Parasitic diseases represent a common issue in fish and, when histozoic forms are present, this elicits a chronic inflammatory reaction leading to granuloma formation. Despite the large knowledge of granuloma formation due to parasites in visceral organs, little is known about the development and the evolutive stages of granulomas in naturally infected fish. Mullets (Osteichthyes: Mugilidae) are a widespread euryhaline fish species that harbor different parasites, thus representing a suitable model for the study of parasite-induced granulomas. Combining histopathology and immunohistochemical tools, we identified three developmental granuloma stages , that ranged from an intact parasite with mild signs of tissue reaction to the formation of a structured granuloma. The identified histological patterns could be reliable tools in the staging of the granulomatous response associated with histozoic parasites and are an attempt to broaden the knowledge of the inflammatory response in different host–parasite systems.Histozoic parasite–fish host interaction is a dynamic process that leads to the formation of a granuloma, a specific chronic inflammatory response with discernible histological features. Mullets (Osteichthyes: Mugilidae) represent a suitable model concerning the development of such lesions in the host–parasite interface. The present work aimed to identify granuloma developmental stages from the early to the late phase of the infection and to characterize the immune cells and non-inflammatory components of the granuloma in different stages. For this purpose, 239 mullets were collected from 4 Sardinian lagoons, and several organs were examined by combining histopathological, bacteriological, and immunohistochemical methods. Granulomas associated with trematode metacercariae and myxozoan parasites were classified into three developmental stages: (1) pre-granuloma stage, characterized by intact encysted parasite and with no or mild tissue reaction; (2) intermediate stage, with partially degenerated parasites, necrosis, and a moderate number of epithelioid cells (ECs); and (3) late stage, with a necrotic core and no detectable parasite with a high number of ECs and fibroblasts. The three-tier staging and the proposed morphological diagnosis make it conceivable that histopathology could be an essential tool to evaluate the granulomas associated with histozoic parasitic infection in fish. The chronic inflammatory response is a non-specific reaction against a multitude of factors, such as harmful substances, bacteria, mycobacteria, fungi, and parasites, particularly common in fish ,12,13,14In natural and experimental infection due to bacterial and particularly mycobacterial and oomycetes species, granulomas have been described as a core of macrophages and bacteria associated with central necrosis, surrounded by a fibrous layer mostly evident in the later stages of infective granulomas ,20,21. UIt is well known that also some histozoic parasites  can elicit chronic granulomatous inflammation .More details of the cellular components of granuloma and their distribution have been provided by electron microscopy studies, where ECs have been typically found near or in contact with the parasite body or cyst wall . AlthougRodlet cells (RCs), although not a regular component in parasite granulomas, increase in the course of infectious diseases, parasitic infections, and environmental pollutants ,36.Granuloma formation is a dynamic process, and many times the structure of these specific chronic inflammatory responses can give useful information about the timing, evolution, and prognosis of the disease.Labratrema minimus  metacercariae has been accomplished [Nevertheless, detailed studies on granuloma progression against histozoic parasites have not been carried out, although numerous details of parasite granuloma cellular components have been reported both in saltwater and freshwater fish ,30,37. Amplished . The premplished  allows aThe present work aims to characterize the histological features of the granulomatous inflammatory response in visceral organs of mullets naturally infected with trematode and myxozoan parasites.For this purpose, different developmental stages of granuloma, from the early to the late phase of the histozoic parasite infection, were identified using discernible histological features, and afterward, the immune cells and other non-inflammatory components associated with the different stages of the granuloma were characterized by histochemical and immunohistochemical tools.From July to October 2014, 239 mullets were collected by fishing nets from 4 different coastal lagoons in Sardinia (western Mediterranean Sea): 60 from Calich lagoon , 60 from San Teodoro lagoon , 60 from Mistras lagoon , and 59 from Marceddì lagoon .Mugil cephalus (11), Chelon labrosus (35), Chelon auratus (83), and Chelon ramada (110), variably combined in the different lagoons. All the specimens were identified at species level following Ben-Tuvia (1986) and Bauchot (1987); fish names conform to FishBase [Identification of Mugilidae species revealed a heterogeneous group composed of FishBase ,45,46. SThe mullets were transferred to the Department of Veterinary Medicine, University of Sassari, and the internal organs  of a subsample of 12 fish were analyzed for the presence of tissue parasites to identify the main taxa present. Squashes and scrapings of organs were placed on slides, pressed under a coverslip, and observed under an optical microscope. The parasites found were trematode metacercariae (TM) and myxozoan plasmodia (MP), and they were identified to the lowest taxonomic level possible according to the observable characters, following the specialistic literature ,49,50,51A culture test was carried using the anterior kidney of the 239 subjects on a primary isolation medium (blood agar). Colonies present in the plates were cloned and identified by phenotypic , biochemical , mass spectrometry (MALDI-TOF), and molecular (using generic primers and subsequent sequencing) methods.Fresh tissues from sampled organs (liver and spleen) were homogenized and decontaminated and inoculated in Stonebrink medium tubes and 2 Löwenstein–Jensen medium tubes  for mycobacteria culture. Decontamination of the samples was carried out using 1.5% cetylpyridinium chloride, following the standard protocol aimed at eliminating contaminating bacteria, in order to allow mycobacteria growth on specially sown soils .Samples of the liver, spleen, heart, and kidney of the 239 mullets were collected and immediately fixed in 10% buffered formalin for 48 h, dehydrated with increasing alcohol concentrations and xylene in an automatic tissue processor, and paraffin embedded. Sections of 3 μm thickness were obtained with a microtome  and stained with hematoxylin and eosin in an automatic multistainer . Slides were then evaluated at light microscopy . Lesions found in visceral organs  were evaluated according to the parasite class involved ,54.A total of 75 well-preserved granulomatous lesions from 47 of the 239 fish were included in this study. All granulomas were selected considering different organs and different classes of parasites involved. Lesions were classified into three developmental stages based on the evaluation of selected histological features  and were investigated to evaluate different structural components  by immunohistochemistry (IHC). Serial sections were mounted on positively charged slides  for IHC according to a previously developed protocol ,56,57. BThe presence of collagen, mast cells, and rodlet cells was also evaluated with Masson’s trichrome and Giemsa staining, respectively . To inveGranulomas were further evaluated for their proximity to the blood vessel and assigned to one of the following three categories: close to the vessel , near the vessel , far from the vessel (granuloma distance from the vessel >100 microns or not present in the field of view at 200× magnification). Additionally, macrophage aggregates (MAs) associated with granulomas were also evaluated. The 75 granulomas were assigned to the “present” or “absent” categories based on the presence or absence of macrophage aggregates near the granuloma in a field of view at 200× magnification.https://imagej.net/ImageJ, accessed on 15 May 2020). The quantitative evaluation of the collagen area, associated with the lesions, was accomplished using ImageJ software on microphotographs taken at 200× magnification on slides stained with Masson’s trichrome. In detail, the total area of the granuloma was selected by the “freehand selection” tool, measured, and then isolated from the background with the “clear outside” option. The central area of the lesion, containing parasite or necrotic material, was selected by the freehand tool, measured, and deleted by the “cut” function to avoid erroneous measurements of color values similar to the collagen area. After that, “color threshold” tool settings were adjusted to identify color values corresponding to collagen, and the obtained area was selected and measured. The color threshold measurement was repeated three times, and the final collagen area was calculated as the mean of the three consecutive measurements and expressed as a percentage of the total area of the granuloma.Each lesion was microphotographed and images were analyzed using a Java-based open-source image processing program  was used to correlate the prevalence of parasites in examined organs. Furthermore, categorical and ordinal variables were compared using the Spearman rho (ρ) rank correlation coefficient. A value of ρ approximately equal to 1 indicates a good correlation, a value near 0 indicates a poor correlation, and a negative value indicates an inverse correlation. A p-value < 0.05 was considered significant.Microscopic features of the lesions were analyzed using Stata 11.2 software . Immunohistochemical results were compared using the non-parametric Kruskal–Wallis test with Dunn’s post hoc comparison. The chi-square test  (Myxobolus sp. (Myxobolidae)  A,B and tbolidae) C,D in thbolidae) .The microbiological examination of all samples was negative for the presence of pathogenic bacteria and mycobacteria.Among the 239 sampled fish, 172/239 (71.96%) presented at least 1 parasite, of which 78/239 (32.63%) were affected only by TM and 40/239 (16.73%) only by MP, whereas in 54/239 fish (22.59%), both classes of parasites were detected. Concerning organ distribution, TM were detected in all examined organs, with a higher prevalence in the kidney (19.6%) and heart (14.2%), whereas MP were more frequently detected in the kidney (38.1%) and spleen (18.8%). Most of the fish affected by a single class of parasite presented an infection confined to a single organ (22.1% for metacercariae and 14.6% for Myxosporea).Mugil cephalus showed a higher prevalence of TM in the heart (54.5%) and in the kidney for MP (36.3%) but without any significant statistical association. In C. ramada, TM were more frequent in the heart (12.7%), whereas MP mostly affected the kidney . The kidney was the most affected organ in C. auratus for both parasite classes . In Chelon labrosus, MP were mostly found in the spleen . Details on the prevalence of organs affected by granulomas induced by both groups of parasites are shown in Most of the fish affected by a single class of parasite presented lesions confined to a single organ (35.9% for metacercariae and 40.5% for Myxosporea). Details on the percentages of each fish species affected by each of the parasite classes in one, two, three, or four evaluated organs are shown in Ziehl-Neelsen staining was negative for mycobacteria in all examined organs. The microscopic examination of the organs revealed a multifocal, mild-to-severe granulomatous inflammation around the parasites and three different stages of lesions. Out of 75 lesions, 25 were categorized as stage I, 27 as stage II, and 23 as stage III. Most of the selected fish showed the presence of only one stage of granuloma (68.1%), whereas 23.4% showed the presence of two stages, and in 8.5%, all stages were detectable.Stage I was termed as pre-granuloma stage and characterized by an intact parasite surrounded by a thin cyst wall, in association with scarce inflammatory cells. At this stage, positive labeling for anti-cytokeratin antibody was occasionally expressed by the parasite capsule, whereas immunoreactivity to anti-vimentin antibody was negative A–C.Stage II was termed as intermediate-stage granuloma and characterized by parasites with loss of somatic details or partially destroyed parasites, but that were still recognizable in the center of the lesion, and often intermingled with a scarce to moderate amount of granular eosinophilic material and nuclear debris (necrosis). Outer encircling layers of flattened cells with a moderate to abundant eosinophilic cytoplasm, an ovoid vesicular nucleus with prominent, basophilic, and central nucleolus were detected around the degenerated parasite and necrosis. Due to these morphological features and to their strong and diffuse positive cytoplasmic signal to anti-cytokeratin CKAE1-AE3 antibody, these flattened cells were identified as epithelioid cells D–F. MoreStage III was designated as late-stage granuloma and characterized by a central eosinophilic core of necrosis, with parasite remnants only occasionally visible, and increased layers of epithelioid cells. The external wall was constituted by increased layers of fibroblasts strongly positive to anti-vimentin antibody G–I.Scattered macrophages were detected near parasite cyst walls in stage I lesions and/or intermingled with necrotic material or scattered in the surrounding parenchyma in later stages. Aggregates of macrophages, frequently showing pigment-laden cytoplasm, were observed in association with the external layers of granulomas in 25/75 granulomas H,I.At histological examination, parasites were described based on characteristic features of the parasite classes, clearly discernible at stage I. Trematode metacercariae were detected in all examined organs, ranging from 150 to 400 microns in diameter, without body cavity and surrounded by a light eosinophilic capsule. Oral and ventral suckers, identifiable by the presence of a radially arranged musculature, were often recognizable, as well as a cuticle with a thickness of 2–4 microns with occasionally detectable cuticle spines that appeared as distinct eosinophilic linear structures A.Histozoic plasmodia of myxozoans were found encysted in the parenchyma of the heart, liver, spleen, and kidney. Plasmodia, containing a variable number (until hundreds) of mature spores, were round to oval, ranging from 100 to 450 μm, and were delimited by a thin eosinophilic wall B.In granulomas, collagen fibers were recognizable as blue filaments with Masson’s trichrome staining in the external layer of intermediate- and late-stage granulomas C.Mast cells (MCs) appeared as ovoid cells with distinct borders and abundant cytoplasm containing numerous eosinophilic granules, brightly evidenced by Giemsa staining, and a central nucleus A.Mast cells also showed a strong cytoplasmic positive signal to anti-vimentin antibody B. In botRodlet cells were observed in scarce numbers in all stages, mainly disposed near the parasite capsule or in the surrounding parenchyma. They were also observed near the main vascular structure both in parasitized and healthy samples D.The results of the structural components of granulomas and associated immune cells, as well as the description of histological parasite characteristics, were combined in a general scheme of possible developmental stages of granuloma formation .2 (corrected for ties) = 62.686, p = 0.00010; Dunn’s post hoc test, pre-granuloma vs. intermediate p = 0.000002; pre-granuloma vs. late p = 0.000000; intermediate vs. late p = 0.000450). In the intermediate stage, ECs covered a mean area of 13.42% (± 7.19% SD) of the total area of the granuloma, whereas in the late stage, they reached approximately 1/3 of the total area of the granuloma (mean area 30.85% ± 14.68% SD).Epithelioid cells showed a statistically significant positive correlation with granuloma progressive stages, showing an increase in parallel to the host immune response progression  = 38.963, p = 0.00010; Dunn’s post hoc test, pre-granuloma vs. intermediate p = 0.000842; pre-granuloma vs. late p= 0.000000, intermediate vs. late p = 0.002460). Fibroblasts covered averagely 2.94% (± 3.85% SD) of the total area of the granuloma in stage II, reaching a mean area of 7.15% (± 5.71% SD) in the late stage.The fibroblast layer increased with granuloma progression  = 36.791, p = 0.00010) . Moreover, collagen showed a statistically significant association with the fibroblast layer in the intermediate-stage granuloma (ρ =0.6257 p < 0.05). The collagen layer covered a mean area of 0.82% (± 1.43% SD) in the intermediate stage and a mean area of 3.58% (± 2.92% SD) in the late-stage granuloma.Statistical analysis evidenced that collagen had a significant increase in intermediate- and late-stage granulomas  =29.531, p = 0.00010 . Moreover, a significant positive correlation between the collagen area and the number of mast cells was observed . Evaluation of the association between the number of mast cells and the granuloma proximity to vascular structures revealed no significant statistical association.Mast cells were observed in all stages of the lesion development, showing an increase from the pre-granuloma stage, with a mean number of 0.68 (± 1.86 SD), to the intermediate- (2.89 ± 3.33 SD) and late-stage granulomas (9.86 ± 18.98 SD). Statistical analysis of MCs revealed a significant increase in number in the pre-granuloma and intermediate-stage granuloma  in the pre-granuloma stage, 0.28 (± 0.71 SD) in the intermediate stage, to 0.65 (± 1.19 SD) in the late-stage granuloma. Moreover, conversely to MCs, no statistically significant difference in the number of the RCs was observed in the different stages of host immune response.Macrophages aggregates were found associated in 16% of the pre-granuloma stage, 44.4% in the intermediate stage, and 39.1% in the late-stage granuloma. However, no statistically significant variation was observed between their presence and the different stages of granulomas.p > 0.05).Quantification of granuloma’s proximity to vascular structures gave the following results: 33.3% of lesions were considered in the “close” category, 33.3% fell in the “near” category, and 33.3% in the “far” category. The association between the distance of granulomas from vessels and granuloma stages was not statistically significant . Heterophyids, which have a fish-eating bird or mammals as the definitive host, are widely encountered parasites of mullets in Sardinian brackish waters [Inflammatory lesions associated with TM and MP were detected in (71.9%) of the examined fish, confirming the observation of other authors that reported Mugilidae as a widely parasitized species . The parh waters ,59,60 ash waters ,42,48. Ph waters ,63,64,65Myxobolus spp. infection in many Mugilidae species from the Mediterranean area, which described encysted plasmodia in different tissues [Myxozoan parasites, especially the Myxobolidae family, have been extensively described in mugilids worldwide ,67,68,69 tissues ,73,74,75 tissues .Parasite infection in fish can be the result of a single contact with intermediate hosts or can be due to multiple exposures to parasites, leading to the formation of younger parasite cysts near to older and almost resolved infection . The graThe response of the fish immune system after parasite invasion is variable and reliant on a multitude of factors , with a different outcome in terms of damage extent to host tissues ,77,78.Myxobolus spp., respectively [In the pre-granuloma stage (stage I), parasites were detected encysted in the parenchyma of several organs, generally surrounded by a compact eosinophilic layer, composed of compressed parenchymal cells because of the cyst pressure. The inflammatory response in this phase was absent or mild, and when present, mainly characterized by a limited number of mast cells close to the parasite wall or scattered in the surrounding parenchyma, and by occasional macrophages. This feature has been reported by other authors in early encysted TM ,64 and hectively .The hallmark of the intermediate-stage granuloma (stage II) was the presence of epithelioid cells that encircled the parasites. These cells were discernible with IHC staining by the strong positive labeling to anti-cytokeratin antibody (CKAE1/AE3), as was reported by studies in other fish species ,28. The Lastly, late-stage granuloma (stage III) represented the late phase of chronic inflammation with the highest quantity of immune cells, which led to the resolution of parasite infection. In this stage, due to the presence of central necrosis and the lack of parasite remnants, the results of Ziehl-Neelsen staining excluded the presence of mycobacterial infection.p < 0.001) could be explained by the fact that in this phase, both fibroblasts and collagen were present in similar proportions. However, in stage III, even if both categories were numerically increased, collagen production augmented fourfold compared to the twofold rise in fibroblasts. The biological explanation for this difference could be that in late stages, fibroblasts have accomplished their role and their number start to decrease, whereas collagen, which represents fibroblast’s final product, is at the highest levels. There is evidence that fibroblasts, although not proper immune cells, make an important contribution in the host response against parasites, by crosstalk with other immune cells, as demonstrated in mammals and fish species [Epithelioid cells and fibroblasts, which are recently considered strictly associated with immune response, were found in the largest amount in the late-stage granuloma. Fibroblasts were more prominent and evidenced by a diffuse membrane immunoreactivity to anti-vimentin antibody, a marker for the intermediate filament of mammalian mesenchymal cells that is preserved in some fish species . Fibrobl species ,92. An i species ,33. Mast species ,93. Thisp < 0.05) reinforced the hypothesis that action of MCs could be more specifically addressed to the regulation of fibroblasts in collagen production, rather than to fibroblast recruitment in the site of infection, in intermediate- and late-stage lesions.In this work, microscopic examination of stages II and III granuloma revealed the presence of mast cells grouped around the external layer, adherent to fibroblasts, thus leading to the hypothesis that a transgranulation mechanism of MCs towards fibroblasts could be plausible. The statistically significant association of MCs with the collagen layer  infected by nematode larvae [The results of the present work suggest that the role of mast cells as promoters of collagen production may not be the only mechanism in which MCs are involved against histozoic parasites. Indeed, the low number of MCs observed in the pre-granuloma stage and their distribution around the parasite capsule suggest that their action could be involved, synergistically with macrophages, in promoting host response, perhaps by the recruitment of other immune cells at the site of infection. This mechanism, recognized in mammalian mast cells during parasite infection, has been supposed in teleosts, where MCs are recognized as first effectors during parasitism . Mast cee larvae .Another group of immune cells, known as rodlet cells, that represent a unique element of the teleostean immune system ,96, was Macrophage aggregates were inconsistently found at the periphery of the granulomas, sometimes containing brownish cytoplasmic pigments. Aggregates of pigment-laden macrophages (MMAs) are normally present in the liver and spleen of many teleosts and can vary in number and dimension under the influence of several factors, such as age, stress, diseases, and pollution . MMAs haThe present study underlined that host immune response against the two groups of parasites revealed a substantial superimposability, leading to a common model of development of the immune response against trematode metacercariae and myxozoan parasites.Parasitic disease is a relevant issue for fish health and represents a challenge between the attempt of parasites to survive and the host’s action aimed to prevent the spread of infection.This study, by combining histopathology with immunohistochemical methods, represents an effort to expand the knowledge of inflammatory reactions associated with parasites in fish organs and their staging. This makes it conceivable that the identified histological patterns could be reliable tools to staging the granulomatous response associated with histozoic parasites and represent an effort to classify granulomas of different origins.Further analyses should be conducted to understand mechanisms of granuloma formation induced by different species of parasites in various experimental models, as well as the development of early detection methods, especially in species of great commercial interest for human consumption.


As for any other wave, classical light was often thought to be constrained by the diffraction limit. However, since the pioneering works in near-field microscopy, in which the evanescent components of light localized at the boundaries of matter beat diffraction, spectacular development of optically resolved images has been achieved in recent years, regularly obtaining nanoscale resolution of optical near-fields, as for instance in optical nanoantennas . In thisIn a new twist in molecular microscopy, the research team in Hefei has managed to combine the information obtained from a set of spectrally filtered ultra-resolved maps of Raman photons emitted from a single Mg-porphine molecule in a plasmonic cavity, and create an image of the molecule where particular vibrational modes of single bonds, as well as of ring modes, are unambiguously identified as the underlying modes sustaining a well-defined atomic structure within the molecule  see Fig. This isAn exciting prospect of this proof of principle is the combination of spectral and microscopic information obtained in this technique with the data-handling that artificial intelligence, and in particular machine learning, could offer, by properly connecting the spectral data in the literature, with the details of the energy-filtered maps in thousands of molecular species. It might be a question of time that a set of molecular images from SRP standardly determine its exact chemical composition. The monitoring of the motion of atoms in molecular assemblies might be just at hand, opening direct real-space access to the dynamics of atoms in relevant physical and chemical processes ranging from vibronic transport, to induced reactivity, through ultrasensing. Seeing them dancing is now clearer than ever.Conflict of interest statement. None declared.


It is commonly asserted that agricultural production systems must use fewer antibiotics in food-producing animals in order to mitigate the global spread of antimicrobial resistance (AMR). In order to assess the cost-effectiveness of such interventions, especially given the potential trade-off with rural livelihoods, we must quantify more precisely the relationship between food-producing animal antimicrobial use and AMR in humans. Here, we outline and compare methods that can be used to estimate this relationship, calling on key literature in this area. Mechanistic mathematical models have the advantage of being rooted in epidemiological theory, but may struggle to capture relevant non-epidemiological covariates which have an uncertain relationship with human AMR. We advocate greater use of panel regression models which can incorporate these factors in a flexible way, capturing both shape and scale variation. We provide recommendations for future panel regression studies to follow in order to inform cost-effectiveness analyses of AMR containment interventions across the One Health spectrum, which will be key in the age of increasing AMR. Antimicrobial resistance (AMR) is an archetypally One Health problem, with increasingly profound global consequences for human health ,2. AntimDecision and policy makers now frequently rely on economic analyses when deciding on which intervention to prioritise . In thisThe size and shape of the relationship between food-producing animal AMU and the number of infections with AMR pathogens in humans are uncertain, and there is no consensus on the favourable versus unfavourable consequences of even non-therapeutic animal antimicrobial use ,14. For Additionally, resistant pathogens pass via the human, animal and environmental components of the One Health system , and a hSome resistant pathogens may already be endemic to human communities independently of transmission from food-producing animals ,26. In sAs agricultural AMU can aid rural livelihoods and poverty alleviation in the short term , reducinOne way of quantifying the link between food-producing animal AMU and human AMR would be to examine the effect of real-world interventions to reduce food-producing animal antimicrobial use. To this end, the global health community commonly looks to the findings of a meta-analysis by Tang et al. (2017) , who synThat meta-analysis is a valuable and novel piece of work that has advanced our understanding of AMR. However, while its results are indicative of the existence of a relationship between food-producing animal AMU and human AMR, they do not allow us to quantify that relationship for the purposes of cost-effectiveness analysis. Included studies looking at human AMR outcomes were pooled across all drug–pathogen combinations and intervention types due to the limited number of studies, which will obscure differing resistance responses of microbial pathogens to AMU . Most stMoreover, it is difficult to reconcile this large treatment effect with the body of mathematical models and genomic studies which suggest a much more ambiguous relationship between human AMR and food-producing animal AMU see . This leMore broadly, knowing the pooled effect of a set of past interventions presents a black-box relationship between food-producing animal AMU and human AMR. Given that the AMR system functions like a bath with several taps , and givGiven the difficulty in quantifying the relationship of interest using empirical meta-analyses, we use this section to propose areas of focus for research going forward. We outline appropriate methods for estimating the relationship, and provide guidelines for future analyses. We recommend applying panel regression models to AMR surveillance data, reinforced by transmission dynamic mathematical models of the One Health AMR system .Transmission dynamic models can take a range of forms, for example including difference equation models or individual-based models (IBMs). Most recent work exploring AMR acquisition in the One Health system has used ordinary differential equation models (ODEs) to estimate the relationship between food-producing animal AMU and human AMR ,23.These models track sub-populations over time, with dynamics being a function of the rate of transmission, antibiotic exposure and resistance evolution. For example, Van Bunnik and Woolhouse  created Models such as this have the advantage of mechanistically modelling AMR acquisition and transfer using known epidemiological foundations. A main limit to applying such methods is their reliance on detailed, directly-comparable AMR surveillance data ,33. For Future research should then aim to fit models to longitudinal surveillance data when and where they are available in order to determine the shape and size of the link between food-producing animal AMU (and AMR) and human AMR for policy makers. Because targeting food-producing animal AMU without addressing other factors is unlikely to occur in reality and is potentially of limited efficacy, such models should explore the effect of varying multiple parameters simultaneously .Mechanistic models are limited in their ability to capture and accurately parameterise all the interacting components of a system. Statistical analysis does not rely on knowledge of the mechanisms and instead can determine the contribution of various elements to key dependent variables. For example, panel regression models can be used to combine surveillance data on AMR and AMU prevalence in various countries and/or subnational administrative areas over time, along with other relevant factors such as economic indicators. Here, human AMR prevalence can be regressed against food-producing animal AMU using static methods such as fixed and random effects, difference in difference, or first difference; as well as dynamic methods such as difference and system generalised method of moments (GMM) ; and conOnce panel regression models have been used to determine the link between food-producing animal AMU and human AMR , this relationship can then be fed into mechanistic health economic models for the purpose of cost-effectiveness analysis. For this reason, regression models which use data over time (including panel regression) have been recommended over simpler methods such as correlation coefficients for estimating the economic cost of AMR .Panel regression techniques have been used, for example, to assess the relationship between vaccination and AMU in Indian states , the relSuch work has been elucidating at the national and subnational level, and works such as Collignon et al.  represenA key advantage of panel regression methods is that these models can accommodate flexible functional forms  which can be used to reflect the relationship of interest accurately and comprehensively. They can also control for a diverse range of factors to isolate the effect of each explanator: Noyes, Slizovsky and Singer  note thaBy including geographically and economically diverse countries, and by controlling for relevant factors, the fitted values of such an analysis could be used to predict the relationship between human AMR and food-producing animal AMU in various countries based on their characteristics, including those for which no (or few) data are available. In this way, while data missingness is still problematic, it can be overcome relatively effectively in panel models.Nonetheless, panel models are by no means a magic bullet, nor do the different model types discussed here answer precisely the same question: in fact, panel models can help to quantify the link between AMU and AMR in a way which can subsequently be fed into mathematical models of transmission, as well as cost-effectiveness analyses. The assumptions of panel regression models do not hold when food-producing animal AMU is neither exogenous nor random (as is likely to be the case), and this issue must be addressed carefully . In addiResearchers employing any of the methods discussed here should endeavour to follow (or at the very least consult) appropriate standards and guidelines. For instance, mathematical modelling exercises can make use of the TRACE paradigm , can incAs discussed, panel regression studies have the potential to produce much-needed inputs for cost-effectiveness analysis, but only by applying appropriate methods. We therefore recommend that future panel studies aiming to quantify the relationship between AMU in food-producing animals and AMR in humans aim to achieve the following:Control for social and economic factors as well as epidemiological ones. Collignon et al.  make excUse panel rather than cross-sectional regression models. This includes the use of lagged dependent and independent variables to understand the time to effect of the relationship of interest as well as the time dependency of human AMR. Both static and dynamic panel methods should be used and compared , allowinBe flexible in terms of functional form. The relationship of interest is a complex one, and not reducible to a single parameter. Because we are unsure of the precise shape of the relationship between human AMR and food-producing animal AMU, we should explore the inclusion of various lags, non-linearities, threshold effects, and interaction terms. Supported by the use of LASSO and other machine learning techniques for covariate selection, this can minimise misspecification and allow the data to ‘speak for themselves’ . As ‘biTake statistical potential sources of bias seriously. Regression models can reveal an insignificant or even negative relationship between antimicrobial consumption and AMR . While tInclude data from LMICs and HICs together. Due in part to data availability, studies on AMR focus disproportionately on high-income settings. The relative social and economic homogeneity of these countries can make it difficult to determine the effect of changes in key parameters when data from LMICs are not included . By undeInclude as outcomes of interest as many drug–pathogen combinations as possible, as well as indices of overall resistance. This will better reflect the unique response of bacterial pathogens to changing levels of AMU , in contFinally, present results in a form with a cardinal interpretation. For the purposes of comparability, many studies look at simple correlation coefficients or present the effect of a one-standard-deviation change in covariates of interest , which dThe two methods discussed here are not mutually exclusive, nor exhaustive, and we recommend that the results of both be compared, along with other methods, in order to triangulate the relationship between food-producing animal AMU and human AMR prevalence. These methods could include randomised control trials, quasi-experimental settings and natural experiments, as well as other modelling strategies such as hierarchical risk-assessment models of AMR acquisition .Parameterising and validating models employing the methods discussed in this article will benefit from the presence of stronger surveillance data on AMR and consumption, across all three One Health compartments. For this reason, we underline the importance of continuing to establish and expand AMR surveillance networks globally.Just as it is important to quantify accurately the effect of food-producing animal AMU on human AMR, so it is equally important to continue to quantify accurately the effect of food-producing animal AMU on farm productivity and profitability and on animal welfare, as this outcome represents the other ‘half’ of the trade-off implied by reduced food-producing animal AMU. Randomised trials at the farm level should be the gold standard for estimating this. Outcomes considered should include feed conversion rate, animal morbidity and mortality, the cost (labour and medicine) of treating sick animals, and the effect of replacing antibiotics with alternatives such as probiotics . RandomiIn fact, a complete understanding of the relationship between animal AMU and human AMR will require the syncrasy of AMU and AMR surveillance data, in vitro experiments on antibiotic susceptibility in human and animal isolates, and animal experiments which evaluate the AMR (and other) outcomes of different levels of AMU .AMR is a significant global problem, one which is set to become more important in the near future, and one which may disproportionately affect the Global South . At pres


The survey revealed Covid-19 had increased awareness of AMR amongst 47% of respondents; increased awareness of connected animal and human health amongst 43% of respondents; and increased awareness of animal welfare information on food labels amongst 34% of respondents. A cluster analysis revealed five distinct consumer segments impacted differently by Covid-19. These segments differed in their levels of objective and subjective knowledge of antibiotic use practises in farming, AMR risk perception, and attributions of responsibility for action on AMR. Findings are discussed with respect to future efforts by the agri-food sector to communicate with the public about AMR and responsible antibiotic use in farming, with particular emphasis on the implications for strategies that incorporate front-of-pack labelling.Covid-19 is a OneHealth crisis with far-reaching and unexpected impacts on many aspects of society. Previous OneHealth issues, such as antimicrobial resistance (AMR), have not received a similar level of attention or action from the public despite representing significant public health and economic threats to society. The current study aimed to explore whether the Covid-19 pandemic may act as a catalyst to increase public awareness related to OneHealth issues, in particular, AMR. This short paper presents overview findings from a survey carried out in September 2020 with a representative sample of food consumers on the island of Ireland ( With evidence strongly supporting Covid-19 as a zoonotic, “animal to human disease,” this pandemic reinforces the “OneHealth” concept and the interconnectedness of human and animal health . Antimicspecifically about antibiotic use and farm animal welfare on food labels to consumers. Front-of-pack labels require a baseline level of awareness and understanding by the consumer, and research suggests public awareness about antibiotic use on farms is low  which fail to communicate the complexities of responsible antibiotic use in agriculture—on the one hand, agriculture needs to reduce its' use of antimicrobials, but to eliminate them completely could lead to unintended impacts on farm animal welfare . Participants were encouraged to “think aloud” as they read and responded to survey items. This is a proven method for identifying areas of confusion or misinterpretation, and increasing face validity and relevance  during September 2020. A market research agency recruited participants and administered the survey. A quota sampling procedure achieved a sample representative of gender, age, region  and social class for both regions. Inclusion criteria included: being aged 18+; being a consumer of either meat or dairy; and being at least partially responsible for household grocery shopping. Individuals holding the occupation of “farmer” were excluded from recruitment. Details of the socio-demographic breakdown of the sample are provided in The survey was refined and administered to food consumers in the Republic of Ireland and Northern Ireland (Survey variables used in the current study included: (1) Perceived impact of Covid-19 on awareness of AMR, OneHealth, and animal welfare food labels; (2) AMR risk perception; (3) Objective  knowledge of antibiotic use in farming; (4) Subjective (perceived) knowledge of antibiotic use in farming; (5) Attributions of responsibility for AMR and; (6) Socio-demographics. Detailed information on wording, scoring, and source of all variables can be found in r <0.6 in all cases) and the variance inflation factor (VIF) scores were all less than the recommended cut-off of 10 (range: 0.55–1.83). A hierarchical cluster analysis using Ward's minimum variance method was carried out to determine the optimal number of segments. Squared Euclidean distance was selected as the distance measure. The optimal cluster solution was selected following an investigation of the agglomeration schedule and a visual inspection of the dendrogram. The 3-, 4-, and 5-cluster solutions were profiled against the cluster variates. The profiling demonstrated that the five-cluster solution provided the most distinct and conceptually meaningful clustering of participants  AMR; (2) OneHealth, and; (3) information about animal welfare on food labels. Before beginning the cluster analysis, variables were examined for collinearity. Examination of the correlation matrix found no substantial multicollinearity between the cluster variates (icipants . Based oI am now more aware of antibiotic resistance.” 42.9% of the participants indicated that they agreed (33.4%) or strongly agreed (9.5%) with the statement “I am now more aware of the connection between the management of animal health and impact on human health.” Finally, one in three (33.8%) indicated they agreed (26.3%) or strongly agreed (7.5%) with the statement “I look more at the labelling on food products for animal welfare information.”Almost half (46.7%) of participants indicated that they agreed (38.1%) or strongly agreed (8.6%) with the statement that as a result of the Covid-19 pandemic, “p = 0.1584); age (p = 0.070); country (p = 0.057); urban-rural divide (p = 0.897); grandparent/relatives owning a farm (p = 0.591); and close neighbours/friends owning a farm (p = 0.859). Each segment profile is described in the following sections with results outlined in Comparison of the five clusters showed they differed significantly on socio-demographic variables and on knowledge (objective and subjective) and beliefs (risk perception and responsibility attributions) related to antibiotic use in farming and AMR . No signParticipants in this segment accounted for 16% of the sample. Covid-19 had little impact on increasing their awareness of AMR, connected animal and human health, and animal welfare information on food labels. These participants were more likely to live alone. They were less likely to come from a farming family and perceived they have the least knowledge about the use of antibiotics in farming, signalling a detachment from farming practises. They had the lowest responsibility ratings for almost all stakeholder groups, indicating a lower likelihood of demanding change from any sector or stakeholder on AMR. From the perspective of communicating with the public about the use of antibiotics in farming, this group will be the hardest to reach. Labelling strategies on agricultural antibiotic use would likely be ineffective. For these types of consumers, and as a first step, there is a need to find a way to increase awareness and knowledge about AMR, OneHealth, and the role of responsible antibiotic use in farming. Given that Covid-19, as a OneHealth emergency in itself, failed to raise awareness, this is not likely to be an easy task.This was the largest segment (30%). Covid-19 did not increase participants' awareness of AMR and only moderately increased their awareness of connected animal and human health and welfare information on labels. These participants were more likely to be in a higher social class and have a higher level of education. Participants tended not to come from a farming family and compared to the other clusters, they were less likely to attribute responsibility for AMR to farmers and vets. Considering their profile, it could be that this group knows little about AMR and antibiotic use in agriculture. This segment may be unlikely to expect change from the farming community with respect to action on AMR. For this segment, representing almost a third of the sample, top-down labelling strategies that proactively communicate about responsible antibiotic use in farming may have little impact. What is first required is an engagement effort that makes clear the concept of connected human and animal health, links between agriculture and AMR, and the need for responsible agricultural antibiotic use.Participants in this cluster (21%) were characterised by an increase in awareness of both AMR and the connection between animal and human health but were not likely to increase their awareness of animal welfare information on labels. Participants were more likely to come from a lower social class, have a lower level of education, live with others and come from a farming family. Along with the “highly aware” cluster, this cluster tended to have higher levels of responsibility attribution overall. Their increased awareness and higher responsibility attributions indicate that participants see a need for action on AMR. However, compared to the other clusters, this group attributed more AMR responsibility to medical professionals—both to vets, and in particular, to medical doctors. It is potentially the case that participants in this segment do not make a connection between issues such as AMR and OneHealth and a need for behaviour change at an individual level . It may be useful to raise awareness and motivation around the need for collective action across different types of stakeholders at all levels, including at the individual consumer level .This was the smallest segment in the sample (12%). Covid-19 increased their awareness of AMR strongly, and of animal welfare information on labels moderately, but did not increase their awareness of connected animal and human health. Participants were more likely to be from a lower social class, come from a farming family, and not have a young child. These participants were least knowledgeable about antibiotic use in farming. They held the lowest AMR risk perceptions and tended to have lower levels of responsibility attribution for AMR overall, signalling that although their awareness of AMR may have increased, they did not view it as an issue of concern or warranting widespread action. In particular, they attributed less responsibility for AMR to agri-food stakeholders and more responsibility to stakeholders such as pharmaceutical companies, public health organisations, scientists, and in particular, medical doctors. Accordingly, this group may view AMR as a “human health” issue and be less aware of the OneHealth connection to AMR. These consumers may not understand the contribution of antibiotic use on farms to AMR, or be aware that AMR also can affect the health and welfare of animals. In such a low-knowledge environment, it is hard to anticipate how such consumers would react to antibiotic-use labels. However, given this group's awareness of farm animal welfare labels; they are likely to be at least interested in them. It would be important to ensure a better understanding of the role of responsible antibiotic use in farming for positive animal welfare.perceived that they had the most knowledge about antibiotic use in farming. They were the most concerned about AMR in respect of risk perception and they were the group most likely to attribute high levels of responsibility for tackling AMR to almost all stakeholders. Given their interest in and concern for AMR, and their awareness of the role of antibiotics in farming, this is a primed audience for immediate or future market-level strategies related to responsible antibiotic use in agriculture.Accounting for one-fifth of the sample, these participants (20.88%) were the most heavily impacted by Covid-19. Their awareness of AMR, connected animal and human health, and animal welfare information on food labels had all strongly increased. Participants had a higher level of education, were more likely to live with others, be a parent of a young child, and come from a farming background. They were both the most knowledgeable about the use of antibiotics in farming, and they all stakeholders  to change their behaviour so as to minimise the spread and development of AMR; more responsibly use antibiotics; or alleviate the need for antibiotics in the first place. At the consumer level, this equates to changing food purchasing and consumption practises . The current study provides early evidence that Covid-19 has catalysed increased awareness of AMR and OneHealth. There may be appetite amongst some consumers to support actions for responsible antibiotic use on farms. However, the pathway for consumer change is not straightforward. A number of conditions need to be met to ensure an effective and responsible market-level strategy.Covid-19 has created additional challenges for tackling AMR—not least of which includes concerns over increased indiscriminate use of antibiotics to treat ill Covid-19 patients ; but, itresponsible (rather than eliminated) use of antibiotics at farm level. This is a fundamental condition for an effective labelling strategy, not only to motivate individual consumer action regarding food choices, but also to avoid any unintended impacts or fall-out from consumer confusion regards the meaning of antibiotic-use labels  . The current study provides an indication of likely support; further research exploring consumers' willingness-to-purchase would be of value . Howeverc free”) , 11. Thec free”) . By whatA limitation of the current study was that the survey only measured perceived increases in consumers' awareness, captured through self-report. The increased awareness of AMR, connected human and animal health, and animal welfare information on labelling could signal that the public are increasingly interested in and aware of the potential impact of transfer through the food chain and desire more assurance in food production. However, we do not have any data to support whether this increased awareness has resulted in behaviour change. Future research avenues include exploring changes in purchasing and consumption behaviour including the extent to which consumers may begin to increase their use of welfare-specific food labels or purchase products with welfare-specific value attributes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.AR was responsible for survey design and over-seeing data collection and led data analysis and paper write-up. SS was responsible for survey design and over-seeing data collection and contributed to paper write-up, CM, TB, and MD advised on survey design and contributed to paper write-up. JH advised on data analysis and contributed to paper write-up. 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.


Background: There is a well-established relationship between cell cycle progression and the development of stomach adenocarcinoma. This study aimed to elucidate the molecular mechanism and biological function of APBB2 in gastric cancer.Methods: Gastric adenocarcinoma (GA) data were downloaded from the TCGA-GA and GEO databases and analyzed to explore differentially expressed miRNAs and mRNAs. Moreover, potential target mRNAs were also predicted. The relative level of gene and protein expression in GA cell lines and gastric mucosa cells was detected by q-PCR and Western blot, respectively. Moreover, the influence of APBB2 on proliferation, metastasis, and cell cycle changes in SGC-7901 and BGC-823 cells was evaluated. The binding relationship between the target miRNA and mRNA was confirmed with a dual-luciferase reporter assay.Results: High APBB2 expression was detected in GA patients, indicating that it may be represent a predictive biomarker for poor prognosis. Related experiments confirmed that APBB2 silencing inhibited GA cellular functions, including proliferation, cell cycle progression, migration, and invasion. In addition, to explore the molecular mechanism, our results indicated that the binding sites were located at hsa-mir-30a and the 3′-UTR of APBB2, suggesting that hsa-mir-30a can regulate the expression of APBB2. The biological functions of hsa-mir-30a were also evaluated. Hsa-mir-30a overexpression attenuated the proliferation and metastasis of cancer cells. In rescue experiments, hsa-mir-30a was confirmed to reverse the cell cycle promoting function associated with APBB2 overexpression.Conclusion: Our findings show that hsa-mir-30a can attenuate the development of GA by down-regulating APBB2 expression. Gastric adenocarcinoma (GA) is a subtype of stomach adenocarcinoma (STAD) that represents a significant cause of cancer-related mortality. –3 DespitCell cycle progression plays a key role in cell proliferation and represents an essential feature of cancer development and progression. In bladder cancer, APBB2 plays a dual regulatory role, mediating the cell cycle via the CDK6 and MET pathways  and was The results of this study showed that miR-30a-mediated APBB2 expression was essential for STAD progression. In addition, APBB2 up-regulation was associated with cell cycle progression in STAD cells, whereas APBB2 silencing attenuated GA cell proliferation. Therefore, exploring the molecular mechanism of APBB2 may provide a novel anti-tumor treatment strategy for GA.https://www.ncbi.nlm.nih.gov/geo/). The SVA package was used for batch correction. To obtain the differentially expressed miRNAs, the “edgeR” package  was used to compare the differences between the control group and tumor group. The mRNA profiles  were obtained from the TCGA-GA clinical database . The “edgeR” package  was used to analyze the differentially expressed miRNAs (DEmiRNAs) between the normal group and tumor group. The potential upstream miRNAs of APBB2 were predicted according to the mirDIP and starBase databases. DEmiRNAs were used to explore the binding sites between the miRNAs and target mRNA. A Kaplan-Meier analysis was used to calculate the overall survival (OS) probability associated with the target mRNA and corresponding miRNAs based on the TCGA-GA dataset.GSE112264  datasets were obtained from the GEO database  and a human gastric mucosa cell line (GES-1) were purchased from Bluefcell . Complete medium consisting of Dulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum  was used for cell culture. All cell lines were cultured in an incubator containing 5% COMimic NC, miR-30a-3p mimic, sh-NC, sh-APBB2 , over-expressed (OE) plasmid of APBB2 (OE-APBB2) and OE-NC plasmid were obtained from Genomeditech  and transfected into GA cell lines using Lipofectamine 3000  in accordance with the manufacturer’s instructions.® Premix Ex TaqTM Kit (TaKaRa Biotechnology) master mix and qPCR reactions were performed on a StepOnePlusTM Real-Time PCR System. The following human primer sets were used: human APBB2: forward, 5-ATGGGACTGCGGAAGAGAAA-3 and reverse, 5-GCCCCTGTTTTCGGATGATC-3; human GAPDH: forward, 5-CCAGAACATCATCCCTGCCT-3 and reverse, 5-CCTGCTTCACCACCTTCTTG-3.Total RNA was obtained using an Axygen RNA Miniprep Kit  in accordance with the manufacturers’ instructions. A NanoDrop 2000/2000C spectrophotometer was used to test the RNA purity and concentration at wavelengths of 260 nm or 280 nm. A PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology) was used to reverse transcribe 2 μg of the extracted RNA into cDNA. The resultant cDNA was used as a template in the TB Green3 cells/well were seeded into six-well plates and incubated at 37°C for approximately 9 days. The cell colonies were fixed in 5% paraformaldehyde for 10 min and the cell colonies were stained with 0.5% crystal violet. The number of colonies was calculated using ImageJ software.A colony formation assay was used to estimate the level of cellular proliferation. At 48 h post-transfection, 1 × 10Adherent cells were collected using trypsin and washed twice in PBS. The cells were stained with propidiumiodide for analysis via flow cytometry . The proportion of cells at different time points were calculated by FCM .5 cells were seeded into the upper chamber with DMEM. Complete medium was placed into the lower chambers. After a 10 h incubation, the cells in the upper surface of the upper chambers were wiped off. The upper chambers were stained by crystal violet to observe the migrated cells.The migration and invasion ability of GA cells was evaluated by transwell experiments using transwell chambers . A total of 1 × 10Luciferase reporter vectors based on psicheck2 were constructed including wild-type APBB2 (APBB2-WT) and mutant-type APBB2 (APBB2-MUT) at the miR-30a-3p binding sites. HEK293T cells were seeded into 24-well plates and co-transfected with the APBB2-WT/APBB2-MUT vector, miR-30a-3p mimic, or NC mimic via Lipofectamine 2000. After transfection for 48 h, the cell lysates were collected and luciferase activity was measured by using a dual-luciferase reporter gene assay system (Promega).t test. The results for multiple group comparisons were analyzed using a Scheffe's test and one-way analysis of variance (ANOVA) with SPSS 22.0 software . A Pearson χ2 test was used to analyze the relationship between miR-30a-3p and APBB2 expression. Values were determined to be significant at *P < 0.05, **P < 0.01, and ***P < 0.001.GraphPad Prism 6.0 (Graphpad Software Inc.) was used for all statistical analyses. All values are presented as the mean ± standard deviation (SD). Differences between the experimental and control groups were evaluated using a Student's Kaplan-Meier analysis indicated that high APBB2 expression indicated poor OS . The sexThe level of APBB2 mRNA expression was down-regulated in SGC-7901 and MGC803 cells using sh-APBB2 transfection . The WesTo explore the mechanism of APBB2 expression in GA, an in-depth analysis of the regulatory pathways located upstream of APBB2 was conducted. A total of 89 different miRNAs were obtained to perform a differential analysis . The potThe binding regions between miR-30a-3p and APBB2 were explored using miRDB and starBase . APBB2 pNext, we further explored the effects of miR-30a-3p expression on APBB2-related cell functions. The efficiency of the APBB2 overexpression  and miR-Our results reveal that APBB2 is up-regulated in GA and promotes the biological function  of GA cells. Further investigation into the associated mechanism demonstrated that miR-30a-3p regulates APBB2 expression. Traditionally, APBB2 has been regarded as an Alzheimer disease protein associated with cognitive impairment . Recent It has been found that miRNA plays an essential role in multiple biological functions, including cellular migration, proliferation, autophagy, and transformation . miRNA fIn addition, miR-30a-3p has been reported to play a key role in several types of cancer –16. In gAPBB2 plays an important role in cancer cell proliferation. Our findings reveal that mir-30a-3p can regulate the malignant process of GA via suppressing APBB2-mediated cell cycle progression. Accordingly, this research confirmed that APBB2 may be used as a novel biomarker for the early diagnosis and therapeutic targeting of GA.The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.The study design was approved by the Ethical Committee of Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.


To assess whether the prognostic impact of conventional risk factors and ancillary biomarkers differs across the 2 largest ProMisE molecular subgroups of endometrial carcinoma (EC).POLE exonuclease domain hot spots and immunohistochemistry for MLH1, PMS2, MSH2, MSH6 and p53 were performed on 745 unselected endometrioid ECs to identify mismatch repair deficient  and no specific molecular profile  ECs. Molecular group-specific survival analyses and interaction analyses were performed to determine the prognostic relevance of clinicopathological factors and various biomarkers  within the subgroups.Direct sequencing of Molecular subgroup did not have an independent effect on disease-specific survival after adjustment for conventional risk factors (P = 0.101). High grade (G3) and p16 hyperexpression remained significant predictors of survival in NSMP. Stage II-IV, ≥50% myometrial invasion, lymphovascular space invasion and loss of E-cadherin were independent predictors in the MMR-D group. In the interaction analysis, molecular subclass significantly modified the prognostic effect of high grade and p16 hyperexpression, which showed a stronger negative effect on survival in NSMP as compared to MMR-D (P for interaction = 0.016 for grade and 0.033 for p16).Grade of differentiation and p16 hyperexpression appear to have a stronger prognostic impact in NSMP as compared to MMR-D EC. While these results need to be confirmed in a larger study population, they indicate that differential impact of risk factors needs to be taken into account when developing new molecular class-integrated risk stratification algorithms for EC. The standard treatment of endometrial carcinoma (EC) consists of hysterectomy and bilateral salpingo-oophorectomy completed with lymph node dissection in selected cases. Post-operative risk stratification guides the selection of adjuvant treatment (observation vs radiotherapy and/or chemotherapy). Current treatment algorithms are based on disease stage, tumor histotype and grade of differentiation, depth of myometrial invasion, lymphovascular space invasion (LVSI) and patient’s age .POLE) ultramutated, microsatellite instability (MSI) hypermutated, copy-number low, and copy-number high [POLE sequencing for ultramutated subgroup, mismatch repair immunohistochemistry/MSI for hypermutated subgroup and p53 protein expression/mutational analysis for copy-number high subgroup [As histopathology-based risk factors including tumor histotype, degree of differentiation and LVSI, suffer from limited reproducibility –4, numerber high . Due to ber high  and the ber high . These msubgroup , 7. TumoMolecular classifiers provide information beyond traditional clinicopathological factors and there is evidence that risk assessment methods can be improved by integrating clinicopathologic and molecular factors , 8. The This was a retrospective study of 842 patients who underwent primary surgical treatment for stage I-IV EC at the Department of Obstetrics and Gynecology, Helsinki University Hospital between 2007 and 2012. The study was approved by the Institutional Review Board and the National Supervisory Authority for Welfare and Health. Informed consent was waived because of the retrospective nature of the study. All data were fully anonymized before analysis. A tissue microarray of primary tumor samples was constructed during the year 2013 . Follow-Clinicopathologic data were abstracted from institutional medical and pathology records. Stage was determined according to the 2009 International Federation of Gynecology and Obstetrics (FIGO) guidelines . Stage IThe following monoclonal antibodies were used for chromogenic immunohistochemistry on multicore TMA slides: MLH1 , MSH2 , MSH6 , PMS2 , p53 , L1 cell adhesion molecule , beta-catenin , estrogen receptor alpha , progesterone receptor , p16 , E-cadherin . TMA slides were scanned with 3-dimensional Histech Pannoramic 250 Flash II scanner by Fimmic Oy . Slide images were managed and analyzed with WebMicroscope Software (Fimmic Oy). A pathologist scored virtual slides blinded to clinical data (A.P). Equivocal cases were examined by a second investigator (R.B) and a consensus was reached.Mismatch repair protein status was considered deficient when we observed a complete diffuse or clonal loss of nuclear expression in carcinoma cells of 1 or more MMR proteins . Abnormal p53 staining was defined as strong and diffuse nuclear staining or completely negative (“null”) staining in carcinoma cells. Weak and heterogeneous staining was classified as wild type expression. Cytoplasmic staining of p53 was not considered as the significance of this finding was unclear at the moment of the scoring. Stromal cells and inflammatory cells served as internal control for MMR and p53 stainings. L1CAM expression was scored as reported earlier, with ≥ 10% of membranous staining considered positive . We adopPOLE exonuclease domain hot spot mutations in exon 9, exon 13 and exon 14 [KRAS mutations in exon 2 (codons 12 and 13). Polymerase chain reaction products were sequenced on an ABI3730xl Automatic DNA Sequencer at the Institute for Molecular Medicine Finland, Helsinki. Sequence graphs were analyzed both manually and with Mutation Surveyor .For DNA extraction, representative areas of formalin-fixed paraffin-embedded (FFPE) tumor sections were macrodissected as identified by pathologist assessment. DNA was extracted by proteinase K/phenol-chloroform method. Direct sequencing was performed to identify POLE mutated, p53 abnormal and NSMP  were used for comparison of categorical variables. Survival curves were calculated by the Kaplan-Meier method. A log-rank test was used to test for survival differences. Simple and multivariable analyses for prognostic factors were conducted by the Cox proportional hazard model. To test the statistical significance of eventual interaction effects between molecular subgroup and single risk factors, we separately added each interaction term in a multivariable regression model including conventional risk factors and the main effects of the variables forming the interaction term. Statistical significance was set at P < 0.05. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 25 software .Variables selected for statistical analyses were FIGO 2009 stage, age, grade of differentiation, depth of myometrial invasion, LVSI, molecular subgroup , L1CAM, ER, PR, beta-catenin, p16, E-cadherin, POLE mutated, 36 (6.7%) as p53 abnormal and 206 (38.5%) as NSMP  were classified as MMR-D, 29 (5.4%) as  as NSMP . ClinicoPOLE mutational data was not available for 162 patients with MMR intact tumors. These cases were excluded from further analysis. When comparing the successfully classified NSMP cases and MMR intact–p53 wild type cases that were excluded due to missing POLE data , the distribution of clinicopathological features did not significantly differ between the included and excluded cases with the exception of myometrial invasion . In pairwise comparisons between NSMP and MMR-D, survival difference remained significant (P = 0.005). Survival curves displaying the effect of single risk factors within the molecular subgroups are depicted in Median follow-up time was 83 months (range 1–136 months). Seventy-two patients (15.3%) died of EC during follow-up. Disease-specific mortality rate was 10.2% in the NSMP and 19.3% in the MMR-D group (P = 0.001). Kaplan-Meier disease-specific survival curves according to molecular subgroups are depicted in The results of simple and adjusted Cox regression survival analyses in NSMP and MMR-D groups are shown in In the interaction analysis, the prognostic effect of grade of differentiation and p16 expression differed significantly between the molecular groups  and low-risk features (POLE mutation and absence of CTNNB1 mutation in NSMP) [CTNNB1 mutational data was not available and immunohistochemical positivity for beta-catenin appeared not to have prognostic value. Regarding other potential biomarkers included in our study KRAS mutation did not correlate with outcome and hormone receptor status lost its significance after adjusting for confounding variables.The ongoing PORTEC-4a trial combines TCGA-based molecular subgroups, clinicopathological risk factors and ancillary molecular markers in order to investigate standard versus molecular-based recommendation for radiotherapy in early-stage EC. The trial algorithm subcategorizes ECs according to the presence of high-risk features , intermediate risk (MSI and in NSMP) . L1CAM iin NSMP) , 24–26. POLE data, which was due to DNA not being available, limited yields of high-quality DNA from formalin-fixed tissue and our stringent criteria of inclusion for POLE sequencing data. Given the order of decision steps in our molecular classification algorithm, missing POLE data limited the number of patients in NSMP but not in the MMR-D subgroup, resulting in a higher than expected proportion of MMR-D cases. However, the distribution of clinicopathological characteristics in the NSMP group remained representative. Given the profound pathogenetic and prognostic differences between endometrioid and non-endometrioid carcinomas of the uterus, analyses were restricted to the more common endometrioid subtype of endometrial carcinoma.A strength of this study is our regularly updated database with long follow-up time. Comprehensive reports on causes of death allowed us to investigate disease-specific survival instead of overall survival, which is a less accurate measure of outcome when searching for causalities in cancer patients. A limitation was the relatively high rate of missing Intratumoral heterogeneity of protein expression may lead to decreased sensitivity in TMA studies. Clonal loss of MMR protein expression has been reported but this phenomenon is not common. As the rate of mismatch repair deficiency in our study was not lower than generally reported, our results were unlikely to be compromised by false negative scores , 27. To This proof-of-concept study provides interaction analysis-based data which show that the relative weights of different risk factors vary between molecular subgroups  of EC. Accordingly, novel putative disease markers of EC should be analyzed in a molecular subgroup-specific manner. Larger studies are needed to confirm our findings and to reveal further interaction effects for known risk factors that we may have missed due to limited sample size. Eventually, randomized clinical trials incorporating both molecular and clinicopathological features in treatment algorithms will determine the true value of TCGA-integrated classifiers and optimal algorithms to guide the management of EC patients. S1 Table(DOCX)Click here for additional data file.


Electrospun metal oxide nanofibers, due to their unique structural and electrical properties, are now being considered as materials with great potential for gas sensor applications. This critical review attempts to assess the feasibility of these perspectives. The article in Part 1 discusses the basic principles of electrospinning and the features of the formation of metal oxide nanofibers using this method. Approaches to optimization of nanofibers’ parameters important for gas sensor application are also considered. In recent years, amid increased attention to metal oxide one-dimensional 1D) nanomaterials such as nanowires or nanotubes D nanomat,4,5,6,7,Earlier in ,4,5,6,7,By now, a huge amount of information has been accumulated concerning the development of gas sensors based on metal oxide nanofibers. Moreover, quite a few reviews have already been published devoted to the consideration of electrospinning technology ,18,19,20Taking into account a wide variety of metal oxides prepared in the form of nanofibers and the large amount of information obtained during the development of gas sensors based on them, our review, “Electrospun metal oxide nanofibers and its conductometric gas sensor application”, is divided into two parts: Part 1: Nanofibers and features of their forming, and Part 2: Gas sensors and their advantages and limitations. The first part, i.e., this article first briefly discusses the basic principles of electrospinning used for the formation of polymer fibers. Further, the results related to the formation of metal oxide nanofibers are considered in detail. This consideration was carried out in terms of the formation of nanofiber-based gas-sensing materials. In the following Part 2 of this article, approaches to the fabrication of gas sensors are considered, as well as the results of analysis of the characteristics of fiber-based gas sensors. It then provides a detailed analysis of the drawbacks that may limit the use of electrospinning technology in the development of gas sensors. Some approaches to solving these problems are also suggested in Part 2. Finally, the summary provides an insight into the future prospects of electrospinning applications for the development of gas sensors.Nanofibers (NFs) can be formed using a variety of approaches ,31,32,33Initially, electrospinning was developed for polymers . A typicAlthough a spinneret with a metallic needle is convenient for applying a charge to the solution, the process also works if a high voltage is applied to the solution using a special electrode with a nonconducting spinneret and needle. When the repulsive force, induced by the charge distribution on the drop surface, is balanced with the surface tension of the liquid, the liquid drop distorts into a conical shape. Once the repulsive force exceeds the surface tension, a charged jet of liquid is ejected from the tip of the cone and moves toward a grounded electrode. Unlike conventional spinning, the jet is only stable near the tip of the spinneret, after which the jet is subject to bending instability. Whether the jet will form a continuous fiber or disperse into droplets depends on polymer molecular weight, polymer chain entanglement, and the solvent used in the process . The liquid jet will stay in a single stream status if molecular cohesion overcomes electrostatic repulsion or break up otherwise. If the resultant is collected as droplets, then the process should be called electrospraying instead of electrospinning, where the liquid jet breaks up into multiple streams. The theory for electrospinning one can find in ,38,39,40Generally, the electrospun fibers are being deposited on a fixed collector having a horizontal or vertical arrangement see . UsuallyThis section may be divided into subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.Although electrospinning could use molten polymers , researcThe vapor pressure of the solvent should be suitable so that it evaporates quickly enough for the fiber to maintain its integrity when it reaches the target but not too quickly to allow the fiber to harden before it reaches the nanometer range. However, very high volatility is not suitable for fiber spinning as the jet can solidify immediately after exiting from the spinneret. If the volatility is too low, the fibers will still be wet when they are deposited on the collector;The viscosity and surface tension of the solvent should be carefully balanced. They must neither be too large nor be too small. It was established that smooth fibers are being produced when the product of intrinsic viscosity (η) and polymer concentration (c), known as Berry’s number, Be = η × c, is greater than a certain critical value, which is characteristic of the polymer used. In the case of low-viscosity and low concentration liquids instead of nanofibers, small droplets are formed as a result of the varicose breakup of the jet. One should note that high solution concentration and large viscosity and surface tension of solvents for the electrospinning process are also not optimal. In this case, the formation of continuous fibers is prohibited due to the impossibility of maintaining the flow of solution at the tip of the needle. In optimal conditions, a solid fiber is generated instead of breaking up into individual drops due to the electrostatic repulsions. As the charged jet accelerates toward regions of lower potential, the solvent evaporates while the entanglements of the polymer chains prevent the jet from breaking up. This results in fiber formation. Typical SEM images of polymer fibers are shown in The power supply must offer a voltage large enough to overcome the viscosity and surface tension of the polymer solution to form and sustain the jet from the pipette;Regardless of the setup configuration used, the collector must be located at a certain distance from the needle. The gap between the pipette and grounded surface should not be too small to create sparks between the electrodes but should be large enough for the solvent to evaporate in time to form fibers. Typically, this distance can be varied from 9 to 25 cm.The experiment showed that for the successful implementation of electrospinning, it is necessary to fulfill several conditions ,45,46:AlAs you can see, the device for electrospinning shown in A side-by-side method of electrospinning offers a different approach to using several polymer-based solutions in the nanofiber forming process . Such dew), molecular weight distribution and architecture  of polymers, polymer concentration, solution viscosity, solution conductivity, and surface tension, flow rate, applied voltage, the working distance between the collector and the needle tip and ambient parameters such as temperature, air humidity and air velocity in the chamber [It is believed that the main advantage of the electrospinning technique is the ability to control the fiber diameter, high surface-to-volume ratio, and porosity of the formed nanofiber mat. However, as a result of extensive research on electrospinning, it was found that it is quite difficult to carry out such control. Nanofiber parameters such as fiber collectability, uniformity of fibers, average fiber diameter, fiber diameter distribution, and fiber porosity are highly dependent on a large number of processing parameters such as solution properties ,62,63,64 chamber ,65,66,67 chamber ,69,70,71From the above, it follows that for each electrospinning application, it is necessary to select suitable materials such as polymer and solvent and to optimize the spinning process in order to obtain nanofibers with a certain morphology and properties corresponding to the desired functions of nanofibers in the expected applications. At that, there are no secondary factors when optimizing the electrospinning process . Even thAccording to Lubasova and Martinova , the useOther advantages of the electrospinning technique include the relatively simple and inexpensive production of a large number of different types of nanofibers ,76,77,782/SiO2 and Al2O3 [2 [2O5/SiO2 [2/ZrO2 [3O4, and NiO [The first attempts to form metal oxide nanofibers by electrospinning showed that it is possible to synthesize metal oxide nanofibers directly in the electrospinning process. This technology is a combination of electrospinning and the sol-gel process. However, it turned out that this process is difficult to implement due to the strict requirements of the viscoelasticity of the solution. This required careful selection of both metallic precursors and solvents and processing parameters ,80,81. And Al2O3 , SiO2 [7Al2O3 [2 , V2O5/Si2O5/SiO2 , SiO2/Zr [2/ZrO2 , Co3O4,  and NiO . The key and NiO . In addi and NiO .w or a significant degree of chain entanglement [In order to reduce the diameter of electrospun fibers and expand the amount of inorganic materials on the basis of which nanofibers can be formed, Li and Xia  suggestenglement . For thenglement ,90. In tnglement . Rapid hnglement . By opti2O3 nanofibers shown in 2O3 nanofibers calcined at T = 500 °C consist of nanoparticles with a primary particle size of 10–20 nm and mesopores with a pore diameter of 10–20 nm , polyvinylpyrrolidone (PVP), dimethylformamide (DMF), and ethanol [The concentration of the metal precursor in solution, the nanofiber diameter, and the interaction of the precursor solution with the environment during electrospinning and subsequent calcination also play an important role in this process . The dat ethanol . The conAs is known, for gas-sensing applications, the size of the grains and crystallites that form the NFs should be minimized. Generally, NFs with smaller grains and crystallites have better sensitivity due to the higher surface area see . Therefo2 [2 [3 [0.8Fe0.2O3-δ [3 [2O3 [2 [At present using mentioned above electrospinning technology, nanofibers of most metal oxides used in the development of gas sensors were subsequently synthesized . For exa2 ,101, SnO2 [2 ,103,104,2 [2 [3 , ZnO [102 [2 [3 ,107, SrTe0.2O3-δ , BaTiO3 2O3-δ [3 , In2O3 [ [3 [2O3 , CeO2 [9 [2O3 [2 , CuO [93 [2O3 [2 , etc. TaAs can be seen from It was found that nanofiber composites and multicomponent nanofibers could also be formed via the electrospinning technique ,17,23,76The experiment showed that nanofibers with hollow and core-shell structures could also be prepared by single-nozzle electrospinning, followed by appropriate post-treatment. The ability to form hollow and core-shell nanostructures is an important factor for gas sensor application of this technology since the use of hollow spheres and nanotubes makes it possible to significantly increase the active surface of gas-sensitive materials .2 [2O3-ZrO2 [2 [12O19 [3O4 and Co3O4 [2O3 [2O4 [2 [Currently, several different mechanisms have been proposed to explain the formation of hollow fibers ,119,120.2 , Y2O3-Zr2O3-ZrO2 , ZnO [12-ZrO2 [2 , BaFe12O2 [12O19 , α-Fe3O4nd Co3O4 , Fe2O3 [2O3 [2O4 , CuO [122O3 [2O4 , and SnO2 hollow fibers by co-electrospinning viscous heavy mineral oil as the core and a mixture ethanol solution of PVP and Ti(OiPr)4 as the shell. They found that rapid stretching of the sheath causes strong viscous stress that stretches the oil phase and lengthens it along with the sheath solution through viscous entrainment and/or contact friction mechanisms. As a result, the heavy mineral oil remains in the shell of the amorphous TiO2/PVP composite. That is why the removal of the mineral oil contributes to the subsequent formation of TiO2/PVP composite tubes, which, after calcination at elevated temperatures (T = 500 °C) in the air, transform into hollow TiO2 fibers. The oil can be extracted by immersing the sample in octane. The wall thickness and inner diameter of the hollow nanofibers could be varied in the range from tens of nanometers to several hundred nanometers by controlling the processing parameters such as the applied voltage and the injection rate for the oil phase  chloride tetrahydrate, dissolved in ethanol and PVP dissolved in a DMF:ethanol solution was used, while for the shell region, the solution of iron (III) nitrate nonahydrate dissolved in DMF and PVP was used. For metal oxide crystallization and removal of residual polymer, as-electrospun NFs were heat-treated at 500 °C for 5 h.Using the coaxial co-electrospinning technique shown in 2–SnO2 fibers  ,137 and Phase separation during electrospinning can be applied to introduce a porous structure by transforming one phase as the pores while the other as the nanofiber matrix. In this case, phase separation can occur (i) between the polymer and solvent or (ii) between the polymer and a nonsolvent . Phase sSome aspects of the formation of porous nanofibers, such as the effect of temperature, air humidity, and solvent volatility, were discussed earlier in The experience gained in the development of conventional gas sensors has shown that pristine metal oxides cannot always meet all the requirements arising in the development of sensors designed for a specific application. As a rule, to solve this problem, these metal oxides have to be modified to provide them the desired properties ,144,145.The blending method refers to the process that is to modify the properties of nanofibers by adding modifying additives directly to the electrospinning solution. For example, for the modification of metal oxides with noble metals, noble metal NPs or their precursors are mixed with the spinning solutions to form a uniform precursor solution, and then the mixed solution is directly electrospun to form after calcination various materials interfaces. This method has advantages such as easy preparation and high yield, and it is widely used to improve the sensitivity and selectivity of gas-sensitive materials . However2O3 nanofibers. Mg-doped In2O3 NFs were prepared using InCl3·4H2O and Mg(NO3)2·6H2O precursors mixed with PVP and DMF. Calcination was performed at 600 °C. Other doping additives used in the optimization of gas sensor parameters are listed in The blending method, in addition to decoration, also allows the doping of metal oxides. For example, using this approach, Lin et al.  fabricat0–SnO2 composite-based nanofibers, proposed the process with the following stages: initially, Pd–SnO2 composite was formed by the blending method using SnCl2 and PdCl2 precursors through the electrospinning. After that, the composite fibers were heat-treated to obtain Pd2+-loaded SnO2 composite fibers, which were finally transformed in Pd0–SnO2 under the reduction by hydrazine hydrate.In post-modification, metal oxide nanofibers are prepared by electrospinning, and only after that are they subjected to processing, which affects the properties of the formed nanofibers. For example, to decorate the surface of metal oxides with clusters of noble metals, metal NPs can be adsorbed from solutions or deposited by various physical and chemical methods onto metal oxide nanofibers to obtain the metal NP-NF-based interface. In particular, for these purposes, you can use the physical dip-coating method, which is one of the simplest methods to endow nanofibers with active sites for target interaction. This process can also include post-treatment, such as calcining. For example, Wang et al. , to obtaThe principles of post-modification of nanofibers can also be used to form hollow and core-shell metal oxide structures. To date, several approaches have been developed for the manufacture of hollow metal oxide nanofibers using the principles of post-treatment. For example, electrospun nanofibers can be used as sacrificial templates for making tubular fibers ,154. In 2O3 hollow nanofibers using a layer-by-layer assembly technique and CNTs as a template. A diagram of this process is shown in 2 at 550 °C during 3 h, hollow In2O3 nanofibers were fabricated. These In2O3 hollow nanofibers had a diameter from 30 to 60 nm [2O3 hollow nanofibers. This property of the nanofibers formed was of great importance for the gas-sensing effect [Various coating techniques, such as chemical and physical vapor deposition, sol-gel process, layer-by-layer assembly, electrochemical deposition, etc. ,199,200,to 60 nm . It is ig effect . Due to 3 nanofibers prepared by deposition of W on SWCNT template with the following annealing in an oxygen-containing atmosphere [It should be borne in mind that when using physical methods of coating template fibers, as a rule, it is not possible to form hollow nanofibers. These methods cannot provide the coating of all sides of the template. Therefore, after removing the template, we obtain ordinary nanofibers, the morphology of which depends on the morphology of the original template and the coating modes. mosphere .2 film. The surface morphology of the template depended on sputtering time. After removing the template, the SnO2 NFs had uniformly distributed branches all over the stem.Fan et al.  showed t2 nanofibers as an example, Zhang et al. [4·5H2O composite fibers before calcination gives a significant increase in their porosity, which is retained even after annealing (see Previously, Zhang et al.  also useg et al.  demonstrling see .2 sensors to ethanol. At 340 °C, the detection limit to ethanol was <1 ppb. Most likely, the high sensitivity is a consequence of the small size of the SnO2 crystallites in the nanofiber, which did not exceed 7 nm. The highly porous structure of nanofibers, due to a decrease in the number of contacting crystallites, hindered the growth of crystallites during annealing. Oxygen plasma etching also increased sensor response of CuO/SnO2 sensors to H2S [It is seen that, after annealing at 500 °C, no obvious changes in the fiber morphology occur. The plasma reaction system worked at a frequency of 13.56 MHz. Zhang et al.  found ths to H2S .To increase the porosity of metal oxide nanofibers, one can use other methods developed for polymer nanofibers . For exaAs for the formation of core-shell metal oxide structures using post-treatment principles, the approaches that allow the creation of such structures does not differ in any way from the approaches described earlier when discussing the processes of formation of hollow nanofibers. The only difference is that instead of polymer or carbon nanofibers, metal oxide nanofibers formed by electrospinning are used as templates, and various chemical methods of forming metal or metal oxide coatings are used as post-treatment. If metals are deposited on the surface of metal oxide nanofibers, then subsequently, the structures undergo thermal oxidation. The most common methods used in the formation of core-shell structures are electrochemical deposition and hydrothermal, sol-gel, sonochemical, and microwave assistance synthesis .The analysis showed that electrospinning technology really presents great opportunities for the formation of gas-sensitive materials with a unique combination of parameters. Electrospinning makes it possible to form a gas-sensitive matrix from small crystallites while maintaining a very high gas permeability of the matrix due to the ultra-high porosity of the structure. This, on the one hand, should provide ultra-high sensitivity of the sensors under optimal conditions, and on the other hand, guarantee a fast response and recovery since such a structure has no diffusion restrictions for gas penetration into the gas-sensitive matrix. This technology is also effective for the manufacture of hollow tubes and core-shell structures, the use of which provides additional opportunities for improving the parameters of conductometric gas sensors. The nanofiber-based conductometric gas sensors are discussed in more detail in the second part of the article.