A network of nanofibers is formed in situ through solid-state deformation of disentangled ultra-high molecular weight polyethylene (dis-UHMWPE) during compounding with a polyolefin elastomer below the melting temperature of dis-UHMWPE crystals. Dis-UHMWPE was prepared in the form of powder particles larger than 50 μm by polymerization at low temperatures, which favored the crystallization and prevention of macromolecules from entangling. Shearing the blend for different durations and at different temperatures affects the extent to which the grains of dis-UHMWPE powder deform into nanofibers. Disentangled powder particles could deform into a network of nanofibers with diameters between 110 and 340 nm. The nanocomposite can be further sheared for a longer time to decrease the diameter of dis-UHMWPE nanofibers below 40 nm, being still composed of ≈70 wt.% of crystalline and ≈30 wt.% of amorphous components. Subsequently, these thinner fibers begin to melt and fragment because they are thinner and also because the amorphous defects locally decrease the nanofibers’ melting temperature, which results in their fragmentation and partial loss of nanofibers. These phenomena limit the thickness of dis-UHMWPE nanofibers, and this explains why prolonged or more intense shearing does not lead to thinner nanofibers of dis-UHMWPE when compounded in a polymeric matrix. All-polymer nanocomposites are still unique and usually prepared by dispersing ready-made polymer nanofibers or nanodroplets in a polymer matrix. There are several techniques for the production of polymer nanofibers or nanodroplets, for instance, hard template synthesis and soft template synthesis ,2. Hard Electrospinning is an effective approach to fabricating long polymer fibers with diameters from micrometers down to even a few nanometers by taking advantage of strong electrostatic forces . This prVoznyak et al.  developeCa, the ratio of viscous drag forces versus surface tension forces acting across an interface, the more efficient the stress transfer from sheared polymer matrix to inclusions.η is the viscosity, U is the characteristic velocity of the material, and σ is the interfacial tension.Another method for fabrication of fibrillar nanocomposites, developed several years ago, comprises an addition of a crystalline polymer in a powder or flake form to another thermoplastic polymer prior to its processing, with the thermoplastic polymer selected from a group consisting of polyolefins, polyacetals, vinyl polymers, polyamides, or polyesters ,22,23. TIn order to deform polymer crystals embedded in a viscous medium, critical shear stress, resolved in a slip plane of the respective crystal slip systems, must be reached and exceeded. In the past, the possibility of deformation of a crystal embedded in a viscous matrix under shearing flow was assessed . ShearinThe stress in a molten polymer matrix is simple, being nearly pure shear stress. The shear stress component resolved in (100) planes of polyethylene crystals can cause crystallographic chain slip (less efficient transverse slip) if its value in the chain direction is sufficient. No other slips are allowed because slip planes cannot cross macromolecular chains. At room temperature, the critical shear stress value for PE (100)<001> chain slip is around 7 MPa. However, crystallographic slips are thermally activated, and at compounding temperature, their level is only 1–2 MPa.However, for fibrous shape, the deformation of polymer crystals to large strains is needed. This is possible when the density of entanglements persisting in the surrounding amorphous phase is drastically reduced. One of the means is polymerization at a lower temperature to allow crystallization during polymerization ,38. The The above introduction is aimed at explaining the possibility of effective deformation of UHMWPE crystals by shearing, especially in the extruder. That was estimated based on the knowledge of plastic deformation of PE crystals and on the shear field strength. The drag forces exerted on the polymer powder grains were compared with the shear stresses needed to deform polyethylene crystals at a higher temperature. The constraints from the surrounding amorphous PE material were drastically reduced due to disentangling of macromolecules. The transformation of powder grains into fiber-shaped entities was predicted due to the easiest crystallographic slips in the chain direction due to PE crystal plasticity. Finally, the possibility of forming a network of nanofibers through solid-state deformation of nascent powder of ultra-high molecular weight polyethylene (UHMWPE), with disentangled macromolecules, is suggested.In the present work, the possibility of forming a network of nanofibers through solid-state deformation of nascent powder of ultra-high molecular weight polyethylene (UHMWPE), with disentangled macromolecules, is explored. The nanofibers are formed in situ during compounding with a polyolefin elastomer at a temperature below that of the melting of the nascent UHMWPE. The influence of shearing time and temperature on the resulting nanofibers is studied. The decrease in fiber diameters and crystal sizes results in a decrease in the melting temperature of the crystals and is interpreted according to the modified version of the Gibbs–Thomson equation.2CH2)x [CH2CH[(CH2)5CH3]] y was used as a polymer matrix. This polyolefin elastomer (EOC) with the trade name Engage 8842 is an ultra-low-density copolymer combining exceptional properties of an ultra-low-density elastomer with the potential possibility of handling this polymer in pellet form. This EOC, with a density of 0.857 g cm−3, octene content of 45 wt.%, a melting temperature of 44 °C, and a melt flow index of 1 g/10 min  was chosen to deform the powders of disentangled UHMWPE to nanofibers in the solid state. Disentangled ultra-high molecular weight polyethylene (dis-UHMWPE), with a molecular weight of 1.4 million g mol−1, melting peak temperature (Tm) of 140 °C, and melting enthalpy of 200 J g−1 , was used as synthesized and described by us earlier [A commercial grade of ethylene–octene copolymer  with the formula  were prepared in situ by compounding EOC with dis-UHMWPE powder at a temperature higher than the melting temperature of the EOC matrix but lower than the melting temperature of the UHMWPE crystals to allow the formation of UHMWPE nanofibers in the solid state. The 5 wt.% dis-UHMWPE concentration was chosen to ensure the formation of a continuous network structure of nanofibers  and to mThe morphology of in situ generated composites was investigated with a JSM-5500 LV scanning electron microscope ). The internal structure of the samples was exposed after a fracture in liquid nitrogen. The cryo-fracture surfaces of samples were coated with 20 nm thick gold layers by ion sputtering  and examined with SEM in a high vacuum mode at the accelerating voltage of 10–20 kV.−1. The entire thermal treatment was performed under nitrogen flow. The melting temperature (mT), the onset temperature of crystallization (oT), the endset temperature of crystallization (eT), and melting enthalpy (Δmh) were determined based on the thermograms. The crystallinity (cX) of UHMWPE in the composites of EOC/UHMWPE was calculated as [Φ is the weight fraction of UHMWPE in the EOC/UHMWPE composites and Δmh0 is the heat of fusion of a 100% crystalline UHMWPE taken as 293 J g−1 [The melting and non-isothermal crystallization of the disentangled UHMWPE powder, EOC, and EOC/UHMWPE composites were examined by employing an indium-calibrated differential scanning calorimeter DSC Q20 . Samples weighing 6–10 mg were heated to 180 °C, annealed for 3 min, and cooled down to crystallize EOC, at the constant heating/cooling rate of 10 °C minlated as :Xc (%) =93 J g−1 .mT of the employed EOC is a specific feature facilitating the solid-state deformation of dis-UHMWPE into nanofibers by simple compounding. The easy deformation ability of dis-UHMWPE is illustrated in The effect of shearing time and temperature on the in situ solid-state formation of UHMWPE nanofibers was scrutinized by compounding the solid dis-UHMWPE powders with the molten EOC in a mini twin-screw extruder. The low −1, were equal to 1693 Pa·s and 1044 Pa·s, for 75 °C and 115 °C, respectively. The viscosities of EOC were measured using a strain-controlled rheometer, ARES LS2, TA Instruments, for shear rate of 350 s−1. The characteristic velocity of the material in the extruder was at the perimeter of the extruder screws which is equal to the screw circumference times its rotation rate, 2 × π ×10 mm ×30 rev min−1 = 31.42 mm s−1. Assuming the interfacial tension at the level of 11.8 mN m−1 for polyethylene crystal lateral face [The morphology of dis-UHMWPE powder is presented in ral face ,48, the ral face ,49 have −1. mT of the nascent powder, the thus obtained fibers could melt at a decreased temperature.An increase in the temperature from 75 to 115 °C results in the formation of much thinner fibrils with diameters varying from 110 to 340 nm. It should be noted that in this case, a physically entangled nanofibrous network is formed e. This i−1, were equal to 1693 Pa·s and 1044 Pa·s, for 75 °C and 115 °C and for the shear rate of 350 s−1, respectively.  The characteristic velocity of the material in the extruder was the perimeter of the extruder screw times its rotation rate, 30 rev min−1 × 2 × π × 10 mm = 31.42 mm s−1. Assuming the interfacial tension at the level of 11.8 mN·m−1 for polyethylene crystal lateral face [The deformation of dis-UHMWPE grains into nanofibers was primarily caused by the viscous drag from the sheared EOC matrix. As mentioned in the Introduction, the best parameter describing the action of viscous drag against interfacial forces and further against shear plastic resistance of dis-UHMWPE crystals is the capillary number. The complex viscosities of the EOC matrix, that was sheared in the extruder at 350 sral face ,48 the ce, but also by the interfacial free energy of (hk0) planes in contact with the surrounding polymer melt, σsl,. Both of these components influence the Gibbs free energy (ΔG) of the crystalline phase of the fibers:fg is the bulk free enthalpy of the volume unit of a nanofiber without surface effects, D is the diameter of nanofiber, and L is the crystal thickness. Melting occurs when ΔG = 0. Taking into account that Δfg = Δfs ΔT = Δfh0 ΔT/mT0, where Δfh0 is the heat of fusion per unit volume, one obtains:The melting of polymer crystals is usually described by the Gibbs–Thomson equation. The in situ generated nanofibers are characterized by a large surface area, which can influence their melting behavior. It seems reasonable to assume that UHMWPE crystals in the highly deformed nanofibers are aligned with their c-axes parallel to the fiber axes. Hence, the melting process is influenced not only by the interfacial free energy of lamellae basal planes in contact with the amorphous phase, σD >> L ΔT2 = 0, hence Equation (4) takes the form of the Gibbs–Thomson equation, whereas for L >> D ΔT1 = 0, and the modified version of the Gibbs–Thomson equation is obtained [For lamellar crystals obtained .slσ, can be considered similar to that between the lateral face of crystal and the rubbery amorphous phase of polyethylene, which was assessed to be 11.8 mN m−1 [mT due to interfacial free energy of dis-UHMWPE crystals in the form of nanofibers embedded in EOC, according to Equation (6), that is when the effect of the basal planes of the crystals is not taken into account. For the crystals with finite thickness, the additional decrease in mT should occur by ΔT2 expressed by Equation (5), according to the Gibbs–Thomson equation. It is evident that the mT of crystalline nanofibers with a diameter in the range of a few tens of nanometers is significantly reduced, and could occur during compounding, even at a temperature below mT of the nascent dis-UHMWPE. Considering that EOC has a chemical structure very close to that of polyethylene, we can assume that the crystal surface free energy in molten EOC is very close to that in polyethylene in the molten state. Moreover, it is reasonable to assume that the polymer chain axes of dis-UHMWPE nanofiber, which was formed by the plastic deformation of chain-extended crystals, are parallel to the fiber axis. Thus, the interfacial tension between the cylindrical lateral face of dis-UHMWPE nanofiber and the EOC matrix, 8 mN m−1 ,48. FiguA very strong depression of melting temperature for high interfacial free energy indicates that the defects on the nanofiber surfaces, such as chain folds, amorphous fragments, etc., could decrease the melting temperature locally, leading to local nanofiber melting and fragmentation. It must be remembered that the total crystallinity of dis-UHMWPE is at the level of 70 wt.%, and 30% of amorphous fragments are then dispersed along the nanofibers, also decreasing the crystal thickness of a nanofiber.−1, of dis-UHMWPE powder, EOC, and EOC/UHMWPE composites are shown in The first heating and the second heating thermograms, measured at 10 °C minm, melting onset (oT), and endset (eT) temperatures and the degree of crystallinity (cX) calculated based on the first and second heating thermograms.mT of UHMWPE powder recorded during the first heating was at 140 °C and decreased to 136 °C during the second heating, which corresponds to the crystal thickness, calculated based on the Gibbs–Thomson Equation (5) (with σe of 90 mN m−1 [The 0 mN m−1 ) of 57 nmT of the dis-UHMWPE powder, clearly showed two distinct melting regions, i.e., below 70 °C and from 128 °C to 150 °C. The first region with a distinct peak at 46 °C is attributed to the melting of EOC crystals and remains almost unchanged independently of processing conditions, and is visible on the thermogram of neat EOC. During the second heating, the melting endotherm of EOC was different but still visible on all thermograms in the low-temperature range. The main effect of composite processing conditions and the resulting structure is on the melting of dis-UHMWPE crystals, showing different values of mT and Δmh. The melting peaks of nanofibers in the range of 100–200 nm, in the nanocomposites, which had been processed at 115 °C, with mT of 144 °C, were featured with shoulders at ca. 140 °C. oT and eT were near 136 °C and 149–150 °C, respectively. mT of 144 °C corresponds to the lamellar thickness of 513 nm, according to Equation (5). mT increased whereas the low-temperature shoulder decreased with increasing heating rate, and only a trace of the latter remained during heating at 30 °C min−1. The increase in mT evidences that its high value did not result from annealing during heating, as faster heating limits reorganization processes in the crystalline phase. Although the high mT could partially result from the effect of constraints on the melting process, it undoubtedly shows that the crystal thickening and perfection occurred in dis-UHMWPE crystalline nanofibers during processing upon shearing at 115 °C. The crystal thickening should be accompanied by an increase in cX. However, after 10 min processing at 115 °C, cX was below that of dis-UHMWPE powder, 38%, and decreased to 34% after an additional 20 min of shearing. This evidences that in parallel to the thickening and perfection of dis-UHMWPE crystals another process could occur. All the composites, processed for different durations and at different temperatures well below cX. mT lower by ca. 20 °C than the thick fibers containing crystals of the same thickness. Considering that on heating at 10 °C min−1 the melting of the nascent UHMWPE powder began close to 125 °C, and that temperature during processing could increase due to shearing, the melting of the thinnest fibers during processing seems to be highly probable.We hypothesize that the thinnest fibers formed upon shearing were unstable, melted during the processing, and crystallized during subsequent cooling with lower mT of 139 °C, which mainly corresponds to the melting of polyethylene crystals with an approximate thickness of 30 nm, as estimated by the Gibbs–Thomson Equation (5), and Δmh corresponding to cX of 62%. Shearing the composite at the same temperature but for longer times of 30 and 90 min changed neither the dis-UHMWPE melting peak temperature range nor mT, but cX decreased to ca. 40%.In turn, the thermogram of the composite processed at 75 °C for 10 min is characterized by a single sharp melting peak with the The decrease in crystallinity could be also due to the formation of very thin fibers, which melt during processing. However, due to the low processing temperature, the decrease in crystallinity was rather related to the transformation process of UHMWPE powder into fibers. Unlike in the case of processing at 115 °C, the process was not accompanied by the thickening of crystals, which is reflected in the melting behavior similar to that of dis-UHMWPE powder. We note that others  have obsmT is 5–9 °C lower than mT during the first run. −1 during the first heating of the EOC/UHMWPE composite, the melting peak is featured with a low-temperature shoulder. This evidences that the shoulder is related to the melting of a fraction of UHMWPE fibers rather than to recrystallization phenomena in the crystalline phase.It is shown in The disentangled UHMWPE obtained directly from a polymerization reactor is characterized by a low density of entanglements in the amorphous phase, which allows easy solid-state deformation. As a result, the powders undergo plastic deformation in a solid state and deform into micro and nanofibers. However, the initial morphology of crystals seems to be influenced during this deformation, which results in different thermal behavior upon melting. The melting of deformed disentangled UHMWPE powders in the range of 112 to 148 °C see a,b assurIn the case of solid-state deformation at a higher temperature of 115 °C, stacked lamellar crystals ease both the local mobility and the diffusion of UHMWPE chains between neighboring crystals. This crystal thickening leads to the formation of thicker crystals and a shift in the melting of the new crystals to higher temperatures. Once the amorphous chain ties become elongated, the folded chains of thick crystals may be tilted to form chains of extended crystals. The formation of a double melting peak in DSC results reveals that the population of chain-extended crystals, attributed to the higher melting peak, is remarkable compared to the initial chain-folded crystals, i.e., shoulder peak. The existing chain-folded crystals still pose a melting peak at 138 °C while chain-extended crystals melt at a higher temperature of 144 °C due to the thickened lamellae  could locally decrease the melting temperature leading to nanofiber local melting and fragmentation. The condition required for the melting of in situ solid-state generated nanofibers of dis-UHMWPE is in good agreement with the prediction of the Gibbs–Thomson equation. The peak at a lower temperature is attributed to the melting of nanofibers, formed at 115 °C, having thinner chain-folded crystals. The crystalline nanofibers with thickened chain-extended crystals persist further with the heating and show a melting peak at higher temperatures, where the melting temperature recorded via DSC goes beyond the equilibrium melting temperature of linear polyethylene due to the transfer of the macroscopic stress to molecular length scale . The ela


Pre-clinical models, postmortem and neuroimaging studies all support a role for muscarinic receptors in the molecular pathology of schizophrenia. From these data it was proposed that activation of the muscarinic M1 and/or M4 receptor would reduce the severity of the symptoms of schizophrenia. This hypothesis is now supported by results from two clinical trials which indicate that activating central muscarinic M1 and M4 receptors can reduce the severity of positive, negative and cognitive symptoms of the disorder. This review will provide an update on a growing body of evidence that argues the muscarinic M1 and M4 receptors have critical roles in CNS functions that are dysregulated by the pathophysiology of schizophrenia. This realization has been made possible, in part, by the growing ability to visualize and quantify muscarinic M1 and M4 receptors in the human CNS using molecular neuroimaging. We will discuss how these advances have provided evidence to support the notion that there is a sub-group of patients within the syndrome of schizophrenia that have a unique molecular pathology driven by a marked loss of muscarinic M1 receptors. This review is timely, as drugs targeting muscarinic receptors approach clinical use for the treatment of schizophrenia and here we outline the background biology that supported development of such drugs to treat the disorder. Signifibilities . This walidation . These f–/– mice  is essentially abolished, and agonist-stimulated phosphatidyl inositol (PI) hydrolysis is reduced by >60%, in primary cortical cells from newborn CHRM1–/– mice . Going b–/– mice . This efctioning . The cha–/– mouse has a two-fold increase in levels of extracellular dopamine in the striatum  following N-methyl-D-aspartate (NMDAR) receptor blockade by phencyclidine . This approach has shown that those with MRDS have no increased association with any sequence variation within the CHRM1 gene or its promotor , exposurq/11 in the dorsolateral prefrontal cortex of people with schizophrenia  gene that are associated with poorer cognitive ability  and N-methyl-4-[123I] iododexetimide (123I-IDEX) that have high affinities for both the CHRM1 and CHRM4  over CHRM4 (nanomolar range) and was preferentially displaced by CHRM1 agonists and antagonists (Molecular imaging studies measuring CHRMs in people with schizophrenia have been possible using two non-selective single photon emission compute tomography (SPECT) tracers 3-quinuclidinyl-4-[nd CHRM4 . The disstriatum . Importaagonists . Recogniagonists . In addiagonists  which haThe need for neuroimaging ligands has become critical to study CHRM target engagement because of recently reported clinical trials in people with schizophrenia. These trials included phase II and III trials showing the efficacy of KarXT, which is predicted to having its therapeutic benefits from the inclusion of CHRM1/CHRM4 dual agonism, against the positive and negative symptoms of schizophrenia . In addi123I-IQNB SPECT to show there were lower levels of CHRM1/CHRM4  in the CNS of drug-free people with the disorder compared to matched control subjects  state in people with schizophrenia, but this limitation may be overcome with the development of 11C-MK6884 (CHRM4 PAM) and 11C -LSN3172176 (bitopic CHRM1 agonist) PET tracers.There are now data that argue that the molecular pathology of schizophrenia extends beyond the CNS  and the –/– mouse has changes in bronchoconstriction (–/– mouse has developmental abnormalities in the auditory cortex (In considering potential roles of the CHRM1 and CHRM4 in comorbid central and peripheral disorders, it is significant that mutations in the CHRM1 may be involved in the genesis of epilepsy . In relatriction  and a patriction . In additriction . Finallyy cortex  which raThe successful transition of the drug KarXT, a coformulation containing the CHRM1/CHRM4 agonist, through a large phase 2 trial  and a phImportantly, the discovery of the antipsychotic properties of chlorpromazine precede understanding the critical role of its action at the dopamine D2 receptor being critical to its antipsychotic effects . This coBD prepared portions of the review and integrated individual contributions into a final version for review. GB and HU prepared specific sections of the review based on their specialist knowledge. RK provided the specialist clinical oversight. All authors contributed to the conceptualization of the scope of this review and involved in preparing the final manuscript submitted for publication.


Although a general consensus that vaccines could be a complementary strategy against antimicrobial resistance (AMR), there is still the need for studies investigating the relationship between childhood vaccination coverage and AMR proportions in the overall population.We performed an ecological analysis of available Italian data  to evaluate the relationships between vaccination coverages in children and AMR proportions in the last 2 decades.E. coli resistant to fluoroquinolones and third generation cephalosporins, K. pneumoniae resistant to carbapenems and third generation cephalosporins, and P. aeruginosa resistant to piperacillin and tazobactam. Polio vaccination coverage was inversely related to proportions of E. coli and K. pneumoniae resistant to third generation cephalosporins.After adjusting for covariates, we showed that AMR proportions decreased with increasing vaccination coverages, especially for some combinations of vaccines, pathogens, and antimicrobials. Vaccination coverages for pertussis, diphtheria, and tetanus were inversely related to proportions of These results, however, should be interpreted cautiously due to the ecological nature of our analysis. For this reason, further studies designed ad hoc should be encouraged to measure the impact of increasing childhood vaccination coverage on AMR.The online version contains supplementary material available at 10.1186/s13756-022-01173-0. Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus strains resistant to antimicrobials [Antimicrobial resistance (AMR) continues to constitute a significant issue for public health. According to the first report published jointly by the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe, in fact, there were approximately 670,000 antimicrobial-resistant infections and 33,000 deaths in the EU/EEA in 2020 . This recrobials . Considecrobials . The reacrobials . For allHemophilus influenzae Type B and Pneumococcus) [Escherichia coli and Klebsiella pneumoniae [There is no doubt that vaccination can play a complementary role in combating AMR, but its application has been largely underestimated to date. Yet, vaccines could counteract AMR by reducing the incidence of infections caused by susceptible or resistant pathogens, viruses, fungi and parasites . Indeed,ococcus) . Others ococcus) , 11, 13.eumoniae .Taken together, these results generated broad consensus on the potential role of vaccination in fighting AMR. However, current evidence is generally vague due to the difficulties of measuring the magnitude and direct effect of vaccines on AMR. Different study designs can be applied to evaluate the efficacy and effectiveness of vaccination on AMR. Even though intervention studies produce the highest level of evidence, rarely they are specifically designed to focus on AMR. At most, changes in AMR proportion are assessed as part of the overall vaccine evaluation. Ecological studies, instead, are important to monitor AMR proportions over the years, and to evaluate their relationship with vaccination coverages. The results thus obtained could be useful to generate new hypotheses and then confirm them through a multi-level approach. In particular, there is still a need for assessing whether childhood vaccination might be—at least in part—related to AMR in the general population. With this rationale, the present ecological study aimed to evaluate the relationship between vaccination coverages in children and AMR proportions over the last 2 decades in Italy.www.epicentro.iss.it/vaccini/dati_Ita. These data are routinely collected through immunization registries implemented at regional level and then transmitted at national level [Pertussis vaccineDiphtheria vaccineTetanus vaccinePolio vaccineHaemophilus influenzae Type B (Hib) vaccineMeasles, Mumps, and Rubella (MMR) vaccineVaricella vaccineWe used data on vaccination coverages in children (≤ 24 months), from 2000 to 2020 in Italy. This information, expressed as the proportion of vaccinated subjects per 100 inhabitants, was reported by the Italian National Institute of Health  and made publicly available on the website al level . The curwww.epicentro.iss.it/vaccini/dati_Ita).It is worth mentioning that data on vaccination coverage for pertussis, diphtheria, and tetanus vaccines were reported in the aggregate  until 2013, and then by specific antigen. Since there were slight differences in vaccination coverages for specific antigens, we conducted separated analyses for pertussis, diphtheria, and tetanus vaccines. Even the data on vaccines against measles, mumps, and rubella were initially reported in the aggregate and then by specific antigen. However, there were no differences in specific vaccination coverages and thus we analyzed data for the MMR vaccine ;Streptococcus pneumoniae resistant to penicillins or macrolides;Enterococcus faecium resistant to aminopenicillins, vancomycin, or gentamicin;Enterococcus faecalis resistant to aminopenicillins, vancomycin, or gentamicin.We also used data on AMR proportions in Italy, provided by the Italian AMR surveillance project of the ISS (AR-ISS) for selected isolates and specific periods throughout the Italian country. The AR-ISS, based on a network of local clinical laboratories testing the antimicrobial susceptibility of invasive isolates of eight key bacterial species, reports AMR proportions to the to the European Antimicrobial Resistance Surveillance Network (EARS-Net) of the ECDC . These dFluoroquinolones (J01MA);Carbapenems (J01DH);Third generation cephalosporins (J01DD);Combinations of penicillins (J01CR);Data on antimicrobial use in the community were obtained from the European Surveillance of Antimicrobial Consumption Network (ESAC-Net) database . The ESAWe first used summary statistics  to describe vaccination coverages, AMR proportions, and antibiotic use in Italy. Next, we evaluated correlations between annual vaccination coverages and AMR proportions, using the Spearman's rank correlation coefficient (rho). Correlation coefficients were displayed as a heat-map for all combinations of vaccine, pathogen, and resistance to antimicrobials. For combinations showing statistically significant correlations after Bonferroni correction for 168 multiple comparisons (adjusted-α = 0.0003), we applied linear regression analyses to evaluate the relationship between vaccination coverage (independent variable) and AMR proportion (dependent variable), adjusting for the number of isolates tested and antimicrobial use. The latter data were obtained from the Italian National Institute of Statistics  . Outputswww.epicentro.iss.it/vaccini/dati_Ita. With respect to AMR, the overall number of isolates tested ranged from 6014 for S. pneumoniae to 58,468 for S. aureus. In general, the number of isolates tested annually increased over the years. AMR proportions exhibited peculiar trends over the years, also in relation to different pathogen-antimicrobial combinations considered. More details on trends of AMR proportions are reported in the Additional file Overall, the average vaccination coverages were 95.5%  for pertussis vaccine, 95.6%  for diphtheria vaccine, 95.7%  for tetanus vaccine, 95.6%  for polio vaccine, 90.9%  for Hib vaccine, 87.5%  for MMR vaccine, and 74.5%  for varicella vaccine, with some variations from 2000 to 2020. While pertussis, diphtheria, tetanus, and polio vaccination coverages slightly decreased over the years, Hib and MMR vaccination coverages increased. No statistically significant trend was evident for varicella vaccination coverage. More details on trends of vaccination coverages are reported in the Additional file E. coli resistant to fluoroquinolones (rho =  − 0.780) and third generation cephalosporins  and third generation cephalosporins (rho =  − 0.795); P. aeruginosa resistant to piperacillin and tazobactam (rho =  − 0.815). As expected, we also found similar correlation coefficients when analyzing the relationship between AMR proportions and coverages of diphtheria and tetanus vaccines. Polio vaccination coverage exhibited a similar pattern of correlation with the different combinations of pathogen and antimicrobial. In fact, it inversely correlated with proportions of: E. coli resistant to third generation cephalosporins (rho =  − 0.825); K. pneumoniae resistant to carbapenems (rho =  − 0.819), third generation cephalosporins (rho =  − 0.845), and fluoroquinolones (rho =  − 0.837). Vaccination coverages for Hib, MMR and Varicella, instead, showed less marked correlations with AMR proportions, which were not significant after Bonferroni correction.Figure Next, we tested the linear relationship between vaccination coverage and AMR proportion for each combination of vaccine, pathogen, and resistance to antimicrobials with statistically significant correlation.E. coli, K. pneumoniae, and P. aeruginosa. After adjusting for the number of isolates tested and for antimicrobial use, the analysis confirmed the inverse relationships of pertussis vaccination coverage with the proportions of E. coli resistant to fluoroquinolones  and third generation cephalosporins , as well as with the proportions of K. pneumoniae resistant to carbapenems  and third generation cephalosporins . The adjusted analysis also confirmed the inverse relationship of pertussis vaccination coverage with the proportion of P. aeruginosa resistant to piperacillin and tazobactam . All the results obtained by applying linear regression models are reported in the Additional file Figure E. coli, K. pneumoniae, and P. aeruginosa. After adjusting for the number of isolates tested and antimicrobial use, diphtheria vaccination coverage was inversely related to the proportions of: E. coli resistant to fluoroquinolones  and third generation cephalosporins ; K. pneumoniae resistant to carbapenems  and third generation cephalosporins ; P. aeruginosa resistant to piperacillin and tazobactam . All the results obtained by applying linear regression models are reported in the Additional file Similar results were obtained for the diphtheria and tetanus vaccines, since data on vaccination coverage showed only minor differences with pertussis vaccine. Figure E. coli, K. pneumoniae, and P. aeruginosa. In particular, tetanus vaccination coverage was inversely related to the proportions of: E. coli resistant to fluoroquinolones  and third generation cephalosporins ; K. pneumoniae resistant to carbapenems  and third generation cephalosporins ; P. aeruginosa resistant to piperacillin and tazobactam . All the results obtained by applying linear regression models are reported in the Additional file Figure E. coli, K. pneumoniae, and P. aeruginosa. Adjusted linear regression analysis confirmed the inverse relationships of polio vaccination coverage with the proportions of E. coli resistant to third generation cephalosporins , and K. pneumoniae resistant to third generation cephalosporins . All the results obtained by applying linear regression models are reported in the Additional file Figure Although formal studies investigating the impact of vaccination on reducing AMR are still scarce, it is plausible to hypothesize an important contribution of vaccines by reducing antibiotic use and therefore selection pressure on pathogens. Thus, in the present study, we performed an ecological analysis of Italian data to evaluate the relationship between vaccination coverage in children and AMR proportions. Our findings proposed an inverse relationship between vaccination coverage and AMR proportion, especially for some combinations of vaccines, pathogens, and antimicrobials. The relationship was more pronounced for pertussis, diphtheria, tetanus, and polio vaccines; the rest of the vaccines  also showed some less marked correlations, which however were not significant after correction for multiple comparisons.This means that AMR proportions decreased with increasing vaccination coverage. However, the ecological nature of our investigation, as well as the unmeasured impact of other factors, suggest caution when interpreting these findings. In fact, the observed relationships did not satisfy the well-known Hill's criteria: a set of nine principles to be satisfied when establishing epidemiological evidence of a causal relationship . Thus, vaccination coverage does not directly cause AMR, but it may be related to other factors affecting it. Our results, however, supported—at least partially—the hypothesis that the proportions of antimicrobial-resistant pathogens could be reduced by increasing vaccination coverages in childhood. At the moment, however, this remains a concept that should be further investigated by designing and conducting specific analytical and experimental studies.There are several mechanisms by which vaccines can prevent AMR of non-target pathogens, including the ability to reduce inappropriate antibiotic use and the lower probability of selecting resistant genes than antibiotics. If for the first mechanism, there are a lot of examples showing the reduction of inappropriate antibiotic prescriptions by increasing viral vaccination coverage , 21–24, S. pneumoniae, Hib, Salmonella Typhi, Bordetella pertussis, tuberculosis, and Neisseria meningitidis can prevent infections from antimicrobial-resistant forms of these pathogens [E. coli and K. pneumoniae. In fact, pertussis vaccination coverage was inversely related to the proportions of E. coli resistant to fluoroquinolones and third generation cephalosporins, and with the proportions of K. pneumoniae resistant to carbapenems and third generation cephalosporins, even considering the effect of covariates . A strong inverse relationship was also evident with the proportion of P. aeruginosa resistant to piperacillin and tazobactam. It should be noted, however, that similar results were obtained for diphtheria, tetanus, and polio vaccinations as well. This is one of the motivations that still makes it challenging to measure the magnitude and direct effect of vaccines on AMR. Vaccines mentioned above, in fact, are often administered together , resulting in similar vaccination coverage. For this reason, it is difficult to understand which of them could be helpful in the fight against AMR.Despite the lack of clinical evidence on the impact of childhood vaccination on AMR, some general consideration can be done. According to the Action Framework Leveraging Vaccines to Reduce Antibiotic Use and Prevent Antimicrobial Resistance, published by the WHO, the most direct way by which vaccination could prevent and control AMR is by reducing the incidence of infections from antimicrobial-resistant pathogens . In partathogens . In our During a recent survey, Italian experts expressed their opinions on the impact of existing vaccines in limiting AMR: about half of them agreed that vaccinations play a role against AMR, attributing the greatest importance to bacterial vaccination, such as pertussis and meningococcal vaccines . DespiteFor some of the abovementioned vaccines, the specific impact on AMR has been estimated, for example S. pneumoniae and Hib . The latOverall, our results—along with what has been shown previously—support the hypothesis that AMR proportions could be reduced by increasing vaccination coverage in children. However, caution is required due to the ecological design and additional weakness of our study. Our analysis, in fact, did not focus on individual data, but only on Italian indicators over the last 2 decades. Thus, results are prone to biases and misinterpretations that do not allow us to evaluate a causal effect between childhood vaccination and AMR . MoreoveBased on these considerations, our study represents a first step in the path of confirming the complementary role of childhood vaccination coverage against AMR. Given the paucity of scientific data on this topic at present, it is still important to investigate how AMR proportions change with changing vaccination coverage. Due to the main limitations described above, it was not our intention to estimate the proportion of AMR that could be prevented by increasing vaccination coverages in children. However, evaluating the role of children vaccination is still an emerging field of research that could be crucial to identify alternative strategies to fight AMR. This becomes even more important if considering the current Italian epidemiological scenario, which requires that all potential interventions against AMR should be evaluated and considered. Although Italy is among the countries most affected by AMR in Europe, further analysis of data from other European countries should be encouraged given the importance of AMR globally. Another future direction in the field, instead, should be the research on mechanisms underpinning the observed relationships, as well as the influence of potential confounding and mediating factors. Since our results do not prove that childhood vaccination can reduce AMR proportions, further studies—especially with analytical and/or experimental designs—are urgently needed in order to understand and predict how increasing childhood vaccination coverage will affect AMR.Our analysis of Italian data suggested an inverse relationship between vaccination coverage in children and AMR proportions in the overall population. Although these findings are generally in accordance with the hypothesis that high vaccination coverages reduce antimicrobial prescriptions and inappropriate antimicrobial use,further efforts should be encouraged to elucidate mechanisms supporting the observed relationships. Where justified by studies designed ad hoc for this purpose, this evidence could be used to inform global and national actions plans dedicated to AMR control, by including vaccination as an additional strategy against AMR.Additional file 1. Trends of vaccination coverages in Italy from 2000 to 2020. Data are publicly available on the website www.epicentro.iss.it/vaccini/dati_Ita.Additional file 2. Trends of AMR proportions for specific combinations of pathogens and antibiotics. Data are publicly available from the Surveillance Atlas of Infectious Diseases of the ECDC.Additional file 3. Linear regressions of the association between pertussis vaccination coverage and antimicrobial resistance, adjusted for number of isolates tested and antimicrobial use.Additional file 4. Linear regressions of the association between diphtheria vaccination coverage and antimicrobial resistance, adjusted for number of isolates tested and antimicrobial use.Additional file 5. Linear regressions of the association between tetanus vaccination coverage and antimicrobial resistance, adjusted for number of isolates tested and antimicrobial use.Additional file 6. Linear regressions of the association between polio vaccination coverage and antimicrobial resistance, adjusted for number of isolates tested and antimicrobial use.


We found that administration of 2000 mg/kg of OJS was able to mitigate mechanical somatic and visceral nociception via Erk signaling without affecting symptom score and polyp number. Moreover, we discovered that OJS has sedative properties and elicits prolonged total sleeping time in AOM/DSS mice. Our in vitro experiments showed that OJS has the capacity to reduce TNFα gene expression in LPS-stimulated BMDM, but no changes were observed in DRG spike number and CT-26 cell proliferation. Taken together, these data suggest that OJS ameliorates nociception in mice and warrants further examination as a potential CAM therapy to promote analgesia.Cancer patients can develop visceral, somatic, and neuropathic pain, largely due to the malignancy itself and its treatments. Often cancer patients and survivors turn to the use of complementary and alternative medicine (CAM) to alleviate pain and fatigue. Thus, it is necessary to investigate how CAM therapies work as novel analgesics to treat cancer pain. Ojeok-san (OJS) is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects and mechanism of action of OJS in a preclinical model of colitis-associated colorectal cancer. Male and female C57BL/6J mice were exposed to the carcinogen, azoxymethane  and a chemical inflammatory driver, dextran sulfate sodium (DSS1-2%), to promote tumorigenesis in the colorectum. OJS was given orally  to determine its influence on disease activity, tumor burden, nociception, sedation, Erk signaling, and behavioral and metabolic outcomes. In addition,  In the United States there are approximately 1 million colorectal cancer survivors . This isIn vitro studies utilizing OJS have found an increase in apoptosis and reduction of inflammation via the activation of caspase-3 and a decrease in cytokine and macrophage-derived chemokines, respectively [The medicinal herb, Ojeok-san (OJS) is used in Asian countries to treat pain, gastrointestinal problems, and depression . In factectively ,28. Caspectively –31. For ectively ,33. Resuectively ,33. Whilin vitro platform system was used to determine the mechanism by which OJS promotes analgesia.The purpose of this study was to investigate the analgesic properties of OJS with respect to colitis-induced colorectal cancer. We first sought to determine the dose at which OJS promotes analgesia without affecting tumorigenesis. Subsequently, to examine referred somatic and visceral nociception, we assessed mechanical threshold and monitored intracolonic pressure changes during colorectal distension. We also studied the possible sedative effects of OJS using the rotarod test and automated behavioral cages. Lastly, a high-throughput ad libitum access to food and water. Animals were maintained on a 12:12-h light/dark cycle, 22°C, and 50% humidity.All procedures and experiments involving animals were approved by the Institutional Animal Care and Use Committee at the University of South Carolina. Mice (maximum of 5 per cage) were housed in ventilated cages with wood bedding, nesting material, and J male and female mice  were purchased from Jackson Laboratory . Mice (10 weeks of age) were separated into four groups following stratified random sampling for sex, body weight, and baseline pain threshold: AOM/DSS-Vehicle , AOM/DSS-OJS 500 mg/kg , AOM/DSS-OJS 1000 mg/kg  and AOM/DSS-OJS 2000 mg/kg . Azoxymethane  was administered to AOM/DSS mice (10–11 wks of age) followed by administration of 2% dextran sulfate sodium  in the drinking water for 7 days at 1 and 4 weeks of treatment. Symptom scores were evaluated every week as described [C57BL6/escribed . ReferreJ mice  were obtained from Jackson Laboratories at 8 wks of age. Mice were divided in four groups following stratified random sampling for sex and body weight: Control non-cancer Vehicle (water), Control non-cancer OJS (2000 mg/kg via gavage), AOM/DSS-Vehicle, and AOM/DSS-OJS (2000 mg/kg via gavage). AOM (10 mg/kg of BW) or saline was i.p. injected. DSS (2%) was administered in the drinking water for 7 days as mentioned in Experiment 1. OJS or vehicle was administered a total of five times over the course of the experiment. Symptom score was evaluated as described [A total of 40 C57BL/6escribed . ColorecJ mice  were divided into two groups following stratified random sampling for sex and body weight. Mice were injected with AOM or saline as described above and received 1% DSS for 3 cycles. DSS was dissolved in filtered drinking water and administered to mice for seven days at 1, 4, and 8 wks post AOM administration. Exploratory behavior, locomotor activity, and neuromuscular function were assessed using place preference, Promethion behavioral/metabolic cages, and rotarod testing, respectively. OJS was administered via oral gavage (2000 mg/kg of body weight) during exploratory behavior paradigm (1 time gavage with OJS) and rotarod test (1 time gavage with OJS). OJS was administered in the drinking water  for 10 days.Another group of C57BL6/Atracylodes lancea D.C.,7.5 gr), Ephedrae Herba , Citri Unshius Pericarpium , Magnoliae Cortex , Platycodonis Radix  Aurantii Fructus Immaturus , Angelicae Gigantis Radix , Zingiberis Rhizoma , Paeoniae Radix , Poria Sclerotium , Angelicae Dahuricae Radix , Cnidii Rhizoma , Pinelliae Tuber , Cinnamomi Cortex , Glycyrrhizae Radix et Rhizoma , Zingiberis Rhizoma Recens , Allii Fistulosi Bulbus  were mixed, extracted with water, filtered, and freeze-dried to form the OJS powdered formula. The purity and chemical profile of the OJS used in these experiments has been published by Kim et. al., [All mice were fed a purified AIN-76A diet  at least two weeks prior to the start of the experiment and throughout the finalization of the studies. OJS was provided by Dr. Hyeun-Kyoo Shin . Briefly, seventeen herbal medicines: Atractylodis Rhizoma . Briefly, mice were placed on a raised transparent plastic box (30x20x15 cm) with a bottom made of wire mesh (5 x 5 mm apertures) for 30 min during 3 days of habituation and prior weekly tests. Von Frey filaments  0.008, 0.02, 0.04, 0.07, 0.16, 0.40, and 0.60 grams were applied five times for 5 seconds in ascending order to avoid windup effects of desensitization. A nociceptive response was defined as: sharp withdrawal of the abdomen, licking or scratching the touched area, and/or jumping. The pain threshold was defined as the force in grams at which any von Frey filament elicited three nociceptive responses out of five. Exclusion criteria were defined as a pain threshold higher than 0.4 grams at week three in cancer mice. The same investigator performed all RSH tests (blinded to treatments groups) during the beginning of the light cycle 7-9am. Additionally, the experimenter analyzing the data was blinded to group assignments.CRD was evaluated as described . BrieflyExploratory behavior was assessed in female and male mice. Mice were habituated for 30 min for three days to a plexiglass box . During the behavioral test, mice were placed in the center of the box (25 x 25 x 30 cm) and time to step up to the platform inside the box (25 x 12 x 5 cm) and/or time to enter to a black enclosing inside the box were recorded in seconds. Each mouse performed ten trials with a cut-off of 120 sec if the mouse remained in the center of the box. Percent time mice favored the center, step-up, and black enclosing space were calculated.https://www.mmpc.org/shared/regression.aspx).Behavioral and metabolic phenotyping were assessed using the Promethion multichannel continuous measurement indirect calorimetry system . Control (non-cancer) and AOM/DSS mice  were singly housed for 3 days of acclimation prior to 7 days of data collection. Food intake, water consumption, body mass, total activity, energy expenditure, respiratory quotient, animal ambulatory locomotion, and sleeping patterns during a 12-h light and 12-h dark cycle were collected and analyzed. Body composition was measured using dual-energy X-ray absorptiometry . Then, tet. al., 1993 [Rotarod test was performed to assess OJS’ sedative properties as described by Martin l., 1993 . This teThe symptom score was evaluated once a week starting at the end of the first cycle of DSS exposure in the drinking water. The symptom score was calculated by adding the recorded scores of weight loss (WL), fecal consistency, and blood in the stool. The following numbers were used to score WL , fecal consistency , and blood in the stool . The higher the symptom score, the worse the animal’s health, which is indicative of cancer progression.DRG cells were purchased from Cell Applications  and used according to the company’s instructions. Briefly, rat DRGs  were seeded on cytoview microelectrode array (MEA) plates  coated with rat neuron coating solution II and plating medium . Cells were incubated at 37ºC, 5% CO2 in rat ganglion neuron culture medium . The medium was changed every 3–4 days and spontaneous activity of the DRGs  were evaluated approximately two weeks after seeding using a Maestro Edge with the impedance and neuro module . Inflammatory soup (IS: 1 μM bradykinin (cat. no. B3259), 10 μM Prostaglandin E2 (cat. no. P5640), 10 μM Histamine (cat. no. H7125), 10 μM serotonin (cat. no. H9523), and five μM ATP (cat. no. A2384) from Sigma-Aldrich, Atlanta, GA) was used to mimic nociceptive stimuli [CT26 cells  were used due to their molecular resemblance with human colorectal carcinoma cells . Cells wBMDM were isolated from the femur and tibia bones. Bone marrow was flushed from the bones with PBS + 2% FBS. RBC lysis buffer  was used to get rid of erythrocytes. Isolated cells were centrifuged and resuspended in DMEM medium containing 1% Pen/Strep, 10% FBS, and 20% L929 conditioned media. Cells (0.5x10^6) were allowed to differentiate for 7 days and then were treated with OJS  for 24 hrs. OJS was removed from the media and LPS (100 ng/mL) was incubated for 24 hrs. Trizol, chloroform, and isopropanol were used to isolate RNA from BMDM cells. Applied biosystem’s high-capacity cDNA kit  was used to convert RNA into cDNA. TNFα and 18s Taq probes (Applied Biosystems) were used to determine gene expression using quantitative real-time PCR.The spinal cord (T13-L5) was homogenized in Mueller buffer containing a protease inhibitor cocktail  and total protein was quantified using the Bradford method . Western blot analysis of 50 μg of spinal cord homogenate was fractionated on 10% SDS-polyacrylamide gels and used to determine Erk activation. Gels were transferred to PVDF membrane at 22 V for 1hr using the Genie electrophoretic transfer system from Idea Scientific. Membranes were stained with Ponceau to verify equal loading and efficiency of the transfer. Membranes were blocked with 5% BSA  and 5% milk in Tris-buffered saline and 0.1% Tween 20 (pErk) for 1 hr. Primary and secondary antibodies: Erk , phosphorylated Erk , and anti-rabbit  were purchased from Cell Signaling. Primary antibodies were incubated overnight at 4°C and secondary antibodies were incubated at room temperature. An enhanced chemiluminescent substrate for detection of horseradish peroxidase (Thermo Scientific) and autoradiography film  were used to visualize the antibody-antigen interaction. All samples were run in the same gel . Films wA complete blood count (CBC) analysis was performed using VetScan HMT . Briefly, blood was collected from the inferior vena cava at sacrifice using a heparinized needle and was placed on ice in a 1.5 ml tube. Approximately 50 μL of whole blood was used for a three-part differential analysis that included white blood cells (WBC), lymphocytes, monocytes, neutrophils, platelets, red blood cells (RBC), hematocrit, and hemoglobin.in vitro experiments). A repeated measures Two-Way ANOVA with a Fisher’s LSD post-hoc analysis was used to assess colorectal distension and rotarod outcomes. A mixed-effects model followed by a Sidak post hoc analysis was used to determine differences among cancer and control mice treated with vehicle and OJS in the exploratory behavior test. TNFα gene expression was analyzed using a one-way ANOVA with a Fisher’s LSD post-hoc test. Any statistical data that did not pass the equal variance test (Barlett’s test) were logarithmically transformed and reanalyzed. Data are presented as means ± SE, and level of significance was set at p<0.05.Data were analyzed using Prism 8 software (GraphPad). Data for the symptom score, colon length, and polyp number was analyzed using an unpaired Student’s t-test (AOM/DSS-vehicle versus AOM/DSS-OJS). Analysis of covariance was used for metabolic parameters. A Kruskal-Wallis test was used to analyze the pathological profile. A one-way ANOVA followed by a Tukey’s multiple comparisons test was used to analyze the nociceptive threshold, disease activity, and polyp number (mice experiments) as well as impedance in CT26 cells, and number of spikes produced by DRGs  weekly after the first cycle of DSS, and polyp number at the end of the experiment. In addition, we assessed cell proliferation n groups .n vitro impedance assay in CT26 mouse colon carcinoma cells. Treatment groups were assigned 24 hrs after cell seeding in order to avoid any well-to-well variability with respect to cell resistance/impedance. Impedance in CT26-NTC (non-treatment control) increased over time due to cell proliferation  between non-cancer Control-Vehicle, and Control-OJS during CRD at 10, 25, 40, 65, and 80 mmHg . To compAOM/DSS mice treated with OJS showed a significant (p<0.05) reduction in mechanical responses at 40, 65, and 80 mmHg CRDs when compared to AOM/DSS-Vehicle mice. Control non-cancer Vehicle, Control non-cancer OJS, and AOM/DSS-Vehicle mice displayed a progressive increase in BalP from 25–80 mmHg . HoweverPrevious studies have reported activation of extracellular signal-regulated kinases (Erk) 1/2 in the spinal cord of rodents with visceral hyperalgesia ,43. ThusTo confirm the safety of OJS, a complete blood count was performed. No differences were detected between groups with respect to WBC and RBC .We assessed exploratory behavior utilizing a custom acrylic box with two differential compartments  or OJS . No statTo better understand the impact of OJS on animal behavior, we utilized the integrated Promethion System consisting of behavioral/metabolic cages. The first week mice were in the behavioral/metabolic cages no treatment was given in the drinking water (pre-OJS). On the second week, mice received OJS in the drinking water (post-OJS). OJS did not impact total energy expenditure, oxygen consumption, or carbon dioxide production . HoweverSince sleep has been shown to be a strong predictor of pain , we deciNon-cancer Control mice ate an average of 1.7 grams during the light cycle and 0.8 grams during the night cycle . MeanwhiWe used DRG sensory afferent nerves in primary cell culture to study the possible cellular and molecular mechanism by which OJS decreases nociception. Previous studies have reported that inflammatory mediators can evoke firing of the DRG sensory afferent nerves ,48. MeanTo characterize the effects of OJS on spontaneous DRG activity, we performed a dose response experiment with OJS. At baseline, no difference in spike number was observed between groups (data not shown). As previously observed on Given that we used an animal model of colitis-induced colorectal cancer and that previous studies have shown that OJS decreases cytokine and macrophage-derived chemokines, we decided to evaluate the impact of OJS on inflammation in macrophages. We used LPS-stimulated bone marrow derived macrophages (BMDM) to assess the anti-inflammatory potential of OJS. Pre-treatment of OJS at 100 and 200 μg/mL was able to reduce TNFα expression, but not at the lower OJS dose of 50 μg/mL .Pain is among the most described symptoms in cancer patients and cancer survivors –7. Due tTrichinella spiralis [To establish OJS’ analgesic properties in a pre-clinical model of colitis associated cancer, we used von Frey filaments and CRD to determine if OJS can decrease somatic and visceral nociception in response to noxious stimuli. These two modalities have been previously used to assess visceral sensitivity caused by colitis and psychosocial stress in rodents ,50–53. Ispiralis  and ethaspiralis , but notspiralis ,63. Likespiralis . This suspiralis .in vitro study is that we only tested OJS 15 min pre-IS and we do not know if changes in duration of OJS treatment can impact neuronal activation. Consequently, more studies need to be done to determine peripheral and central effects of OJS in visceral pain.In many instances, referred somatic hyperalgesia and visceral pain signal through Erk activation ,43,65. Tin vivo and proliferation in vitro. We found no differences in symptom score, polyp number, and colon length between AOM/DSS-vehicle and AOM/DSS-OJS mice. In addition, no changes were observed in the proliferation rate between CT26-NTC cells and CT26 cells treated with OJS at concentrations varying from 10–500 μg/mL. Contrary to these findings, a study conducted in human uterine myomal cells reported that OJS increases the number of dead cells proportionally to OJS concentration [in vivo.Given that OJS has shown anti-tumor activity which can alter nociception and analgesia, we evaluated multiple outcomes associated with disease progression ntration . One expntration . In geneIn the present study, we were also interested in examining behavioral and metabolic phenotyping of mice treated with OJS. OJS shifted mice preference from the black box compartment to the center enclosure during exploratory behavior assessment. This result was likely due to OJS’ effects on neuromuscular function as AOM/DSS mice treated with OJS showed a decrease in time spent on the accelerating rod. Additionally, AOM/DSS-OJS mice were less engaged in ambulatory activity and spent more time sleeping. Collectively, these results suggest that OJS possesses sedative (motor impairment) effects. Nevertheless, we cannot abandon the idea that OJS might also be anxiolytic. Interestingly, we did not observe significant differences in motor impairment in Control non-cancer mice. However, Control non-cancer mice treated with OJS reduced locomotor activity during the night cycle. In humans, studies examining the interaction between celecoxib, acetaminophen, and OJS in healthy volunteers reported high tolerability and the most frequent adverse events included feeling hot, sore throat, dry mouth, and headache ,33. SincIn general, OJS administration at a concentration of 2000 mg/kg was able to attenuate both referred somatic hyperalgesia and visceral nociception in AOM/DSS mice. Extrapolation of this dose of OJS (2000 mg/kg) that yielded a positive effect on analgesia to a human equivalent dose based on body surface area , a commoIn summary, we establish that OJS has anti-nociceptive effects, which may be mediated, in part, by Erk signaling. Moreover, we show that OJS possesses sedative effects and prolongs the total sleeping time in mice with colitis-induced colorectal cancer. Since OJS has been vastly understudied, further research is necessary to uncover its potential to treat chronic pain. It should be noted that because OJS is a complex formula consisting of 17 herbal medicines, we presume that multiple mechanisms likely contribute to the analgesic properties of OJS.S1 Fig(TIF)Click here for additional data file.S2 Fig(A) Comparison of intracolonic pressure to CRD in control non-cancer mice and AOM/DSS mice. (B) The relationship between BalP response to CRD and colon histopathology.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigLean mass (A), bone mineral density (B), fat mass (C), and (D) percent fat were measured using dual-energy X-ray absorptiometry. Black dots represent male mice and pink dots symbolize female mice. n = 8, control, and n = 8 AOM/DSS.(TIF)Click here for additional data file.