The developments and potential use of hybrid PET/magnetic resonance imaging (MRI) for neurotransmission brain research will also be highlighted.Positron emission tomography (PET) neuroimaging in neuropsychiatry is a powerful tool for the quantification of molecular brain targets to characterize disease, assess disease subtype differences, evaluate short- and long-term effects of treatments, or even to measure neurotransmitter levels in healthy and psychiatric conditions. In this work, we present different methodological approaches ( For example, dynamic changes in the dopaminergic system are known to contribute to a wide range of behaviors including affect, reward, decision-making, and inhibitory control.Many transformative therapies for neurological and psychiatric disease states over the last decades have targeted neurotransmitter systems through serotonin, dopamine, or opioid receptors and transporters. Neurotransmitters play an important role in regulating brain activity at the molecular and neurochemical level and are centrally involved in many brain functions, including, for example, cognition, behavior, sleep, appetite, and mood. Endogenous and exogenous stimuli, including behaviorally relevant stimuli, mood changes, and pharmacological challenges, evoke widespread changes in neurotransmitter systems. These changes are important to understand neural function in health and disease. One of the most and extensively characterized neurotransmitter system is the mesocorticolimbic dopaminergic pathway. Dopamine, both the “in vivo functional molecular positron emission tomography (PET) imaging, we are able to image and quantify with very high sensitivity and specificity the local concentration of a range of neuroreceptor targets in a non-invasive way. PET radiotracers for imaging of neurotransmission had been primarily focused on studying changes in endogenous levels of dopamine in the striatum  represent receptors as static targets that are not dynamically regulated by processes like internalization within post-synaptic membranes , and decay exponentially to baseline thereafter (at rate τ). When any of these assumptions are violated, the estimates of the model parameters could be biased and/or inaccurate.However, LSRRM assumes that effects of the stimuli on endogenous neurotransmitter release are instantaneous, maximal at the start time of stimulation [equal to More flexible kinetic approaches and associated resolution models have been proposed to resolve these limitations, such as the lp-ntPET model . In ordeSpecifically, lp-ntPET is the union of the conventional multilinear reference tissue model (MRTM)  and a tihi(t), where γ describes the response magnitude, and the three implicit parameters  describe the time course of the response , assuming values incremented over finite intervals. The efficiency of lp-ntPET makes it practical to perform a voxel-by-voxel analysis of the whole brain or for localized activation patterns. The model has been successfully applied to 11C-raclopride data to estimate the temporal dynamics of dopamine release in the mesolimbic circuit during smoking  (unpublished results). In this way, the kinetic parameters are estimated more accurately, independently from the magnitude of the endogenous neurotransmitter release, and the macroparameter, the displacement ratio, seems to allow better detection of neurotransmitter discharge.An optimization of the lp-ntPET displacement modeling method, called 2-step lp-ntPET, has been proposed by 11C-raclopride  have been shown to occur in response to changes in neuronal activity and during neurotransmission , althoug2/D3 receptor occupancy through neurovascular coupling mechanisms (L that binds to D2 receptors and displaces dopamine (DA), a linear relationship between hemodynamic changes (ΔH) and receptor occupancy (θ) is expressed as:Several PET/MRI studies have been performed that have investigated the effects of time-varying neurotransmitter modulations  on fMRI and receptor-specific PET signals simultaneously. Much of this line of work has focused on establishing contributions of the dopaminergic system on the fMRI response, demonstrating that the hemodynamic response can be directly linked to Dchanisms . The relchanisms , 2015. CND2 is a neurovascular coupling constant , Bmax,D2 is the total concentration of D2 receptors, θL(t) is the time-varying occupancy of an exogenous ligand (if present), and ΔθDA(t) is the time-varying occupancy of dopamine. In a more general framework, the functional hemodynamic response ΔH can be expressed as the sum of receptor occupancies by different ligands, L, (endogenous and exogenous) and receptors, R, as:where 2/D3 antagonism and agonism (in vivo and requires further expansion of these model frameworks (This model incorporates the possibility of any number of receptors and neurotransmitters (or ligands) that may contribute to the hemodynamic response. Experimental evidence for this relationship has been demonstrated using D agonism , yet remameworks .11C-diprenorphine  have been proposed to signify the dynamic nature of the system function in health and disease.JC and CS revised the literature and wrote the first draft of the manuscript. HL, KV, and EM revised it critically for important intellectual content. All authors contributed to the article and approved the submitted version.EM is a half-time employee of the company Invicro LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 . The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- ( Therefore, it is reasonable to assume that pairing up to a local network of binding partners at sites of preferential presence of a distinct glycoconjugate will likely occur. When having disclosed such well-structured patterns of expression for glycosaminoglycans by bio- and histochemical mapping, the characterization of modes of molecular recognition with tissue lectins is warranted as the next step toward clarifying the issue on functional significance, both for the lectin and for the glycan. Here, we exemplify this hypothesis-driven approach by analyzing the interaction between bovine corneal keratan sulfate (KS) with human adhesion/growth-regulatory lectins present in the cornea, i.e. galectin-3  and Gal-7, by using NMR spectroscopy and also molecular dynamics (MD) simulations with two other galectin family members present in the cornea  to depict bound-state conformation(s) and to calculate and compare binding energies.As a key aspect of their (patho)physiological functionality, the sugar code concept ascribes the nature of molecular messages to glycans that are ‘read’ and ‘translated’ into cellular effects by tissue lectins8. It was biochemically defined as “sulfated mucopolysaccharide, composed of equimolar quantities of glucosamine, acetyl, galactose, and sulfate, for which we propose the name keratosulfate”9. In structural terms, KS belongs to the family of glycosaminoglycans: its disaccharide building block N-acetyllactosamine  can be sulfated at the galactose C6′ position and the N-acetylglucosamine (GlcNAc) C6 position16. The “sugar code” for KS is relatively simple compared to heparin, heparan sulfate, chondroitin sulfate and dermatan sulfate, due to its more homogeneous sulfation pattern that is basically composed of the four major disaccharides . This leads to fewer micro-heterogeneities in the KS chain compared to those from other sulfated GAGs and therefore, much lower variations for protein interactions . Thus, positional diversity of sulfation is less than that seen in e.g. heparan sulfate, directing interest to the backbone as the contact site. With respect to possible pairing partners, it is inspiring that the sulfate-free disaccharide unit is the canonical ligand for the family of ga(lactose-binding)lectins that are broad-impact effectors in cell physiology by bridging (in trans and in cis) glycoconjugate-based counter-receptors20. Providing substantial incentive for our study is the report that 6-O-sulfated LacNAc and a trisulfated LacNAc dimer  are indeed potent ligands for galectins, as detected for Gal-1, -3 and -923.KS was first discovered in extracts from cornea, where it is abundantly present22, which is fitting for an in situ interaction where expression of this lectin is strong and phylogenetically conserved in cornea28. In addition, Gal-3 is known: (i) to bind to LacNAc oligomers with relatively high affinity that increases with chain length34 and, as a hint to functional interplay, (ii) to contribute to the integrity of the ocular surface epithelial barrier35. Cross-linking spatially accessible polyLacNAc chains by a bi- or oligo-valent galectin that then acts like a molecular glue, can thus provide meaning to the development of the respective enzymatic machinery for this type of glycosaminoglycan37 that may help explain local co-expression of KS-presenting proteoglycans and galectins. Since other members of the galectin family with cross-linking capacity are also present in the corneal epithelium , we broadened the scope of our study beyond Gal-3 to include these galectins as well.Giving our study a clear direction is a comparative analysis within the galectin family that has identified Gal-3 as a major binding partner for KS15N–1H] HSQC NMR studies with 15N-labeled Gal-3 [full-length (FL) and its carbohydrate recognition domain (CRD)] and Gal-7 with KS and KSDS as ligands. It is noteworthy that KSDS resembles embryonic KS prior to the onset of developmental sulfation40 that is a potential docking site for developmentally expressed galectins as noted in work on the anterior segment of the adult chicken eye28. Mapping the galectin-binding epitope was also performed using compounds akin to the building blocks of KS, i.e. the disaccharide LacNAc and the tetrasaccharide lacto-N-tetraose (LNT). To extend this empirical approach, we also ran MD simulations with KS-based oligosaccharides  in order to visualize the contact profile and to facilitate calculation of binding energies. Due to the well-defined capacity to accommodate polyLacNAc chains by the N-terminal CRD of human Gal-9  and the detection of different contact sites for Gal-1  vs Gal-3  on these oligosaccharides42, we also investigated Gal-1 and -9N using the same approach.To advance from simply detecting interactions between galectins and KS (and its desulfated form KSDS) to elucidating molecular details of their binding, we performed 1/2. Intensity changes (∆Intensity) were calculated as (1 − Inti/Into), where Into is the initial resonance intensity and Inti is the intensity upon addition of KS or KSDS or another saccharide. Using this equation, a value of 1 means that that resonance is no longer observable, and 0 means the absence of resonance broadening, with a negative value indicating increased resonance intensity. In chemical shift or resonance broadening maps, the most perturbed resonances are defined as > 2SD above the average or > 1SD above the average. The 50% bound mark was taken from titration curves at 50% bound.Chemical shifts, δ, were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid, and chemical shift differences (∆δ) were calculated as [ dissolved in the same buffer was added at increasing concentrations as molar ratios of [Gal-3]/[KS]. PFG NMR data were acquired at 30 ◦C on a Bruker AVANCE 700 spectrometer, essentially as previously described51. For unrestricted diffusion of molecules in isotropic solutions, the PFG NMR signal amplitude, A, normalized to the signal in the absence of gradient pulses, is related to the diffusion coefficient D by Eq. (For pulsed field gradient (PFG) NMR self-diffusion measurements, a solution was made up with KS (4 μM) in 20 mM potassium phosphate buffer with γ is the gyromagnetic ratio, g is the magnitude of the magnetic field gradient pulse, ∆ is the time between gradient pulses51. Here, we set g = 1–75 G/cm, ∆ = 34.2 ms, with a longitudinal eddy-current delay of 100 ms. Each D value was determined from a series of 14 1D PFG NMR spectra (256 scans each) using different g values. To derive D values, the gradient-induced change in amplitude of KS resonances at 3.64 and 3.7 ppm was used. In this spectral region, KS resonance intensity was considerably greater than that from Gal-3. In order to decrease Gal-3 resonance intensity further, 13CH and 15NH resonances from labeled Gal-3 were not decoupled.where 93. Briefly, the Amber 14SB force field was applied for proteins, and GLYCAM06 was used for oligosaccharides. KS and KSDS oligosaccharides were initially dock on the galectin structure using the program AutoDock Vina94. Complexes were solvated by TIP3P water models with a box size of 10 Å and were then subjected to energy minimization  in order to optimize the complexes and remove close contacts. Subsequently, a position-restrained phase of MD simulations was carried out for 500 ps by first slowly heating up the systems from 0 to 300 K for 100 ps and then maintaining the temperature at 300 K for another 400 ps. During this phase, a soft-force constraint  was applied to restrain the complexes. Finally, free MD simulations were performed for 50 ns.Similar approaches and protocols were used as previously reported95 that is optimized for sulfated glycans was employed, and results followed the same trends as with the program AutoDock Vina94. Using Glycotech Vina, derived binding free energies were relatively low  compared to those from use of AutoDock Vina .Essential parameters (e.g. temperature (300 K), pressure (1 bar) and time steps (2 fs with SHAKE constraint)) were set at standard values as indicated during the free MD simulations. Energy minimization and MD simulations were employed by using AMBER16 program. BFE calculations were performed by extracting trajectories during the 30–50 ns period of MD simulations (100 snapshots), and the molecular mechanics/ generalized Born surface area (MM/GBSA) approach  was used for this purpose. The 3D structures of galectins were obtained from the Protein Data Bank: Gal-1 (PDB code 1W6N), Gal-3 (PDB code 1A3K), Gal-7 (PDB code 5H9S), Gal-9C (PDB code 3NV3), and Gal-9N (PDB code 2EAL). The Uni-Prot IDs used for sequence alignment by the “clustal Omega” program of the UniProtKB are P09382 , P17931 , P47929 , O00182 . In some instances, the program Glycotech VinaIn all simulations, we used a pH value ~ pH 7 where histidine can have three different protonation states: protonation at the NE atom (HIE), at the ND atom (HID), and a double protonation at both NE and ND atoms (HIP). His158, for example, is located at the CRD binding site, and its protonation state could influence binding to KS and KSDS. However, because we focused this study on relative binding free energies, we set the His protonation state to the same value (HIE at pH 7 by default), and all systems remained stable during the simulations.Supplementary file1Supplementary file2Supplementary file3Supplementary file4Supplementary file5Supplementary file6Supplementary file7Supplementary file8Supplementary file9Supplementary file10


Varied drug ratios of 5FUR−W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR−W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR−W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR−W and 5FUR−W Cholesterol was purchased from Millipore Sigma . Doxorubicin (DOX) was purchased from LC labs . 5-fluorouridine with a tryptophan conjugated by ester bond linkage on the primary alcohol (5FUR−W) was a prodrug based on 5-fluorouracil synthesized by Pharmaron . 5-flurouridine with tryptophan conjugated to each alcohol (5FUR−W3) was a prodrug synthesized by Dr. Reddy’s Laboratories. Doxorubicin Hydrochloride Liposome Injection (DLI) was also provided by Dr. Reddy’s Laboratories .The lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-4T1 murine breast cancer cells (ATCC CRL-2539) were purchased from ATCC . 4T1 cells were grown in RPMI-1640 cell growth media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. SKOV-3 cells were grown in McCoy’s 5a Medium Modified supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cellular inhibition assays used 3--2,5-diphenyltetrazolium bromide (MTT) to quantify cell viability. All aforementioned cell culture materials were purchased from ThermoFisher Scientific . Cell culture flasks and tissue culture-treated well plates were purchased from Corning .Liposomes were fabricated through the conventional thin film hydration method . 40 µmolLiposomal size and zeta potential were quantified by measurement of the number size distribution and zeta potential distribution by a Malvern Zetasizer. Liposomes were diluted 100-fold in milliQ water prior to analysis. Liposomal drug content was quantified by reverse phase HPLC. An example of the free drug HPLC chromatogram is given in n = 3) of both monoester 5FUR−W and triester 5FUR−W3 were prepared in milliQ water and kept at 65 °C with stirring. At each timepoint  a 10 µl sample was taken from the stock solutions and diluted 100-fold in milliQ water. The samples were run on HPLC using the previously described method for drug quantification.1 mM solutions (R=2.5 (n = 3) was stored in PBS at 25 °C for five days, and at 4 °C for six days. A 10 µL sample was taken each day from the DAFODILR=2.5 staored at 25 °C, and on day 0, 1, 2, 4, and 6 from the DAFODILR=2.5 stored at 4 °C. The samples were run on HPLC using the previously described method for drug quantification.To quantify the storage stability of DAFODIL, DAFODIL4T1 cells were plated in 96-well plates at a seeding density of 500 cells/well, and SKOV-3 cells were plated at a density of 5000 cells/well. Cell culture media containing treatment were administered onto six wells per treatment. Treatment was incubated on the cells for 72 h before the removal of the cell media and administration of 0.5 mg/mL MTT reagent in media. After 5 h, formazan crystals were dissolved in DMSO and absorbance was measured by plate reader.5 4T1 cells in 50 µL of PBS into the fourth abdominal mammary fat pad of female BALB/c mice (age 50–56 days) purchased from Charles River Laboratories . This method has proven to produce uniform breast cancer tumors that grow rapidly and metastasize to the lungs [L and W represent the length and width of the tumor, as determined through caliper measurements. Treatment with liposomal formulations was initiated when tumors reached 50 mm3 in size. Treatment consisted of 100 µL of liposomes administered intravenously every other day a total of four times. Liposomes were adjusted to a dose of 3 mg/kg DOX, and the accompanying 5FUR−W dosage was determined by the molar ratio of 5FUR−W: DOX encapsulated in liposomes  in healthy female BALB/c mice. Blood was collected by mandibular puncture at 5 min (~20 μL) and by cardiac puncture at 2, 6, and 24 h (~200 μL) after injection and stored in heparin-coated collection tubes from BD . Blood collected by mandibular puncture was diluted five-fold with PBS and blood collected by cardiac puncture was diluted two-fold in PBS. 100 μL of the blood and PBS mixture was centrifuged at 7000 g for 10 min to remove the red blood cells. The supernatant was then 10-fold diluted in 1:1 methanol: acetonitrile with 0.05% formic acid for drug extraction. The sample was centrifuged to remove serum proteins and the resulting supernatant was filtered with 0.2 μm syringe filters from Waters  and run using the drug quantification protocol on LC-MS. The mass spectrometer was used to determine if the metabolite forms of the drugs were present in the plasma. Finally, pharmacokinetic parameters for the one-compartment model were calculated using the PK Solver 2.0 Microsoft Excel plugin developed by Zhang et al. .Liposomal biodistribution was performed by intravenous injection of liposomes at a dose of 1.5 mg/kg DOX into BALB/c mice. Mice were euthanized 24 h after injection, and the tumor, liver, lungs, and kidneys were extracted and weighed. The samples were cut into small pieces and enzymatically digested for one hour at 37 °C in 5 mg/mL of collagenase and 50 U/mL of DNAse in HBSS buffer. Samples were further broken down with a homogenizer probe and centrifuged at 20,000 g for 5 min. Drug content was extracted from the samples by 5-fold dilution in 1:1 methanol: acetonitrile with 0.1% formic acid. The resulting mixture was sonicated for 15 min and centrifuged to remove the homogenized material. The supernatant was filtered using 0.2 µm syringe filters from Waters  and analyzed using the aforementioned reversed phase HPLC method.p < 0.05, ** p < 0.01, and *** p < 0.001.Statistical significance was determined through unpaired t-tests and one-way ANOVA with Tukey’s Multiple Comparison Test in Graphpad v5 . Statistical significance was defined as * 3 facilitated active loading into liposomes. However, multiple hydrolysis products complicated the characterization of the formulation, and 5FUR−W was developed to provide better stability and higher loading. The thermal stability of the 5FUR−W monoester   . Liposom3 were compared in vitro on the 4T1 murine breast cancer cell line. Free drugs and free drug combinations of 1:5, 1:1, and 5:1 of prodrug to DOX were evaluated. Free 5FUR−W had a similar dose-response to 5FUR−W3, although free DOX had approximately 10-fold lower IC50 than both prodrugs , a clinically equivalent generic form of Doxil (AB-rated). All formulations were injected in equal doses and observed over a 24 h period. The gradual decrease in DOX concentration of DLI and DAFODIL was similar, with half-lives of 14.79 ± 4.11 and 10.47 ± 2.02 h respectively  made using the same protocol as DAFODIL. DOX-L were also compared against DLI to determine if differences in fabrication procedure contribute to any significant differences in efficacy. No significant difference between DOX-L and DLI was found. DAFODILR=10 exhibited exceedingly effective tumor volume control compared to both DOX-L and DLI  (R=6 group was significantly different (p < 0.05) from the control group at 20 days   C. No gro 20 days D. DAFODI< 0.001) .3 at the encapsulation temperature, 65 °C. Through in vivo optimization, we have concluded DAFODILR=2.5 improved efficacy while also remaining nontoxic. Next, we evaluated the storage stability of 5FUR−W in DAFODILR=2.5. At 25 °C, approximately 40% of initial 5FUR−W was hydrolyzed to 5FUR by the end of five days (from 1.64 mM to 0.99 mM 5FUR−W) (We have confirmed 5FUR−W remains stable longer than 5FUR−W 5FUR−W) A. Howeve 5FUR−W) B. Overal 5FUR−W) C. This i®, Marquibo®, Onivyde®) and albumin nanoparticle-bound paclitaxel (Abraxane) nanomedicine has emphasized the efficacy of long-circulating formulations with sustained release [Liposomal doxorubicin, better known as Doxil, was among the first clinically approved nanocarriers for cancer therapy . With th release . These f release ,37,38, b release ,40,41,42 release .Our approach to improving liposomal doxorubicin was to incorporate a fluoropyrimidine analog, 5-fluorouridine, which metabolizes to the same active compounds as 5-fluorouracil (5FU), a drug used with doxorubicin and cyclophosphamide in the treatment of breast cancer ,45. We dWhile synergy was not present in the drug combination of 5FUR−W and DOX on either the triple-negative 4T1 murine breast cancer cell line or the SKOV-3 human ovarian cancer cell line, previous work has reported synergy of the DAFODIL formulation on the BT-474 cell line, which belongs to the Luminal B subtype of breast cancer ,47. Howe3, the new single ester 5FUR−W enables the formulation of combination liposomes with R > 1. Our resulting formulations, DAFODILR=10, DAFODILR=6, and DAFODILR=2.5, were compared against an AB-rated generic liposomal doxorubicin formulation, DLI. As a commercially used drug, DLI was the most stringent control formulation to represent liposomal doxorubicin. 5FUR−W-L was not included as a control due to the lack of a clinical equivalent. The purpose of this preliminary animal study was to evaluate if DAFODIL could leverage the incorporation of the 5FUR−W prodrug to achieve improved tumor control over the closest clinical liposomal formulation, represented by DLI. All formulations were dosed four times at 1 mg/kg of DOX, with an accompanying amount of 5FUR−W as determined by ratio. The cumulative dosages are far below the reported doses of PEGylated doxorubicin liposomes (25 mg/kg) [In typical clinical combination regimens, 5FU is given in large excess of DOX due to the respective clearance profile and mechanism of each drug ,23. Whil5 mg/kg)  and free5 mg/kg) .R=10 demonstrated remarkable tumor control, but the incorporation of a high amount of 5FUR−W proved toxic. As shown in the pharmacokinetic studies, 5FUR−W has a similar circulation half-life to doxorubicin when administered in DAFODIL. A high ratio, an aggressive dosing regimen, and protection from enzymatic degradation likely led to the toxic buildup of 5FUR−W. Two formulations with lower 5FUR−W:DOX ratios, DAFODILR=6 and DAFODILR=2.5, were tested against DLI. DAFODILR=2.5 had similar efficacy to DAFODILR=6. Additionally, no toxicity was shown in mice treated with DAFODILR=2.5. This indicates that there is likely a window of ratios that are both effective and nontoxic. In our preliminary study, the DAFODILR=2.5 formulation was able to observe a 62.6% reduction in tumor size compared to the untreated control group, and a 33% reduction when compared to the DLI-treated group.DAFODILAn improvement in 5-fluorouridine prodrug design led to increased stability and widened the range of drug loading ratios. We were able to identify that ratios greater than 6:1 5FUR−W: DOX in liposomes led to weight loss in mice, while a ratio of 2.5:1 5FUR−W: DOX had increased therapeutic efficacy in the 4T1 murine breast cancer model while maintaining mice body weight. Subsequent evaluation of efficacy and survival rates in more clinically relevant animal models, such as patient derived-xenograft models, would greatly inform on the clinical translatability of DAFODIL. Further safety evaluations are also recommended, including cardiac toxicity and tissue H&E staining, as well as closer analysis of liposome morphology.


Textile-based Scaffolds preparation has the attractive features to fulfill the stated and implied needs of the consumer but there are still challenges of stability, elongation, appreciable bio-compatibility, and stated hydrophilic behavior. To overcome these challenges, the authors tried to fabricate a scaffold by blending of two highly biocompatible polymers; polyvinyl alcohol and poly through co-electrospinning. The resultant scaffold by the stated innovative approach evaluated from different characterizations such as dimensional stability/morphology was evaluated by scanning electron microscopy, chemical interactions by that Fourier transmission infrared spectra, wetting behavior was analyzed by a static angle with a contact angle meter from drop method, elongation was examined by tensile strength tester and in-vitro assessment was done by MTT analysis. Based on verified results, it was concluded that PVA/PICT scaffold has a potential for dual nature of hydrophilicity & hydrophobicity and appreciable cell culture growth, stated dimensional stability and suitable elongation as per requirements of the nature of scaffold. Different techniques have been used to produce nanofibers. The drawing technique is only used for the viscoelastic polymeric material, usually, the tip of the micropipette dipped in the polymeric solution and drawn mechanically from the solution results in the formation of nanofibers10. In template synthesis, nano-porous tubular templates (molds) are used to produce solid or hollow nanofibers. Usually, polymeric materials drew through these molds by some mechanical action result in the formation of nanofibers. The advantage of this technique is that it can be used to produce nanofibers from any material like metals, conductive polymers, and semiconductor polymers. The drawback of this technique is that continuous nanofibers cannot be produced12. Another simple but time taken technique is the preparation of nanofibers by using the phase separation method. Usually, a gel of the polymer solution is formed by storing the solution at gelation temperature, this gel is socked into the distilled water for exchange of solvent, removal of water results in the formation of nanofibers matrix. This method is beneficial at the lab scale13. In the self-assembly technique, pre-existing components arranged themselves into a specific pattern. As similar to the phase separation technique, the self-assembly technique is a time-consuming process.Nanofibers are those fibers that have a diameter in the nanometric range that is, less than 100 nm. These nanofibers possessed different properties as compared to the microfibers. When the diameter of the polymeric fiber’s changes from micrometric scale to the nanometric scale, there is a significant change in the properties of the fibers such as surface to volume ratio increased, surface functional properties changes, mechanical properties such as stiffness, the strength of the fibers changed. These properties make nanofibers superior from the other fibers. Due to these superior properties nanofibers are extensively used in different fields for different application purposes, such as medical applications  show excellent superhydrophobicity with the contact angle of 160° when the diameter of the fibers was in the range of 150 nm to 40 nm22.Similarly, electrospinning of lots of hydrophilic drugs can be done with liquid phase PVA or PEO. Liu et al. found that polyimide nanofibers having a diameter in the range of 300 nm to 400 nm show excellent hydrophobic properties at with contact angle of 140°. They have found that these nanofibers can be used for self-cleaning surfaces, self-cleaning solar panel surfacesApart from the above discussion, this study is focused on the development of nano-composite by using co-electrospinning of PVA and PICT polymers. The resultant nano-composite material will have both hydrophobic and hydrophilic properties because PVA polymer is hydrophilic in nature and PICT is hydrophobic in nature so when these two polymers electrospun together the nanofibers will possess dual properties. Such a nano-composite can be used for the scaffold and filtration.Polyvinyl alcohol (PVA)  (PICT) was kindly supplied by SK chemicals, the Republic of Korea as pellet type. Trifluoroacetic acid (99.9%) and chloroform (99%) were purchased from Wako Pure Chemical Industries, Ltd, Japan, and deionized water was used.−1.To prepare the spinning solution, PICT 10% wt was dissolved in trifluoroacetic acid and chloroform in ratios of 1:3 and PVA solution was prepared in which PVA powder 10% wt was dissolved in deionized water and stirred at 70 °C for 6 h. The scaffold was prepared by co-electrospinning as mentioned in Fig. 24.The surface morphology of the scaffold was analyzed by SEM  accelerated with a voltage of 12 kV. The average diameter of nanofibers was determined from 200 measurements of the random nanofibers using image analysis software −1 with a resolution of 4 cm−1 and the addition of 128 scans24.The chemical interactions between PICT and PVA nanofibers were studied by Fourier transform infrared (FT-IR) spectra achieved by IR Prestige-21 . The spectra were recorded from 400–4000 cm24.The tensile strength test was performed to check the mechanical behavior of nanofibers by using the Universal Testing Machine , the 5 specimens for each sample were prepared under a cross-head speed of 1 mm/min at room temperature and the values of stress and strain were calculated by the following formulas , was usedThiazolyl Blue Tetrazolium Bromide  was added to each well and incubated in 37 °C with 5% CO2 for 12 h. After this, the medium was removed, and samples were washed with PBS. Formosan crystals were dissolved in 400 µl DMSO. The amount of 200 µl of each solution was transferred into the new 96 wells and the absorbance was measured at 570 nm. To measure the dye absorption by the scaffolds, all samples without any cells were immersed in a medium culture with the addition of MTT, then washed with DMSO and absorbance was read at 570 nm. For the estimation of the number of cells adhering to the scaffolds, the absorbance of scaffolds was subtracted from the absorbance in the presence of cell line L92926.To investigate the cell’s attachment behavior of resultant scaffolds, an MTT study was done for seven days in line L929 with a two-time repeat in triplicate for each sample and cell line. MTT {3--2,5-diphenyltetrazolium bromide} is a dye compound that is used as an indicator for assessing the growth of the cells. Due to the high light sensitivity of MTT reagent, the MTT assay is done in the dark. The darker solution corresponds to a higher number of cell growth. After sterilization, the samples were cultured for up to 7 days in DMEM/F12 with 10% FBS for L929. The cell viability was measured in the percentage of the negative control. After each day, 15% of medium culture containing The Surface morphology of all the electrospun nanofibers was analyzed by using a scanning electron microscope (SEM) as shown in Fig. Elemental analysis of pure PVA, pure PICT, and Blend (PVA: PICT) electrospun nanofibers was carried out with the help of energy-dispersive X-ray spectroscopy (EDS). In Fig. The results confirm that there is no interference of any impurity in the prepared samples.−1. FTIR spectra of pure PVA and blended nanofibers are almost similar to each other. In the case of pure PVA and blended nanofibers, a broader band can be seen in the range of 3000 to 3500 cm−1 which indicates the presence of hydroxyl (–OH) group due to the hydrophilic nature of PVA polymer1. The peaks at 1541.12 cm−1, 1558.48 cm−1 (in case of PVA) while peaks at 1539.1 cm−1, 1556.5 cm−1, 1575.8 cm−1 (in case of the blend) indicates C=C bond stretching due to conjugated alkene. The peaks at 1652.9 cm−1 in both spectra (PVA and Blend) indicated C=C bond stretching due to cyclic alkene. The peaks at 1716.64 cm−1, 1732.07 cm−1 in the case of PVA indicate C=O bond stretching due to the presence of carboxylic acid and aldehydes group. The peak at 1681.92 cm−1 in the case of blend nanofibers indicates C=O bond stretching due to secondary amide linkage. The peaks at 2337.72 cm−1, 2358.94 cm−1 (in case of PVA) and the peaks at 2339.65 cm−1, 2358.94 cm−1 (in case of the blend) indicate O=C=O stretching due to the carbon dioxide. The peaks at 2916.36 cm−1, 2941.51 cm−1 (in case of PVA) and 2912.51 cm−1, 2941.44 cm−1 (in case of the blend) indicate the C-H bond stretching due to the alkane. The peaks at 3647.39 cm−1, 3734.18 cm−1, 3851.84 cm−1 (in case of PVA) and the peaks at 3628.1 cm−1, 3734.18 cm−1, 3851.84 cm−1 (in case of the blend) indicates O–H stretching due to the formation of alcohol. On the other hand, the spectra of PCIT nanofibers is quite different from that of the PVA and blended nanofibers. The highest and sharp peak in the case of PICT spectra can be seen at 1714.7 cm−1 which indicates C=O bond stretching due to carboxylic acid. The peaks at 1541.12 cm−1, 1558.48 cm−1, and 1577.77 cm−1 indicate the presence of C=C bond stretching due to conjugated alkene. It can be seen in the spectra that there is no peak of O–H is present between 3000 cm−1 to 3500 cm−1, like that of indicated in the case of PVA and blend nanofibers, this is due to the hydrophobic nature of PICT polymer as it contains a cyclic structure which is very stable against the moisture. PCIT spectra the peak at 1714.7 cm−1, 2370.51 cm−1, indicated C=O and O=C=O stretching respectively. Small peaks of –OH stretching appears in all three spectra indicated the presence of an alcoholic group24.Figure 25. So proposed PVA/PICT nanofibers have the ideal level of absorbency as per requirements of scaffolds.To investigate the hydrophilic/hydrophobic behavior of neat PVA nanofibers, PVA/PICT nanofibers and PICT nanofibers, a state water contact angle test was performed as shown in Figs. 25. The behavior of PICT/PVA nanofibers is appropriate for the scaffold application because there is need of appropriate elongation and stiffness which was obtained by co-electrospinning of PVA and PICT polymers26.To investigate the stress and strain behavior of resultant nanofibers, samples were evaluated by a universal testing machine as shown in Fig. 26.To investigate the toxicity of PVA nanofibers, PICT nanofibers and PICT/PVA nanofibers, MTT analysis was done as shown in Fig. Herein, successfully innovation of scaffold by co-electrospinning of super hydrophilic and superhydrophobic polymers was done. The resultant PICT/PVA nanofibers scaffold revealed the appreciable results of in-vitro, morphology, chemical interactions, wetting behavior and stress and strain behavior which were evaluated by concerned characterizations. PVA/PICT scaffold showed the cell growth and it was gradually increased as no day increased and the growth rate of cells in PVA/PICT nanofibers was higher than others up to 3 days but lower than neat PVA nanofibers up to 7 days but was higher than neat PICT nanofibers. Similarly, wetting behavior was appreciable as compare to PVA  and PICT nanofibers . Hence, it was concluded based on characterization results that PICT/PVA nanofibers can be used scaffold for tissue engineering to fulfill the stated and implied needs of the investors.


In blunt traumatic superficial femoral arterial (SFA) injuries, hemorrhage from the branches without injury to the main artery is rare, but can lead to serious complications, such as compartment syndrome affecting the clinical outcomes. Although open surgical repair has been the standard approach to peripheral vascular injuries, endovascular treatment has become more refined and is now an alternative to open surgery, which potentially involves lower morbidity and mortality rates. However, management of arterial injuries, especially when they involve simple bleeding from small branches of the main artery, can be challenging, and the best treatment options for such injuries remains controversial.Three cases suffered blunt trauma that resulted in hemorrhage from branches of the SFA.All patients underwent selective angiography, which demonstrated active extravasation from branches of the SFA.n-butyl cyanoacrylate (NBCA).All patients were treated using embolization with A post-embolization angiography demonstrated successful hemostasis, with no complications.Superselective catheterization using a coaxial technique with a 5-F curved catheter and the smallest caliber microcatheter, and using a permanent liquid embolic agent, such as NBCA, increases the success rate of embolization for cases of hemorrhage from SFA branches. A trauma lesion of the main artery in the lower limb can be treated with placement of a bare metal stent or a stent graft, whereas distal branch bleeding can be embolized with coils, gelfoam particles, liquid embolic agents , or a combination of these approaches. However, embolization for bleeding from small branches of the SFA is technically more challenging and sometimes even impossible, due to the unfavorable vascular anatomy or small caliber of the vessels, and subsequently the best treatment option for such injuries remains controversial.Endovascular repair has been widely employed in the management of traumatic arterial injuries, and with advances in technology, it is now associated with better in-hospital mortality and lower rates of sepsis than open surgery.,6 Herein, we report 3 cases of hemorrhage from branches of the SFA after blunt trauma, all of which were successfully treated with embolization by NBCA using a coaxial technique with a 5-F curved catheter and the smallest caliber microcatheter.There is little evidence about the efficacy and safety of endovascular methods for hemorrhage from branches of the SFA, and there are a few reports where embolization failed, necessitating open surgery.The 2 survivors have provided informed consent for publication of the case. Meanwhile, we could not obtain consent from the deceased patient, but the Institutional Review Board (IRB) of the Chungbuk National University Hospital in Cheongju, Republic of Korea considered exempt of approval for publication of the case.22.1A 74-year-old man was admitted to our hospital after a car accident, with multiple traumas that included a comminuted fracture of the right distal femur Fig. A. Active2.2A 69-year-old woman presented to our hospital with multiple traumas after being hit by a car. A tense, tender swelling had developed in the right thigh with systolic blood pressure dropping to 70 mmHg. Therefore, a vascular injury caused by blunt trauma was strongly suspected. The patient was referred directly for emergency angiography without any imaging evaluation. The right SFA was catheterized via left femoral artery access and selective angiography demonstrated active extravasation from the muscular branch of the right SFA Fig. A. Select2.3A 73-year-old man was admitted to the trauma center after being hit by a car, with multiple traumas. The patient was hemodynamically unstable and complained of severe pain in the left leg. CT scan revealed active bleeding from the second portion of duodenum and the patient underwent open surgery on admission. That evening, acute swelling and worsening pain developed in the left thigh; therefore, a vascular injury was strongly suspected. We proceeded to conduct angiography and right femoral arterial access was achieved with a 5-F sheath. Angiography of the left SFA was performed and revealed active extravasation from a muscular branch of the left SFA (Fig. 3,7 Historically, open repair has been the mainstay of surgical management for arterial vascular trauma.,9 However, surgery usually necessitates general anesthesia and it may be challenging for surgeons to work in a field that is already distorted by injury, especially if the affected vessel is located deep in an obese patient. Endovascular repair yields a shorter hospital stay and requires a shorter period of inactivity than surgery. With improvements in medical technology and evolution of less invasive methods, transcatheter arterial embolization has been widely used in approaches for dealing with lower extremity arterial injury, as an alternative to open surgery.,5,11 Nevertheless, the efficacy of embolization in hemorrhage from branches of the SFA has infrequently been reported; these reports involved a few patients where embolization has failed or could not be catheterized, and required open surgery.,6Traumatic vascular injuries involving the extremities are rare, and in fact, only 26% of extremity traumas involve vascular lesions; among these, hemorrhage from small branches of SFA after bunt trauma is extremely rare.,11 However, covered stents could have disadvantages for younger patients with a long life expectancy, because the long-term consequences of implanting covered stents are not known. Conservative treatment could also be considered in cases of simple arterial bleeding from small branches, especially when patients are hemodynamically stable on admission. However, a conservative approach may result in complications, such as continuous bleeding and compression of the surrounding structures by the expanding hematoma,7 and acute compartment syndrome of the thigh may result in blunt trauma patients. In 1 case report of conservative treatment of a patient with SFA branch bleeding, the patient eventually developed deep vein thrombosis and subsequent pulmonary embolism due to venous compression from the worsened hematoma. Therefore, embolization should be considered rather than conservative treatment, if it is technically possible, and it is not associated with a risk of ischemia distal to the lesion.Although the injury pattern to the vessels has been suggested to play a role in determining the type of repair employed, regardless of the injury mechanism, the best treatment options for simple arterial bleeding from small branches remain controversial. Covered stents are attractive options for the management of SFA hemorrhages, particularly in areas that are difficult to expose surgically.,8,11,12 A selective technique is essential for avoiding technical failure or procedure-related complications. Therefore, we recommend that the following points be considered when embolization is performed in patients with hemorrhage from branches of the SFA. First, when selecting bleeding branches, a 5-F curved catheter, such as the Cobra II catheter, should be used. It is difficult to select the bleeding branches of the SFA directly with the microcatheter because the tip of the microcatheter is difficult to move in the desired direction, especially in wide diameter arteries such as the SFA. On the other hand, with a curved catheter, we can control the direction of the catheter tip, and consequently, it can be located as close to the bleeding branches as possible, or the orifice of bleeding branches can be selected directly, as shown in our cases (Figs.  Other potential advantages of NBCA include its capacity to obliterate small distal vessels, and its effective occlusion of bleeding sites that have complex anatomical structures. However, the danger of damaging a limb's circulation due to reflux of the agent should be considered. To prevent this complication, an appropriate volume, speed of injection, and adjustable ratio of iodized oil to NBCA mixture should be used. In our cases, we used 1:2 mixtures of NBCA and iodized oil and 0.2 to 0.4 mL of the mixture, which was carefully injected under fluoroscopic monitoring. To avoid adhesion of the catheter tip to the vessel wall, the microcatheter was quickly removed after injection. Fortunately, there was no inadvertent embolization, which is associated with a risk of ischemia distal to the lesion (Table With advances in equipment and the introduction of new catheters and microcatheters, the frequency of unsuccessful catheterization has decreased.es Figs. B. It als4In conclusion, arterial embolization can be a feasible, safe, and effective option for treating traumatic vascular injuries involving the extremities, allowing for timely and minimally invasive management. Hemorrhage from branches of the SFA after blunt trauma is extremely rare, but can potentially lead to fatal complications, such as compartment syndrome. Embolization could be used for managing SFA branch bleeding if the vessels can be safely sacrificed without any anticipated ischemic sequelae. Therefore, performing directly selected catheterization of the orifice of the bleeding branches with a 5-F curved catheter, and then introducing a small caliber microcatheter using the coaxial technique, and using a permanent liquid embolic agent, such as NBCA, may increase the success rate of embolization.XXX


We define a value as a quality or standard which a CE project is aiming for, whilst a principle is an objective which underpins the value and facilitates its achievement. The values of Clarity, Creativity, (being) Evidence-led, Equity, Interdisciplinarity, Sustainability and Flexibility were identified by a network of 40 researchers and practitioners who utilise CE approaches to tackle complex One Health challenges including, but not limited to, AMR. We present our understanding of these seven values and their underlying principles as a flexible tool designed to support stakeholders within CE for AMR projects. We include practical guidance on working toward each value, plus case studies of the values in action within existing AMR interventions. Finally, we consider the extent to which CE approaches are appropriate to tackle AMR challenges. We reflect on these in relation to the tool, and current literature for both CE and AMR research. Authors and co-producers anticipate this tool being used to scene-set, road map and trouble shoot the development, implementation, and evaluation of CE projects to address AMR and other One Health challenges. However, the tool is not prescriptive but responsive to the context and needs of the community, opening opportunity to build a truly collaborative and community-centred approach to AMR research.This paper presents seven  Antimicrobial resistance (AMR) is the process by which microbes  change or evolve to survive the drugs used to destroy them. Although naturally occurring, AMR is accelerating on a global scale due to the overuse, misuse, and inappropriate disposal of antimicrobials. It is considered a One Health issue because it impacts humans, animals and the environment and requires cross-sector collaboration to tackle . WithoutThere is a growing literature that discusses the potential of CE in tackling Health challenges in LMICs . Howevervalues which should be considered when developing, implementing, and evaluating CE interventions within One Health. The tool also presents key principles, which act as indicators as to whether each value is being realised. By utilising similar language to existing frameworks this tool could be implemented alongside them. However, it also provides a holistic One Health view of the CE method through discussion of specific values and principles implicated in AMR research. We anticipate users of the tool to be those working on either AMR or another complex One Health challenge who wish to utilise methods which truly engage the focal community they are aiming to support.This article discusses the creation of a flexible tool to guide the application of CE methods to AMR or broader One Health research. The tool was compiled based on workshop discussions by a range of researchers, practitioners and government officials. It draws on the collective experience of these co-producers, summarising this into seven values and their underpinning principles which should facilitate and support the successful application of CE to AMR. We also exemplify these values in action through four Case Studies.In June 2019 a group of 40 researchers and practitioners met in Kathmandu, Nepal for a three-day workshop designed to discuss the role of community engagement in tackling AMR. The group was convened by a partnership between HERD International and the University of Leeds, and aimed to bring together teams utilising CE methods in AMR and wider health research. Invited delegates represented 20 projects delivered by 18 organisations, including Universities, NGOs, and local government, with interventions spread across 10 LMICs. From here on we refer to these delegates as our co-producers. While some were experts in various dimensions of AMR, others brought insights from other One Health issues. Disciplinary representation was broad and included those in the field of medicine, social science, the arts, and animal health. What bound co-producers were their interests and expertise in community-level research across One Health challenges which ranged from the use of participatory theatre to improve mental health, to discussing AMR with children via grassroots comics. Their diversity provided rich discussions around what constitutes successful CE and how it can be applied to AMR. This paper synthesises discussions into a practical tool detailing seven key A major step in the process of creating the tool was to clarify our co-producers’ shared definition of CE. From our perspective CE is a specific type of intervention within the wider community-based participatory research (CBPR) continuum ,19. It i‘A participatory process through which equitable partnerships are developed with community stakeholders, who are enabled to identify, develop and implement community-led sustainable solutions using existing or available resources to issues that are of concern to them and to the wider global community.’Community Engagement: This definition is important because, although CE appears in cross-disciplinary literature the extent to which the community is engaged in research can vary dramatically from filling in a questionnaire to co-producing an output, such as a policy brief or piece of art ,19,21–24values and principles underpinning community-based research, using an inductive thematic approach [what determines how you work when developing a CE project or activity? This could be resources such as money, time, and place, challenges to the working environment such as gender and intersectionality issues, but also ways of working such as interdisciplinary collaboration and co-production’.The workshop sought to understand what co-producers considered as key approach  to analywho we work with’, ‘institutions’, ‘scalability’, ‘creativity’, ‘power’, ‘evidence’ ‘interdisciplinary.’ Groups of approximately six co-producers discussed how each area influenced their research, and what challenges and opportunities they posed. Six individuals volunteered to facilitate these discussions and remained on a single table whilst all others had the freedom to move between tables (but could remain at one if they so wished). After approximately 40 minutes of discussion facilitators fed back these reflections to the group. As such co-producers essentially began a thematic analysis because discussions were summarised and shared with the entire room inviting feedback and comments on this summary. Two note-takers recorded discussions and the whole event was filmed providing the data sources for the next stages of the analysis to determine the key values of community engagement research to tackle AMR  were asked to produce short case studies to exemplify the values and principles in action.From this analysis, seven ventions . The proClarity: Throughout discussions, plenary sessions and presentations, the issue of clarity was of paramount concern. From a research perspective, co-producers stressed the importance of focusing on clear questions and communicating these openly with other stakeholders to avoid over- or false-promising on outcomes. From the practitioner perspective, there was a focus on the use of simple, locally-relevant language to communicate with wider stakeholders for which AMR may be a novel term. Finally, in the community, the onus was on clarifying the needs and expectations of the project based upon lived experiences and the local context of the AMR challenge. Linking to the value of flexibility, co-producers felt it was important to create space within the project timeline for discussions. This ensures knowledge can be shared between stakeholders whilst keeping focus on the project’s aims and everyone’s roles within it.Creativity: Co-producers were keen for project design to be question-focused and to utilise methodologies that are familiar to stakeholders. However, there was also acceptance that certain methods may be better suited to answering certain questions and so this value does overlap with the next (being evidence-led). There was agreement on the huge benefit to having artistic practitioners  in our co-production team with respect to this value. The group stressed the need to ensure that, where creative outputs are developed, value is placed on the artistic form being used. This extended from the project-planning phase where discussion should focus on why a specific form  is appropriate, through to dissemination. For example, the film outputs of Case Study 1 [ Study 1  have intEvidence-led: Understanding previous, current and emerging work on AMR was a common theme of co-producer discussions. Many stressed the need to look beyond academic publications for evidence, and to value the expertise and lived experience of one’s community who are experts in their own lives, particularly their health seeking and hygiene behaviours which likely influence AMR. Preliminary dialogue with the community and wider stakeholder network was seen as crucial to formulating AMR research questions and deciding on methodology, as exemplified by Case Study 4 [ Study 4 . Pilot aEquity: Co-producers overwhelmingly stated that within CE the voices of all stakeholders should be appreciated, but this may not always be equally weighted hence the value of equity. Although researchers will be seeking to co-develop AMR solutions with their community partners, clear and accurate health-based information on AMR must be provided and inaccurate comments corrected by the research team. This must be handled fairly so as to balance power dynamics between stakeholders. Many co-producers stated that involving the community in project planning was a successful way of ensuring equitable partnerships developed from the offset as the community, research team and wider stakeholders could share existing knowledge and have their assumptions challenged within a safe space. Consideration of social norms in the community is also important with regards to equity, for example, Case Study 1 [ Study 1  realised Study 1  this cauInterdisciplinarity: AMR impacts on human, animal and environmental health. Thus, it is almost impossible to address via a single discipline. Case Study 3 considered the use of antimicrobials in rural Kenyan farming communities but, because humans and animals share the same water sources in these locations, it was difficult to attribute AMR at sampling sites exclusively to livestock. Through creative engagement with people (comic book development and interviews) the team began to understand the behaviours which underpinned their biological data. Co-producers also stressed that community knowledge represented interdisciplinarity. For example, is it appropriate to advocate for prescription-only antibiotic use in a community that lives several days walk from a medical centre? Community stakeholders are best-placed to answer this question. Academic interdisciplinarity was seen as actively encouraged by research councils via their development of cross-cutting funding calls. Understanding the experience of different team members in terms of the value it brings to the project’s research questions, rather than hierarchy or standing within a given professional research community, was considered a key approach to facilitate project-wide interdisciplinarity. Considering the Sustainable Development Goals could also drive interdisciplinary focus, particularly in terms of troubleshooting problems arising from presumed solutions. Finally, linking to the value of being evidence-led, co-producers stressed the need to evaluate projects in a way that was relevant to multiple disciplines so that learnings can be shared more widely.Sustainability: A key challenge for co-producers was the ethics around what happens to a project when the funding ends. One Health issues are rarely solved by a silver bullet and CE, in particular, is invariably a slow-burning solution realised through incremental changes in behaviour. Co-producers discussed the need to ensure communities have strong ownership of a project, allowing them to visualise how resources and skills could be used beyond the funding lifespan. The development of equitable partnerships built upon joint interests also appeared in the discussion of this value, as did utilising existing stakeholder networks to ensure that solutions are taken up and embedded in the longer-term strategy of community-based organisations. As researchers, it was felt that this approach to CE projects sowed the seed for continued relationships beyond the funded phase of the project and facilitated long term evaluations of the work. This value resonates with that of equity and being evidence-led, since for a community to take ownership of a project and its outputs, they must be fully invested in the project and not view it as a tokenistic opportunity.Flexibility: Flexibility was deemed essential to managing expectations and ensuring positive outcomes for all stakeholders. AMR is driven by multiple dynamic factors, many of which are poorly understood, including the environmental burden of AMR. As such research questions relating to AMR have the potential to change mid-project with no regard for planned outcomes and impacts. CE is a flexible approach which is iterative in nature, allowing stakeholders to modify methods and outputs as they learn throughout the project. However, communication of this flexibility was seen as paramount from the researchers’ side. A community may be looking to researchers or other stakeholders for clear guidance and defined answers. Challenging this expectation early on in the project was seen as important to allowing flexibility to be accepted across stakeholders facilitating the iterative development of the project itself and supporting the values of clarity and equity.values to life and actively assist the design, development and evaluation of successful CE projects, we have created a practical tool. principles and additional guidance on how to achieve them. This tool is intended to be used by all stakeholders as a roadmap to ensure each CE project is upholding the seven values of Clarity, Creativity, being Evidence-led, Equity, Interdisciplinarity, Sustainability and Flexibility.To bring these values and principles of community engagement in action we discuss four case studies of CE for AMR projects, dissecting how each value (or principle) is achieved  Evidence-Led, and wider literature which considers CE to value local knowledge, foster a sense of trust between stakeholders, and build specific solutions which hinge on changing behaviours at very local levels [values and principles presented here will allow projects to capitalise on the potential of CE to tackle AMR challenges, and support them to consider context and disciplinary reach when deciding on evaluative methods. In combination this should allow robustly designed and evaluated projects which best serve their community’s needs, but also evidence the scope of CE to address AMR.Because AMR is often a locally specific and complex problem, co-producers strongly agreed that CE has the potential to build contextually appropriate solutions. This is reflected within the l levels ,18,33. Hl levels . Co-prodl levels . Additiol levels . It is hvalues and principles tool has been co-produced by over 40 researchers and practitioners who utilise community engagement approaches to tackle One Health issues. It was created in response to both the growing threat of AMR, and the growing realisation of AMR as a social issue which can, at least in part, be addressed by community-based interventions. Unfortunately, as discussed by co-producers, language barriers and limited methodological support for CE means it is currently under-utilised within the AMR sphere. To bridge this gap, we propose this tool be used to guide the development of clear, creative, evidence-led, interdisciplinary, equitable, sustainable and flexible research which can support multiple stakeholders to tackle an AMR problem collaboratively and through a locally meaningful intervention. However, this tool is not prescriptive and the methods by which a team decides to approach each principle and value are entirely plastic and should be driven by local context. Our co-producers have exemplified this by providing Case Studies of the tool in action, in so doing reflecting upon their current CE for AMR projects. Case studies demonstrate the different ways each value and principle can be achieved but also the key challenges a project team can face. CE is popular and appropriate within the broader sphere of Health interventions and has the potential to revolutionise AMR research. However, it is no silver bullet and it cannot be used formulaically. All stakeholders must respond to the local context around their problem and be prepared to listen, learn, and reflect throughout the process. It is hoped this tool can encourage and provide practical support for high quality community engagement interventions which positively impact the complex challenge of AMR.This


OTUD6A leads to lower Drp1 levels and suppresses mitochondrial fission, and the affected cells are consequently less prone to tumorigenesis. Conversely, the overexpression of OTUD6A increases Drp1 levels and its protein half‐life and enhances cancer cell growth.OTUD6A deubiquitylates and stabilizes Drp1, thereby facilitating regulation of mitochondrial morphology and tumorigenesis. The depletion of  OTUD6A leads to lower Drp1 levels and suppressed mitochondrial fission, and the affected cells are consequently less prone to tumorigenesis. Conversely, the overexpression of OTUD6A increases Drp1 levels and its protein half‐life and enhances cancer cell growth. Therefore, our results reveal a novel upstream protein of Drp1, and its role in tumorigenesis that is played, in part, through the activation of mitochondrial fission mediated by Drp1.Dynamin‐related protein 1 (Drp1) is a cytosolic protein responsible for mitochondrial fission and is essential in the initiation and development of several human diseases, including cancer. However, the regulation of Drp1, especially of its ubiquitination, remains unclear. In this study, we report that the ovarian tumor‐associated protease deubiquitinase 6A (OTUD6A) deubiquitylates and stabilizes Drp1, thereby facilitating regulation of mitochondrial morphology and tumorigenesis. OTUD6A is upregulated in human patients with colorectal cancer. The depletion of  Drp1dynamin‐related protein 1DUBsdeubiquitinasesHis‐ubHis‐ubiquitinIBimmunoblotIHCimmunohistochemistryIPimmunoprecipitationMARCH5membrane‐associated ring‐CH‐type finger 5Mfn1mitofusin 1Mfn2mitofusin 2NIKNF‐κB‐inducing kinaseOpa1optic atrophy 1OTUsovarian tumor‐associated proteasesOTUD6Aovarian tumor‐associated protease deubiquitinase 6A1Dynamin‐related protein 1 (Drp1), the ‘master regulator’ of fission, is involved in several important aspects of mitochondrial structure, and it functions by governing the processes of mitochondrial dynamics and providing necessary adenosine triphosphate to cells , 3. Addiet al. [et al. [et al. [The post‐translational modifications of Drp1 have been discovered to affect different aspects of Drp1 function, which is associated with cellular function , 12, 13.et al.  showed t [et al.  explaine [et al.  showed t [et al. . The ACP [et al. . Moreove [et al. .It is well documented that the ubiquitination of many proteins can be reversed by deubiquitinases (DUBs), which play an important role in cell growth, apoptosis, and cancer , 20. AppHere, we investigate OTUD6A is a novel upstream of Drp1 and upregulated in patients with colorectal cancer, which suggested a potential mechanism linking OTUD6A to mitochondrial fission in cancer. These results uncover the importance of OTUD6A in tumorigenesis and new therapeutic strategies that attenuate mitochondrial fission through inhibition of the OTUD6A‐Drp1 pathway.22.1−1 streptomycin. HeLa‐Drp1−/− cells were constructed by CRISPR/Cas9 and kindly given by T. Zhang. All cell lines were previously measured for mycoplasma contamination.Multiple cell types, namely HEK293, HEK293T, HCT116, DLD1, and HeLa cells, were cultured in Dulbecco's modified Eagle medium  containing 10% fetal bovine serum (FBS), 100 units of penicillin, and 100 mg·mLFlag‐OTUB1, Flag‐OTUB2, Flag‐OTUD3, Flag‐OTUD5, Flag‐OTUD6A, Flag‐OTUD6B, and Flag‐OTUD7B constructs have been described previously . The len2.2μ filter, added with 4 μg·mL−1 polybrene , and used for infecting the cells at 50% confluence. Forty‐eight hours postinfection, the cells were passaged and selected using 1 μg·mL−1 puromycin (Sigma) for 72 h to eliminate the uninfected cells before harvesting and subsequent western blotting analysis.For cell transfection, cells with 80% confluence were transfected using Lipofectamine  in Opti‐MEM medium (Invitrogen). Forty‐eight hours post‐transfection, the cells were harvested for analysis. For viral infection, the HEK293T cell line was used for packaging and amplifying the lentivirus. The media containing the viruses were collected at 48 and 72 h after transfection. Then, the media were filtered through a 0.45‐μ2.3The antibodies used in this study are listed in Table 2.4m Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, and 0.5% NP‐40), subjected to SDS/PAGE, and further incubated with the indicated antibodies.Cell lysates were collected according to a standard method described previously . For imm2.5A total of 100 tissue samples, including 10 samples of normal tissues and 90 of colorectal cancer tissues, were purchased from Kexin Biotech Company  and stained with human Drp1 or OTUD6A (dilution 1 : 200) for the immunohistochemistry (IHC) assay. Unfortunately, eight of colorectal cancer tissues were damaged during the IHC assay, the rest of 82 cancer tissues could be used for statistical analysis. The stained sections were scanned using a panoramic viewer . Detection and analysis of the staining were performed as described previously . The sta2.64 cells per well were plated in 6‐well plates. The cell number was calculated among 6 days. For colony formation, the cells were plated in 6‐well culture dishes (500 or 1 000 cells/well) and approved to growth for 10–14 days. The cells were stained with crystal violet, and the colony numbers were counted.For cell growth, 1 × 102.7m) overnight and then performed according to the previous description. The pull‐down proteins were subjected to SDS/PAGE for immunoblot (IB) analysis [Cells were transfected with His‐ub, Drp1, OTUD3, OTUD6A, OTUD6B, and OTUD7B. After transfection for 48 h, the cells were treated with MG132 . Manders' colocalization coefficients were chosen for the colocalization ratio. Manders' M1 (M2) represents the proportion of red (green) fluorescence overlapped with green (red) fluorescence in total red (green) fluorescence. For fluorescence microscopy of mitochondria, cells with 80% confluence were incubated with MitoTracker Red (100 nm) for 30 min at 37 °C and then washed with PBS followed by image acquisition using an inverted fluorescence microscope (Olympus IX71). The mean mitochondrial length was determined by measuring mitochondrial from cells obtained by fluorescence microscopy using the imagej software (NIH). Fifteen cells from three independent experiments were analyzed for quantification.The cells were seeded in a 12‐well plate with prepaved glass coverslips. For immunofluorescence, cells were incubated overnight at 4 °C with anti‐Drp1 and anti‐OTUD6A antibody and measured with Alexa Fluor 555‐labeled donkey anti‐mouse IgG  and FITC‐conjugated goat anti‐rabbit IgG (Invitrogen). Cells were subsequently stained with DAPI (Invitrogen) as a nuclear staining containing Antifade reagent  followed by image acquisition using an inverted fluorescence microscope . The colocalization was analyzed by the 2.96 OTUD6A−/− HCT116 cells as previously described [L × W2 × 0.52 . After 20 days, mice were sacrificed, xenografted solid tumors were dissected, and then tumor weights were measured. BALBc_nu female nude mice (4–5 weeks of age) were purchased from the SLAC Laboratory Animal Co., Ltd , following the principles of the National Institutes of Health on the use of laboratory animals, with a protocol that was approved by the University of Xi'an Jiaotong Institutional Animal Care and Use Committee.BALBc_nu mice were injected with 5 × 10escribed . Tumor s2.10graphpad software  was used to analyze data. Unpaired, two‐tailed Student's t‐test was used to evaluate data from two groups. One‐way or two‐way ANOVA was used to assess other data, followed by a Bonferroni post hoc test. P < 0.05 was considered to indicate statistical significance.The results are presented as the mean ± SD. 33.1in vitro using Flag IP, while OTUD3, OTUD6A, OTUD6B, and OTUD7B interacted with Drp1 in vitro using HA IP. However, OTUD6A both interacted with Drp1 using the two agarose beads . We constructed the mutant C152A OTUD6A and found that the mutant C152A lost its ability to interact with Drp1 , mitofusin 1 (Mfn1), and mitofusin 2 (Mfn2), the other key regulators of mitochondrial dynamics, were not affected (Fig. OTUD6A (Fig. To test the effect of OTUD6A on Drp1 stability, we measured the expression of Drp1 in cells infected with OTUD6A shRNA by western blotting. The depletion of ted Fig. . On the ted Fig. . This inD6A Fig. A,B. AddiD6A Fig. C,D. ThesTo investigate whether OTUD6A could remove the ubiquitin chain of Drp1, the OTUD subclasses were determined by ubiquitination assays in vitro. The result showed that OTUD6A could obviously deubiquitinated Drp1 Fig. . Interes3.3The previous studies showed that an increase in the fission protein Drp1 was found in breast, lung, and oncocytic thyroid cancers compared with matched nonmalignant tissues , 6, 7. O3.4OTUD6A could dramatically attenuate cell proliferation (Fig. in vivo (Fig. To assess the biological roles of OTUD6A in cancer, we inhibited the expression of OTUD6A in cells with shRNA Fig. A. We obsion Fig. A and colion Fig. B, C in vivo Fig. D–F. Convivo Fig. A could aivo Fig. B and colivo Fig. C,D. Overivo Fig. A–D, suggMitochondrial dynamics are related to the initiation and development of cancer, and Drp1 has a critical role in mitochondrial morphology. As OTUD6A regulates Drp1 stability which has been revealed above, we next suppose that OTUD6A could affect mitochondrial dynamics. To investigate whether OTUD6A affects mitochondrial dynamics, we examined mitochondrial morphology in cells knocked down with OTUD6A. Fluorescence microscopy revealed that the depletion of OTUD6A resulted in less fragmented mitochondria. The length of the mitochondria was also significantly increased in HeLa cells transfected with OTUD6A shRNA Fig. G, H. The3.5Drp1 could markedly attenuate mitochondrial fission, cell proliferation, and colony formation (Fig. Given that the Drp1 protein is changed in OTUD6A‐mediated tumors, we next examined whether Drp1 was involved in the molecular signaling pathway. It has been revealed that Drp1, a mitochondrial fission protein, plays a crucial role in cancer cell growth, migration, and invasion , 32. Conion Fig. . Alignedion Fig. . These f4The current study reveals that OTUD6A might play an oncogenic role in tumorigenesis and that OTUD6A regulates Drp1 stability and mitochondrial fission. Our results implicate a potential role for OTUD6A dysregulation in mitochondrial dynamics and in tumor cell growth Fig. .Numerous studies have revealed a connection between ubiquitin and protein , 34. Simet al. discovered that Drp1 was dramatically reduced in colon and lung cancer tissues, whereas no change in Drp1 was observed in breast or prostate tumors, a finding contrary to other findings [Drp1‐mediated mitochondrial fission is essential for the completion of cytokinesis and for the proper distribution of mitochondria into daughter cells , 39. Furfindings , 7. In ffindings .et al. [The post‐translational modifications of Drp1 regulate its activity and mitochondrial function. Gu et al.  have repet al. , suggestet al. . A recenet al. , which iOTUD6A reduces mitochondrial fragmentation, inhibits tumor cancer cell proliferation, and impairs xenograft growth. Accordingly, small molecule inhibitors against OTUD6A might be potential drugs in the field of anticancer therapies. Interestingly, the diterpenoid derivative 15‐oxospiramilactone showed that it could inhibit DUB USP30, resulting in the elevated Mfn protein levels that promote mitochondrial fusion [Recent combinatorial studies have indicated the great potential of DUBs as novel targets in strategies to combat cancer. We provide evidence that the deletion of l fusion . However5Mitochondrial dynamics have an crucial effect on the abnormal of mitochondrial function and can increase cell oxidative stress. As the key signaling protein regulating mitochondrial fission, Drp1 has been found to be tightly related to various cancers. To explore, the regulation of Drp1 signaling pathway will help understand the initiation and progression of cancer and improve the development of new targeted drug. Our study demonstrates a novel OTUD6A/Drp1 axis, and its role in tumorigenesis, partly via regulating Drp1 stability and mitochondrial fission. However, future studies are needed to deeply explore the mechanism of OTUD6A regulating Drp1 and determine the physiological functions of OTUD6A in tumorigenesis, for example, those using OTUD6A‐knockout mice.The authors declare no conflict of interest.LS, JGL, and JKL designed the study; LS performed the experiments with the help of JL, YHP, and SXZ, YW; LS, and JGL analyzed the data and wrote the manuscript; and LS, JGL, JKL, JFZ, and XPD revised the manuscript.Fig. S1. The stability of Drp1.Click here for additional data file.Fig. S2. Overexpression of OTUD6A affects cell growth.Click here for additional data file.Fig. S3. Overexpression of Drp1 affects cell growth.Click here for additional data file.Supplementary MaterialClick here for additional data file.


Pyoderma gangrenosum is a rare inflammatory condition with varying clinical presentations and severity. It is commonly seen in association with an underlying condition, most common of which is inflammatory bowel disease. We report a case of a 26-year-old male who came to the emergency department with increasing lower extremity ulcers, intermittent hematochezia, and pain in the small joints of his hands. After excluding a broad list of differentials for lower extremity ulcers, the diagnosis of pyoderma gangrenosum was made. He was also found to have erosive changes at multiple proximal interphalangeal joints and jug-like syndesmophytes at T12 and L1 on CT scan. Although there was evidence of a spondyloarthropathy, there was no evidence of inflammatory bowel disease on colonoscopy, psoriasis, or sexually transmitted infections. After multiple failed trials of medications including azathioprine and sulfasalazine, 4 weeks of Adalimumab resulted in rapid healing of pyoderma gangrenosum lesions and improvement in his synovitis. Coupled together, this suggests a diagnosis of pyoderma gangrenosum associated with undifferentiated spondyloarthropathy and erosive inflammatory arthritis. This case is suggestive of spondyloarthropathy going underdiagnosed and untreated in other patients with pyoderma gangrenosum as lower extremity ulcerations can be the primary complaint for seeking treatment. Although rare, axial spondyloarthropathy associated with pyoderma gangrenosum should be kept as an associated differential diagnosis when faced with pyoderma gangrenosum. Pyoderma gangrenosum is a rare inflammatory condition with varying clinical presentations and severity. Typically, it presents as a papule or pustule that rapidly evolves to an ulcerative lesion with irregular, erythematous to violaceous edges. Pathergy is a characteristic feature of pyoderma gangrenosum as lesions often occur at sites of trauma. It has an estimated annual incidence of 3–10 cases per million people . AlthougA 26-year-old Hispanic male with no significant past medical history came to the emergency department complaining of multiple wounds that gradually developed over the past 3 years. He reported a wound at the vertex of his scalp, posterior aspect of his neck, and on bilateral lower extremities. He reported the wounds initially started as small pimples, with progression in size and pain severity. The patient also reported serosanguineous drainage from his wounds, hematochezia, and pain in the small joints of his hands bilaterally, 9/10 in severity, associated with swelling and stiffness. The patient reported that his scalp lesions started 3 years prior to this admission and remained the same size since then. The lesions over his lower extremities started within 3 months of this admission and progressed so rapidly that it prompted him to go to the emergency department and seek help. He reported that his joint pain started approximately the same time as his scalp lesions and has progressively gotten worse over the past 3 years. He denied diarrhea, back pain, uveitis, iritis, sexually transmitted infections, abdominal pain, fever, or night sweats.nd and 4th proximal interphalangeal (PIP) joints and the left 5th PIP joint. There was associated fusiform swelling of the second and fourth right digits, suggestive of dactylitis.Physical exam was significant for a morbidly obese male in mild distress due to pain. Triage vitals were significant for mild tachycardia of 103 but otherwise were unremarkable. He had multiple wounds with serosanguineous drainage: ulcer at the vertex of the scalp measured 7 cm × 3 cm, posterior neck ulceration 5 cm × 2 cm, and multiple ulcers, of varying stages, circumferentially on bilateral lower extremities . Further3 μL (reference range 4.5–11.0 × 103) with 74% neutrophils, hemoglobin of 12.4 g/dL (reference range 13.0–17.0), ESR of 43 mm/hr (reference range 0–20), and CRP 54 mg/L (reference 0.0–5 mg/L). X-ray of bilateral hands revealed marginal erosions at the right second and fourth PIP joints and left 5th PIP joint , anticyclic citrullinated peptide (CCP), antinuclear antibodies (ANA), anti-Ro/SSA, anti-La/SSB, anti-RNP, complements levels, double-stranded DNA, anti-smith antibodies, anti-neutrophil cytoplasmic antibodies, HLA-B27, HIV, hepatitis B, hepatitis C, Quantiferon, and blood cultures were all negative. Punch biopsy of the left lower extremity edge revealed mixed dermal infiltrate with neutrophils and vasculitis. The tissue culture was positive for c joints . The gasAs there was no improvement of the ulcerative lesions with antibiotics, and in conjunction with syndesmophytes, enthesitis, and dactylitis, we started treatment for undifferentiated spondyloarthropathy associated with pyoderma gangrenosum. The patient was initially started on solumedrol 120 mg intravenously for 3 days followed by tapering doses of prednisone in combination with sulfasalazine. There was poor response after 6 weeks; thus, prednisone was discontinued and azathioprine was added to sulfasalazine. Azathioprine 100 mg daily and sulfasalazine 1000 mg twice daily were continued for 8 weeks with minimal response. In fact, the lower extremities ulcerations coalesced and increased in circumference. Due to lack of response to corticosteroids, sulfasalazine and azathioprine, he was subsequently started on adalimumab 40 mg subcutaneously every 14 days. Within four weeks, there was significant improvement . UnfortuPyoderma gangrenosum can have many different clinical presentations with varying degrees of severity. Clinically, it can present as classic or ulcerative subtype, bullous, pustular, vegetative, drug-induced, postsurgical, or peristomal types . Our patThe exact pathophysiology of pyoderma gangrenosum is poorly understood but is believed to be a combination of genetics, irregular activation of inflammasomes, and both innate and adaptive immune systems . There iThe diagnosis of pyoderma gangrenosum is challenging, as it is ultimately a diagnosis of exclusion. Su et al. have proposed diagnostic criteria for pyoderma gangrenosum and require two major and two out of four minor criteria to establish the diagnosis . Major cTreatment of these cutaneous ulcerations is based upon the severity and the associated medical conditions. Although a direct association between severity of an associated condition and pyoderma gangrenosum has not been established, it is important that medications ideally be used that are beneficial for both conditions. Limited disease can be managed with wound care, topical, and intralesional therapy, whereas aggressive disease requires systemic immunomodulatory agents. However, no gold standard treatment exists as there are limited randomized controlled trials . TopicalInitially, sulfasalazine was started for our patient's pyoderma gangrenosum as there was suspicion for an associated inflammatory bowel disease, and colonoscopy had not been completed at this point. Subsequently after minimal response, azathioprine was added as a mild immunosuppressant because our patient was considered high risk for infection with his large, open wounds. However, he did not respond. Finally, adalimumab was started instead of other longer-acting biologics such as infliximab. We preferred adalimumab as it is dosed every two weeks and thus has a shorter half-life, in case the medication had to be discontinued in the future due to an infection. Our indication for an anti-TNF agent was our patient's refractory pyoderma gangrenosum and enthesitis seen on ultrasound examination. The final diagnosis for our patient was pyoderma gangrenosum with superimposed pseudomonas infection associated with undifferentiated spondyloarthropathy.Traditionally, pyoderma gangrenosum is commonly associated with inflammatory bowel disease, but this was not the case in our patient. He initially sought out treatment for ulcerations from pyoderma gangrenosum and then was subsequently found to have erosive inflammatory arthritis and undifferentiated axial spondyloarthropathy. This is suggestive of spondyloarthritis possibly going underdiagnosed and subsequently untreated in other patients with pyoderma gangrenosum. Thus, even though axial spondyloarthropathy may be rarely associated with pyoderma gangrenosum, it should be kept as an associated differential diagnosis when faced with pyoderma gangrenosum.


The availability of an accurate continuous cuffless blood pressure (BP) monitor would provide an alternative to both invasive continuous BP and 24-h intermittent cuff-based BP monitors. We investigated the accuracy of a cuffless beat to beat (BtB) device compared to both invasive BP (iBP) and brachial cuff BP (cBP) measurements.Patients undergoing clinically indicated coronary angiography (CA) and/or percutaneous coronary intervention (PCI) were recruited. After calibration to an initial cBP reading, BP was measured simultaneously using a BtB device (SOMNOtouch NIBP), brachial artery iBP, and cBP at two time points.The study was terminated early due to a significant bias. Recordings from 14 participants  were analysed. Readings from BtB BP were higher than iBP. The bias between BtB BP and iBP was 34.3 mmHg  and 23.6 mmHg  for SBP and DBP respectively. A similar bias was seen between BtB BP and cBP, but cBP and iBP were largely in agreement.In patients undergoing CA/PCI, significant differences were detected between BtB BP and both invasively measured and cuff BP. The non-invasive BtB BP measurement device tested is not suitable for clinical or research use. Short-tThe concepts and methods of cuffless BP measurement have been discussed for decades . However22.1This was a prospective study including patients >18 years old undergoing clinically indicated coronary angiography (CA) and/or percutaneous coronary intervention (PCI). Patients with atrial fibrillation (AF) or vascular lesions affecting the upper limb arterial tree were excluded from this study. Informed consent was obtained from all participants. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the University of Otago Health Ethics Committee (reference: H19/024).2.2The device consists of three ECG leads and a peripherally placed photoplethysmogram (PPG), both of which are connected to a watch-like unit that is placed at wrist level. The device was placed around the patient's left wrist during CA/PCI. PPG is an optic-based technique used in pulse oximetry to measure arterial oxygen saturation (SpO2) ,14. ThisThe PPG sensor was placed around the index finger. The device manufacturer provided different sizes of the PPG sensor; appropriate sensor size ensures proper contact of the sensor with the patient's finger without having high contact pressure. The ECG was connected according to the manufacturer's instructions. The device estimates BP based on beat to beat measurement of PTT. PTT is calculated as the time interval between the R wave on the ECG detected through the connected ECG leads and the pulse wave detected by the PPG on the patient's finger. SBP and DBP are estimated using a nonlinear model . BP was 2.3The brachial cuff was placed around the left arm. BP was measured using the oscillimetric cBP method integrated into the cardiac catheterisation system .2.4BP was measured using an invasive BP line; pressure transducer was placed in a position that was at the approximate level with the heart and was set to zero by obtaining atmospheric pressure. The line was connected to a 5 French catheter inserted via the right radial artery with the tip placed at the level of the brachial artery.2.5Two measurement points were taken; with the time stamp defined, measurements of both cBP and iBP were simultaneously recorded while corresponding BtB BP reading defined by the time stamp was later extracted and recorded. The first measurement point was taken after radial artery access while second measurement point was taken after the completion of the procedure.2.6For each measurement modality, two variables were analysed: SBP and DBP. Both SBP and DBP variables contained BP measurements at the two time points (before and after the CA/PCI procedure). To assess agreement between iBP and BtB BP measurements, we used Bland-Altman analysis. A univariate linear regression model was fitted to assess proportional bias between measurements. For the comparison of the differences in the mean between measurement modalities, paired two-tailed Student's t-test was used. Pearson correlation was used to assess correlations between variables. Statistical analyses were conducted using R . Alpha of 0.05 was used as the cut-off for significance. Based on an expected mean of differences of 10 mmHg with a standard deviation of the mean of 5 mmHg, a sample size of 50 participants was estimated to provide at least 80% power to define difference at this level of significance.3The study was terminated early after enrolment of 19 patients, due to larger than expected differences being identified. BtB BP measurements were satisfactorily acquired for 14 patients; five BtB recordings were excluded due to unsatisfactory quality or technical failure. Characteristics of the study participants are presented in t-test) and 6.6 mmHg  for SBP and DBP respectively.The bias between BtB SBP and cBP SBP was 30.6 mmHg  with upper limit of agreement 70.2 mmHg  and lower limit of agreement −9.1 mmHg ; The bias between BtB DBP and cBP DBP was 15.2 mmHg  with upper limit of agreement 45.7 mmHg  and lower limit of agreement −15.2 . The Pearson correlation coefficients between BtB BP and cBP were 0.76 and 0.82 for SBP and DBP respectively. There was minimal bias between cBP and iBP; the bias was 0.1 mmHg  against iBP during CA/PCI and oscillimetric cBP. The accuracy of SOMNOtouch NIBP BtB BP monitor was limited in comparison to simultaneously recorded iBP measured at the level of the brachial artery and cBP. This was noticed for both SBP and DBP measurements. The cBP readings agreed to a large extent with the iBP.The need for an accurate non-invasive continuous BP monitor has driven research into cuffless BP monitors. One of the concepts underpinning those monitors is PTT . While tSOMNOtouch NIBP PTT-based BtB BP monitoring has been previously investigated in a number of clinical settings with inconsistent results . The devWe found a large difference between BP measurements recorded by SOMNOtouch NIBP and recorded by both iBP and cBP measurements. In their recent study, Armstrong et al. showed that invasively measured radial BP and brachial BP were different .We showeThis study had some limitations that should be considered when interpreting its results. The measurement of iBP was taken from the contralateral brachial artery. However, the cBP measurement was taken from the ipsilateral side as the BtB BP, and was highly accurate compared to iBP. In addition, most of our patients (71%) had ischemic heart disease which could reflect abnormal vascular elasticity with potential impact on the correlation between PTT and estimated BtB BP . As suchIn conclusion, cuff-less continuous BtB BP monitoring has the potential to provide both patients and clinicians with a tool that could directly influence patient management. However, the accuracy of the technology tested in this study is insufficient for clinical or research use.10.13039/100008235School of Medicine/Basic Medical Sciences Joint Award, University of Otago, New Zealand.This research was funded by the Dunedin Mohammed Moharram: Methodology, Investigation, Formal analysis, Visualisation, Writing - original draft. Luke Wilson: Writing - review & editing. Michael Williams: Supervision, Investigation, Writing - review & editing. Sean Coffey: Conceptualization, Methodology, Supervision, Writing- Reviewing and Editing.The authors have no conflicts of interest to declare.