with WT animals from the βArr1 strain, MDL also normalized the LSD-disrupted PPI in the WT βArr2 mice. Thus, the LSD effects on PPI in the βArr mice are complex, with restoration of PPI with MDL in both strains of WT mice, normalization of PPI with haloperidol in βArr1-KO animals, and without any discernable effect in βArr2-KO subjects.LSD-induced states share many similarities with the early acute phases of psychosis1 and these responses have been attributed to 5-HT2AR agonism17. We observed LSD to stimulate motor activity, head twitches, grooming, retrograde walking, and nose-poking in both βArr strains of WT mice and in βArr1-KO animals. LSD also disrupted PPI in these same genotypes. The LSD-elicited responses in βArr2-KO mice were either significantly attenuated or completely absent. In conditions where LSD produced changes in behavior, these alterations were blocked with the 5-HT2AR antagonist MDL. While these results suggest that the 5-HT2AR is an essential component for all these responses, it should be recalled that LSD exerts a plethora of actions at many GPCRs10 and, aside from HTRs, other behaviors are inconsistently affected by hallucinogens17. Hence, it is likely that LSD’s effects on the 5-HT2AR are involved in a cascade of many GPCR-signaling events mediating these varied responses. In this regard, arrestins are known to serve as scaffolds for many signal transduction molecules54. Future work will examine some of these mechanisms across our behavioral tests.LSD and other psychedelics are well-known for their hallucinogenic actions55. Since the glutamatergic neurons project to multiple subcortical brain areas, actions on 5-HT2ARs in these neurons could exert varied effects on behavior. Within 5-HT2AR-containing neurons, agonist actions at this receptor can lead to G protein-dependent and -independent signaling, the latter of which involves βArr24. Disruption of the Arrb1 or Arrb2 genes would leave G protein signaling intact, while affecting respective βArr2 or βArr1 mediated signaling and desensitization. Zurkovsky and colleagues48 have proposed a model of arrestin actions that may apply to our results with LSD. Both βArr1 and βArr2 are co-expressed, with few exceptions throughout the adult rodent brain. However, expression of βArr1 mRNA is much higher than that for βArr2–-except in selected brain areas56. While the 5-HT2AR binds to both βArr proteins in vitro and is complexed with these βArrs in cortical neurons in vivo24, there is some evidence that the affinities of βArr1 and βArr2 for different GPCRs can vary in vitro57. Moreover, in the few systems that have been studied, signaling in the presence of βArr2 is more efficacious than with βArr147. In our experiments, the LSD-elicited responses were largely intact in the βArr1-KO than in the βArr2-KO mice, because in the βArr1-KO animals βArr2-mediated signaling is still retained. In this regard, it is especially intriguing that LSD-induced HTRs were much more robust in both WT strains and in the βArr1-KO animals, than in the βArr2-KO mice. Our results with LSD suggest that βArr2 may be essential for the expression of hallucinogenic-like actions at the 5-HT2AR.Our immunohistochemical results show that the 5-HT2AR protein is expressed in several areas of the brain, especially the cortex. In this brain region the 5-HT2ARs are localized primarily to pyramidal cells and, to some extent, to interneurons31. All mice had been backcrossed onto a C57BL/6J genetic background. Heterozygotes were used to generate the respective WT and KO animals. The mice were housed 3–5/cage in a temperature- and humidity-controlled room on a 14:10 h (lights on at 0600 h) light–dark cycle with food and water provided ad libitum. All experiments were conducted with an approved protocol from the Duke University Institutional Animal Care and Use Committee and all experiments and methods were performed in accordance with the relevant regulations and ARRIVE guidelines.Adult male and female WT and βArr1-KO, and WT and βArr2-KO mice were used in these experimentsThe drugs consisted of (+)-LSD-(+)-tartrate , MDL 100,907 , haloperidol , and (-)-1--2-aminopropane hydrochloride . The vehicle was composed of N,N-dimethyllacetamide  that was brought to volume with 5% 2-hydroxypropoyl-β-cyclodextrin (Sigma-Aldrich) in water . All drugs were administered (i.p.) in a 5 mL/kg volume. All studies used groups that were administered the vehicle and the 5-HT2AR antagonist, MDL100907 as controls.45. All behaviors were filmed. Mice were injected with the vehicle or different doses of MDL and placed into the open field. Thirty min later, they were administered the vehicle or LSD and were immediately returned to the open field for 90 min. Motor activity was monitored using Fusion Versamax 5.3 Edition software  for locomotor activity (distance traveled), rearing , and stereotypical activities (repetitive beam-breaks less than 1 s) in 5-min blocks or as cumulative activities.Motor activities were assessed in an open field  illuminated at 180 luxThese behaviors were filmed during assessment of motor activity. The responses were scored over the first 30 min following injection of the vehicle or LSD after collection of baseline activity. Observers who were blinded to the sex, genotype, and treatment conditions in the experiments scored the video recordings. The data are expressed as the numbers of head twitches, duration of grooming, and incidences of retrograde walking.58. Each chamber had five LED-illuminated 1.24 cm2 nose-poke apertures with infrared diodes to register nose pokes. No food or liquid reward was available. Mice were injected with the vehicle or different doses of MDL and returned to their home-cages. Thirty min later, the animals were injected with the vehicle or LSD and were placed immediately into the operant chambers for 30 min. The data are depicted as the numbers of head pokes.Nose-pokes were monitored in a 5-choice serial reaction-time apparatus 45. Mice were injected with vehicle or different doses of MDL or with 0.1 mg/kg haloperidol and returned to their home cages. Fifteen min later the animals received the vehicle or LSD and were placed into the apparatus. After 10 min of habituation to a white noise background (64 dB), testing began. Each test consisted of 42 trials with 6 null trials, 18 pulse-alone trials, and 18 prepulse-pulse trials. Null trials comprised the white noise background, pulse trials consisted of 40 ms bursts of 120 dB white-noise, and prepulse-pulse trials were composed of 20 ms pre-pulse stimuli that were 4, 8, or 12 dB above the white-noise background , followed by the 120 dB pulse stimulus 100 ms later. Testing commenced with 10 pulse-alone trials followed by combinations of the prepulse-pulse and null trials, and it terminated with 10 pulse-alone trials. PPI responses were calculated as %PPI = [1 − ]*100.PPI of the acoustic startle response was conducted using SR-LAB chambers  as reported3H]-ketanserin  as the radioligand53 with varying concentrations of unlabeled DOI (Sigma-Aldrich) and 75 μg protein from brain. Binding was analyzed by GraphPad Prism . The 5-HT2AR immunofluorescence study was performed as described59 with a validated 5-HT2AR-specific antibody60. Mice were intracardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were harvested, post-fixed overnight in 4% PFA, and dehydrated in 30% sucrose. Brains were sectioned at 40 µm by cryostat. Brain sections were washed 3X with 0.4% Triton X-100 in PBS (TX-100/PBS) before incubating for 1 h with blocking buffer . Next, they were incubated for 48 h at 4 °C with the anti-5-HT2AR antibody . Subsequently sections were washed 3X with 0.1% TX-100/PBS and incubated for 2 h with the secondary antibody . The sections were imaged under a 20X objective using an Olympus VS120 virtual slide microscope .Binding experiments on mouse brains were conducted as described using 2.3 nM [post-hoc analyses. A p < 0.05 was considered significant. All results were plotted using GraphPad Prism.All statistical analyses were performed with IBM SPSS Statistics 27 programs . The data are presented as means and standard errors of the mean. No sex effects were detected in any experiments. Hence, this variable was collapsed. All data were normally distributed. One- or two-way ANOVA, repeated measures ANOVA (RMANOVA), or analyses of covariance (ANCOVA) were used to analyze the data, followed by Tukey or Bonferroni Supplementary Information 1.Supplementary Movie 1.Supplementary Movie 2.Supplementary Movie 3.Supplementary Movie 4.Supplementary Movie 5.Supplementary Movie 6.Supplementary Movie 7.Supplementary Movie 8.Supplementary Movie 9.


Beyond static assessment, functional techniques are increasingly applied in magnetic resonance imaging (MRI) studies. Stress MRI techniques bring together MRI and mechanical loading to study knee joint and tissue functionality, yet prototypical axial compressive loading devices are bulky and complex to operate. This study aimed to design and validate an MRI-compatible pressure-controlled varus–valgus loading device that applies loading along the joint line.Following the device’s thorough validation, we demonstrated proof of concept by subjecting a structurally intact human cadaveric knee joint to serial imaging in unloaded and loaded configurations, i.e. to varus and valgus loading at 7.5 kPa (= 73.5 N), 15 kPa (= 147.1 N), and 22.5 kPa (= 220.6 N). Following clinical standard (PDw fs) and high-resolution 3D water-selective cartilage (WATSc) sequences, we performed manual segmentations and computations of morphometric cartilage measures. We used CT and radiography (to quantify joint space widths) and histology and biomechanics  as references.We found (sub)regional decreases in cartilage volume, thickness, and mean joint space widths reflective of areal pressurization of the medial and lateral femorotibial compartments.Once substantiated by larger sample sizes, varus–valgus loading may provide a powerful alternative stress MRI technique.The online version of this article (10.1007/s10334-020-00844-6) contains supplementary material, which is available to authorized users. Magnetic resonance imaging (MRI) is clearly the most powerful and versatile technique for musculoskeletal imaging. Providing the reference standard imaging modality for the non-invasive assessment of intra- and periarticular structures of the human knee joint, current clinical-standard morphological MRI studies are usually performed with the patient in a supine position and their joints in an unloaded and, consequently, unphysiological configuration.B0 ≤ 0.5 T), signal-to-noise ratios, and image resolutions  and the lateral tibia  are less related to degeneration, while changes in the lateral femoral condyle  are considerably less related to loading . In practical terms, we positioned the load cell between the pressure applicator (without padding) and both counter-bearings that were clamped by a rigid PVC plate. We then determined resultant forces transmitted to the load cell through the pressure applicator for increasing pressure levels set at intervals of 0.3 bar. We repeated these measurements three times and calculated mean forces as a function of set pressure levels. For data analysis, we determined Pearson’s correlation coefficient n 3.6.5)  . Of note, local Institutional Review Board approval  and written informed consent by the body donor were available at study initiation.In preparation of the MRI measurements, the human cadaveric knee joint was positioned in slight flexion of approximately 20° by placing appropriate positioning aids underneath the joint. Additionally, we used mechanical positioners such as sandbags to reduce joint motion in response to loading. In varus or valgus loading, the medial or lateral joint line was brought in line with the centre of the padded pressure applicator. Both counter-bearings were adjusted to the anatomy of the knee joint at the greatest possible distance from each other. Attention was paid that the loading components were in loose contact with the knee joint, i.e. the counter-bearings with the lower and upper thirds of the femur and tibia and the pressure applicator with the medial or the lateral joint line.B0 and B1 mapping sequences  acquired with a water phantom with and without the loading device in position. Moreover, we used a T2*-weighted 2D gradient-echo sequence  to assess the presence of image artefacts because of the loading device. Once the connections and system operability were checked, we performed the MRI measurements in seven distinct configurations: (1) unloaded (δ0), (2) valgus load of 7.5 kPa , (3) valgus load of 15 kPa , (4) valgus load of 22.5 kPa , (5) varus load of 7.5 kPa (δvar1), (6) varus load of 15 kPa (δvar2), and (7) varus load of 22.5 kPa (δvar3). Of note, pressure levels were set by digital control. Throughout the study, equilibration periods of 5 min were observed after each change in pressure level. For each loading position, fat-saturated 2D proton density-weighted (PD-fs) sequences and 3D water-selective cartilage scans (WATSc) were acquired. Please see Table δ0) and the axial and coronal planes , respectively. The WATSc sequence was acquired in the coronal plane and used to perform cartilage segmentations and morphometric measurements [The VVL device was loaded with the human cadaveric knee joint and centrally positioned in the bore of a clinical 3.0-T scanner  Fig. c. Imaginδ0, δvlg1, δvlg2, δvlg3, δvar1, δvar2, and δvar3) using the pressure-controlled VVL device. An equilibration period of 5 min was observed after each change in pressure.Analogously, the human cadaveric knee joint was scanned sequentially in the craniocaudal direction using a multidetector-row clinical CT scanner  and the following scan parameters: tube voltage 120 kV, tube current 800 mAs, slice thickness 0.6 mm, rotation time 1 sec, increment 3 mm, pitch 0.8. After adapting the field of view (158 mm × 158 mm) to the joint outline, the chosen matrix (512 × 512) resulted in a pixel size of 0.31 mm × 0.31 mm. Scan duration was 5.28 s per joint configuration. For image reconstructions, the Siemens kernel Br64s was used and axial, coronal and sagittal reconstructions were obtained. As for MR imaging, the CT measurements were carried out in all joint configurations  is not possible with this device.Stress radiography was performed using the commercially available Telos GA-III/E device (Telos GmbH) at 20° of knee flexion. Using standard settings for clinical radiography of the knee , anteroposterior projection radiographs of the knee were obtained in the following configurations: MR images of the human cadaveric knee joint were processed separately and in each configuration. Morphometric measurements of the medial and lateral femorotibial cartilage were based on manual segmentations and computations using Chondrometrics software  as published before, e.g. , 27. On For CT analyses, sagittal and axial reconstructions were used to identify the coronal reference plane as the coronal imaging slice that centrally bisected the weight-bearing region of the MFTC and LFTC at the centre of the anteroposterior distance between the deepest point of the trochlear groove and the posterior condylar line. To this end, the joint space widths, i.e. the vertical distances between the subchondral bone contours of the tibial and femoral condyles, were measured at three locations per compartment, i.e. at the inner, central, and outer third. Similarly, joint space widths were measured at three locations on the projection radiographs. Mean joint space width was calculated for each loading position, compartment, and modality.In addition, potential positional changes of the knee joint in response to loading were determined in terms of joint flexion and rotation. Flexion angle was determined between the femoral and tibial shafts on sagittal reconstructions. Rotation was measured as the angle between the device’s horizontal base plate and a line that joined both corners of the patella on axial reconstructions. One reader  who was blinded to the joint configuration performed these measurements for each joint configuration using the inbuilt calliper tool of the clinical-routine picture archiving and communications system . For visualization purposes, shaded CT volume renderings were generated using IntelliSpace Clinical Applications (Philips). Because of variable flexion and rotation at the different loading configurations, the knee joint (as the volume-of-interest) was defined and subsequently rolled and rotated to allow for unobstructed visualization of both joint spaces.2 to map maximum pressures of up to 103.42 bar (= 10342 kPa). Due to its flexible and thin configuration (thickness: 0.1 mm), the area conforms to the individual knee joint anatomy. After accessing the joint via a medial parapatellar approach, the sensors were inserted into the medial and lateral compartments covering the femoral articular surfaces. Care was taken to avoid crinkling of the sensor areas and to cover the medial- and lateral-most portions of the compartments. However, due to limitations in sensor area, only the anterior two-thirds of the tibiofemoral articular surface were covered. To gain access to the joint, the collateral and cruciate ligament complex had to be partially cut. Prior to the measurements, sensors were preconditioned and calibrated in line with the manufacturer’s instructions. Then, the knee joint was placed into the VVL device and loaded in six configurations, while equilibration periods of 5 min were observed after setting each loading position: 7.5 kPa varus (δ1var), 15 kPa varus (δ2var), 22.5 kPa varus (δ3var), 7.5 kPa valgus (δ1vlg), 15 kPa valgus (δ2vlg), and 22.5 kPa valgus (δ3vlg). Reasoning behind the choice of these loading intensities involves the most commonly used device for stress radiography, i.e. the Telos device , which uses 15 kPa (= 147.2 N). The raw data obtained from these measurements were imported into Matlab  and analysed using customized analysis routines.Immediately after completion of imaging studies, femorotibial pressure levels as a function of gradually increasing VVL were spatially mapped in the human cadaveric knee joint using digital electronic pressure-sensitive sensors . This system has been used widely in static and dynamic pressure mappings of the human knee joint , 29. ItsNext, the central weight-bearing femoral and tibial joint surfaces were identified and the peripheral femoral and tibial cartilage areas as well as the meniscus body regions were sampled to undergo histological and biomechanical reference assessment as published before –34  between set pressure levels and resultant weights or forces , 46.6 N (δvlg2), 50.3 N (δvlg3), 13.1 N (δvar1), 37.7 N (δvar2), and 38.6 N (δvar3).Validation of the pressure–force relationship revealed a close-to-perfectly linear association ; 1.558 mm (δvar3); 1.724 mm (δvlg3). Corresponding findings were made for the LFTC, where ThC of the lateral femur was slightly decreased in response to valgus loading and increased in response to varus loading, e.g. mean ThC (cLF): 1.637 mm (δ0); 1.735 mm (δvar3); 1.591 mm (δvlg2); 1.622 mm (δvlg3). Generally, changes in VC and ThC of the LFTC in response to valgus loading were less clearly associated with loading intensity than the corresponding changes in the MFTC in response to varus loading.Quantitative evaluation of cartilage morphometric measures, i.e. VC and ThC, were reflective of these changes throughout the various loading configurations Table . In the Increasing the loading intensity in the high-loading range, i.e. 15–22.5 kPa, produced only smaller—and partially inconsistent—changes in VC and ThC as compared to the low-loading range, i.e. unloaded to 15 kPa. Changes in AC were inconsistent and not clearly related to type and intensity of loading.Overall, analysis of the distinct subregions confirmed the above-mentioned findings Table , Fig. 4.δ0) to 4.0 ± 0.6 mm (δvar1), 3.5 ± 0.8 mm (δvar2), and 3.4 ± 0.6 mm (δvar3). Correspondingly, in response to valgus loading, mean JSWs of the LFTC decreased from 7.2 ± 1.5 mm (δ0) to 5.2 ± 0.6 mm (δvlg1), 4.8 ± 0.6 mm (δvlg2), and 4.4 ± 0.9 mm (δvlg3). For the unloaded joint compartments, i.e. the LFTC under varus loading and the MFTC under valgus loading, gradually distended JSWs were found, e.g. LFTC under varus loading: 7.2 ± 1.5 mm (δ0), 9.1 ± 2.0 mm (δvar1), 10.5 ± 0.9 mm (δvar2), 11.5 ± 0.3 mm (δvar3); mean JSWs of the medial femorotibial compartment under valgus loading: 4.6 ± 0.6 mm (δ0), 7.3 ± 2.1 mm (δvlg1), 8.3 ± 2.6 mm (δvlg2), 8.7 ± 2.7 mm (δvlg3).For CT studies, qualitative and quantitative evaluation confirmed the findings above. Figure δ0) to 3.7 ± 1.0 mm (δvar1), and 3.0 ± 0.6 mm (δvar2) in response to varus loading. Correspondingly, mean JSWs of the LFTC decreased from 6.5 ± 0.8 mm (δ0) to 5.1 ± 0.1 mm (δvlg1), and 4.6 ± 0.2 mm (δvlg2) in response to valgus loading.For radiography, similar observations were made Fig. b. Mean Jδ0) to 29° (δvar1), 45° (δvar2), and 48° (δvar3) as well as to 23° (δvlg1), 25° (δvlg2), and 28° (δvlg3). Similarly, rotation of the knee joint gradually increased, too, from 13° external rotation (δ0) to 17° (δvar1), 25° (δvar2), and 29° (δvar3), while it remained unchanged in response to increasing valgus loading.Flexion of the knee joint gradually increased in response to loading, from 20° , 0.34 MPa (LF), 1.88 MPa (MT), and 1.92 MPa (LT). Figure The most important findings of the present study are that (1) VVL of the human knee joint induces efficient compartmental compression, thereby providing an alternative loading mechanism for functional joint assessment and that (2) intra-tissue adaptations in response to VVL were reliably detected using high-resolution morphological MRI techniques.Based on the developed MRI-compatible VVL device, pressure may be actuated along the joint line in exactly defined varus or valgus configurations as controlled by pressure. The relation of set pressure levels and resultant forces generated at the padded actuator was close-to-perfectly linear. Most likely because of unavoidable friction and leakage within the pressurized infrastructure, i.e. the pressure lines and the pneumatic mechanism, this association was not perfectly linear. Longitudinal force measurements confirmed that the pneumatic mechanism maintains constant forces over extended time periods, i.e. at least 30 min, which is a prerequisite for combining loading with advanced MR imaging techniques that are time-consuming and necessitate constant loading conditions to reduce movement artefacts. Even though reference pressure mappings indicated effective pressurization of the MFTC and LFTC, VVL induced considerably lower intraarticular pressures than compressive axial loading along the joint’s mechanical axis with ≥ 50 % body weight (thoroughly reviewed in ). Of couIn terms of cartilage deformation, our findings are well in line with previous stress MRI studies. Subjecting a healthy volunteer’s knee joint to a compressive axial load of 50% body weight, Subburaj et al. determined decreases in ThC of 0.074 mm (MFC), 0.003 mm (LFC), 0.071 mm (MT), and 0.061 mm (LT) based on mean (unloaded) ThC values of 1.42 mm, 1.49 mm, 1.35 mm and 1.71 mm, respectively , 47, ourWhile stress radiography of the knee has been performed with variable loading intensities ranging from 3 to 30 kPa , our resOur study and others , 46, 47 Whenever absolute cartilage deformation is quantified, the degree of pre-existent degeneration needs to be considered as degenerative cartilage is more compliant , 52 whicIn our study, morphometric analysis was performed on the basis of a high-resolution WATSc sequence that is commonly applied for cartilage segmentation  and was Our study has a number of limitations. First and foremost, we included only one human knee joint specimen. However, the present study’s primary objective was to demonstrate proof of concept for joint functionality assessment based on the validated pressure-controlled VVL device. Of course, future studies investigating larger specimen sizes are necessary to corroborate our findings and to define the physiological response to loading across the joint and its pathological aberrations. Second, we only assessed knee joint functionality within the framework provided by the static stabilizers, i.e. capsule, menisci, and cruciate and collateral ligaments, while the dynamic stabilizers, i.e. muscles, could not be assessed. Hence, when it comes to the in vivo translation of our findings, studies including healthy (non-OA) individuals and OA patients are required to bridge this translational gap. Similarly, once regulatory and medicolegal issues have been solved, this proof-of-concept study has to be followed by a comprehensive feasibility study that includes sufficient numbers of volunteers (without OA or knee pain) and patients (with OA and/or knee pain). Aspects related to the concept’s in vivo translation such as patient comfort, joint positioning, stabilization, and fixation, device safety and handleability, convenience of operation as well as measurement validity and reproducibility will then be addressed with the device in clinical operation. Future efforts need to be directed at keeping examination times as short as possible, possibly by including abbreviated MRI protocols , and loaIn conclusion, the MRI-compatible pressure-controlled VVL device provides a new and innovative way of assessing knee joint functionality based on pressurization of the MFTC and LFTC. In this proof-of-concept study, effective compartmental pressurization is reflected by distinct loading-induced patterns of subregional cartilage deformation and indicates the methodology’s potential application in basic, translational and clinical research questions. Once substantiated by more refined imaging, biomechanical, and compositional cross-references, this approach may improve handleability, decrease logistical demands, and provide a safe and powerful alternative technique for stress MRI in the context of functional joint and tissue assessment.Supplementary file1 (DOCX 25 kb)Below is the link to the electronic supplementary material.


Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury. The characterization of neuropathic pain is continuous and/or paroxysmal pain associated with dysesthesia or allodynia. It has been previously established that peripheral neuropathies caused by lesions in the peripheral nervous system are extremely difficult to treat as multiple underlying mechanisms can be involved and effective interventions are dependent on the cause. Drug development for treating neuropathic pain often focuses therefore on the inhibition or relief of certain underlying causes or symptoms.Receptor-interacting serine/threonine protein kinase 1 (RIPK1), a member of the serine-threonine protein kinase family, is a well-known and critical mediator of necroptosis and a regulator of the responses to cellular stress and inflammation ,3,4. PreTumor necrosis factor (TNF)-α is regarded as a proinflammatory cytokine with a central function in regulating pain communication between the immune system and the brain . GeneralWe hypothesized that the TNF-α-mediated activation of the RIPK1 pathway plays a key role in trigeminal neuropathic pain following inferior alveolar nerve injury. For this purpose, we examined changes in RIPK1 expression and TNF-α concentration in the trigeminal subnucleus caudalis (TSC) following inferior alveolar nerve injury in a rat model. We also used this model to examine the changes in the air-puff thresholds and TNF-α concentration after blockade of the RIPK1 pathway by intracisternally administered Nec-1, an RIPK1 inhibitor. Finally, we evaluated the cellular localization of RIPK1 and TNF receptor in the TSC of these animals by double immunofluorescence analysis.p < 0.05, F(2.17) = 800.124, p < 0.05, (2.18) = 44.791, p < 0.05, (2.19) = 53.625, p < 0.05, (2.19) = 13.4, p < 0.05, (2.18) = 172.249, p < 0.05, p < 0.05, Alterations in the air-puff thresholds and RIPK1 expression in naïve rats following the intracisternal administration of rrTNF-α protein are illustrated in p < 0.05). As shown in p < 0.05, F(2.18) = 48.416), which appeared 2 h after the 20 μg injection and 4 h after the 2 μg injection. These effects had dissipated by 24 h post-injection. Vehicle administration did not alter the mechanical allodynia induced by the malpositioned dental implantation. Western blotting indicated that the intracisternal administration of TNF-α antibody, but not vehicle, attenuated the upregulated RIPK1 expression in the TSC, resulting from the inferior alveolar nerve injury on POD 3 . These findings indicate that the RIPK1 pathway is activated by the actions of TNF-α in the TSC. This TNF-α-mediated RIPK1 pathway has been studied by the cell death cellular signaling mechanism. In TNF-α-stimulated cells, RIPK1 was reported to be activated intracellularly through the TNF receptor  and to rTNF-α has two receptors and both TNFR1 and 2 are known to be involved in neuropathic pain ,27. TNFRThe animal experiment followed the protocols of the Committee of the School of Dentistry, Kyungpook National University (No. 20210034). Adult male Sprague Dawley (SD) rats were used. A total of 269 SD rats were purchased from Jung-Ang experimental laboratory . All animals, weighting between 200 and 220 g, were maintained in a pathogen-free environment, in a 12:12 h light-dark cycle, and maintained at 23 ± 1 °C. Food and water were provided ad libitum. All experiments followed the ethical guidelines set for the investigation of experimental pain in conscious animals by the International Association for the Study of Pain (IASP). Each rat was used only once and all rats were handled at least 5 days before the surgery or behavioral testing to minimize stress. All of these experiments were conducted in a blinded fashion by a single researcher and the animals were randomized during the experiments.In the trigeminal neuropathic pain model, SD rats were anesthetized with a ketamine/xylazine  mixture. After anesthetization, the second molar of the left mandibular was extracted and a mini-dental implant was placed  to injure the inferior alveolar nerve, as previously described . SimilarWhile under anesthesia, intracisternal administrations were performed in the rats using a stereotaxic frame, and a polyethylene tube 10 (PE10) was implanted into each animal, as previously described ,31,32,33We measured mechanical allodynia using previously described methods ,37,38. FFor immunofluorescence analysis, the rats were sacrificed and perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer . The caudal medulla was dissected and a portion of this tissue then underwent additional fixation using the same fixative solution for 2 h at 4 °C. Following 2 h fixation, the sample was then placed in 30% sucrose in 0.1 M PB solution overnight. Prior to processing for immunofluorescence analysis, transverse frozen sections  were cut using a cryostat. All tissue was blocked with a 10% normal donkey serum in phosphate buffered saline  for 30 min at room temperature and for the double immunofluorescence, the sections were incubated overnight at 4 °C with a mixed solution of rabbit anti-RIPK1  or a rabbit anti-TNFR1  with a mouse anti-NeuN , a goat anti-Iba-1 , or mouse anti-GFAP . Following these primary antibody incubations, samples were than incubated with a mixture of anti-rabbit Cy3  and anti-mouse or anti-goat FITC . Images were collected with a fluorescence microscope  or a confocal laser scanning microscope  to observe the immunofluorescence signals.For Western blotting analysis, the ipsilateral caudal medulla was collected and homogenized as described previously by Yoon et al. . Total pAt the indicated time points, protein supernatants were collected and evaluated for cytokine levels using commercial ELISA kits, in accordance with the manufacturer’s protocol. The TNF-α ELISA kit was purchased from R&D Systems  and the optical density (OD) was measured spectrophotometrically at wavelengths of 420–570 nm. The data were expressed relative to a standard curve prepared for TNF-α.Nec-1 was purchased from Selleckchem  and dissolved in a combination of 45% PEG 300, 5% DMSO, and 50% DDW. TNF-α antibody (R&D systems) was dissolved in normal saline. Recombinant rat TNF-α (rrTNF-α) protein was obtained from R&D systems and dissolved in sterile PBS.n = 7 per group). Mechanical allodynia was determined at 2 days prior to the operation and at 1, 2, 3, 5, 7, 10, 14, 21, 30, 35, 40, 45, and 50 days after the inflicted injury to the nerve. Western blotting analysis was conducted to evaluate RIPK1 expression following inferior alveolar nerve injury (n = 6 per group) on POD 3, 7, 21, and 50. Neuropathic mechanical allodynia was assessed after a blockade of RIPK1. The dose of Nec-1, an RIPK1 inhibitor, was based on prior reference [n = 7 per group).Prolonged nociceptive behavior was observed following inferior alveolar nerve injury in several previous studies ,38. Hereeference . Nec-1 (n = 7 per group). RIPK1 protein expression changes in the TSC were determined by Western blot analysis 2 h after treatment with rrTNF-α protein (n = 8 per group). To assess the participation of RIPK1 in TNF-α-induced mechanical allodynia, the effects of Nec-1, an RIPK1 inhibitor, on mechanical allodynia generated by the administration of rrTNF-α protein were monitored in naïve rats. Nec-1 (10 µg/10 µL) was administered 4 h before the rrTNF-α protein (200 ng/10 µL) injection and mechanical allodynia was evaluated at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 24 h after rrTNF-α protein injection (n = 7 per group).To evaluate the effects of rrTNF-α protein on air-puff thresholds, the rrTNF-α protein (20 or 200 ng/10 µL) was injected intracisternally into naïve rats. Mechanical allodynia was then measured at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 24 h after treatment with the rrTNF-α protein or vehicle (n = 10 per group) on POD 1, 3, and 5 to evaluate changes in the TNF-α concentration subsequent to inferior alveolar nerve injury. We also investigated changes in neuropathic mechanical allodynia and RIPK1 expression after a blockade of TNF-α in the rats with inferior alveolar nerve injury. On POD 3, TNF-α antibody (2 or 20 µg/10 µL) was administered intracisternally followed by saline flushing. Mechanical allodynia was then measured at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 24 h after treatment with TNF-α antibody or vehicle (n = 7 per group). Western blotting was used to evaluate expression changes in RIPK1 within the TSC at 6 h after treatment with TNF-α antibody in the rats with inferior alveolar nerve injury (n = 8 per group).ELISA analysis was performed (n = 6 per group) was completed on POD 3 for RIPK1 and TNFR1 to evaluate their subcellular localization within the ipsilateral TSC, using markers for neurons (NeuN), microglia (IBA1), or astrocytes (GFAP).Double immunofluorescence staining .Repeated measures analysis of variance (ANOVA), followed by the Holm–Sidak post hoc test, was utilized to statistically analyze all behavioral data. For qualitative data obtained by Western blotting or ELISA, changes were evaluated using a Student’s Our results provide a connection between trigeminal neuropathic pain and TNF-α-RIPK1 pathway. Inferior alveolar nerve injury results in a significant upregulation of RIPK1 expression in the TSC and a blockade of the RIPK1 pathway effectively relieves mechanical allodynia. A single intracisternal administration of TNF-α antibody attenuates trigeminal neuropathic pain and suppresses the upregulation of RIPK1 expression. Double immunofluorescence analyses revealed the colocalization of RIPK1 and TNFR1 with astrocyte markers. Hence, an astroglial TNF-α-mediated RIPK1 pathway is involved in important pathogenic mechanisms underlying our rat pain model. Modulation of this pathway may be a viable therapeutic strategy to alleviate neuropathic pain.


This paper attempts to examine how successful this intervention has been in improving air quality for both passengers and workers within the station. New air pollution data in 2020 has enabled comparisons to the 2016/2017 monitoring campaign revealing a 23–42% decrease in measured NO2 concentrations. The new levels of NO2 are below the Occupational Health standards but still well above the EU Public Health Standards. This reduction, together with a substantial decrease (up to 81%) in measured Particulate Matter (PM) concentrations, can most likely be attributed to the new fan system effectiveness. Carbon Monoxide levels were well below Occupational and Public Health Standards at all times. The COVID-19 pandemic “initial lockdown” period has also allowed an insight into the resultant air quality at lower rail-traffic intensities, which produced a further reduction in air pollutants, to roughly half the pre-lockdown concentrations. This study shows the scope of improvement that can be achieved through an engineering solution implemented to improve the ventilation system of an enclosed railway station. Further reduction in air pollution would require additional approaches, such as the removal of diesel engine exhaust emissions via the adoption of electric or diesel-electric hybrid powered services.High NO Despite the known health , PM10, PM2.5, and carbon monoxide (CO) for comparison with the results below.When combined with the low enclosed environment created by the concourse level (marked by grey shading), it becomes apparent that there is limited potential for wind dispersion from outside of the station. With platforms up to 240 m long, it is common for multiple diesel trains to be entirely within the enclosure, highlighting the requirement for the installed bi-directional fan system. Platforms 10 and 11 were noted in the Hickman et al., 2018 study [2 and PM. Their locations within the station on Platform 10B and 11B are represented by the blue triangle shown in Two South Coast Science Praxis monitors , installed by Emission Solutions Ltd. (EMSOL) in January 2020, displayed in 2 dome atrium 25 m high and 50 m diameter [diameter . At the Air pollution sensors at Birmingham New Street have previously been mounted at 1.5–2.0 m above platform level to simulate height of mouth and nose for human respiration. The Praxis monitors utilised in this study were mounted within the roof panelling at 3 m above the platform a,b. This2, CO and particulates PM1, PM2.5, and PM10—atmospheric particles with aerodynamic diameter <1.0 µm (PM1) <2.5 µm (PM2.5) and <10 µm (PM10), respectively—were acquired from EMSOL for the period January to June 2020 [3 for NO2, <±0.6 µg/m3 for CO. For PM, the monitors utilise Alphasense optical particle counters (OPCs) capable of measuring particulates of aerodynamic diameter of 0.35 to 40 µm [Fixed monitor data for the pollutants NOune 2020 . Air quato 40 µm .2, all the sensors were certified to be temporally stable by Alphasense preventing any drift in measurements or need for manual recalibration over the monitoring period. The sensors incorporate a temperature sensor to recalibrate microscale measurement deviations due to temperature change [All sensors utilised in the Praxis monitors were operated within their optimal pollutant and environmental ranges minimising the possibility of erroneous readings. Furthermore, with the exception of NOe change ,15.Before monitoring began all data were zero referenced by EMSOL following the manufacturer’s instructions. An acclimatisation period during January 2020 was established, which featured intermittent gaps within the data, and thus February 2020 was deemed the first month of reliable data. Data included continuous monitoring at a ten-second sampling rate from 1 February–30 June for Platform 10B and 1 February–25 April for Platform 11B. A 68-day monitoring period was established (1 February–8 April) for comparisons with the Hickman et al., 2018, study, which utilised a monitoring period of identical duration [Unfortunately, due to COVID-19 restrictions, the UK was placed in a nationwide lockdown on 23 March 2020. This resulted in the final 15 days of the 68-day monitoring period being under different rail operating conditions. However, monitoring data from both platforms did allow assessment of pollution levels post lockdown and additionally the Platform 10B monitor, whose measurements continued until June 2020, provided data as restrictions were eased throughout May and June.2, PM2.5, and PM10 could be determined to again compare to pre fan upgrade conditions. Subsequent TWAs of 8 h and 15 min were established to discern the current working conditions within the station in accordance with EH40/2005 WELs (Workplace Exposure Limits) and STELs (Short Term Exposure Limits).A 24 h TWA (Time Weighted Average) was established to discern daily pollutant conditions during the 68-day monitoring period and to allow comparison to the daily conditions measured under the previous fan system operation, during the Hickman et al., 2018 study [At a station such as Birmingham New Street, in Birmingham, UK, the station owner and the train operators, who have employees working there, have a legal duty, so far as is reasonably practicable, to manage the risks to health of their employees and users of the station (including passengers) from exposure to hazardous substances as specified by the Control of Substances Hazardous to Health Regulations 2002 (COSHH) (as amended) .2 24 h concentrations of between 42% for platform 10b and 23% for platform 11b. Even so average NO2 concentrations of 224 µg/m3 and 293 µg/m3 at Platform 10B and 11B, respectively, are still of concern, given EU and Daily Air Quality Index (DAQI) Public Health 1 h regulations of 200 µg/m3 and Annual average of 40 µg/m3.The results of the 68-day monitoring period displayed in 3 (listed as 10 mg/m3) at 307 µg/m3 and 457 µg/m3 on Platforms 10B and 11B, respectively. Maximal 24 h values followed the same trend of higher concentrations measured at Platform 11B than Platform 10B with 604 µg/m3 against 453 µg/m3.CO was measured well below EU 8 h limit values of 10,000 µg/m10 averages of 20 µg/m3 and 14 µg/m3, respectively. PM2.5 and PM1 also displayed the same observed trend with averages of 12 µg/m3 and 4 µg/m3 against 8 µg/m3 and 3 µg/m3. The maximal 24 h PM10 value measured was 52 µg/m3, which is in exceedance of the EU 50 µg/m3 air quality standard for ambient air, although this was only exceeded a total of 5 times between both platforms over the course of the 68-day monitoring period. Maximal 24 h PM2.5 concentrations were similar with 31 µg/m3 and 34 µg/m3 at Platform 10B and 11B, respectively. Finally, maximal PM1 concentrations were 11 µg/m3 and 17 µg/m3, respectively. As a whole, PM concentrations were at an order of magnitude lower scale than the gas pollutants and thus were plotted separately (1 and PM2.5) from total particulates measured over the 68-day monitoring period were calculated at 57% at Platform 10B and 60% at Platform 11B. This suggested Diesel Engine Exhaust Emissions (DEEE) to be the likely source due to known fine particulate production from high temperatures and pressures in combustion processes [Particulates followed the observed trend of higher concentrations at Platform 11B than at 10B with PMparately  to approrocesses . The prerocesses ,20 increrocesses ,22,23,242, consistent with concentrations measured at the Automatic Urban and Rural Network (AURN) stations  measured in this study  with maximal 15 min concentrations reaching 953 µg/m3 on Platform 10B (50% of STEL) and 1436 µg/m3 on Platform 11B (75% of STEL)  and 488 µg/m3 on Platform 11B (51% of WEL).In spite of the high concentrations of gas pollutants measured, and regular EU air quality standard regulation exceedances discussed previously, no gas pollutants exceeded limits from EH40/2005. NOof STEL) . The NO23 STEL value nor the 23,000 µg/m3 WEL value throughout the entire study period.Finally, CO exceeded neither the 117,000 µg/m2, PM10, and PM2.5 from 1 February 2020, through the COVID-19 lockdown period of 23 March–11 May and into eased restrictions until the end of June 2020. Note the use of a dual y-axis due to the discrepancy in scale of gas pollutant concentrations against PM concentrations. As a reference, pre-pandemic average concentrations for each pollutant were plotted using 1 min data from 1 February to 23 March, which equalled 268 µg/m3 for NO2, 16 µg/m3 PM10, and 9 µg/m3 PM2.5.2 concentrations averaged 135 µg/m3 during this period whilst PM10 and PM2.5 averaged 9 µg/m3 and 5 µg/m3, respectively. These averages equated to 50%, 56%, and 55% of the pre-lockdown averages. The easing of lockdown restrictions on 11 May enabled journeys previously deemed non-essential during lockdown. This appeared to have little immediate effect on pollutant concentrations with values staying within 15% of averages over April and May, consistent with the mobility trends reported by Apple Mobility and CityMapper in Birmingham , which has only been in operation since June 2021. Further monitoring and research are needed at New Street station to follow up on the results of this research to examine the impact of ventilating the station’s NO