New Statesman about the G7 Summit and antimicrobial resistance (AMR) [In June 2021, I wrote a strongly worded opinion piece published in the The G7 Summit took place in Carbis Bay, Cornwall, UK on the 11–13 June 2021 and the UK took Presidency of the G7 group of nations .Antimicrobial resistance  is an insidious and silent pandemic that is already happening. It is not a future pandemic and the microbiology community are well aware of this. For those unfamiliar with AMR, it is one of the most urgent global healthcare challenges for the human species, alongside climate change. Micro-organisms such as fungi, parasites, bacteria and viruses that can cause infections have evolved and are evolving to become resistant to the antimicrobial drugs that we use to treat them. Overuse of antimicrobials in healthcare, animals and the environment has significantly accelerated AMR globally . The latstill broken and this requires investment and long-term planning to address effectively. A recent World Health Organization (WHO) report indicated that ‘none of the 43 antibiotics in development target the most antibiotic resistant bacteria’, which is worrying long term [The main approach to tackle AMR has relied on antimicrobial discovery and development. While there is merit to this approach, the antibiotic discovery economic pipeline is ong term . Howeverong term . The UK ong term . Microbiong term . Howeverong term .Diagnostics, however, are a feasible alternative solution to tackle AMR by enabling clinicians to rationalize critical antimicrobial use and increase antimicrobial stewardship. However, there has been limited global funding to accelerate diagnostics development for AMR infections . The devCurrent diagnostic tests employed at point of care often rely on the detection of inflammatory markers, such as C-reactive protein, for indication of bacterial and viral infections . While tHence, investment into novel diagnostics for AMR, ideally for use at point of care, could help to rapidly diagnose patients with AMR infections without relying on empirical methods, assist in tailoring effective antibiotic treatment regimens and promote antibiotic stewardship. Rapid identification of AMR infection would reduce patient mortality, transmission of infection and costs to healthcare providers. The benefits of diagnostics as an alternative approach are huge. There is also an urgent need to develop low-cost and portable diagnostics for use in low-to-middle income countries (LMICs), where access to medical care is limited and patient suffering/mortality is high .Another approach is infection prevention and control (IPC). IPC solutions in the context of AMR appear to have been overlooked in recent years despite their obvious and important links to water sanitation and hygiene (WASH) . HoweverIn LMICs the burden of AMR is high, partly because of the lack of access to healthcare facilities, infrastructure and WASH. If we consider that the world population is predicted to increase to 9.7 billion by 2030, that also puts additional strain on sanitation infrastructure in highly populated countries . The majAn important point to note here is that UK Overseas Development Aid cuts have severely affected the AMR research community globally, and many collaborative UK research projects in LMICs have had to be abandoned . FinallyThe outcome of the 2021 G7 Summit has actually been hopeful for AMR. While we still have significant hurdles to overcome in developing sustainable solutions to tackle AMR long term, the acknowledgement of AMR within the G7 documents is an important step forward. Investing in multiple solutions is perhaps the best strategy to ensure we stay ahead of the microbes. Solutions such as antimicrobial development pipelines, subscription models and alternatives such as AMR stewardship, vaccines, clinical education, diagnostics and IPC all require research and development. It is said that ‘the proof of the pudding is in the eating’; well I, amongst others in the AMR sphere, are pretty interested in what that pudding tastes like! Will the resilience strategy survive the true test of AMR? Only time will tell.


This clock effect controls electrical synapses between rods and cones so that rod-cone electrical coupling is minimal in the day and robust at night. The increase in rod-cone coupling at night improves the signal-to-noise ratio and the reliability of very dim multi-photon light responses, thereby enhancing detection of large dim objects on moonless nights.Conversely, maintained (30 min) bright illumination in the day compared to maintained darkness releases sufficient dopamine to activate low-affinity dopamine D1 receptors on cone-bipolar cell dendrites. This non-circadian light/dark adaptive process regulates the function of GABAA receptors on ON-cone-bipolar cell dendrites so that the receptive field (RF) surround of the cells is strong following maintained bright illumination but minimal following maintained darkness. The increase in surround strength in the day following maintained bright illumination enhances the detection of edges and fine spatial details.The vertebrate retina, like most other brain regions, undergoes relatively slow alterations in neural signaling in response to gradual changes in physiological conditions , or in response to gradual changes in environmental conditions . As occurs elsewhere in the brain, the modulatory processes that mediate slow adaptation in the retina are driven by extrinsic signals  and/or by intrinsic signals such as those of the circadian (24-h) clock in the retina. This review article describes and discusses the extrinsic and intrinsic modulatory processes that enable neural circuits in the retina to optimize their visual performance throughout day and night as the ambient light level changes by ~10 billion-fold. In the first synaptic layer of the retina, cone photoreceptor cells form gap junctions with rods and signal cone-bipolar and horizontal cells (HCs). Distinct extrinsic and intrinsic modulatory processes in this synaptic layer are mediated by long-range feedback of the neuromodulator dopamine. Dopamine is released by dopaminergic cells, interneurons whose cell bodies are located in the second synaptic layer of the retina. Distinct actions of dopamine modulate chemical and electrical synapses in day and night. The retinal circadian clock increases dopamine release in the day compared to night, activating high-affinity dopamine D A major goal of research on the retina and other brain regions is to understand the mechanisms that underlie the modulation of neuronal properties , synaptic communication, and neural network activity in response to changes in the environment or physiological conditions. Most regions of the central nervous system including the retina undergo relatively slow alterations in neural signaling in response to gradual changes in physiological conditions, as when activity changes to rest, or in response to gradual changes in environmental conditions, as when day changes into night. The retinal response to daily changes in the visual environment due to rotation of the Earth are particularly intriguing because the ambient (background) light level changes a great deal (~10 billion-fold) and because visual performance needs at midday are very different than those on a moonless night. Survival requires high acuity at midday and sensitivity to very dim objects on moonless nights. As occurs elsewhere in the brain, the modulatory processes that mediate slow adaptation in the retina are driven by extrinsic signals  and/or by intrinsic signals such as those of the circadian (24-h) clock in the retina.This review will focus on extrinsic and intrinsic signal modulation in the first synaptic layer (or outer plexiform layer) of the retina. In this portion of the retina, cone photoreceptor cells initiate visual processing when the ambient (background) illumination is bright and rod photoreceptor cells initiate visual processing when it is very dim or dark  increases adenylyl cyclase activity, whereas activation of the D2 receptor family (Gi receptor) decreases adenylyl cyclase activity , which function as dopamine autoreceptors, i.e., activation of D2Rs decreases dopamine release  surround. Specifically, it has been shown recently that dopamine D1Rs mediate light-dark modulation of the strength of ON-cBC receptive field surrounds by regulating GABAARs on the dendrites of the cells . In the second system, the extracellular concentration of dopamine, by activating low-affinity D1Rs, constitutes a response to extrinsic changes in the visual environment . We focus for the remainder of this review, on these dopamine-mediated extrinsic and intrinsic neuromodulatory processes, which involve these two dopamine receptor systems.These above observations of how dopamine acts as a neuromodulator in the retina thus provide evidence for the notion that there are two dopamine receptor systems in the retina. In one of these systems, dopamine, by activating high-affinity DLimulus, inhibited each other’s light responses . Under these conditions, ON-cBCs produced center and surround light responses, including surround activation and surround antagonism  when dopamine was in the bath but not when D1Rs were blocked or when dopamine was not added to the bath  and during hyperpolarizing surround light responses  and that hyperpolarizing responses to surround stimulation can be attributed to reduced GABAAR excitation. It is worth noting that these results are inconsistent with the idea that surround stimulation evokes a GABA-mediated conductance that inhibits the cells  is such that EGABA is more positive than the resting membrane potential, possibly due to activity of the chloride cotransporter NKCC on the dendrites of the cells  changing the membrane potential of HCs modulates the membrane potential of nearby BCs and the spiking of nearby ganglion cells; and (2) artificially hyperpolarizing HCs, as occurs when they respond to light stimuli, antagonizes the center light responses of nearby BCs and ganglion cells  HCs express GABA  and during hyperpolarizing surround light responses  and that hyperpolarizing surround responses can be attributed to reduced GABAAR excitation. Moreover, it is well documented that bright light uncouples HCs by activating their D1Rs , which are on cones but not HCs  or dopaminergic amacrine cells  activates cone Dunctions . In conthen cHCs .It is important to emphasize that cones (and cHCs) are able to respond to very dim scotopic light stimuli at night in the dark due to the action of the retinal clock. In other words, cones and post-synaptic neurons in cone pathways respond to very dim (low scotopic) light stimuli at night because the conductance of the rod-cone electrical synapse is high. This phenomenon was observed by maintaining retinas under dark-adapted conditions with no illumination present brighter than low mesopic  on photoreceptors, there is downstream activation of cAMP and PKA and hence increased rod-cone coupling  and intrinsic (D4R) dopamine systems function in a complementary manner and unfold slowly, i.e., .they only need to keep pace with the rotation of the Earth. In the outer retina, D1Rs and D4Rs, which have opposite effects on intracellular cAMP/PKA when activated  are located on rods and cones, and D1Rs (but not D4Rs) are located on the dendrites of cone-connected BCs and HCs (Witkovsky, via D4Rs (Ribelayga et al., 1Rs on the dendrites of cBCs and HCs in goldfish and rabbits regulates GABAAR expression and activity on the dendrites of these cells (Witkovsky, 1Rs and D4Rs in the outer retina discussed in this review can be generalized across species that contain both rods and cones.Finally, it is important to note that the modulatory effects of dopamine in the outer retina are likely conserved across vertebrate species, including mammals and non-mammals. Specifically, circadian pathway components and effects and the location of DMG and SM wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clinical pathology developed from the study of macroscopic organ and tissue changes at autopsies . The firThe diagnosis of neoplastic diseases is the main focus of clinical pathology. Reproducible and internationally comparable diagnostics need standards for the interpretation of morphology, immunohistochemical stains, molecular tests, and nomenclature. The World Health Organization (WHO) and the International Agency for Cancer Research (IARC) established with the aid of international experts a classification of tumors that has meanwhile been published in a series of reference books . In 2001HER2 amplification in breast carcinoma, KRAS mutation in colorectal cancer, and EGFR mutation in lung adenocarcinoma. Indicative of the proclaimed era of precision medicine is also the formation of molecular tumor boards, in which molecular pathological findings, especially in patients resistant to standard therapy, are discussed.Pathology has become more integrated into the clinic over the past decades, in particular through the development of targeted therapies and the associated need to determine predictive biomarkers. First historical examples for the molecular pathological determination of biomarkers for targeted therapies in solid tumors are the However, molecular pathological investigations serve not only the purpose of identifying therapy targets or resistance mechanisms but also that of diagnosing and classifying tumors that are characterized by recurrent genetic aberrations. The advancement of molecular pathology has been driven not only by the increase in knowledge derived from cancer research but also through technical innovations, in particular, the development of “next-generation sequencing” (NGS). NGS has from about approximately 2010 gradually and progressively found wider implementation in clinical pathology departments and is at present the workhorse of molecular pathology.BRCA 1/2 mutations in ovarian cancer [ALK, ROS1, and NTRK genes, although overall rare in cancers, are frequently effectively inhibited by targeted drugs and addressed in this compendium for glioma [BRCA 1/2 [KIT genes [This special issue entitled “Molecular Pathology of Cancer: The Past, the Present, and the Future” encompasses 12 publications from researchers working on a diverse range of neoplastic diseases and addressing various research questions. However, a theme common to most of the contributions is the elucidation of biomarkers for targeted therapies, such as n cancer  and homon cancer , which ar glioma  and non-r glioma . In veryr glioma . PathogeBRCA 1/2  and KIT IT genes . FurtherIT genes . MoleculIT genes .The analysis of DNA that is released from dying tumor cells and present in body fluids, the so-called “liquid biopsy,” is an emerging new diagnostic tool in molecular pathology that may aid in the identification of therapeutic targets and resistance mechanisms, in disease monitoring and the detection of minimal residual disease. In patients with brain tumors, the acquisition of diagnostic tumor tissue is often hampered by the risks of biopsy complications and deterioration of patients with advanced disease. Particularly in such scenarios, liquid biopsy could offer an alternative to the analysis of tissue as described by Baumgartner A. et al. and Madlener S. and Gojo J. ,11. The Cytology, the morphological analysis of single cells isolated from body fluids, such as pleural effusion or ascites, is the second morphological diagnostic pillar besides histology in clinical pathology. However, the sensitivity of traditional cytology is often limited by the paucity of malignant cells and the difficulty of differentiating them from normal or reactive cells. Grigoriadou et al. present data that support the molecular profiling of malignant pleural effusion with NGS .The further development of molecularly defined personalized medicine poses many challenges to the education and training of physicians and an increasing need to inform and advise patients on molecular testing and the interpretation of results .The future of clinical pathology is morpho-molecular. Traditional histopathological findings will increasingly be combined with molecular pathological results. Nevertheless, it is to be hoped that clinical pathology can again broaden its specialist spectrum beyond the currently dominant tumor pathology and that it perpetuates the great tradition as the “science of diseases.”


These findings demonstrate the untapped potential of molecular necklaces as cross-linkers and open the door to extend their advanced applications in intelligent supramolecular materials.Molecular necklaces have attracted much research attention due to their unique topological structures. Although numerous molecular necklaces with exquisite structures have been constructed, it remains a major challenge to exploit the functions and applications associated with their fascinating architectural and dynamic characteristics. Herein, we report a class of mechanically interlocked networks (MINs) cross-linked by a molecular necklace, in which multiple crown ethers are threaded on a hexagonal metallacyclic framework to furnish a cross-linker with delicate interlocked structures. The molecular necklace cross-linker possesses multiple peculiar advantages: multivalent interactions and rigid metallacycle framework guarantee robust features of MINs while the motion and dissociation of the interlocked structures bring in notable mechanical adaptivity. Moreover, the MINs could respond to the stimuli of K Constructing cross-linked networks with different topologies is attractive but challenging. Here the authors present mechanically interlocked networks cross-linked by a molecular necklace whose peculiar architectural and dynamic features endow the materials with robust yet mechanically adaptive properties. The typical features of such biomacromolecular cross-linkers could be summarized as delicate structures, multivalent interactions, and dynamic characteristics. Inspired by nature, it can be expected that synthetic cross-linkers possessing these merits would also be able to endow materials with sophisticated functions and emergent properties. Although numerous novel cross-linkers have been developed in the past decades13, the construction of polymeric networks in which the cross-linkers, simultaneously have the advantageous features of delicate structures, multivalent interactions, and dynamic characteristics, is still an enormous challenge.The implementation of many sophisticated functions in living organisms relies on their highly complex but organized architectures which are oftentimes formed via dynamic cross-linking of corresponding constituents. For example, the sliding motion of the thin filament during muscle contraction depends on a strong polymeric network wherein the thick filament serves as a cross-linker precisely and reversibly forms multiple cross bridges with thin filaments20, are derived from catenanes, in which a large macrocycle is threaded by a number of small rings (at least three). A molecular necklace consisting of (n − 1) small rings threaded on a large ring  is denoted as [n]MN21. The first molecular necklace was discovered by Sauvage et al. during the synthesis of [3]catenane22. Since then, molecular necklaces with diverse structures have been constructed33. For example, Kim and coworkers prepared a [6]MN based on intermolecular charge-transfer interactions stabilized by cucurbit[8]uril hosts  Molecular necklaces bearing multiple small rings would enable multivalent cross-linking to form robust MINs. (ii) Multiple interlocked structures along with inherent noncovalent interactions in molecular necklaces would endow the MINs with abundant dynamic properties towards external stimuli, such as force and addition of chemicals. (iii) Molecular necklaces, especially those with bulky and rigid frameworks, could act as reinforcing components to strengthen the MINs. (iv) The delicate and complicated structures of molecular necklaces would give rise to cross-linked networks with novel topologies, thereby enriching the library of MINs. Nevertheless, mechanically interlocked networks cross-linked by molecular necklaces have yet to be achieved.These exquisite architectures indicate that the structural complexity and synthetic chemistry for molecular necklaces have reached a high level. However, the exploitation of functions and applications associated with their unique structural characteristics is severely lagging behind their frameworks themselves, which represents an urgent issue for promoting the further development of molecular necklaces. From the point of view of topological structures, we envision that the molecular necklaces hold great potential as a bioinspired cross-linker for the construction of mechanically interlocked networks (MINs)2 showed notable mechanical performance including stiffness (Young’s modulus = 47.3 MPa), strength (breaking stress = 12.1 MPa), and toughness (49.3 MJ/m3). Moreover, we took advantage of K+-responsiveness of the host–guest recognition and Br−-responsiveness of the metal-coordination to modulate the mechanical properties of the MINs via dethreading the DB24C8 rings from the metallacycle and decomposition of the metallacycle, respectively.Herein, we report a de novo chemical design of MINs cross-linked by a molecular necklace in which the multivalent interlocked structures and the hierarchical organization of dynamic interactions endow the resultant MINs with mechanically robust yet stimuli-responsive properties. In specific, the molecular necklace consists of multiple dibenzo-24-crown-8 motifs  and a hexagonal metallacycle (large ring) formed by coordination-driven self-assembly Fig. , wherein42 , respectively.The polymer adopted in this work is a DB24C8-functionalized polynorbornene derivative prepared by ring opening metathesis polymerization (ROMP)42 Fig. . The mol42 Fig. , which w1H NMR spectra were measured to prove the formation of MINs. As shown in Supplementary Fig. 1−H4 signals of the hexagonal metallacycle underwent significant shifts compared with those of the precursor of 1,2-bis(pyridinium)ethane  test was performed to disclose detailed information of the molecular necklace 47. The model fit to the SAXS data gave Rg and Rs with the values of 7.01 and 1.02 nm, respectively, which were close to the diameters of metallacycle (6.70 nm) and DB24C8 (0.83 nm)  ligand  ligand  has been utilized to cross-link the DB24C8-functionalized polymer through the formation of [2]pseudorotaxane moiety  of MIN-2 and control-2 were always larger than their loss moduli (E″) over the entire frequency range, showing high resistance to flow. On the contrary, the control-1 had comparable elastic and viscous moduli in a broad frequency range with a distinct frequency dispersion. As mentioned above, the formation of a coordinate bond was not possible in the structure of control-1, and the frequency dispersion might stem from a slight entanglement of the polymer chains. In addition, the elastic moduli of the three samples showed the following order: MIN-2 > control-2 > control-1, indicating the high stiffness of MIN-2. Furthermore, stress relaxation was employed to reveal the structural features of the three samples. As shown in Fig. 1 was released instantaneously and almost completely relaxed in about 100 s, whereas the stress relaxation for MIN-2 and control-2 were much slower, and finally, part of stress was kept. These results of frequency sweep and stress relaxation experiments further proved the effective cross-linking role of a molecular necklace in MIN-2, and also confirmed the structures of the two controls: control-2 is a cross-linked network but no significant cross-linking exists in control-1.To better showcase the peculiar cross-linking role of molecular necklace, two control samples were also designed. In the structure of control-med Fig. . The sto2, 3.02 MPa for control-1, and 29.9 MPa for control-2  was also much higher than those of control-1 (2.64 MPa) and control-2 (7.09 MPa). The multivalent interactions and the reinforcing effect of the rigid skeleton may still be responsible for the superior strength of MIN-2, but the unique dynamicity of the molecular necklace made a contribution at the same time (vide infra). Besides, in terms of toughness, MIN-2 had the best performance as well, which may be also strongly affected by the force-induced dynamicity of the molecular necklace as discussed in the following section.More striking contrasts were shown by the stress−strain curves of the three samples Fig. . The Youl-2 Fig. , which w2 at different deformation rates were displayed in Fig. 54. As for MIN-2, the dynamic bonds should be attributed to the supramolecular interactions of host–guest recognition and/or metal-coordination in molecular necklaces (vide infra). Subsequently, the force-responsive behaviors of the two kinds of supramolecular interactions and even the whole molecular necklace are discussed below.Among the multiple roles of molecular necklace cross-linker, its dynamic behavior upon force is a crucial feature and has a significant effect on the mechanical properties of MINs. Stress–strain curves of MIN-56. Such an estimation was also supported by the stress relaxation experiments at different temperatures for MIN-2 and control-2. As shown in Fig. 2 which remained notable applied force even at a temperature of 120 °C, and complete relaxation occurred until the temperature was up to 160 °C. By contrast, a rapid stress relaxation was observed for control-2 at the temperature of 120 °C  ligand . In this stage, the wheel DB24C8 reaches the site of platinum which bears two triethylphosphine groups and is bulky enough to serve as a stopper. And this terminal stage is an enthalpic regime where bond deformation takes place. After the culmination of this regime, dissociation of metal-coordination occurs. Therefore, the dynamic process could be summarized as that the molecular necklace successively undergoes dissociation of host–guest recognition, sliding of DB24C8 wheel, and dissociation of metal-coordination in a synergistic fashion upon stress, which could efficiently equalize force and dissipate energy, and thereby imparts mechanical robustness and adaptivity to our MINs. In addition, the advantages of such a synergistic dynamicity were further highlighted through its comparison with control-2. The elongation curve for the cross-link in control-2 was roughly equal to the first and second stages in that of molecular necklace, thus possessing shorter stretched distance and lower dissociation energy  simulationMore information about the mechanical responsiveness of molecular necklace to external stress was provided by creep and recovery experiments under different applied loads Fig. . A typic2 are much superior to those of control-2 in all aspects including stiffness, strength, stretchability, and toughness  ligand to be recognized by the DB24C8 moieties. The 1H NMR spectra showed that the signals assigned as the complexed peaks of bis(pyridinium) ligand and DB24C8 completely disappeared after being treated by KPF6, and the bis(pyridinium) ligand signals were roughly similar to those of the pure metallacycle , the sample had very poor mechanical performance in light of stiffness (0.21 MPa), strength (0.05 MPa), and toughness (0.85 MJ/m3), exhibiting a sharp contrast with the virgin one . In addition, it is disclosed that the hexagonal metallacycle could be separated from the molecular necklace by the introduction of competitive K+, and also be completely decomposed in the presence of Br−, exhibiting abundant dynamic and responsive properties. We believe that the molecular necklace cross-linked MINs hold great promise for the development of mechanically robust yet adaptive supramolecular materials, particularly, for those with precise structures and functions.In this work, we have designed and constructed a class of mechanically interlocked networks cross-linked by a molecular necklace in which multiple DB24C8 moieties are threaded on the metallacyclic core. Due to the unique dynamic interactions of the molecular necklace, the MINs are prepared in a convenient method by simply mixing hexagonal metallacycle and polymer decorated with DB24C8 moieties. Molecular necklace as a cross-linker integrates multiple advantages into the MINs to improve their mechanical properties. Multivalent interactions based on the multiple interlocked structures and the role of skeleton originated from the rigid and bulky metallacycle guarantee the stiffness and strength of the network. Furthermore, the force-induced motion of the interlocked structures and dissociation of the dual supramolecular interactions provide a unique and effective energy dissipation pathway to further reinforce the robustness of MINs. As a result, the MINs exhibit good mechanical performance in terms of stiffness (Young’s modulus = 47.3 MPa), strength (breaking stress = 12.1 MPa), and toughness (49.3 MJ/mSupplementary InformationPeer Review File


Atractylodis macrocephalae Rhizoma (AMR) is a famous classical Chinese traditional medicine (CTM), which has been used as a tonic for many diseases for thousands of years. In ancient China, it was used as a supplementary food for beauty in the palace. In preliminary studies, the function of whitening skin and the significant inhibiting effect on tyrosinase (TYR) which is the reactive enzyme in the composition of melanin of AMR were discovered, and the relevant research was rarely reported. In this study, high-performance liquid chromatography (HPLC) along with partial least squares regression analysis (PLS) was applied to survey the coherence between the chemical constituents and the inhibiting activity of 11 batches of AMR on TYR activity. The results of PLS showed that the chromatographic peaks 11 (atractylenolide III) and 15 could be important effective ingredients of the inhibition TYR activity as ascertained by spectrum-activity relationships. Furthermore, TYR inhibitory activity of atractylenolide III was validated by in vitro test by β-arbutin served as a positive control drug. The results of the in vitro test and the molecular docking showed that atractylenolide III has high TYR inhibitory activity and could link to the residues in TYR catalytic pocket. Therefore, bioassay, molecular docking, and spectrum-activity relationships are appropriate for linking the quality of samples with pharmaceutical-related active ingredients. And our studying would lay a theoretical foundation for applying the water extracts of AMR in whitening cosmetics. The reaction solutions were prepared according to X), and TYR inhibition rate was set as the dependent variable (Y).The software “Chinese traditional medicine chromatographic fingerprint similarity evaluation system 2012 A Edition” was used to adjust the retention times of each peak, and the peak area (PA) was processed by equalization. Then, the quantitative data were obtained. The PLS regression equation was set up with the software SIMCA14.1; the peak area was taken as the independent variable  was taken as the 3D structure of TYR [x, y, z) =  and the grid center was designed to  = . In each simulation procedure, progress with default parameters operates from AutoGrid and AutoDock. Lamarckian genetic algorithm (LGA) was adopted to ascertain the most appropriate ligand binding orientations.The AutoDock4.2 program was applied in docking simulations. The crystal structure of e of TYR . We perfPrecision, repeatability, and stability were validated for the analytical method. In precision testing, precision of relative retention times (RRTs) and relative peak areas (RPAs) did not exceed 0.02% and 4% in RSD, respectively; the similarity was 1. Repeatability of RRTs and RPAs was less than 0.11% and 4% in RSD, and the similarity was greater than or equal to 0.998. The stability was estimated by testing one sample solution preserved at lab environmental temperature after 0, 6, 8, 12, and 18 h. The RSDs of RRTs and RPAs of the common peaks were less than 0.1% and 4%, respectively; the similarity was more than 0.999. These results suggested that the AMR experimental system for fingerprint analysis is steady and dependable.The fingerprint of Atractylodes III reference substances is exhibited in HCA was applied to identify AMR from different production areas based on different clusters and the similarity of fingerprints. The common peak area in 11 batches of AMR was used as an indicator, and the cluster analysis was performed by software SIMCA14.1. The results are shown in In this study, the PCA of 11 batches of AMR was calculated; in 16 principal components, the cumulative contribution of the variance of the first five principal components was 91.3%. The score matrix of the first two components would be used for analysis as the cumulative variance exceeded 65.9%. Therefore, in the absence of some information, construct a two-dimensional plane of the principal component that the abscissa was a principal component and the ordinate was another principal component. Then, the 11 samples were projected onto the 2D plane so that their natural gathering was observed . It was Each dot in the load diagram represented a chromatographic peak, which represents the contribution of each chromatographic peak to the comprehensive effect of the principal components. The weight value of the variables can reflect the correlation between the chemical composition and the sample to the greatest extent. The farther away from the origin of the load diagram, the greater the variable weight. The result is shown in R2X and R2Y characterize the explanatory rate of the model to x and y matrices, individually, and Q2 characterizes the predictive ability of the model. The results showed that the values of R2X, R2Y, and Q2 were 0.939, 0.992, and 0.583, respectively, all of which were greater than 0.5, indicating that the model could distinguish the two groups of samples and had a better fitting and predictive ability in the data processing. It could be used to distinguish AMR from different production areas  in the OPLS-DA model can reflect the contribution of each chromatographic peak to the sample. The VIP values of the 16 chromatographic peaks reflected the influence power on every AMR sample. Based on the selection principle that the VIP value was more than 1.0 and the error bar was less than the origin , the 4 iThe analysis results of the load diagram are shown in The inhibitory effects of AMR from different producing areas on TYR activity were determined by the biochemical enzyme method. The effects of samples on TYR activity are listed in In this article, PLS was applied to analyze the spectrum-activity relationship. The partial regression coefficient and VIP value are shown in Figures β-arbutin as a positive control. The results showed that the inhibition rates of Atractylodes III and β-arbutin on TYR activity were 63.68 ± 2.36% and 94.85 ± 0.35%, respectively, when the samples' concentration was 1 mg/mL.The inhibitory effect of Atractylodes III on TYR activity was determined by biochemical enzyme method in vitro with 2+) each stabilised by three histidine residues  at the enzyme's active center. In this study, Atractylodes III is a small molecule compound located in the active center of the enzyme and interacts with the surrounding residues . Remarkably, Atractylodes III formed a stable hydrogen bond to Asn 260 with a bond length of 1.9 Å.Molecular docking was used to study the binding mechanism of Atractylodes III interacting with TYR Figures . The binIn this study, a systematic method was established connecting the HPLC fingerprints with chemometric analysis to distinguish AMR samples from different origins, and the whitening effect of AMR was proved by the biochemical enzyme method. Spectrum-effect relationships indicated that the chromatographic peaks 1, 2, 5, 6, 9, 10, 11, 15, and 16 were positively correlated with AMR inhibition effect on TYR activity, among which peaks 11 (atractylenolide III) and 15 might be important active constituents. Meanwhile, atractylenolide III has high TYR inhibitory activity in vitro test, and the result of molecular docking showed that its mechanism might be related to the binding of amino acid residues in TYR catalytic capsule. Therefore, these results proved that bioassay, molecular docking, and spectrum-activity relationships are appropriate for linking the quality of samples with pharmaceutical-related active ingredients.


The nanofibers were then modified with silver nanoparticles (AgNPs) via the dip-coating method. The silver-containing nanofiber meshes showed good cytocompatibility and interesting immunomodulatory properties in vitro, together with the capability of stimulating the human beta defensin 2 and cytokeratin expression in human keratinocytes (HaCaT cells), which makes them promising materials for wound dressing applications.Wound dressings are high performance and high value products which can improve the regeneration of damaged skin. In these products, bioresorption and biocompatibility play a key role. The aim of this study is to provide progress in this area via nanofabrication and antimicrobial natural materials. Polyhydroxyalkanoates (PHAs) are a bio-based family of polymers that possess high biocompatibility and skin regenerative properties. In this study, a blend of poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxyoctanoate- Nanofibers have received much attention in biomedical applications lately. These materials have a high surface area, porosity, versatility in material choice and morphological similarities to the naturally occurring extracellular matrix, which enhances cell attachment and proliferation, transport of nutrients, gas exchange and waste excretion. Furthermore, the physical and/or chemical properties of the electrospun nanofibers can be modified by encapsulation and or immobilization of bio-active species to receive specific biological reactions . The uniNanofibers are most frequently produced by the electrospinning technique, as it is an inexpensive and reliable method to create fibers with diameters ranging from the nanometer scale to a few micrometers ,6. Many co-3HO). Scl-PHAs typically have a higher molecular weight, crystallinity and brittleness, while mcl-PHAs are more elastomeric. Mcl-PHAs and their copolymers are preferred for biomedical applications where flexible biomaterials are required, such as heart valves or controlled drug delivery [g), which limits their spinnability and causes sticky and fused fibers. Several groups have investigated the electrospinning of different scl-PHAs, including P(3HB), poly P(3HB-co-3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(HB-co-3HHx) and poly P(3HB-co-3HV-co-3HHx) [co-3-hydroxyhexanoate) P(3HO-co-3HHx) nanofibers by blending with a scl-PHA, P(3HB-co-3HV) [Polyhydroxyalkanoates (PHAs) are fully biodegradable polymers which are biosynthesized with high purity by a wide range of Gram-positive and Gram-negative bacteria from inexpensive waste carbon sources ,15,16,17delivery ,22. The co-3HHx) ,27,28,29-co-3HV) ,31. Elec-co-3HV) .2, silver nanoparticles (AgNPs) have been investigated most intensively in the medical field due to their excellent chemical stability, high conductivity, catalytic activity, localized surface plasma resonance as well as their antibacterial and anti-inflammatory properties and efficacy against a broad range of bacteria found in chronic wounds [co-3HV) suspensions [co-3HV) nanocomposites [co-3HV) film coated with P(3HB-co-3HV)/AgNPs nanofibers [In recent years, metal nanoparticle-containing electrospun nanofibers have attained widespread attraction for various applications such as bio/gas sensing, catalysis, and antimicrobial wound dressings ,34. Amonc wounds ,36. AgNPc wounds ,38. Sevepensions , in situmposites  and an anofibers , in whicAntimicrobial biomaterials are very useful in a number of biomedical applications related to many diseases, especially those concerning or deriving from skin damage ,43. SomeThe biological mediators of innate immunity and inflammation are cytokines, biological molecules that act as soluble mediators of natural immunity and the immune response . The mosin-situ reduction of silver nanoparticle precursor  to silver nanoparticles in a polymer solution [in-situ reduction process, N,N dimethylformamide (DMF) is used as a reductant, which is a very harmful solvent, and the process itself takes a long time, approximately 5–10 days. Moreover, it is not possible to achieve a 100% successful reduction of silver precursor to silver nanoparticles. In addition, in the physical reduction processes, UV-light is used and it is not a very cheap technique in terms of the consumed energy and time. However, dip-coating method enables the obtaining of silver nanoparticle containing nanofibers via a simple, cheap, environmentally friendly technique for biomedical applications. To the best of our knowledge, PHA-based nanofibers were not functionalized with silver nanofibers via dip-coating. For this purpose, we proposed an alternative method for AgNP deposition via dip-coating technique using a colloidal silver solution that allows a timesaving, simple and reagent-free way to produce antimicrobial P(3HB)/P(3HO-co-3HD) membranes. We first examined the electrospinning of elastomeric P(3HO-co-3HD) using an eco-friendly solvent mixture, but wet fibers were obtained due to the low Tg of the polymer. In order to tune the electrospinnability, elasticity and mechanical properties, we prepared blends of the elastomeric mcl-PHA, P(3HO-co-3HD) with scl-PHA, P(3HB) which has a higher Tg and brittleness. Since scl-P(3HB) could not be dissolved with the previously used eco-friendly solvent system, a chlorinated solvent mixture was used for the electrospinning of P(3HB)/P(3HO-co-3HD) blends. The electrospun P(3HB)/P(3HO-co-3HD) nanofibers were then modified with AgNPs via a dip-coating method. The antimicrobial properties of the modified nanofibers were investigated and were found to be promising for wound dressing applications. Conventional wound dressings such as acrylic, polyethylene or polypropylene-based pads usually suffer from a lack of recycling—they are either incinerated or landfilled. In our study, the bio-based material of the nanofibrous mats offer organic composting as an option for waste management. Moreover, the high polarity of the PHAs and the porosity of the fibrous meshes will provide advantages such as easy and proper impregnation of the agents, odor suppression, efficacy of treatment and easy removal of the dressing from the skin. Having a properly functional bioactive and biocompatible wound dressing which combines a reduced environmental impact with remarkable antibacterial properties would greatly improve the portfolio of wound dressing products.The goal of this paper was to design nanofibrous wound dressings from bio-based polymers  in order to contribute to reducing environmental pollution and to provide antimicrobial features to the PHA membranes using AgNPs. For the production of nanofibers with silver nanoparticles, there have been suggested some chemical and physical methods such as solution ,47,48, asolution ,50, etc.co-3HD) was created within 15 L bioreactors  containing a 10 L mixture of Pseudomonas mendocina CH50 and 20 g/L glucose. Subsequently, a two-stage batch fermentation was applied such as described in Basnett et al., 2020 [Bacillus subtilis OK2 was used with single-stage batch fermentation. The P(3HB) produced exhibited a Mw of 796 ±40 kDa, 2–4% elongation at break, Tg = 2.9 °C, and the mcl-PHA P(3HO-co-3HD) exhibited a Mw of 350 ± 60 kDa, 440% elongation at break, and Tg between 48 °C and 44 °C. A specific purification protocol was applied to obtain ultra-pure PHAs as described in Basnett et al., 2020. Aqueous colloidal silver solution  was purchased from Argenol Laboratories . Lithium bromide (LiBr), ethanol (EtOH), and Dulbecco’s phosphate-buffered saline were purchased from Sigma-Aldrich . The HaCaT cell line was provided by CLS-Cell Lines Service, Eppenheim, Germany. Dulbecco’s Modified Essential Medium (DMEM), L-glutamine, MgCL2, penicillin, streptomycin (penstrep) and fetal calf serum were bought from Invitrogen . Alamar Blue was obtained from Thermo Fisher Scientific . LC Fast Start DNA Master SYBR Green kit was provided by Roche Applied Science . The solvents used for solubility studies and electrospinning include acetone, anisole, 2-butanol, butyl acetate, chloroform, dichloromethane, diethyl ether, dimethyl carbonate, ethyl acetate, and methyl ethyl ketone, which were purchased from Sigma-Aldrich and have a purity above 98%. PanCK monoclonal antibody, hydrogen peroxidase, citrate buffer, standard avidin–biotin conjugated complex and DAB staining were provided by Dako . Formalin was bought from Bio-Optica .The P. Spectra were recorded in the range of 200–800 nm with a reflection data interval of 4 nm. The obtained reflection was converted into Kubelka–Munk (K–M). Electrospinning of P(3HO-co-3HD) was performed in a climate chamber (Weiss WK-340/40) with temperature of 10–40 °C and relative humidity (RH) of 10–40%. The nanofibrous membranes were dip-coated in an aqueous colloidal silver solution at room temperature. This coating was performed at a speed of 170 mm/min with the aid of computer-regulated dip-coating equipment (KSV Instruments). The entire coating process happened in a clean room . The samples were dip-coated in a silver solution (4000 ppm) by immersion for 1 min 3 successive times.The nanofibers were analyzed morphologically by an FEI Quanta 200 F FE-SEM and a Phenom ProX SEM . The samples were coated with gold using a sputter coater  prior to analysis. The nanofiber diameters were calculated via ImageJ software (1.51v). Additionally, the histogram graphs of the nanofibers were obtained from the measured diameter values by using the Origin software (Origin 2017). The average nanofiber diameter and the standard deviation were based on 50 measurements per sample. In addition, the weight percentage (wt%) and the atomic percentage of silver nanoparticles on the silver nanoparticle-coated samples were determined by an FEI Quanta 200F FE-SEM-EDX. An attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrometer  from Thermo Scientific was used to record the IR spectra of the samples. The spectra were collected after 32 scans for each sample between 4000 cmco-3HD) was electrospun in a solvent mixture containing anisole , acetone  and ether  with a flow rate of 0.5 mL/h, tip-to-collector distance of 40 cm and voltage of 40 kV in a humidity (relative humidity of 10–40%) and temperature-controlled (10–40 °C) climate chamber. A quantity of 0.002 g/mL LiBr was added into the electrospinning solution to increase the conductivity.P(3HO-co-3HD) is elastomeric and has a low Tg, which creates difficulties in spinning such as fused fibers, it was blended with P(3HB) that has higher Tg and tends to produce fibers with better morphology.Since P(3HO-co-3HD)  and a 5 wt% of P(3HB)/P(3HO-co-3HD)  were prepared in a chloroform/2-butanol  solvent system and stirred for 24 h at room temperature. A quantity of 0.002 g/mL LiBr was added into the electrospinning solution to increase the conductivity. The relative humidity (RH) was 38 ± 2.1% and the temperature was 22 ± 1.6 °C during the electrospinning process for the 5 wt% of P(3HB)/P(3HO-co-3HD) solution.Polymeric solutions at 11 wt% and 5.5 wt% of P(3HB)/P(3HO-co-3HD)  polymer solutions, a voltage of 40 kV, a 40 cm tip-to-collector distance and a flow rate of 0.5 mL/h was used. Nanofibers were collected on a chitosan non-woven carrier that was backed by an aluminum foil.For the P(3HB)/P(3HO-co-3HD) was selected as 1/3 (w/w), which was suitable to produce uniform and continuous fibers with low diameters that were also not brittle.The drawback of P(3HB) is its brittleness. As the aim was to produce nanofibers with good morphology without losing the elasticity, the ratio of P(3HB)/P(3HO-co-3HD)  solution, electrospinning parameters of 20 kV applied voltage, 15 cm distance between the needle tip and the collector, 1 mL/h feed-rate were applied, and nanofibers were collected on a flat collector which was covered by aluminum foil.For the P(3HB)/P nanofibers were modified with an aqueous colloidal silver solution (4000 ppm) via a dip-coating technique. The size of the nanoparticles in the solution ranged between 10 and 80 nm. For dip-coating, the nanofibrous scaffolds with a size of 30 mm × 60 mm were prepared, clamped from two sides and hung on the portable holder of the dip-coater. The samples were dipped into the colloidal silver solution and withdrawn with a speed of 170 mm/min. Three different protocols were tried, including dipping and withdrawing immediately, dipping and withdrawing after 1 min and dipping and withdrawing 3 successive times. In all cases, the withdrawing speed was the same and the samples were washed with distilled water and dried.The P(3HB)/P(3HO-co-3HD) nanofibers have been sterilized in EtOH overnight, then DPBS was used to rinse the nanofibers three times before usage. DMEM was used to culture the cell line HaCaT combined with 1% Penstrep, 1% glutamine and 10% serum of calf fetuses at 37 °C, combined with 5% CO2 and humidified air. The cells were plated on the glass slides coated with the fibrous samples and cultured for 6 h and 24 h.Blank and AgNP-containing P(3HB)/P was then determined with the aid of a spectrophotometer  and a reading at two wavelengths . At the end, the relative reduction of Alamar Blue (%ABred) was determined by associating the absorbance and the dye’s molar extinction coefficients at the aforementioned wavelengths, following the protocol suggested by the manufacturer. The obtained values are entered in the equation below, in which: c = control, s = sample, and λ = absorbance:The results obtained are expressed as %ABred, which is related to metabolically active cells.co-3HD) nanofibers were determined. The HaCaT cells were seeded within 12-well plates up to a confluence of 80%; therefore, they were incubated for 6 h and 24 h with the glass slides coated by the nanofibers (n = 3). At these respective moments, all the RNA was isolated with the aid of Trizol. Subsequently, 1 µM of RNA was reverse-transcribed into the complementary DNA (cDNA). This was carried out with the aid of random hexamer primers at 42 °C for a duration of 45 min such as stated within the guidelines of the manufacturer. A LC FAST START DNA Master SYBR Green kit was used to carry out the quantitative reverse transcriptase polymer chain reaction (qRT-PCR). A quantity of 10 ng of total RNA and 2 µL of cDNA were used in a final volume of 20 µL with 3 mM MgCl2 and 0.5 µM sense and antisense primers, such as depicted in The skin immunomodulatory properties of Ag-loaded and plain P(3HB)/P. The antigenicity was retrieved by treating the slides at 100 °C in a steamer with 10 mMol citrate buffer (pH 6.0) for 60 min. Subsequently, the sections were immersed for 20 min in a methanol solution with 0.3% hydrogen peroxidase to stop the activity of the endogenous peroxidase. Afterwards, nonspecific binding was reduced by incubation in a 2.5% blocking serum. The specimens were incubated for 90 min at 37 °C with the primary antibody PanCK. Both the processes of the standard avidin–biotin conjugated complexing and the subsequent DAB staining were automated according to the surgical pathology guidelines . The immunopositive epithelial cells were highlighted by the brown precipitate on the cell membranes and in their cytoplasm. Negative controls were obtained by subtraction of primary antibody during the standard IHC procedure.co-3-hydroxydecanoate) P(3HO-co-3HD) is a biocompatible and biodegradable polymer with high ductility (440% elongation at break), which makes it an ideal candidate for body contact medical applications such as wound dressings [g (below −40 °C) to its medium chain length alkane side-groups and its copolymeric nature [The non-toxic biosourced poly nanofibers can be obtained from an eco-friendly solvent mixture that contains anisole, acetone and ether  solution in an anisole/acetone/ether (30%/35%/35%) mixture at 10 °C and RH of 40%, fine nanofibers are obtained with an average diameter of 300 nm  solution in three different anisole/acetone/ether solvent compositions (40%/30%/30%), (30%/35%/35%) and (10%/45%/45%) with a RH of 40%.The importance of solvent composition was evaluated using 10% P(3HO-The (30%/35%/35%) anisole/acetone/ether mixture b,e was fco-HD) nanofibers become increasingly fused at longer electrospinning times (>30 min) at the same spot, preventing their applicability as a stand-alone nanofiber membrane, they could also be electrospun onto a chitosan carrier as a thin layer (co-3HD) nanofibers are sufficient. One of the main reasons for the fused morphology of the nanofibers at room temperature electrospinning is the low Tg of P(3HO-co-3HD). In order to eliminate this effect, a blend of P(3HB) and P(3HO-co-3HD) can be used, which is discussed in the subsequent subsection.Although the neat P nanofibrous membranes cannot be electrospun at room temperature conditions and always need a backing carrier structure, the brittle P(3HB) has a proven track record of electrospinnability with halogenated solvents, avoiding fiber fusion altogether [co-3HD) with the standalone and non-fused characteristics of P(3HB) nanofibers.While standalone P. This requires the substitution of typical P(3HB) electrospinning solvents such as chloroform or dichloromethane. For this, we could turn to the literature on P(3HB) extraction  from bacterial cultures, which is, however, mostly based on the same chlorinated solvents [2O (40%/50%/10%), anisole/DMSO (60%/40%) and dimethyl carbonate/DMSO (80%/20%). However, immediate solidification occurs at room temperature, making electrospinning (at room temperature) impossible.Ideally, a low toxicity solvent system is used for blend electrospinning P(3HB) and P mixture, which results in neat but brittle nanofibers with an average diameter of 775 ± 106 nm.Hence, for a successful electrospinning, a chlorinated solvent was included, and pure P(3HB) nanofibers were electrospun from a chloroform/2-butanol  solvent system, the ratio of P(3HB)/P(3HO-co-3HD) should be considered carefully as a high amount of P(3HB) can cause loss of elasticity, leading to brittle fibers, but a high P(3HO-co-3HD) may lead to fused fibers at large membrane thicknesses.When electrospinning a blend of both polymers with the chloroform/2-butanol   solutions onto chitosan carriers as wound dressing candidates, with average diameters of 470 ± 330 nm and 3420 ± 2100 nm, respectively /P blends were continued to obtain nanofibers that are as fine as possible while remaining flexible and bead-free. For this, a 5 wt% of P(3HB)/P(3HO-co-3HD)  was prepared in chloroform/2-butanol  (co-3HD) nanofibers was 410 ± 180 nm with bead-free and homogenous morphology (Optimization studies on the electrospinning of P(3HB)/P c,d. The rphology c. These co-3HD) nanofibers based on the optimum 1/3 (w/w) blends. The P(3HB)/P(3HO-co-3HD) nanofibers were modified with silver nanoparticles (AgNPs) via a dip-coating technique to provide anti-microbial properties to the scaffolds. For this purpose, the nanofibers were first dipped into a water solution with colloidal silver (4000 ppm), containing particles in a size range of 10–80 nm, and subsequently taken out at a speed of 170 mm/min.The modification and characterization processes were conducted on the P(3HB)/P(3HO-co-3HD) nanofibers were dipped in a beaker containing the colloidal silver solution and taken out immediately, followed by washing with distilled water and drying. In the second trial, the nanofibers were immersed into the aqueous silver solution and kept in the solution for 1 min. Afterwards, the samples were taken out, washed with distilled water and left to dry. In the last protocol, the nanofibers were dipped into the aqueous silver solution and taken out immediately for three successive times, followed by washing and drying.Three different protocols were examined in order to achieve the best silver deposition. In the first trial, P(3HB)/P(3HO-co-3HD) nanofibers (co-3HD) nanofibers was clearly observed (co-3HD) nanofibers was confirmed by EDX characterization and determined as 18 wt% and 5.3 atomic percentage, respectively.SEM images showed that the first protocol allowed the immobilization of the AgNPs. However, the distribution of the nanoparticles was not homogeneous and dense . It was nofibers . Additioobserved . Subsequco-3HD) nanofibers and AgNP-loaded P(3HB)/P(3HO-co-3HD) nanofibers are given in −1 and 3250 cm−1 represents the stretching band of the amine groups. The peaks at 2924 cm−1 and 2848 cm−1 can be attributed to asymmetric CH3 and symmetric CH2 vibrations, respectively. The peak at 1730 cm−1 is assigned to the ester carbonyl C = O stretching mode. In addition, the peak at 1279 cm−1 occurs because of C-N stretching and N-H bending. Moreover, as consistent with the literature, nearly the same spectrum was observed for AgNP containing P(3HB)/P(3HO-co-3HD) nanofibers [FTIR-ATR spectra of neat P(3HB)/P nanofibers was also characterized by UV-Vis spectroscopy. While AgNP containing P(3HB)/P(3HO-co-3HD) nanofibers show a strong absorbance peak at 417 nm, neat P(3HB)/P(3HO-co-3HD) nanofibers do not show any peaks between 250–600 nm. This is in line with literature stating that the characteristic silver surface plasmon resonance band is attributed to the maxima between 400 and 420 nm, depending on the size of the particle, stabilizing molecules, and dielectric constant of the medium [The AgNP deposition on the P(3HB)/P nanofibers did not show significant cytotoxic activity, which is in fact indicated by a dye reduction of 100% and 105%, respectively. Keratinocytes, which completely cover the outmost layer of our body, lead the immune reaction  in the skin by the production of pro-inflammatory, anti-inflammatory and antimicrobial  molecules in response to injury and external stimulation. The complexity of immune reactions occurs via the production those cytokines and antibacterial molecules with a different time scale, which ultimately ensures an efficient tissue response to foreign agents.The biological activity of the AgNP-containing nanofibers was further examined. Alamar Blue assay was performed after a 24 h presence of HaCaT cell culture on the substrates. This shows that both the plain and the AgNP-containing P(3HB)/P(3HO-co-3HD) nanofibers, which did not show appreciable differences in the basal production of these molecules by HaCaT cells in culture, the AgNP-containing P(3HB)/P(3HO-co-3HD) nanofibers were able to upregulate the most important proinflammatory cytokines after 6 h, namely, IL-1(α and β), IL-6 and IL-8, in order to initiate the healing process (co-3HD) nanofibers can induce the mRNA production of HBD-2, thus stimulating the innate defense capability of the skin from harmful entities. This aspect is very relevant in the wound repair processes. In addition, such an indirect antimicrobial activity is desirable as the need for antibiotic drugs is reduced. The TGF β expression was insignificantly downregulated.Different from the plain P(3HB)/P(3HO- process . Later oco-3HD), as shown in In addition, HaCaT cells were able to express cytokeratin upon culture for 7 days onto the plain and Ag-loaded P(3HB)/P(3HO-co-3HD) scaffolds could be prepared from the eco-friendly solvent system, as the fibers were fused due to the low Tg and elastomeric nature of the polymer. The morphology and elasticity of the nanofibers were optimized by blending P(3HO-co-3HD) with P(3HB), which has a higher Tg but lacks appropriate mechanical properties required for wound dressing applications due to its more brittle nature. Since P(3HB) had poor solubility in eco-friendly solvents, a chlorinated solvent-based system consisting of chloroform and 2-butanol was utilized for the electrospinning process, and P(3HB)/P(3HO-co-3HD) nanofibers with an average diameter of 410 ± 180 nm could be obtained. Afterwards, the surface of the fabricated P(3HB)/P(3HO-co-3HD) nanofibers was modified with AgNPs via a dip-coating method. The amount of AgNPs on the P(3HB)/P(3HO-co-3HD) nanofibers was determined as 18.05 wt% and 5.29 atomic % by EDX characterization. Metabolic activity, immunomodulatory, indirect antimicrobial activity and cytokeratin expression of both the modified nanofibers were examined in vitro using HaCaT cells, revealing the potential ability to effectively contribute to the wound healing process. In fact, also in presence of AgNPs, the metabolic activity of the cells was above 100%. Moreover, the most important ILs were modulated in 24 h, and HBD-2 was upregulated with respect to the basal condition of HaCaT cells specifically in Ag-loaded P(3HB)/P(3HO-co-3HD). Finally, a proper keratinocyte function was confirmed by the expression of cytokeratin at a protein level after 7 days in culture. These preliminary findings highlight the suitability of the produced AgNP-functionalized PHA-based nanofibers for biomedical applications, particularly as wound dressings.The aim of this study was to produce wound dressing prototypes from bio-based polymers, namely PHAs. Only very thin layers of pure P(3HO-


The most widely used commodity polymers in the rigid packaging industry are polypropylene (PP) and high-density polyethylene (HDPE). For example, blow molding grade of HDPE as a bottle and injection molding grade of PP as a cap are often used to produce detergent bottles. Therefore, the recycled HDPE bottles from post-consumer waste include PP as a contaminant originated from PP bottle caps. To simulate mechanical recycling of bottle waste, the mechanical properties of HDPE-rich-HDPE/PP virgin model blend were studied. For compatibilization, ethylene-based olefin block copolymer, propylene-based olefin block copolymer, ethylene propylene random copolymer, and styrene-butadiene-styrene triblock copolymer were chosen as potential compatibilizer candidates. Contact angle measurements, morphological analysis, adhesion tests of compatibilizer candidates to polymer blend components and the tensile as well as tensile impact properties of the ternary blends were studied. It was found that the ethylene-based olefin block copolymer was the most effective compatibilizer resulting in a return of mechanical properties to those of neat vHDPE due to its ability to encapsulate dispersed vPP particles in a vHDPE matrix (core-shell morphology) and the best adhesion to polymer blend components. Polypropylene (PP) and polyethylene (PE) are the most abundant thermoplastic resins with a share of 49.2% of European plastic demand in 2019 ,4,5,6,7.A method for the improvement of interfacial adhesion between two phases and thereof mechanical properties of immiscible blends by the addition of proper pre-made block or random copolymer is known as compatibilization . This prThe addition of compatibilizer to a binary blend results in a ternary blend. The morphology determines final mechanical properties. According to the literature, ternary blends morphology can be estimated using spreading coefficients calculated by interfacial tensions ,12,13,14According to the literature, a typical PP contamination range in HDPE is 3–12 wt% for bottle waste stream from blow molding applications . TherefoIn order to determine the most effective compatibilizer for the compatibilization of 10 wt% vPP (injection molding grade) contaminated vHDPE (blow molding grade) model virgin blend (HDPE-rich-HDPE/PP blend), a set of methods based on estimation of morphology from contact angle measurements, morphological analysis, adhesion tests of compatibilizer candidates to polymer blend components and tensile as well as tensile impact properties of ternary blends with compatibilizer candidates have been applied.Blow molding grade of HDPE homopolymer  with a melt flow rate (MFR) of 0.4 g/10 min (190 °C/2.16 kg) was purchased from Lyondellbasell. Injection molding grade of isotactic PP (HF 7005A) with a melt flow rate (MFR) of 21 g/10 min (240 °C/2.16 kg) was purchased from Borealis. The HDPE and PP were used for binary blend preparation. For the compatibilization of binary HDPE rich HDPE/PP blend, various types of compatibilizer candidates were used. All used materials and compatibilizer candidates are listed with their corresponding MFRs as well as short description of chemical structures in The six compatibilizer candidates used for the model HDPE rich HDPE/PP blend are an ethylene-based olefin block copolymer (OBC) (C1), a propylene-based olefin block copolymer (OBC) (C2), two propylene-based ethylene propylene random copolymers (EPR) (C3 and C4) and two styrene-ethylene-butylene-styrene triblock copolymers (SEBS) (C5 and C6). The propylene-based ethylene propylene random copolymers differ from each other in their ethylene contents as well as MFRs. Furthermore, one of the styrene-ethylene-butylene-styrene triblock copolymers (C5) has 13% polystyrene content, whereas another styrene-ethylene-butylene-styrene triblock copolymer (C6) has 20% polystyrene content.The samples with 25 mm diameter as well as 1.2 mm thickness were cut out from compression molded sheets for dynamic rheology measurements. The rheological analysis of the samples was carried out in parallel-plate-mode using Anton Paar MCR 301 rheometer equipped with CTD 450 heating chamber under nitrogen at 240 °C with 1 mm gap size. The deformation raised logarithmically from 1% to 2% with a frequency sweep from 628 to 0.01 rad/s during measurements.The measured shear rate dependence of melt viscosity curves of materials is provided in 1–103 shear range at 240 °C. Furthermore, the ethylene propylene random copolymer (C4) with a melt viscosity of 1000 Pa.s has the lowest melt viscosity at 240 °C.It is worth underling that the high molecular weight of HDPE has a similar viscosity to that of ethylene-based olefin block copolymer (C1) between 10The vHDPE and vPP were first ground into smaller particles with a diameter of 3 mm in a Fritsch granulator “Pulverisette 16”. The vHDPE, vPP, blend including 90% vHDPE and 10% vPP without compatibilizers, and the blends with 5% wight amount of six different kinds of compatibilizer candidates were separately extruded at 240 °C in Haake Minilab 2 twin screw extruder with 30 s residence time and 100 rpm screw speed. The generated strands from extrusion were cut into small pieces and then compression molded into sheets using the Collin P 200 P heating press at 190 °C. The following parameters were adjusted for compression molding: preheating at 150 °C under 8 bar pressure for 10 min, heating from 150 °C to 190 °C at 22 bar for 8 min, compressed by the melting temperature noted above (190 °C) at 30 bar for 5 min and then cooled with 10 K/min from 190 °C to 30 °C at 35 bar for 20 min. The sample specifications and abbreviation are summarized in 2O) and diiodomethane (CH2I2) using the sessile drop (3 µL) methods. The used materials, as shown in θ is the contact angle, γ is the surface energy (mN m−1). The polar component and the dispersed component are pγ and dγ, respectively. The indices A, B and C symbolize the components in ternary blends. In this case, the ternary blend components are HDPE, PP and compatibilizer candidates.The contact angle measurements were performed by using contact angle meter (Krüss DSA 30). The static contact angle of used materials  were determined relative to water . The samples were first cut at −120 °C using rotation microtom (Mikrom HM360). Consequently, the fractured surfaces were etched with n-heptane at 80 °C for 5 h and then sputtered with gold layer.The adhesion of compatibilizer candidates to HDPE and PP were measured using peel testing. The sheets of HDPE with geometry of 140 mm × 80 mm × 0.6 mm, PP with geometry of 140 mm × 80 mm × 0.6 mm and compatibilizers with geometry of 140 mm × 60 mm × 0.1 mm were hot pressed at 190 °C by using a mold with a geometry of 140 mm × 80 mm, then sandwiched together (HDPE/Compatibilizers(C)/PP) and consequently once again hot pressed at 240 °C 30 s to simulate extrusion process. The hot press machine used was a Collin P 200 P heating press. The schematic representation of samples was shown in For the peel tests, the sandwiched sheets by compression molding were cut with a sample width of 10 mm. The obtained sandwiched sheets were placed in tensile tester (Zwick 050) equipped with a 1 kN load cell. The samples were pulled apart at 10 mm/min. The peel force was measured as a function of displacement. The peel strength (N/mm) was displayed as the peel force divided by the sample width.For the tensile test specimens, approximately 22 g of small pieces from extruded strands were cut, weighted, and pressed into sheets with 1.8–1.9 mm thickness at 190 °C. The molded sheets were then punched out into tensile test specimens according to ISO 572-2 type 5A. Seven tensile test specimens were punched out for tensile test measurements to ensure reproducibility. Tensile tests were conducted at 23 °C using a test machine (Zwick 050) equipped with a 1 kN load cell and an extensometer with 10 mm/min test speed.For the tensile impact test specimens, approximately 12 g of small pieces from extruder strands were cut, weighted and pressed into sheets with 1.1–1.2 mm thickness at 190 °C. These sheets were then punched out into tensile impact test specimens according to the ISO 8256 method A. Afterwards, the test specimens were notched using a Notch-Vis  on both sides. The tensile impact tests were performed by an Instron CAEST 9050  impact pendulum equipped with 2 J hammer and 15 g of a cross head mass. For the reproducibility, seven tensile impact test specimens were measured.To predict the morphology of ternary blends, the contact angles of vHDPE, vPP, and compatibilizer candidates were measured. According to Equations (1)–(3), the polar component (p) and dispersed component (d) of surface tension were calculated. The obtained results are shown in However, the surface tension strongly depends on temperature as well as molecular weight of polymer . TherefoABγ, ACγ and BCγ, were calculated using the well-known harmonic mean Equation [After the calculation of surface tension of ternary blend components at process temperature (240 °C), the interfacial tension of possible component pairs, such as Equation :(5)γAB=γFurthermore, the adhesive energy can be calculated using Equation (6).ABW) at 240 °C are summarized in The calculated interfacial tension as well as adhesive energy  as a ternary blend component leads to a decrease in interfacial tension from 3.67 mN/m to 2.29 mN/m and an increase in adhesive energy from 61.86 mN/m to 64.75 mN/m. As can be seen in λ) can be calculated using Equations (7)–(9):The interfacial tension plays an important role in ternary blend morphology. The Hobbs spreading coefficient theory describes the relationship between interfacial tension and ternary blend morphology . To predABλ, BCλ and CBλ represent the spreading coefficients A over B, B over C and C over B, respectively. Based on the spreading coefficients, the phase morphology of ternary blends can be estimated. For the estimation of ternary blend morphology, a dispersed phase diagram –(7). The spreading coefficients of component couples of six ternary blends with corresponding morphologies are determined according to dispersed phase diagram  and showC1/vPP > 0, λvPP/C1 < 0 and λvHDPE/C1 = λvHDPE/vPP < 0. This result indicates that the dispersed vPP particle in vHDPE matrix is encapsulated by the ethylene-based olefin block copolymer (C1) as a compatibilizer, as schematically illustrated in According to the spreading coefficient theory, the ternary blend of vHDPE/vPP with ethylene-based olefin block copolymer as a compatibilizer (C1) has a unique core-shell morphology due to λC1/vPP < 0, λvPP/C1 > 0 and λvHDPE/C1 = λvHDPE/vPP < 0, as illustrated in Conversely, the ternary blend of vHDPE/vPP with other compatibilizer candidates, such as propylene-based olefin block copolymer (C2), ethylene-propylene random copolymers (C3 and C4) and styrene-ethylene-butylene-styrene triblock copolymers (C5 and C6), is encapsulated by vPP according to spreading coefficient theory due to the λThe encapsulation of PE in PP matrix by propylene-based olefin block copolymer ,32,33, eConsequently, the ethylene-based olefin block copolymer (C1) can only encapsulate the dispersed vPP phase in vHDPE matrix to form core-shell morphology, whereas the other compatibilizer candidates are encapsulated by dispersed vPP in vHDPE matrix. The predicted two different morphologies a,b in teThis methodology can be used for morphology prediction of compatibilized recycled rHDPE-rich-rHDPE/rPP blends from post-consumer detergent bottle waste. However, it should be noticed that the detergent residues in recycled bottle waste could affect the results of contact angle measurements and thereof the predicted morphology.The SEM images of 10 wt% vPP contaminated vHDPE/vPP blend with 5 wt% of ethylene-propylene random copolymer (C3) are shown in The adhesion of compatibilizer candidates to vHDPE as well as vPP is an important factor that determines the mechanical performance of compatibilized blends by means of their contribution to the enhancement of stress transfer between phases. The adhesion is correlated with peel strength. The peel strengths of six trilayer films (vHDPE/C/vPP) with six compatibilizer candidates as tie layers are shown in As seen in Furthermore, the ethylene-propylene random copolymer (C3) and styrene-ethylene-butylene-polystyrene triblock copolymer (C6) have the lowest peel strengths, whereas the propylene-based olefin block copolymer (C2), ethylene-propylene random copolymer (C3) and styrene-butylene-polystyrene triblock copolymer (C5) have peel strengths in between. The significant difference between compatibilizer candidates are viscosity curves  and therAccording to the literature, the copolymers with insufficient molecular weights have the worse adhesion due their low interfacial entanglements . ConversThe effects of adding 5 wt% of various compatibilizer candidates to 10 wt% vPP contaminated vHDPE (vB10 model blend) on tensile properties were investigated. The tensile test results of vHDPE, vPP, vB10 and vB10 with 5 wt% of six different compatibilizer candidates  are provided in The compression molded vHDPE homopolymer demonstrates 600% of elongation at break, whereas the compression molded vPP homopolymer shows the lowest elongation at break at about 2.1%, the highest E-Modulus as well as tensile strength at about 2200 MPa and 25 MPa, respectively. The compression molded vPP (injection molding grade) is brittle because of the high MFR. The addition of 10 wt% of vPP to vHDPE (vB10) results in significant reduction of the elongation at break from 600% to 200%. The reduction in elongation at break is approximately 67%. The brittle behavior of vB10 is due to the immiscibility between vHDPE and vPP homopolymers. The immiscibility between vHDPE and vPP leads to phase separated morphology. According to the literature, the reasons for deterioration in elongation at break of blends are immiscibility, phase separated morphology, insufficient number of interfacial entanglements at the interface between blend components and therefore high surface tension as well as weak adhesion ,20,40,41The E-Modulus and tensile strength of vHDPE are also influenced by the addition of compatibilizers, as seen in In addition to the tensile testing, the tensile impact testing provides important information regarding impact resistance under dynamic load. The tensile impact strength results of vHDPE, vPP, vB10 and vB10 blended with 5 wt% of different compatibilizers are shown in 2 due to the high MFR, low viscosity and low molecular weight of vPP. The addition of 10 wt% of vPP to vHDPE deteriorates the tensile impact strength of vHDPE from 72 kJ/m2 to 52 kJ/m2. The reduction in tensile impact strength is of approximately 30%. The deterioration in impact strength with the addition of PP to PE or PE to PP has been reported in the literature [Here, one can see that the compression molded vPP (injection molding grade) shows brittle behavior with a tensile impact strength of 15 kJ/mterature ,48,49,50terature ,32,38,42The increasing in elongation at break as well as impact strength is a significant evident for enhancement of phase adhesion and successful compatibilization, as reported in the literature ,50,51,52Selection of an effective compatibilizer within the industrially available compatibilizers for the recycling of PE-rich PE/PP blends can be challenging. This study investigated the compatibilization of vHDPE contaminated with 10 wt.% vPP and proposes a new methodology for rapidly identifying optimal compatibilizers based on basic surface properties, such as static contact angles, spreading coefficients, adhesion strength, tensile and tensile impact properties and surface morphology. The proposed methodology comprises two distinct stages, initially, prediction of ternary blend morphology using contact angle derived spreading coefficients, and secondly, evaluation of the adhesion strength of the compatibilizer to ternary blend components. The morphology of six ternary blends modelled using their spreading coefficients revealed that only ethylene-based olefin block copolymer (C1) exhibited core-shell morphology, with C1 as a shell, vPP as a core and a vHDPE matrix. This core-shell morphology is critical for successful compatibilization. The other investigated compatibilizers were encapsulated by vPP in HDPE matrix. C1 also provided the best adhesion strength of the candidates due to its high viscosity, which matched vHDPE in processing temperature and shear rate. PE/PP blends compatibilized with as little as 5 wt.% C1 exhibited 67% higher elongation at break and increases in tensile impact of 25%. Future research should further investigate the role of compatibilizer adhesion and core-shell morphology in the mechanical properties of blends. The identification of C1 as an optimal compatibilizer supports its widespread adoption for improving PP contaminated HDPE recycled material into virgin grade material, a move which could improve recycling yields to meet increasingly ambitious EU targets by allowing the use of more deteriorated post-consumer waste as feedstock and improving consumer confidence in the quality and properties of recycled materials. The proposed methodology can also be widely used for the screening and accelerated identification of optimal compatibilizers for other polymer blends and recycling streams, which will be absolutely critical in the revolution and expansion of the recycling sector as it moves to become the central player in the circular economy of the future.


Sus scrofa) population has rapidly expanded over the years, raising public health concerns over the species reservoir of several pathogens, including Mycobacterium microti (Mm), a Mycobacterium tuberculosis complex member. In this study, we aimed to investigate the Mm natural infection in WB in Lombardy and Emilia Romagna Italian regions by statistically evaluating the granulomatous lesions' histological features and Mm microbiological isolation. We analyzed 103 WB retropharyngeal and submandibular lymph nodes (LNs) for Mm identified by gyrB PCR-restriction fragment length polymorphism, and were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and Ziehl–Neelsen stained slides were evaluated. Considered histological variables were: the number of granulomas, size and maturational stage of granulomas, granulomas completeness within the section, number of multinucleated giant macrophages (MGMs), and acid-fast (AF) bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and granuloma incompleteness and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The results indicate that granuloma maturation should ensures an efficient containment of Mm infection in the WB, suggesting that the intra-species transmission of the disease might be an unlikely event.The European wild boar (WB) ( Sus scrofa) population has rapidly expanded over the last decades (The European wild boar (WB)  have been identified in wild animals, and for this reason, several countries engage in wildlife monitoring and management. Specifically, M. bovis, M. caprae, and M. microti (Mm) are the MTBC members identified in WB, and the role of the latter as a reservoir for these pathogens is demonstrated or highly suspected in various ecosystems —the adapted host , and generalized diseases are not reported , 120 WB retropharyngeal and submandibular LNs, sampled according to Boniotti et al. .gyrB PCR-restriction fragment length polymorphism analysis. Microbiological isolation was performed after decontamination onto Löwenstein-Jensen and Stonebrink solid media with addition of pyruvate, and into modified Middlebrook 7H9 broth incubated in a Bactec MGIT 960 system . Incubation was carried out at 37°C for 18 weeks, and the isolates were identified as Mm using a gyrB PCR-restriction fragment length polymorphism analysis. Considering histology, after paraffin-embedding, 4 μm thick sections were cut from the paraffin block and were dewaxed, rehydrated, and Hematoxylin-Eosin (HE) and Ziehl-Neelsen (ZN) stained  to each lesion. We also recorded if the lesions were entirely or incompletely depicted in the section.Granulomas were classified into four stages, according to Wangoo et al. . Stage 1The number of MGMs per lesion was counted in physical HE stained slides at 40× magnification.Each lesion was then found in the corresponding ZN-stained section and accurately and wholly viewed at 100× magnification with oil immersion to count AF bacteria.For the statistical data elaboration, two outcome variables of interest were identified:The microbiological status of the LNs (dichotomous variable: Positive/Negative);The histological stage of granulomas .Based on the outcome variable's nature, we adapted specific Bayesian regression models to identify which variables among those measured significantly influenced the outcomes .In the applied model, the statistical unit was the single LN, and predictors used were:a) AF bacilli: the presence of AF bacteria in LN granulomas. This variable was obtained by calculating, for every LN, the average number of AF bacteria present in the various granulomas and then categorizing the LN itself as AF = 1  when the average was >0, and AF = 0  in the case of averages equal to 0;b) MGM: the presence of MGMs in the LN granulomas. The categorization of LNs based on the presence of MGMs in the granulomas was obtained following a method similar to the method performed for AF bacilli. LNs were categorized as MGM = 1 when at least one MGM has been counted in one of its granulomas. On the contrary, an LN was classified as MGM = 0 when no MGMs were observed in its granulomas.c) G1, G2, G3, G4: to understand the influence of the granulomas histological stage on the microbiological status of LNs, we constructed four new variables identifying the proportions of granulomas of different stages in each LN. For each LN, we calculated the number of granulomas of the different histological stages. Then, we divided it by the total number of granulomas per LN, thus obtaining a value between 0 and 1, which corresponds to the proportion of granulomas of different stages. Precisely, G1, G2, G3, and G4 indicate the proportion of granulomas of stages 1, 2, 3, and 4 concerning the total number of granulomas present in an LN.Mm microbiological isolation) outcome variable was modeled using a Bayesian General Linear Model , weakly uninformative priors were selected according to McElreath :The model parameter estimates were obtained by sampling with the Hamiltonian Monte Carlo algorithm. For this model, the brms package was used as the interface of the STAN implemented in R in the rstan package –27. BayeIn the applied model, the statistical unit was the single granuloma, and predictors used were:a) lnGrArea: log of the granuloma area;b) laf: log of the number of AF bacteria per granuloma;c) lmgm: log of the number of MGMs per granuloma;d) micro: microbiological state of the LNs (Positive/Negative);e) Grcompl: status of complete/incomplete granuloma;f) lngr: log of the number of granulomas per LN;g) Idlymph: code identifier of LN.In our case, a Bayesian ordinal regression model was fitted according to Bürkner and Vuorre . In our k, one has to first achieve all lower categories 1 to k − 1. For every category k ∈ {1, …, K} there is a latent continuos variable k + 1th category. We assume that k:In the Sequential Model the dependent variable Y results from a counting process and is truly ordinal in the sense that in order to achieve a category X where β is the matrix of regression coefficients and X is the matrix of the values of predictors.where η is the linear predictor: βSo, we can formulate the Bayesian model as:with these priors:The model parameter estimates were obtained by sampling with the Hamiltonian Monte Carlo algorithm using four chains, with 4,000 iterations of which 1,000 were warmup (excluded after sampling). For this model, the brms package was used as the interface of the STAN language implemented in R in the rstan package. Bayesian posterior estimates of the parameters were then summarized in a table using the posterior distribution median, the 95% credibility intervals, the PD, and the ROPE .To show how a single predictor statistically influences the distribution of the histological stage of granulomas while holding the other predictors constant, we plot the conditional effects of some relevant predictors, as described by Bürkner .Mm was detected directly in fresh tissue samples by gyrB PCR-restriction fragment length polymorphism, were selected from IZSLER electronic archives. Of these, 103/120 were enrolled for histological evaluation and statistical analysis, while 17/120 were excluded due to autolysis artifacts. In 19/103 (18%), LNs Mm was microbiologically isolated, while in 84/103 (82%), the bacteriological examination resulted negative. Most of the LNs contained few granulomas; in some others, hundreds of granulomas were identified. Usually, when only a few granulomas were present in the section, they were classified in the highest maturational stages; conversely, when numerous granulomas were present, they were mainly of the first two maturational stages. For example, 422 is the maximum number of granulomas counted in an LN, and of them, 408, 13, and 1 were, respectively, of the first, second, and fourth maturational stage. On the contrary, in the eight LNs in which only one granuloma was detected, the latter was of the fourth maturational stage. AF bacilli were identified in 64/103 (62%) LN histological sections, while 39/103 (38%) resulted ZN negative. In the ZN positive LN sections, only one or a few granulomas usually contained AF bacilli. In such granulomas, usually <10 AF bacilli were detected. The detailed results of the analyzed variables  are reported in One hundred and twenty WB retropharyngeal and submandibular LNs, in which The statistical model results used to study how the considered variables influence the microbiological status of the LNs are shown in The Bayesian posterior distributions of estimated coefficients for each predictor with respect to the LN microbiological status are shown in Briefly, keeping the value of all other variables constant, as the proportion of stage 1 granulomas per LN increases, there is a 90% probability that the LN is microbiologically positive. There is a similar effect for the variable G2, even if with a probability of 74%. As the proportion of stage 3 and 4 granulomas increases, there is a tendency to decrease the probability that the LN is microbiologically positive with a probability of 91 and 72%, respectively. In LNs where granulomas with AF bacteria are present, there is a greater probability that they are microbiologically positive in 81% of cases. On the contrary, in 70% of cases, the presence of MGMs in granulomas reduces the likelihood of the LNs to be microbiologically positive. However, the predictors' relevance in terms of distance from 0 is not particularly high, and the uncertainty of the estimates also appears to be rather large.The statistical model results used to study how the considered variables influence the histological stage of granulomas are shown in The Bayesian posterior distributions of estimated coefficients for each predictor with respect to the granuloma histological stage are reported in The statistical model suggests that as the granuloma's size increases, the probability that the granuloma is classified in the higher stages increases. On the contrary, as the number of granulomas in the LN increases, the probability that LN's granulomas are of high histological stages decreases. According to the model, the microbiological status does not seem relevant, whereas the probability of granulomas being classified in high stages decreases as the number of MGMs increases. Regarding the number of AF bacilli, the model suggests a positive effect of this parameter on the granuloma histological stage, but a probability of only 70% supports this result. Finally, incomplete granulomas compared to complete ones are more likely to be classified in high histological stages.The marginal effects  of granuloma size, the total number of granulomas per LN, and the number of MGMs per granuloma on the granuloma histological stage are, respectively, depicted in Mm, a member of the MTBC whose epidemiological dynamics in WBs are still largely unknown, probably due to its difficult identification before the advent of diagnostic molecular methods  are also necessary to better establish the association between AF bacteria and the possibility of isolating the pathogen in culture.The statistical analysis also revealed that if AF bacilli are identified in an LN histological section, the considered LN is more frequently microbiologically positive for pulation , 31. Eve members . The poscid wash . The twoMm microbiological isolation. This finding contrasts with the literature where MGMs are generally reported as a negative prognostic factor for tubercular infection containment  was observed. Since MGMs are phagocytic cells with a reduced bactericidal activity that are exploited by the mycobacteria as persistence and replicative niche, our results suggest that granulomas of the highest maturational stages are less permissive to the pathogen compared to the less mature ones . ConsequMm naturally infected WB were histologically and statistically evaluated. The results obtained suggest that granulomas' maturation and organization permit an efficient containment of the infection. This containment can be due to Mm's characteristics, such as RD1mic deletion, and those of the WB, a species known to be generally resistant to tubercular infections (Mm infection. However, further studies considering other parameters influencing the disease transmission such as the duration of the infection, a factor potentially limiting the findings of the present study, are necessary to fully elucidate the effects of the considered parameters on the epidemiological dynamics of Mm infection in the WB.Concluding, in the present study LNs of fections , 12. OurThe original contributions presented in the study are included in the article/Ethical review and approval was not required for the animal study because it did not involve the purposeful killing of animals. Retrospective samples collected in the context of Lombardy and Emilia Romagna regions wildlife monitoring and control plan, which involves post-mortem inspection of hunted wild animals, were used. No ethical approval was deemed necessary.CP, VT, LRG, GS, MC, and VG designed the study. CP, LRG, GS, and VG performed the histological investigation. VT performed the statistics analysis. CP, VT, LRG, and VG were involved in table and figure preparation. LRG, AG, GLA, MP, MZ, and MBB were involved in samples and data acquisition. CP, VT, and VG drafted the original paper. LRG, AG, GLA, MP, MZ, MBB, GS, MC, and VG revised and edited the manuscript. CP, GS, MC, and VG supervised and administered the project. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.