Our results also suggested that BTZ increased the number of regulatory T cell (Treg) and significantly reduced the proportion of T helper (Th17) cell (p < 0.05). Besides, BTZ induced the significant upregulation of anti‐inflammatory cytokines but downregulated pro‐inflammatory cytokines (p < 0.05). After dealing with Atg5 siRNA‐lentivirus, the effect of BTZ alleviating AMR was reversed and Th17/Treg proportions were also significantly modulated. Collectively, these findings show that BTZ slows down the process of AMR and Atg5 may be the key mechanism. Furthermore, Atg5 silencing results may be demonstrated that Atg5 alleviated AMR by modulating the ratio of Th17/Treg.Antibody‐mediated rejection (AMR) is one of the most dominant mechanisms responsible for the loss of kidney grafts. Previous researches have shown that donor‐specific antibodies (DSAs) are the major mediators of AMR. In order to prolong the survival time of grafts, it is vital to reduce the incidence of AMR and inhibit the generation of DSAs. We established an animal model of AMR by performing kidney transplantation in pre‐sensitized rats. Then, we investigated the effect of bortezomib (BTZ) on AMR. We found that BTZ could reduce the serum level of DSAs and alleviate post‐transplantation inflammation in peritubular capillaries (PTCs) and glomeruli, which was demonstrated by the reduction of C4d and IgG deposition in PTCs, and the reduced number of B cell and plasma cell in peripheral blood and the transplanted kidney ( It is well established that Treg is one of the key mechanisms used to regulate inappropriate or excessively strong immune response.Bortezomib (BTZ) was the first clinical proteasome inhibitor and has been approved for the treatment of multiple myeloma, a malignant plasma cell (PC) disorder.22.1All animal experiments were conducted in the Animal Core Facility of Nanjing Medical University and were performed in accordance with National Health Organization and Nanjing Medical University Institutional Ethical Guidelines for animal experiments .2.2BTZ  was injected intravenously into recipient rats every day following kidney transplantation. Before the experiment, we divided BTZ into three concentration gradients for pre‐experiment, and finally confirmed the best concentration. Rat Atg5 siRNA‐lentivirus (Atg5 siRNA) (sc‐41445) were acquired from Santa Cruz Biotechnology. The antibodies used for Western blot (WB)  were purchased from Cell Signaling Technology. The antibodies used for immunohistochemistry (anti‐CD19 and anti‐CD138) were purchased from Abcam. The antibodies used for immunofluorescent staining were anti‐rat C4d antibody , FITC‐conjugated goat anti‐rat IgG antibody (Jackson ImmunoResearch). Several antibodies were used for flow cytometric analysis: FITC‐CD4, APC‐CD25, PE‐FOXP3 and PE‐IL‐17A (Thermo Fisher Scientific); APC‐CD19 ; and APC‐CD138 (Abcam).2.3u) rats were used as donors, and Brown Norway  rats were used as recipients. These animals were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All rats were male, 6 weeks of age and weighed between 180 and 220g.Lewis (LEW)  were taken from the hackles of LEW rats and then transplanted onto the hackles of BN rats. Once the skin was removed from the donor LEW rats, it was wrapped with gauze and placed in ice water containing penicillin (8 × 104 u/ml). Blood samples were then obtained from the BN rats, and serum samples were prepared by centrifugation. We then tested the serum concentrations of DSAs after skin transplantation at 1, 3, 7, 10, 14, 21, 28 and 35 days, respectively. Kidney transplantation was performed when the level of DSAs reached peak values. After anaesthesia, the LEW rats were fixed, disinfected and incisions were made along the middle abdominal line. Next, we fully exposed the abdominal aorta, inferior vena cava, renal artery and renal vein; these vessels were carefully isolated by light microscopy. The kidney of donor animals was perfused, placed in perfusion fluid, and then stored in a refrigerator at 4°C. Similarly, we also visualized and separated the renal artery, renal vein and ureter of BN. The renal artery and renal vein were then anastomosed end‐to‐end, and the ureter was embedded into the bladder. After the removal of the contralateral kidney, the abdomen was closed and disinfected. We obtained further serum and transplant kidney on day 4 after kidney transplantation. Serum samples were frozen at −80°C to await analysis. Every transplanted kidney was divided into two parts. One was partially filled with RNAkeeper (Vazyme Biotech Co. Ltd) solution and then stored at −80°C while another part was kept in 4% paraformaldehyde.2.5Our experiments featured four groups of rats: a normal group (non‐treated BN rats); a skin group (STx) featuring BN rats on day 14 after skin transplantation; a kidney transplantation group (KTx) featuring rats that underwent renal transplantation; and an AMR group featuring BN rats which underwent renal transplantation after skin grafting. Every group have at least 5 rats samples. Kidneys were harvested on day 4 after kidney transplantation. Our aim was to use AMR model to investigate the effect of BTZ. Similarly, in this research, every group have at least 5 rats samples. BN rats were injected with 0.2 mg/kg of normal saline (once daily by tail vein injection) after receiving an open/closed operation; these formed a Con‐Veh group. Another group of BN rats were given 0.2 mg/kg of BTZ (once daily) after receiving an open/closed operation; these formed a Con‐BTZ group. The AMR‐Veh group featured pre‐sensitized BN rats that were given 0.2 mg/kg of normal saline (once daily) following kidney transplantation. While the final group, the AMR‐BTZ group, featured pre‐sensitized BN rats that were given 0.2 mg/kg of BTZ (once daily) following kidney transplantation.2.6TM MESF Kit (Bangs Laboratories) is supplied with one blank microsphere population and four microsphere populations that are surface‐labelled with increasing concentrations of a specified fluorochrome. These populations are calibrated in MESF units. The five microsphere populations were tested by the Beckman DxFLEX FCM; data were analysed by downloading specific software from www.bangslabs.com. MFI was then converted to MESF units; this practice added internal parameters to the MFI data and increased reliability.Spleen lymphocytes were extracted from normal LEW and counted under aseptic conditions. Serum samples of BN rats were incubated with spleen lymphocytes at room temperature for 30 min. Cells were then washed and incubated with fluorescein isothiocyanate (FITC)‐labelled anti‐rat IgG antibody (BD Biosciences) at room temperature for 30 min. Cells were then analysed by flow cytometry (FCM) , and mean fluorescence intensity (MFI) was measured to determine the serum level of DSA. The Quantum2.7Kidney grafts were taken from the recipients at a fixed time point after transplantation. Allografts were harvested, formalin‐fixed, embedded in paraffin and stained in haematoxylin and eosin (H&E) and periodic acid‐Schiff (PAS). Pathological sections were then observed using a Nikon E100 microscope (Nikon) and investigated for the characteristic features of AMR, including PTC inflammation, glomerulitis and intimal arteritis, as described by the Banff 2013 guidelines.2.8For immunohistochemistry, renal tissue was fixed with 4% paraformaldehyde, embedded in paraffin and then sectioned. Tissue sections were placed into a box and then heated in a microwave oven for antigen retrieval. Then, we blocked endogenous peroxidase by treating the sections with 3% hydrogen peroxide. Sections were then incubated with a primary antibody followed by an appropriate secondary antibody. Antibody binding was then visualized by DAB treatment. The nuclei were then stained, and the sections were dehydrated. Finally, the sections were mounted on glass slides for analysis. For immunofluorescent analysis, renal tissue was frozen and then embedded in paraffin. In order to observe the deposition of C4d, frozen sections were incubated with rabbit an anti‐C4d antibody (diluted 1:50 in PBS) for 30 min at room temperature. The sections were then washed four times in PBS and then incubated with FITC goat anti‐rabbit IgG. To investigate the deposition of IgG in tissue grafts, we stained 4 mm cryosections with FITC‐conjugated goat anti‐rat IgG. Image‐pro Plus software (version 6.0) was then used to evaluate the expression CD19 CD138 C4d deposition and IgG deposition.2.9Samples of serum and supernatant were acquired, processed and immediately frozen at −80°C to await analysis. The level of cytokines in these samples was then determined using a range of commercial ELISA kits: TNF‐α, IL‐17A, TGF‐β and IL‐10 ; and IL‐35 (Bioswamp). All assays were carried out in accordance with the manufacturer's guidelines. ELISA data were detected using a Tecan Sunrise infinite M200 PRO platereader .2.10First, Red Blood Cell (RBC) Lysis Buffer  was added to whole blood samples to lyse the RBCs. For CD19 and CD138 flow analyses, CD45R also needed to be labelled; appropriate antibodies were incubated with the lyzed blood samples and then detected by a Beckman DxFLEX Flow cytometry (FCM). A CD19 antibody was added and incubated on ice for 45 mins prior to fixation and permeabilization (Thermo Fisher Scientific). Cells were then washed and detected by a Beckman DxFLEX FCM. For the analysis of CD4 and CD25, antibodies were added and incubated on ice for 45 mins prior to fixation and permeabilization (Thermo Fisher Scientific). In addition, 2% normal Sprague‐Dawley (SD) rat serum was used to block cells for 15 mins before incubation for 30 min with an antibody raised against FOXP3. Cells were then washed and analysed by a Beckman DxFLEX FCM. For the analysis of Th17, a CD4 antibody was added and incubated on ice for 45 mins. A Cell Treatment Reagents  Kit was used to deal with for 5–18 h and a Cell Fixation & Permeabilization Kit  was used to fix and permeabilize cells. An IL‐17A antibody was added and incubated for 15 mins at room temperature. Cells were then washed and detected by a Beckman DxFLEX FCM.2.11Kidney tissue was used to extract proteins from transplanted kidney tissue. Protein concentration was then determined using a BCA Protein Assay Kit (Pierce). All samples were then separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene fluoride  membranes. Membranes were then blocked for 60 min with 5% skimmed milk at room temperature and were then incubated overnight at 4°C with antibodies against Atg5 and β‐actin. Membranes were then washed three times in TBST buffer , and then incubated for 1h at 37°C with a peroxidase (HRP)‐conjugated secondary antibody. Immunoblotted proteins were then carried out by ImageJ .2.125 × 10^6 CD4+ T cells were isolated from the spleens of rats and treated with Atg5 siRNA or BTZ for 48 h. Then, the cells injected from caudal veins into Rag1−/− rats 30 min before being subjected to kidney transplantation.2.13p < 0.05 were considered to be statistically significant.Each experiment was repeated at least three times. GraphPad Prism 8.0 software was used for all data analysis. Data are presented as means ± standard deviation. The Student's t test was used to compare differences between groups. The log‐rank test was used to compare graft survival. Differences in which 33.1As shown in Figure 3.2p < 0.001, log‐rank (Mantel‐Cox) Test). In addition, serum creatinine level in rats from the AMR‐BTZ group was significantly higher than in the Con‐Veh group, but improved significantly in response to BTZ treatment from day 1 when compared with the AMR‐Veh group (p < 0.05)   Figure . Most im) Figure . Collect3.3p < 0.05)  and plasma cell (CD45R+CD138+) when comparing the Con‐Veh group with Con‐BTZ group. However, the number of B cell and plasma cell was significantly reduced after BTZ treatment (p < 0.05)  Figure . In conc3.4+CD25+FOXP3+) and Th17 (CD4+IL‐17A+) by flow cytometry. There was no significant difference in the ratio of Treg and Th17 when comparing the Con‐Veh group with the Con‐BTZ group. However, the ratio of Treg and Th17 in the AMR‐BTZ group showed significant changes when compared with the AMR‐Veh group; in the AMR‐BTZ group, BTZ treatment led to a significant increase in the level of Treg but a significant reduction in Th17 (p < 0.05)    Figure . Further) Figure . However) Figure . Therefo3.5Since BTZ was found to alleviate AMR by modulating the expression of Th17/Treg, we next explored the potential mechanism underlying the action of BTZ. Atg5 is a well‐known gene related to autophagy and has been widely proven to facilitate the differentiation of Treg.4p < 0.05).In this study, we constructed and verified a rat model of AMR following kidney transplantation. On this basis, we used model to investigate the effect of BTZ on AMR. We found that BTZ slowed down the pathological process of AMR and reduced the deposition of C4d and IgG. In addition, BTZ reduced the number of B cell and plasma cell, and improved both renal function and graft survival in AMR rats. Our results also found that BTZ increased the number of Treg and significantly reduced the proportion of Th17  was significantly increased after BTZ treatment, while level of pro‐inflammatory cytokines (IL‐17A and TNF‐α) were significantly reduced in response to BTZ treatment. Next, we carried out a preliminary exploration of the mechanisms underlying the action of BTZ on AMR. We found that BTZ might alleviate AMR by facilitating the expression of Atg5 to modulating the differentiation of Th17/Treg. Atg5 is a well‐known autophagy gene and the silencing of this gene is known to activate T cell and fail to upregulate Foxp3 when compared to wild‐type cells.BTZ was the first clinical proteasome inhibitor and has been approved for the treatment of multiple myeloma. BTZ also is a relatively new medication in the transplantation field, and while there is no research providing a clear indication for its indication in transplant. Although additional clinical trials are needed to support BTZ as an effective alternative medication for desensitization with fairly good short‐term success rate, its utility for treating AMR appears to be more uncertain. In this research, we investigated the effect of BTZ on AMR following kidney transplantation using a rat AMR model. Our results demonstrated BTZ alleviated AMR by reducing antibody deposition in tissues and serum. However, these effects were reversed when Atg5 was silenced. Our findings provide a new theoretical basis for the prevention and treatment of AMR after renal transplantation.In this research, we only found that BTZ modulating the differentiation of Th17/Treg and alleviate AMR through Atg5. There was no direct inhibition of Treg to observe the effect of BTZ on AMR. We found that BTZ can alleviate AMR by regulating Atg5 expression rather than induce immune tolerance. Furthermore, our discovery is just one way that BTZ act on AMR, and indirectly proved that BTZ alleviates AMR may modulate the proportion of Th17/Treg. Therefore, more experiments are needed to verify our hypothesis that BTZ inhibits AMR by regulating Th17/Treg.In summary, we found that BTZ was able to reduce the level of DSAs and slow down the pathological process of AMR in rat model. Atg5 plays a vital role in the mechanism of BTZ for AMR and Atg5 may alleviate AMR by modulating the expression of Th17/Treg. This research provides support for the inhibition of AMR by BTZ and Atg5 siliencing may be an effective therapeutic regimen for AMR.The authors confirm that there are no conflicts of interest.Hong Cheng: Conceptualization (lead); Methodology (lead); Writing‐original draft (lead). Bin Xu: Methodology ; Resources (lead); Supervision (lead); Writing‐review & editing . Lijie Zhang: Project administration ; Software ; Validation ; Visualization . Yi Wang: Conceptualization ; Data curation ; Investigation ; Software . Ming Chen: Data curation ; Formal analysis ; Funding acquisition ; Investigation (lead); Project administration ; Visualization ; Writing‐review & editing . Shuqiu Chen: Conceptualization (lead); Data curation (lead); Funding acquisition (lead).


The patient was a 33-year-old woman with inflammatory bowel disease presenting for worsening lower leg lesions with significant pain recalcitrant to oral doxycycline.Pyoderma gangrenosum is a rare ulcerative skin condition with significant pain that is often associated with other systemic diseases typically treated with immunosuppressive medications aimed at the underlying cause. The patient was a 33-year-old female with a history of untreated ulcerative colitis who presented to the emergency department for evaluation of painful leg lesions. The lesions had developed four days prior and she presented to an outside facility where incision and drainage was performed on one of the lesions. She was prescribed doxycycline on discharge. The lesions continued to spread and were associated with worsening pain. On exam she had multiple, tender violaceous nodules with surrounding erythema on the legs and one similar lesion on the right antecubital fossa at the site of a prior intravenous catheter from the outside facility  and 2.Laboratory results were notable for elevated inflammatory markers. A wound culture was negative. The patient was admitted for further workup and pain control following treatment with intravenous methylprednisolone. A biopsy later performed ultimately supported the suspected diagnosis of pyoderma gangrenosum.Pyoderma gangrenosum is a rare ulcerative skin condition frequently associated with systemic disease, most often inflammatory bowel disease.This patient exhibited several classic features of pyoderma gangrenosum, including a history of inflammatory bowel disease, rapidly progressing painful ulcerative skin lesions with surrounding erythema, and evidence of pathergy (appearance of lesions at sites of trauma). Treatment of pyoderma gangrenosum is aimed at addressing the underlying disease process.What do we already know about this clinical entity?Pyoderma gangrenosum is a rare, painful ulcerative skin condition often associated with other systemic diseases typically treated with immunosuppressive medications.What is the major impact of the image(s)?This visual representation of pyoderma gangrenosum may assist with recognition for emergency providers.How might this improve emergency medicine practice?We review the presentation and management of pyoderma gangrenosum to aid providers in delivering effective and timely treatment.


In the past decades, cellulose , in the form of nanofibers, has received special attention. The nanofibrous morphology may provide exceptional properties to materials due to the high aspect ratio and dimensions in the nanometer range of the nanofibers. The first feature may lead to important consequences in mechanical behavior if there exists a particular orientation of fibers. On the other hand, nano-sizes provide a high surface-to-volume ratio, which can have important consequences on many properties, such as the wettability. There are two basic approaches for cellulose nanofibers preparation. The top-down approach implies the isolation/extraction of cellulose nanofibrils (CNFs) and nanocrystals (CNCs) from a variety of natural resources, whereby dimensions of isolates are limited by the source of cellulose and extraction procedures. The bottom-up approach can be considered in this context as the production of nanofibers using various spinning techniques, resulting in nonwoven mats or filaments. During the spinning, depending on the method and processing conditions, good control of the resulting nanofibers dimensions and, consequently, the properties of the produced materials, is possible. Pulp, cotton, and already isolated CNFs/CNCs may be used as precursors for spinning, alongside cellulose derivatives, namely esters and ethers. This review focuses on various spinning techniques to produce submicrometric fibers comprised of cellulose and cellulose derivatives. The spinning of cellulose requires the preparation of spinning solutions; therefore, an overview of various solvents is presented showing their influence on spinnability and resulting properties of nanofibers. In addition, it is shown how bottom-up spinning techniques can be used for recycling cellulose waste into new materials with added value. The application of produced cellulose fibers in various fields is also highlighted, ranging from drug delivery systems, high-strength nonwovens and filaments, filtration membranes, to biomedical scaffolds. Cellulose is the most abundant natural polymer on Earth . The ambThe emergence of polymeric nanofibers spinning dates back to the late 20th century, with techniques adapted for thermoplastic polymers. The most utilized and famous technique, which is widely used for polymer nanofibers preparation, is electrospinning ,4. OtherHistorically, the first-ever man-made fiber of macroscopic diameter in the beginning of the 20th century was regenerated cellulose fiber, so-called viscose rayon, using the conventional wet spinning technique ,17. InteMany review articles are focused on the extraction and purification of CNFs or CNCs using a top-down approach. However, in this review, those are not included; instead, CNFs are mentioned as a precursor for spinning. Readers are encouraged to read high-quality reviews regarding top-down approaches for cellulose nanofibrils and nanocrystals isolation and preparation ,20,21. MThe publication’s analysis has been focused on using the database Scopus up to September 2021, using the keywords “cellulose” and “nanofibers” . The seaElectrospinning, ES, is a technique that was utilized for the first time to process cellulose-based derivative, cellulose acetate, in 2002 , followeCurrently, a high number of studies have presented the latest achievements, mostly in the electrospinning technique, for cellulose nanofibers production ,27,28,29Since cellulose has limitations regarding its solubility in various solvents and the impossibility of being melted, the production or spinning of cellulose nanofibers is very challenging. However, in recent years, many researchers have been breaking barriers and pushing the technology further to find out ways to first dissolve the cellulose and then successfully spin the nanofibers from a solution. One of the breakthroughs is the use of ionic liquids ,31, whicThe main objective of this study is to identify and present major techniques for cellulose nanofibers production, based on the source of cellulose , parameters of the used method, especially solvent for the preparation of spinning dope, and the potential application of prepared nanofibrous materials.Cellulose is a linear polymer comprised of D-glucopyranose units linked via β-1-4 glycoside bond . It is aDue to the high number of hydroxyl groups, cellulose can also be transformed in other derivatives from common reactions such as esterification, producing esters, the most important being cellulose acetate (acetyl cellulose) and nitrocellulose.Moreover, cellulose can be converted to corresponding ethers as well, by reactions of etherification, thus producing important derivatives such as alkyl celluloses and hydroxyalkyl celluloses.Cellulose does not melt, but it degrades at high temperatures. Therefore, when processing cellulose, it is important to have a deep knowledge of its solubility as well as the properties of its derivatives since they might be completely different from those of the cellulose itself as a consequence of hydroxyl group substitution.The solubility of cellulose is dependent both on the molecular weight and polydispersity . Long moOn the other hand, when dissolution of cellulose is considered, certain discrepancies appear in terms of supramolecular structure and dispersion of macromolecules. Liquid penetrates the cellulose structure, causing intermolecular hydrogen bonds to break and macromolecules to disperse. According to a specific interaction of cellulose with a particular solvent, the classification of cellulose solvents can be made on the basis of derivatizing and non-derivatizing solvents, as stated by Heinze and Koschella .N, N—Dimethylacetamide (LiCl/DMAc). It is frequently used in cellulose analytics ..53].Endo et al.  used wet2. The solvent for microcrystalline cellulose was 1-allyl-3-methylimidazolium chloride (AMIMCl) ionic liquid. Silica particles were of 80 nm diameter. For spinning, 5 wt% MCC solutions were used with different ratio of nano SiO2 (0.2–0.6%). The air gap was 3 cm and the coagulation bath contained deionized water. After, the spinning fibers were vacuum dried for 48 h. Authors reported good dispersion of SiO2 in cellulose fibers and an increase in thermal stability for the composite fibers.Dry-jet wet spinning was used by Song et al.  to produ2 solution was used in the coagulation bath. Spinning dope consisted of 2 wt% suspension of CNFs prepared from TEMPO oxidized hardwood bleach pulp. The eventual cellulose crosslinking with the Ca2+ ions of the coagulation bath enabled the production of very-high-strength cellulose fibers.Kafy et al.  used theInterfacial polyelectrolyte complex (IPC) spinning of cellulose nanofibers  is basedThe successful preparation of multicomponent fibers of circular shape was reported. The importance of IPC spinning, as the authors stated, is the fact that, in IPC, there is no shear stress induced upon the CNF gel, contrary to what occurs when gel flows through a needle or other type of capillary. In this way, fibrils are oriented by lateral contraction during drying and by self-assembly at the interface. However, the authors proposed that the combination of IPC with shear-induced orientation prior to stretching might lead to further anisotropy of mechanical properties.For biological application and mimicking muscle filaments, IPC proved to be very effective, especially for spinning CNFs with antimicrobial chitosan into ordered structures that might be used as biomaterials . As anotCellulose acetate is the most important ester of cellulose. Commercial cellulose acetate has substituted OH groups in cellulose by acetyl groups, and its degree of substitution is approximately 2.5. The derivative for which all cellulose hydroxyl groups are substituted by acetyl groups is called triacetate (CTA). Partial hydrolysis up to DS 2.4–2.5 after acetylation yields to commercial cellulose diacetate, also simply referred to as acetate ,75. CommCommercial CA fibers are spun using conventional dry-spinning technology where a spinning dope made of 25% CA solution in acetone is used and the filament from spinneret is passed through a 4–6 m long heating column. On the other hand, CTA is usually spun from the solvent system methylene chloride/methanol, even though CTA is more often used to make films than fibers  since itCellulose acetate nanofibers are usually prepared using electrospinning, rotary jet spinning, wet spinning, and solution blow spinning, as outlined in For some techniques such as solution blow spinning, pure CA nanofibers preparation is still a challenge and, up until now, CA nanofibers were successfully spun only alongside other polymers such is PAN . On the Electrospinning was utilized in cellulose acetate for the first time in 2002 . Two decIn addition to the polymer molecular weight, the solvent system highly influences the production of cellulose acetate nanofiber. It has been shown that the solvent system can be crucial for the spinnability of the polymer solution ,110, notLee et al.  studied There are several reports of using system comprising of three components of solvent ,119,122.Recently, Yang et al.  reportedApplication of electrospun cellulose acetate is very diverse, ranging from the already-mentioned drug delivery to many more, as outlined in Hydrolysis can be performed using aqueous NaOH or NaOH/ethanol solutions ,109,141.Additionally, after hydrolysis, the nonwoven membrane can be further treated with acetone  in orderA significant decrease in deacetylation times can be achieved by using ultrasonication during hydrolysis in aqueous NaOH and NaOH/ethanol solutions, reducing the times of the deacetylation process from 30 h to just 1 h . RegenerNeedleless electrospinning has becoming more popular in recent years due to the fact that the production rate with needleless ES is much higher compared to classic needle electrospinning . During v/v) with a processing rate of 10–20 mL/h. The concentration of CA in solution varied from 14 to 18 wt%, obtaining the best-quality smooth nanofibers when using 16 wt%. For comparison, a common solution processing rate in classical electrospinning with a single needle for some polymers was used (0.05 mL/h). In the work by Wu et al. [Wu and coauthors  proposedu et al. , the prou et al. .Apart from the number of advantages of producing cellulose acetate nanofibers using electrospinning, it should also be highlighted that it can be used for recycling, which is important from the point of view of environmental challenges and reducing waste. For instance, Huang et al.  used cigWith the same idea of recycling, Liu et al.  used oldBesides widely used electrospinning, there are few other techniques for acetate nanofiber production, as indicated in For the preparation of cellulose acetate nanofibers, a hybrid technique was also used: electrostatic induction-assisted solution blow spinning . A 17 wtThe production of cellulose acetate nanofibers using only SBS without the assistance of electrostatic force remains a challenge which should be addressed in the future to simplify the process as much as possible. The first step toward processing cellulose acetate using SBS was taken by Dadol et al. , althougConventional wet spinning of cellulose usually results in thick fibers within the micrometer range (>20 μm) ,141, butCellulose ethers are another class of cellulose derivatives which have found its use in the preparation of nanofibers for different purposes. The etherification of cellulose is an important derivatization route which yields a very broad class of derivatives with many potential applications . Among mCarboxymethyl cellulose (CMC) is commercially the most important cellulose ether. Commercial CMC, with DS of less than 1 (from 0.3–0.9), is biodegradable, non-toxic, and usable as a food additive and in pharmaceuticals. In fact, the use of CMC in the particular form of nanofibers, especially for food packaging or medical application, is very common .For hydroxyalkyl ethers, such as hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC), it is necessary to consider the so-called molecular substitution, MS  ,173. In Yu et al.  used a sIn a work presented by Esmaeili and Haseli , a drug In this work, a comparison, in terms of drug release ability, between the core-shell fibers and fibers obtained from the polymer blend was made. A longer sustained release was observed over a 72 h period with core-shell fibers; however, both types of fibers exhibited excellent antimicrobial activity against Gram-positive and -negative bacteria.Cellulose and cellulose derivatives in the form of nanofibers can have many applications such as in food packaging, drug delivery, biomedical materials, sensors, and filtration membranes, among others. Although there are other polymers that are more easily processed in the form of nanofibers, cellulose can be considered special for that purpose because it is a natural, biodegradable, biocompatible, and renewable polymer. Having in mind the much-needed sustainability, cellulose is a perfect candidate for use in material manufacturing. Electrospinning is currently a superior technique for cellulose-based nanofiber processing, which includes a great variety of cellulose sources, many solvents already utilized in this technique, and high-quality fibers, with many applications. On the other hand, very low production rates during ES and the use of a high-voltage electric field drives the research further to address these issues. Solution blow spinning, as an alternative to electrospinning and needleless ES, has the potential to bring the necessary qualities of the production technique to a level appropriate for industrial use. Therefore, research should be directed in exploring these techniques for cellulose-based nanofibers preparation. Additionally, it was shown that very interesting and novel IPC spinning has promising potential for cellulose- and polysaccharide-based fibrous materials, especially for biomedical applications.In terms of the cellulose source, it was shown that there is very high potential is the utilization of cellulose ethers in the preparation of drug loading systems for controlled release. The utilization of these derivatives is driven by the fact that they are biodegradable, biocompatible, and mostly water-soluble derivatives. Additionally, the use of CNCs or CNFs combined with other cellulose or cellulose derivatives can provide all-cellulose materials, an ecofriendly solution to decrease the use of synthetic polymers.One very important aspect was highlighted in this review, the possibility to use bottom-up spinning technologies for recycling cellulosic waste into new value-added products. In this way, much needed sustainability could be achieved.


Coffea arabica), cacao (Theobroma cacao), and poplar (Populus trichocarpa). Moreover, somatic embryogenesis can speed up the regeneration of mutants, especially for slow-growing plant species. However, various plants of economic value  are recalcitrant to somatic embryogenesis and a better understanding of this process could therefore help engineering tailored solutions for plant regeneration  AGAMOUS-like 15 (AGL15) is a MADS-box-containing protein known to directly bind DNA  and orchestrate transcription of its targets, including a cluster of three core genes all necessary and sufficient for the process of somatic embryogenesis . As a consequence, AGL15 overexpression leads to an increase in somatic embryogenesis events  able to promote somatic embryogenesis to various extents. Many of them are redundant and involved in the same regulatory circuits. Among these TFs, the Arabidopsis  led to a 10% increase in somatic embryogenesis events, as already previously observed for AGL15 . Vice veAGL18- or AGL15-overexpressing lines allowed a comprehensive mapping of AGL15 and AGL18 binding loci and target genes. Interestingly, the authors found 9729 and 3446 genomic loci associated to AGL15 and AGL18 overexpression, respectively. The 1254 shared loci were mostly associated with gene ontology categories related to post embryonic development and gene expression regulation, while AGL18-specific loci were mainly linked to seed development and plant ovule morphogenesis categories and AGL15-specific loci were mainly linked to hormonal responses.ChIP-Seq and RNAseq approaches on AGL18 overexpression, but less than 10% of these genes were also physically associated with the AGL18 protein, according to ChIP-Seq analyses. This discrepancy between direct (or indirect) promoter binding and transcriptional regulation might be due to the technique used  but also to the high redundancy of the regulatory circuits that govern somatic embryogenesis, which likely require a plethora of other regulatory components to drive (or repress) transcription besides AGL15/AGL18.Interestingly, RNAseq results revealed that several hundred genes were differentially regulated by AGL18 overexpression as well as physical association of AGL18 to the promoter region of a subgroup of candidate genes, including the gibberellin biosynthesis gene GA3OX2  and ChIP-qPCR experiments confirmed both the transcriptional regulation in response to e GA3OX2 . Notably mutants . By analAGL15 itself. A deeper analysis of cross-transcriptional regulation between AGL15 and AGL18 using overexpressing lines and loss-of-function mutants revealed that AGL15 represses the transcription of AGL18, while AGL18 promotes AGL15 transcription. Thus, AGL18 transcription could regulate itself by a negative feedback loop by promoting AGL15 transcription that in turn represses both AGL15 (AGL18 transcription (Another direct target of AGL18 also confirmed at the transcriptomic level was th AGL15  and AGL1cription .In conclusion, this work shed important light on AGL15/AGL18 roles in orchestrating somatic embryogenesis at the genomic scale. To further extend these results and boost applications of somatic embryogenesis for the regeneration of recalcitrant crops, it will be important to better characterize the transcriptional regulatory complexes of AGL15/AGL18 by CoIP followed by proteomics and by structural approaches for protein supercomplexes, such as cryoEM, on a variety of plant species.Conflict of interest statement. None declared.


Acute lung injury (ALI) and acute respiratory distress syndrome, which is a more severe form of ALI, are life-threatening clinical syndromes observed in critically ill patients. Treatment methods to alleviate the pathogenesis of ALI have improved to a great extent at present. Although the efficacy of these therapies is limited, their relevance has increased remarkably with the ongoing pandemic caused by the novel coronavirus disease 2019 (COVID-19), which causes severe respiratory distress syndrome. Several studies have demonstrated the preventive and therapeutic effects of molecular hydrogen in the various diseases. The biological effects of molecular hydrogen mainly involve anti-inflammation, antioxidation, and autophagy and cell death modulation. This review focuses on the potential therapeutic effects of molecular hydrogen on ALI and its underlying mechanisms and aims to provide a theoretical basis for the clinical treatment of ALI and COVID-19. The lungs not only are the primary location where gas exchange occurs in mammals but also have the largest epithelial surface in that is direct contact with the external environment. Therefore, the lungs are the primary target for many airborne pathogens, toxicants, and allergens that cause pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) Kumar . ALI andIn recent times, treatment to alleviate the pathogenesis of ALI has improved to a great extent, and existing therapies can be divided into supportive intervention and pharmacological treatment. Supportive intervention, such as lung-protective mechanical ventilation, together with adjunctive approaches, such as prone positioning and the use of neuromuscular blocking agents, has decreased mortality  and peroxynitrite (ONOO−), which are highly strong oxidants, in cells, thereby suppressing brain injury in ischemia/reperfusion (I/R) injury and stroke in a rat model  for inhalation yields better results than oxygen alone , moderate (100 mm Hg < PaO2/FiO2 ≤ 200 mm Hg), and severe (PaO2/FiO2 ≤ 100 mm Hg) ; the nucleotide-binding oligomerization domain-, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3); mitogen-activated protein kinase (MAPK); and Toll-like receptor (TLR) signaling. NF-κB is considered the master regulator of inflammatory responses. It activates a series of proinflammatory transcriptional programs to shape the inflammatory response. Animal studies have established that this transcription factor is an important mediator in ALI/ARDS -1β -induced ALI, LPS binds to Toll-like receptor 4 (TLR4) and stimulates the TLR4-dependent inflammatory responses, including the activation of the TLR4/MyD88/NF-kB and TLR4/TRIF/IRF3 pathways , and non-free radical species such as singlet oxygen (1O2) and hydrogen peroxide (H2O2). They are generated inside the body by aerobic organism as a byproduct of energy metabolism through oxidative phosphorylation (Sies Reactive oxygen species (ROS) include free radicals, such as ion Sies . Normallion Sies . The dysion Sies . During ion Sies . Recent ion Sies .The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a major regulator of the cytoprotective antioxidative protein expression products. The activation of Nrf2 signaling plays an essential role in preventing injury induced by oxidative stress to cells and tissues. The redox balance maintained by Nrf2 is important for the airways, and Nrf2 activation confers protective effects on various lung disorders including ALI/ARDS  Table .Table 1TMolecular hydrogen exhibits anti-inflammatory activities in various injury models. In animal experiments, it downregulates the expression of proinflammatory and inflammatory cytokines, such as IL-1β, IL-6, TNF-α, HMGB-1, and intercellular cell adhesion molecule-1 (ICAM-1)  impaired autophagy. By activating the autophagy pathway, molecular hydrogen alleviated ERS and mitigated inflammation and organ injury in mice , caspase-3, and caspase-8, and upregulating the expression of antiapoptotic proteins, such as B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)  levels, respiration control ratio, mitochondrial–respiration complex activities, and Mfn2 expression along with a decrease in the histological score and dynamin-related protein 1 levels  levels in a PMVECs lung transplantation model and a rat lung transplantation model (Liu et al. Molecular hydrogen could also considerably inhibit apoptosis in rat lung transplantation and inhibit the expression of proapoptotic proteins caspase-3 and caspase-8 in lung grafts, but activate the antiapoptotic proteins Bcl-2 and Bcl-xL, thus stabilizing the mitochondrial outer membrane and terminating the release of cytochrome c into the cytosol via the intrinsic apoptotic pathway (Kawamura et al. The administration of oxygen in high concentration is required to maintain sufficient blood oxygenation in some critically ill patients, but prolonged exposure to it can result in HILI, even leading to respiratory failure. HILI is mainly caused by excessive ROS generated during hyperoxic conditions (Altemeier and Sinclair To further understand the molecular mechanisms underlying molecular hydrogen effects in HILI, quantitative proteomics in vitro revealed that molecular hydrogen can protect AECIIs from hyperoxic injury by modulating a range of protein expression levels and biological processes, such as VEGFA, PDGFB, IGFBP3, EDN1, NADPH oxidase levels, and the coagulation cascade (Lu et al. Ventilator support is often required in the intensive care unit for critically ill patients with respiratory failure, but it can also induce lung injury and even exacerbate it (Tremblay and Slutsky Radiation is used in medical diagnosis and cancer treatment. However, damage can also occur during radiation therapy. ROS generated in therapy are one of factors contributing to the side effects of radiation therapy Ward . ExtensiMajor burns can cause massive tissue destruction and activate a cytokine-mediated inflammatory response, which leads to dramatic pathophysiological effects at sites both local to and distant from the burn (Bittner et al. In an extensive rat model for burns, severe burns with delayed resuscitation caused impaired oxygenation and lung edema. Intraperitoneal HRS ameliorated these effects, substantially attenuated pulmonary oxidative products, and reduced the levels of pulmonary inflammation mediators and myeloperoxidase (Fang et al. SIRT1 expression in rat lung after burn injury presented an increasing trend after a short period of suppression. SIRT1 might provide protection from burn-induced remote ALI by attenuating PMVEC apoptosis via p38 MAPK signaling; thus, SIRT1 exhibits a potential therapeutic effect on burn-induced ALI (Bai et al. In seawater instillation-induced ALI in rabbits, molecular hydrogen inhalation alleviated histopathological changes and cell apoptosis; decreased the malondialdehyde content and MPO activity in lung tissues; decreased the levels of TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid; and markedly improved lung endothelial permeability. These protective effects may be related to the activation of the Nrf2 pathway (Diao et al. In addition, molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked ALI (Terasaki et al. Molecular hydrogen is easily applicable because it exhibits remarkable efficacy on nearly all pathogenic states that are involved in inflammation and oxidative stress. In this study, we reviewed the protective effects and underlying mechanism of molecular hydrogen in ALI caused by different pathogenetic factors. The anti-ALI effects of molecular hydrogen were mainly attributed to its anti-inflammatory, antioxidative, antiapoptotic, and autophagy-modulating effects. However, the potential molecular mechanisms still need further exploration, and some results remain controversial, warranting further research. Although none have been found till date, it is unlikely that there are no adverse effects of molecular hydrogen. Additional research is required to confirm whether molecular hydrogen has undetected potential adverse effects to provide better clinical guidance.Most current research is based on animal experiments; additional clinical trials are needed. Until now, inhalation of molecular hydrogen at a concentration of 1.3–4% has been employed as a form of treatment in clinical trials; however, the optimal dose of molecular hydrogen varies depending on the type of expected biological effect. Recently, a prospective, open-label, rater-blinded clinical pilot study in patients experiencing ST-elevation myocardial infarction demonstrated that molecular hydrogen gas inhalation during percutaneous coronary intervention is feasible and safe and may also promote left ventricular reverse remodeling at 6 months after ST-elevated myocardial infarction (Katsumata et al. 2/O2 inhalation for the treatment of COVID-19 in more than 2000 patients and yielded positive and effective outcomes (Alwazeer et al. 2/O2 inhalation therapy can be performed in general wards or at home using portable H2/O2 generating and inhalation devices. This will ease the pressure on hospitals, reduce hospitalization time, and prevent severe illness caused by COVID-19. Currently, several clinical trials of molecular hydrogen on COVID-19 are registered in the Clinical Trial Registry (Alwazeer et al. For COVID-19, which has led to an urgent demand for therapeutic agents to alleviate and prevent the pandemic, many therapeutics and interventions have been investigated to combat the viral infection-induced inflammation and oxidative stress that contribute to its etiology and pathogenesis; however, these have not yielded any significantly improved treatment outcomes, In China, Nanshan et al. applied HTill date, the reported benefits of molecular hydrogen therapy in patients with ALI are still limited to symptomatic descriptions. To expand the utility of molecular hydrogen therapy in ALI, further understanding of molecular hydrogen pharmacokinetics and its biological mechanisms of action will facilitate the clinical applications of this important molecule. We believe that this will generate standard clinical practice in the near future, and molecular hydrogen may provide strategies for ALI.


Pasteurellaceae species obtained from the infected lungs of Pyrenean chamois (Rupicapra pyrenaica) and domestic sheep found in a National Hunting Reserve from Spain. We have found evidence of the presence of AMR in both animal species. Furthermore, some isolates were resistant to cephalosporins and fluoroquinolones, which are antimicrobials of critical importance in human medicine. Further research is needed to discern pathways of AMR dissemination within natural environments and assess its persistence in wildlife in order to prevent further AMR spreading at a global scale.Antimicrobial resistance (AMR) is a public health concern. Increasing resistance has arisen due to the inappropriate use of antimicrobial drugs in both, human and veterinary medicine. Although AMR is mainly linked to direct and indirect human activities, little is known about the presence and impact that AMR has on wild animals, especially for those bound to habitats subjected to little human pressure. Our study analyzed the AMR profiles of different Rupicapra pyrenaica), and nasal swabs from sympatric domestic sheep were also collected. The swabs were cultured in agar plates to obtain bacterial isolates from the Pasteurellaceae family. The presence of AMR was assessed in a total of 28 Pasteurellaceae isolates from 45 Pyrenean chamois, and 9 isolates from sympatric domestic sheep found in the National Hunting Reserve of Freser-Setcases . The isolates belonged to one of the following three species: Pasteurella multocida, Mannheimia haemolytica and Bibersteinia trehalosi. Some P. multocida and M. haemolytica isolates tested positive for AMR. The statistical analysis revealed no differences between the AMR levels from chamois and domestic sheep isolates. However, one P. multocida of chamois origin presented resistance to cephalosporins and fluoroquinolones, which are antibiotics of critical importance for human health. Further studies are required to elucidate potential routes of dissemination of AMR genes in natural environments and assess any significant persistence in wildlife to design risk mitigation actions.Antimicrobial resistance (AMR) has spread worldwide due to the inappropriate use of antimicrobial drugs in human and veterinary medicine, becoming a public health problem. However, little is known about its occurrence and maintenance in wild animals, and very few studies have been carried out in ecosystems subjected to low human pressure. In our study, nasal and lung swabs were collected from hunted Pyrenean chamois ( Antimicrobial resistance (AMR) is a natural phenomenon; most antimicrobial agents are produced by free-living fungi, plants and bacterial species that are present in all environments, enabling them to fight infections and directly compete for ecological niches . The genThe emergence of AMR in both Gram-positive and Gram-negative bacteria has resulted in difficult-to-treat or untreatable infections, an increase in morbidity and mortality, and an increase in the costs of treating infectious diseases ,8. TheseDespite the importance of AMR, its occurrence and maintenance in wild animals is poorly understood. Antibiotic-resistant bacteria have already been described in different species ,13. AMR The study of AMR occurrence in remote habitats, or in ecosystems subjected to limited human influence, can help to identify levels of AMR in natural environments and assess how anthropogenic activities are influencing AMR in bacterial populations worldwide ,15. TherEnterobacteriaceae [Campylobacter spp., Listeria monocytogenes [Staphylococcus aureus [Pasteurellaceae.Up to date, most of the studies on AMR and wildlife have focused on Enterococci , Enteroberiaceae ,20, Camps aureus . Yet, vePasteurellaceae family, isolated from sympatric Pyrenean chamois (Rupicapra pyrenaica) and domestic sheep that graze in alpine meadows. These studies are vital to establish baseline data of AMR occurrence and assess their extent in an area with a priori low human influence.The aim of this study was to assess the antimicrobial susceptibility profile of the bacteria involved in respiratory infections of the Ovies aries musimon), roe deer (Capreolus capreolus), red deer (Cervus elaphus) and wild boar (Sus scrofa) also occur in much lower numbers. Domestic ruminants, including domestic sheep, cattle and horses, also dwell with the wild ungulates during the grazing period from May to November [The present study was performed in the Freser-Setcases National Game Reserve (FSNGR) in the Pyrenees, Northeastern Spain. This area is a high mountain habitat with strong seasonal influence and is mainly composed of alpine and subalpine ecosystems. Pyrenean chamois is the most abundant ungulate species, although mouflon (November .Pasteurellaceae isolates were obtained from pneumonic lesions in Pyrenean chamois (n = 45) legally hunted in FSNGR during the regular hunting season (August–November), and from nasal swabs of domestic sheep (n = 24) in two flocks that seasonally graze at FSNGR.Between 2015 and 2017, a total of 37 2 [Pasteurellaceae were replated to obtain a pure culture and were further identified using a 16S rRNA PCR, following previously described protocols [P. multocida isolate Sanger-sequenced from our strain collection. Isolates that were confirmed to belong to the family Pasteurellaceae were then stored in 20% glycerol/brain heart infusion  at −80 °C until further analysis.For each sampled Pyrenean chamois, both nasal and lung swabs with Amies transport medium were collected. In the case of domestic sheep, a single nasal swab with Amies transport medium was also collected. All swabs were carefully labeled and stored at 4 °C and processed in the lab within the first 24 h post-collection. Amies swabs were cultured on Polivytex agar plates  and incubated for 48 h at 37 °C and 5% CO2 . MacroscTM SensititreTM, Waltham, MA, USA) following the Clinical Laboratory Standards Institute guidelines . Antimicrobials and concentrations tested were as follows: chlortetracycline (0.5–8 μg/mL), oxytetracycline (0.5–8 μg/mL), gentamicin (1–16 μg/mL), spectinomycin (8–64 μg/mL), neomycin (4–32 μg/mL), florfenicol (0.25–8 μg/mL), penicillin (0.12–8 μg/mL), ampicillin (0.25–16 μg/mL), ceftiofur (0.25–8 μg/mL), danofloxacin (0.12–1 μg/mL), enrofloxacin (0.12–2 μg/mL), sulphadimethoxine (256 μg/mL), trimethoprim/sulfamethoxazole (2/38 μg/mL), tylosin tartrate (0.5–4 μg/mL), tulathromycin (1–64 μg/mL), tilmicosin (4–64 μg/mL), clindamycin (0.25–16 μg/mL) and tiamulin (1–32 μg/mL). MIC determinations and clinical breakpoints were used in accordance with criteria provided by the CLSI . Multidrug resistance was defined as resistance to three or more families of antimicrobials [MIC was determined by microbroth dilution method . Bibersteinia trehalosi was not included on the statistical analysis due to a lack of representative samples and absence of data regarding MIC breakpoints. For the statistical analysis, an isolate was considered resistant when it presented resistance to at least 1 antimicrobial family. Factors included in the statistical model were animal species, bacteria species, and resistance profile. The statistical analysis was performed using the R 3.3.3 program [Fisher’s exact tests were applied in order to evaluate statistical differences between resistance found in bacterial species, and whether resistance was related to animal species. Pairwise Test of Independence for nominal data was applied as a post-hoc test. The  program .Pasteurella multocida (19 from chamois and 1 from sheep), 14 Mannheimia haemolytica (6 from chamois and 8 from sheep) and three Bibersteinia trehalosi from chamois were isolated , 10.5% were resistant to ampicillin, 10.5% to penicillin and 5.2% to ceftiofur. Additionally, intermediate resistance was detected for enrofloxacin (5.3%), spectinomycin (5.36%) and florfenicol (5.3%)  were pansusceptible , yet the MIC results obtained for our isolates belonging to this species are displayed on Among the l (5.3%) . One P. ceptible . NeitherThe statistical analysis revealed no significant differences in the AMR profiles between bacterial species or animal species (chamois and sheep).Pasteurellaceae obtained from chamois exhibited similar levels of resistance to several antimicrobial agents used in human and animal health, such as beta-lactams and macrolides. Furthermore, this is the first report of resistance to a third-generation cephalosporin (ceftiofur) in a P. multocida isolate of wildlife origin. The bacteria from the Pasteurellaceae family can acquire resistance to antimicrobials by mutations or horizontal gene transfer from different sources, including other bacteria species. In this regard, mobile genetic elements, such as plasmids or transposons, are the main elements that contribute to the dissemination of AMR, and Pasteurellaceae can act as both receptors and donors of these genetic elements [Enterobacteriaceae, rather than respiratory-related bacteria. In general, AMR levels in Pasteurellaceae from domestic animal species have shown to be low but with an increasing tendency [Monitoring AMR in geographic areas with a relatively low level of human influence provides a suitable system for studying AMR in natural habitats. Surprisingly, the isolates from elements . In factelements . Howevertendency ,29.Enterobacteriaceae. However, there are a lack of studies based in other species and further work should be designed to unravel possible ARGs transmission routes between respiratory bacteria and the environmental pool of ARGs.In our case, interactions between livestock and chamois may occasionally occur in alpine meadows , and seaP. multocida from cattle origin [P. multocida isolates analyzed herein exhibited similar AMR profiles to those described from cattle in Europe [The most common antibiotics used to treat respiratory infections in domestic ruminants include ceftiofur, tulathromycin (macrolide), sulfonamides, tetracyclines, streptomycin and florfenicol ,31. The e origin . P. multn Europe ; in genen Europe  also repP. multocida and M. haemolytica described rising numbers of AMR worldwide in food-producing animals [M. haemolytica typically exhibited more resistance than P. multocida. Although our results did not show statistical significance regarding resistance profiles between M. haemolyitica isolated from chamois and sheep, resistance to danofloxacin and tulathromycin was detected in sheep isolates, probably as a result of antimicrobial drug usage during rearing.Studies focusing on  animals . In agreB. trehalosi obtained in this study is too small to get to any conclusive results. However, considering the lack of information in the literature regarding AMR for this bacterial species and the lack of clinical breakpoints, the results from this study contribute as valuable reference for further studies.Finally, the number of isolates of Pasteurellaceae isolates from wild and domestic animals in an alpine ecosystem from Northeastern Spain are consistent with that described in other livestock from Europe. The similar frequency of AMR detection in isolates from chamois may indicate an indirect anthropogenic influence for the AMR spread in these ecosystems. Furthermore, we report the presence of cephalosporin and fluoroquinolone resistant isolates, which are antimicrobials of clinical importance for human health. Further studies are needed to elucidate potential routes of dissemination of AMR genes in natural environments and assess any significant persistence in wildlife in order to implement risk mitigation actions.In conclusion, AMR levels found in the


Particularly, AIE luminogens (AIEgens) with emission in the range of second near-infrared window  have displayed salient advantages for biomedical imaging and therapy. However, the molecular design strategy and underlying mechanism for regulating the balance between fluorescence (radiative pathway) and photothermal effect (non-radiative pathway) in these narrow bandgap materials remain obscure. In this review, we outline the latest achievements in the molecular guidelines and photophysical process control for developing highly efficient NIR-II emitters or photothermal agents with aggregation-induced emission (AIE) attributes. We provide insights to optimize fluorescence efficiency by regulating multi-hierarchical structures from single molecules (flexibilization) to molecular aggregates (rigidification). We also discuss the crucial role of intramolecular motions in molecular aggregates for balancing the functions of fluorescence imaging and photothermal therapy. The superiority of the NIR-II region is demonstrated by fluorescence/photoacoustic imaging of blood vessels and the brain as well as photothermal ablation of the tumor. Finally, a summary of the challenges and perspectives of NIR-II AIEgens for  Structural and process controls of NIR-II AIEgens realize manipulating of radiative (R) and nonradiative (NR) decay for precise theranostics. Recent years have witnessed the establishment of strong ties between light and life by being able to visualize and detect many previously unknown life processes. Understanding photophysical behaviors is crucial for the design and interpretation of light-based applications. According to the classical photophysical diagram , photoacoustic (PA) imaging, laser resurfacing, desalination of seawater, etc.2,3 Unfortunately, these two critical processes compete against one another: the dominant R will suppress NR and vice versa. So how can we regulate these two processes to attain desirable properties?Light is one of the most fundamental branches of science. It is essential to decipher the interaction between light and matter for promoting the advances in technologies associated with the utilization of light, such as fiber-optic communication, optical storage, optical devices,  diagram , matter in vivo bioimaging.4,5 On the other hand, some flexible molecules, such as tetraphenylethylene (TPE), are almost non-emissive at a molecular level, owing to the dominated NR by active intramolecular motions , the same substance may display predesigned photophysical properties to meet various applications.Among all factors affecting the process of R and NR, the structure is undoubtedly one of the most significant aspects , right.  motions .6–8 Upon9–13 Notably, compared with visible (400–680 nm) and first near-infrared region , fluorescence imaging in the second near-infrared wavelength region  shows salient advantages of deep tissue penetration with high signal-to-background ratio (SBR), owing to the decreased scattering of photons at longer wavelengths  decreases remarkably with wavelength (λ), especially in low energy bandgap NIR-II materials, as intramolecular motions around the single or double bond result in dominant non-radiative relaxation dynamics . To drive emission to the NIR-II window, AIE luminogens (AIEgens) are designed as conjugated donor–acceptor (D–A) structures, where they are being twisted with each other  while maintaining the TICT property (molecular level). Moreover, introducing molecular motors to molecules further suppresses the intermolecular interactions to trigger the AIE effect. Therefore, NIR-II AIEgens with long-wavelength fluorescence and intense brightness can be seized simultaneously.Aggregation-induced emission (AIE) is an inventive fluorescent technique and has the potential to address this issue.Based on the above-mentioned structural design (twisted backbone + molecular motor) and process control (AIE + TICT), many outstanding NIR-II AIEgens have been reported. In this feature review, to understand the structural design, process control, as well as involved mechanisms, we have selected some representative examples to analyze and guide further developments of NIR-II AIEgens. The R and NR channels can be opened or blocked by tuning the structure at multi-hierarchy levels. AIE technology endows molecules with high fluorescence quantum yield (QY), while the TICT state gives molecules with both red-shifted emission and enhanced photothermal property owing to the predominant NR. By controlling structure and process, systems with predominated fluorescence intensity, or predominated photothermal property, or a balance of fluorescence intensity and photothermal property can be obtained. We hope this review will offer valuable guidance on how to design desirable NIR-II theranostic agents by regulating structure and process.55–57 It will be favorable to design fluorophores with inherently longer emission wavelength, as well as much higher QY (>2%) to achieve effective NIR-II fluorescence imaging. Given the diversity of designing D–A conjugated molecules, this may be realized through molecular engineering of electron donors, acceptors, spacers, etc.58,59 On the other hand, AIE features outstanding solid-state photoluminescence.44,60 These AIEgens naturally own twisted architectures dressed up with several molecular motors, which are valuable for suppressing the robust intermolecular interactions. In general, visible light-emissive AIEgens are relatively easy to achieve through the incorporation of twisted molecular motors. However, this method is not fit for NIR-absorbing fluorophores, as they suffer from much stronger intermolecular π–π interactions, which results in fluorescence quenching when dispersed in a physiological environment. To address this issue, the strategy of “backbone distortion + twisted motors” is generally accepted for designing highly bright NIR-II AIEgens because of the efficient restriction of intermolecular interactions.61To date, the majority of the existing NIR-II organic chromophores have low quantum yields between 0.3% and 2%.g]quinoxaline (DPTQ) and benzobisthiadiazole (BBTD), are generally utilized to drive the emission wavelength to the NIR-II region.20 The energy gap can be decreased by anchoring molecular donors such as phenyl or substituted-thiophene. The critical element in molecular design lies in the distortion of the central D–A–D backbone, which is crucial to restrict the intermolecular interactions. Meanwhile, the peripheral twisted motors such as tetraphenylethylene (TPE) and triphenylamine (TPA) are indispensable. On the one hand, molecular motors are beneficial for further restricting of intermolecular interactions. On the other hand, they provide intramolecular mobility to access the TICT state. To date, based on the above molecular design philosophy, Tang, Qian and Ding,61–68 Zheng and Liu,69 Lu and Xiao,70 Hong,71 Chen and Feng,72 Liu,73 Xiao,74 Wu,75 Wang and Jiang,76 and Wu77et al. developed several types of highly bright NIR-II AIEgens with emission peak >1000 nm, with representative chemical structures shown in Accordingly, NIR-II AIEgens are characterized by a motor–donor–acceptor–donor–motor (M–D–A–D–M) structure. Strong molecular acceptors, such as 6,7-diphenyl-thiadiazolo[3,4-61 As shown in ortho-substituted alkylthiophene and BBTD core, AIEgen 9 displayed a significantly distorted backbone . Another critical feature of AIEgen 9 was the presence of twisted molecular motor TPA.To deeply understand the molecular design strategy, Tang and Ding systematically studied the effects of backbone distortion and molecular motor on the solid-state fluorescence efficiency of the dyes.fw) from 0% to 50 vol%, the fluorescence intensity of AIEgen 9 decreased progressively, due to the forming of a dark TICT state with an increase in polarity. Further increase of fw to 90 vol%, the PL intensity showed a rapid enhancement (AIE effect), due to the suppression of intramolecular rotations decisive to the TICT state through molecular aggregations. Therefore, AIEgen 9 possessed a TICT + strong AIE effect with a high QY of 8.4% when in a nanoparticle state. The αAIE (PL intensity ratio of fw = 90 vol% to fw = 0) is also a clear indicator of solid-fluorescence efficiency, with measurements of up to 6.2, which was higher than the other systems (αAIE ≈ 2).71 To demonstrate the molecular motor effect, AIEgen 13 with phenyl motor while maintaining the highly twisted backbone was used as a control. A typical TICT + AIE effect was observed, however, αAIE sharply decreased to 0.54, suggesting a significant impact of the molecular motor on the fluorescence efficiency. On the other hand, the backbone twisting effect was studied by shifting the alkyl chains from ortho to meta position. In contrast to molecule 9, the resultant molecule 14 adopted a planar backbone , owing to the steric distance of meta-positioned alkyl chains to the BBTD core. The PL intensity of molecule 14 decreased with fw because of the TICT effect upon an increase of polarity. Notably, the emission was almost wholly quenched in the aggregate state (fw = 90 vol%), which resulted from the strong intermolecular interactions in the planar D–A–D core. Moreover, removing the twisted TPA motor in molecule 15 further intensified the ACQ effect. Overall, backbone distortion and motor twisting have an equivalent effect on the solid-state fluorescence efficiency of NIR-II AIEgens.Photoluminescence (PL) spectra were performed to detect emission fluctuation upon molecular aggregation using THF/water mixtures . Upon inin vitro fluorescence property was examined. As shown in in vivo fluorescence imaging quality of AIE9 NPs, blood vessel imaging of mouse hindlimb was carried out via intravenous tail injection of AIE9 NPs. Indocyanine green (ICG), a NIR-I dye with clinical approval, is served as a comparison. The imaging quality from AIE9 NPs outperformed that of ICG (Benefiting from high QY of AIEgen 9 nanoparticles (AIE9 NPs) in the NIR-II region, t of ICG , indicatetc. Noninvasive detection of inflammation is crucial for the early diagnosis of pathogenic processes. In particular, the diagnosis of brain inflammation is viewed as the Holy Grail for bioimaging, as it is well-protected by the blood–brain-barrier (BBB). Tang and Ding ingeniously utilized immune cells, neutrophils (NEs), as “living” carriers to deliver AIE9 NPs to penetrate the BBB to arrive at the brain inflammation site.61 As displayed in Inflammation is a sign of many major diseases such as cancer, diabetes, cardiopathy in vivo fluorescence imaging in the NIR-IIb region (1500–1700 nm) can provide higher resolution and deeper tissue penetration in comparison with conventional NIR-II window.78–80 While inorganic NIR-IIb fluorophores have been developed, the study on organic counterparts is still in its infant, due to the lack of long-wavelength materials. To address this dilemma, Tang and Qian proposed a molecular design guideline for the manipulation of TICT and AIE at molecular and agminated levels, respectively, to explore purely organic NIR-IIb fluorophores.66 As shown in ortho-substituted alkylthiophene as the donor, and TPA as the motor. Notably, the keystone of the molecular guidelines is engaged in the selection of secondary carbon-branched alkyl chains, as they supplied a tunable steric barrier not only for restricting intermolecular interactions but also for the activation of intramolecular motions. On a molecular level, the flexible intramolecular rotations of D–A units in AIEgen favored the formation of dark TICT state to yield significantly red-shifted emission wavelength. On the other hand, the NPs formation process (nanoprecipitation with soft amphiphilic polymer DSPE-PEG) partially restricted the intramolecular motions of AIEgens, giving significantly enhanced fluorescence signal. Thereof, dyes with both long-wavelength emission and adequate efficiency can be obtained concurrently by linking ostensibly contradicting individuals. The optimized AIEgen 7 with 2-ethylhexyl unit presented an emission tail extending to 1600 nm with a high NIR-II QY of 11.5% in the nanoparticle state.Due to the reduced photon scattering and almost zero autofluorescence, To glean the advantage of NIR-IIb fluorescence imaging of AIE7 NPs, long-pass (LP) filters with 1100 and 1500 nm were examined. As shown in et al. developed a series of highly bright NIR-II AIEgen 10 (QY = 11.9%) and 11 (QY = 11.7%) with a fluorescence tail extending to the NIR-IIb region. The resultant AIE NPs could be utilized as excellent NIR-IIb imaging agents for superior spatial–temporal resolution of whole-body, cerebral, tumor vasculature, lymphatic drainage systems, etc.74,81 On the other hand, NIR-IIa (1300–1400 nm) window is also appealing owing to the reduced light scattering and decreased water absorption. Wu et al. reported an AIE-active NIR-IIa conjugated polymer with a high QY of 1.7% in aqueous solution, realizing clear through-skull visualization of the cerebral vasculature.77Very recently, based on the mechanism of AIE + TICT, Xiao 70 reported a strategy of tuning AIE and TICT properties to balance the fluorescence and photothermal effect through the coordination of human serum albumin (HSA) with NIR-II AIEgen 6  and photothermal effect (NR) is hard to control. To address this issue, Lu and XiaoAIEgen 6 . In a poAIEgen 6 . On the AIEgen 6 . Thus, tAIEgen 6 . Directevia activating molecular motion in aggregates.82 The molecular design guideline for an efficient photothermal agent is displayed in via strong intermolecular interactions when aggregated. The twisted TPA unit acted as molecular motors to assure intramolecular rotations and as a molecular donor to red-shift the absorption. The critical element of molecular design lies in the adoption of long-branched alkyl chains to provide the necessary room for molecular motions, which are the prerequisites for the formation of the TICT state.83,84 As expected, AIEgen 17 with 2-decylmyristyl unit displayed quenched fluorescence upon molecular aggregation  contributed to not only a narrow energy gap beneficial for light-harvest ability, but also to trigger the TICT state giving a fluorescence quenching effect. Molecule 18 displayed excellent photothermal conversion property with temperatures rising to 81 °C , a molecule, 18, possessing a TPE molecular motor and long-branched alkyl chains in the π-conjugated planar backbone, was designed.to 81 °C . The PCE87 Fan and co-workers boosted nonradiative decay channel through manipulating intermolecular charge transfer, attaining an exceptionally high PCE of 61%.88 Lee and co-workers reported a kind of π-conjugated oligomer F8-IC linked with A–D–A structure, whose intramolecular flexible rotations led to an enhanced non-radiative decay. The resulting F8-IC NPs displayed a high PCE of 82% for high-performance PA imaging-guided cancer therapy.89 Liu and co-workers integrated D–A structure and molecular motors into one molecule, TA1, giving a high PCE of 84.8%. The obtained TA1 NPs could significantly suppress primary breast tumor growth and metastasis.90 Peng and co-workers introduced a “barrier-free” motor to the meso-position of the BODIPY scaffold, allowing efficient dissipation of absorbed photons as heat. The obtained NPs showed a high PCE of 88.3% leading to complete removal of tumors in mice.91 Li and co-workers reported that the activation of intramolecular rotation around C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N double bond enabled a high PCE of ∼90%.92 Altogether, the activation of intramolecular motion within NPs provides a platform for developing efficient photothermal agents.Recently, based on the understanding of the strategies for activation of intramolecular motions within NPs, many excellent PTT/PA systems have been developed. Tang and co-workers reported an AIEgen full of molecular motors, allowing efficient intramolecular motions for PA imaging-guided phototherapy.93–95 Nevertheless, pure organic materials with strong absorption in the NIR-II window have been far less reported, calling for high-performance materials with both long-wavelength absorption and high PCE.Despite high PCEs of above photothermal agents, they mainly rely on NIR-I laser. Owing to the deeper tissue penetration and higher maximum permissible exposure to the laser, photothermal agents in superior NIR-II region have received increasing attention.in vivo NIR-IIb imaging. On the other hand, systems can also display high fluorescence efficiency (AIE) with a tunable photothermal effect (TICT), which is suitable for fluorescence-guided PTT or fluorescence/photoacoustic dual-mode imaging. To further enhance the photothermal properties, a molecular guideline of “backbone planarization + motor/spacer” is proposed. The resultant fluorophores display an ACQ process owing to the intense intermolecular interactions from the planar conformation. However, with the presence of molecular motors and long-branched alkyl chain spacers, a significantly enhanced photothermal effect is detected owing to the activation of intramolecular motions. Overall, from molecule design to aggregate control, AIE research emphasizes the significance of studying materials properties at a higher hierarchical level.In this review, through regulation of hierarchical structures at a molecular level (flexibilization) and aggregate level (rigidification), respectively, organic NIR-II fluorophores with AIE characteristics for efficient fluorescence/photoacoustic imaging and PTT have been summarized. The molecular design strategy of AIEgens, “backbone distortion + motor twisting”, has been demonstrated to be indispensable for improving the QY of NIR-II dyes, due to the suppression of strong intermolecular interactions at aggregates. The flexibilization in molecular structure brings the fluorophores to a TICT state, giving a significantly red-shifted emission with strong NR. By intelligent control of AIE and TICT processes, molecular aggregates with high QY (AIE) and long-wavelength emission (TICT) can be achieved, which finds salient advantages for 96 Last but not least, since many organic NIR-II NPs tend to accumulate in mononuclear phagocyte systems such as in livers and spleens, new NIR-II fluorophores should possess the overall dimension within the renal filtration limit (∼5 nm) for rapid urine excretion.While NIR-II AIEgens hold substantial promise for biological theranostics, several concerns need to be addressed for their further development. First, the overall brightness of NIR-II AIEgens can be further improved by increasing the molecular absorptivity while maintaining the high QY, as the backbone twisting in AIEgens may destroy the absorption. Second, pure organic fluorophores with peak emission exceeding the golden imaging region (>1500 nm) are seldom reported. Third, investigating ways to design NIR-II AIEgens with targeting ability or in response to external stimuli is of crucial importance. For example, Wu and Zeng reported a nitroreductase-responsive NIR-II AIEgen, realizing the detection of breast cancer metastasis.In summary, the development of single AIE NPs with balanced fluorescence and photothermal therapy functions through structure adjustment at different hierarchical levels is of great interest for the advancement of next-generation NIR-II fluorophores.There are no conflicts to declare.


One of the strategic objectives of the AMR national action plan is to improve public awareness and understanding of AMR. Changing people's behaviour is not easy and the older a person is, the more they are set in their ways. Schoolchildren are in their formative years, which is the right time to impart knowledge and best practices that will guide their behaviour in life. Importantly, students have links with families and communities and are future leaders. Roll Back Antimicrobial resistance Initiative (RBA Initiative) has been pioneering the use of youth as agents of change to increase AMR awareness, promote positive behavioural change and thus reduce treatment failure.The main objective was to equip young people (schoolchildren) with knowledge and skills to understand antimicrobial use and resistance and ability to pass the knowledge to their families, other students and community at large.Through AMR clubs, RBA Initiative used different methodologies to engage and educate schoolchildren on AMR. These included classroom teaching, arts and crafts like songs, skits, drama, traditional dances and storytelling and fun videos. Further motivation for active participation was encouraged through competition. The content of the training included topics such as behaviour that fuels AMR, hand hygiene and sanitation, the impact of fake drugs and the One Health approach.In 2020, the project build the capacity of 160 students. Subsequently, these trained students have reached over 1000 fellow secondary school students, over 3000 primary school pupils and over 800 community members including family members with key AMR messages.If engaged and empowered, young people are able to increase community knowledge and awareness regarding AMR as agents of change, contributing to the national action plan on AMR.


Histopathological classification of endometrial carcinoma has evidenced two main groups with different biological behavior: low-grade (G1–G2) and high-grade (G3) endometrial tumors. Moreover, the Cancer Genome Atlas (TCGA) documented four molecular categories with distinct clinical, pathologic, and molecular features: POLE/ultramutated (7% of cases) microsatellite instability (MSI)/hypermutated (28%), copy-number low/endometrioid (39%), and copy-number high/serous-like (26%). The aim of the present paper is to review all endometrial carcinoma histotypes in light of the morphological and molecular prognostic TCGA groups.Endometrial carcinoma represents the most common gynecological cancer in Europe and the USA. Histopathological classification based on tumor morphology and tumor grade has played a crucial role in the management of endometrial carcinoma, allowing a prognostic stratification into distinct risk categories, and guiding surgical and adjuvant therapy. In 2013, The Cancer Genome Atlas (TCGA) Research Network reported a large scale molecular analysis of 373 endometrial carcinomas which demonstrated four categories with distinct clinical, pathologic, and molecular features: POLE/ultramutated (7% of cases) microsatellite instability (MSI)/hypermutated (28%), copy-number low/endometrioid (39%), and copy-number high/serous-like (26%). In the present article, we report a detailed histological and molecular review of all endometrial carcinoma histotypes in light of the current ESGO/ESTRO/ESP guidelines. In particular, we focus on the distribution and prognostic value of the TCGA groups in each histotype. The POLE/ultramutated group included both low-grade and high-grade EECs and showed excellent prognosis with no recurrence regardless of the FIGO grade. The MSI/hypermutated group is characterized by MSI, mostly caused by MLH1 promoter methylation, and a high mutational rate (18 × 10−6 mutations per megabase). The MSI/hypermutated group included both low-grade and high-grade EEC (similarly to the POLE/ultramutated group) and is characterized by intermediate prognosis. The copy-number low/endometrioid group is characterized by low mutational rate (2.9 × 10−6 mutations per megabase), with no MSI or POLE mutations and with a low degree of somatic copy-number alteration (SCNA). The copy-number low/endometrioid group mainly includes low-grade EEC and has been compared to the prototypical Bokhman type I category of endometrial carcinoma; the overall prognosis in this group is intermediate. The copy-number high/serous-like group is characterized by low mutational rate (2.3 × 10−6 mutation per megabase) but extensive SCNA, with TP53 mutation in 90% of cases. The copy-number high/serous like group mainly includes SECs and has been compared to the prototypical Bokhman type II category; this group shows poor prognosis [Endometrial carcinoma is the sixth most commonly diagnosed cancer and the 14th leading cause of cancer death in women worldwide ,3. Historognosis . The outrognosis . Other rrognosis ,16,17.Distribution of endometrial carcinoma histotypes according to TCGA molecular groups, is illustrated in In 2020, the European Society of Gynaecological Oncology (ESGO), the European Society for Radiotherapy and Oncology (ESTRO), and the European Society of Pathology (ESP) published their joint guidelines for the management of endometrial carcinoma, for the first time incorporating the TCGA findings in the risk stratification of endometrial carcinoma. The ESGO/ESTRO/ESP guidelines propose two alternative approaches to stratify the risk in endometrial carcinoma, based on whether molecular classification is available. The two approaches are detailed in EEC is the most common histotype of endometrial carcinoma. EEC often arises from atypical endometrial hyperplasia/endometrioid intraepithelial neoplasia (AEH/EIN), which is its recognized precancerous lesion. EEC is characterized by glandular structures lined by columnar/cuboidal cells with round/ovoidal pseudostratified nuclei and a smooth luminal surface; nuclear atypia is most commonly low-grade. Altered differentiations, such as mucinous, squamous, and morular, are common and are used as confirmatory features of endometrioid histotype. Pathological grading is crucial for the risk stratification and management of endometrioid carcinoma. EECs are subdivided into “low-grade” (FIGO grade 1–2) and “high-grade” (FIGO grade 3), based on the percentage of solid growth pattern (< or ≥50%); one grade is added in the case of marked nuclear atypia ,20. Low-There are other morphologic factors that might have an independent prognostic value in EEC, such as a tumor budding, microcystic, elongated, and fragmented (MELF) pattern of invasion and WT1 immunohistochemical expression ,30,31, aSEC is the prototypical Bokhman type II carcinoma, i.e., mostly arises in postmenopausal women, is not associated with estrogens, and shows poor prognosis ,9. A proCCEC has traditionally been lumped together SEC in the Bokhman type II category . CCEC isThe term “mixed carcinoma” indicates the presence of two different endometrial carcinoma component (one of which is SEC or CCEC), with the minor component accounting for at least 5% of the tumoral area; each component typically shows an immunophenotype that can be superimposed on the correspondent pure histotype. Given the presence of an SEC or CCEC component, mixed carcinomas are considered of high grade by definition ,20, and UEC/DEC is a recently described variant of endometrial carcinoma. According to the WHO classification of gynecological tumors, UEC is characterized by sheets of medium-sized, monotonous discohesive cells with absence of any obvious glandular, nested, or trabecular architecture. UEC is often associated with a low-grade EEC component, configurating the so-called “dedifferentiated carcinoma” (DEC) ,20. ReceUCS is an aggressive biphasic neoplasm composed by a carcinomatous and a sarcomatous component . PreviouNEEC is a rare histotype of endometrial carcinoma, accounting for less than 1% of all cases. NEEC is characterized by the expression of at least one neuroendocrine marker  in at least 10% of tumoral cells; however, this feature is not specific and needs to be correlated with histomorphology. NEEC can be subdivided into small cell NEEC, characterized by hyperchromatic nuclei with high nuclear-to-cytoplasmic ratio, molding, and crushing artifacts, and large cell NEEC, characterized by appreciable cytoplasm, coarse nuclear chromatin with prominent nucleoli, and nested growth with peripheral nuclear palisading. Endometrial NEEC is often found admixed with other components, especially EEC . The proMLEC is a rare entity which shows morphological and immunohistochemical features overlapping with cervical mesonephric carcinoma. MLEC may show different growth patterns, such as tubular, glandular, retiform, sex cord, papillary, hobnail, glomeruloid, and sieve-like, and may easily be misdiagnosed as other histotypes; solid and spindle-cell areas reflect poor differentiation and may show altered immunophenotype. The typical immunophenotype includes positivity for the “mesonephric markers” , negativity  for estrogen and progesterone receptor, patchy p16 positivity, and p53-wild-type expression pattern ,70.At a molecular level, MLEC is associated with KRAS mutations. Unlike mesonephric carcinomas, MLEC may show molecular alterations typical of endometrioid carcinoma, such as PTEN and CTNNB1 mutations, indicating a possible Müllerian origin with a secondary acquisition of a Wolffian phenotype . The fewGTEC is an uncommon mucinous neoplasm which resembles endocervical gastric-type adenocarcinoma. GTEC has also been termed “gastrointestinal-type” carcinoma, given the spectrum of gastric and intestinal differentiation that may be found in these lesions . While mThe emphasis linked to the TCGA molecular groups has led to the hypothesis that molecular data might completely replace the traditional histopathological assessment . However-Geographical loss of MLH1 and PMS2 due to heterogeneous hypermethylation within the tumor.-Geographical loss of MSH6 and/or MSH2 due to a secondary (non-germline) mutation in an MSH6 coding mononucleotide tract or a mutation in POLE.-Weak focal/patchy immunoreactivity for MSH6 can be seen with MSH2 loss of expression/germline mutations.-Subclonal loss of MMR protein expression.-MSS/MSI-Low with loss of MMR protein expression due to MLH1 promoter hypermethylation or somatic MMR variants.-MSI with retained/proficient MMR protein expression due to POLE variants.The correct assignment of endometrial carcinomas to one of the molecular subgroups requires a correct interpretation of molecular and immunohistochemical data. For instance, not all POLE mutations impact on prognosis. A large majority of POLE mutations outside the exonuclease domain are not associated with a ultramutated phenotype, and part of the mutations inside the exonuclease domain are not pathogenetic . RecogniIn the case of simultaneous presence  of two or three molecular signatures , it should be remembered that outcomes correspond to those predicted by the driver molecular subtype; in particular, the POLEmut signature, when characterized by a pathogenic status, prevails over the other signatures, conferring a good prognosis regardless of MMR and p53 status, while the MMRd signature prevails over the p53abn signature .We extensively discussed the impact of molecular groups on the risk stratification and management of endometrial carcinoma. In addition, molecular signatures may predict susceptibility of endometrial carcinoma to specific therapeutic agents. Given their high mutational load, abundance in activated cytotoxic TILs and peritumoral CD8+T-cells, and a greater density of neoantigens, the POLEmut and MMRd groups, defined hot tumors, may benefit from immunotherapy . In partSeveral studies suggested that low-grade, early-stage EECs harboring CTNNB1 exon 3 mutations have worse OS and recurrence-free survival, as also described for MMR deficiency ,97,98, aNowadays, an intriguing challenge is represented by the application and validation of TCGA classification in small diagnostic biopsies/endometrial curetting, in order to decide the surgical approach, since high grade POLE mutated tumors may benefit from reduced extent of surgery. Recent studies suggest highly concordant results in diagnostic biopsies and hysterectomy specimens, in particular for MMR loss, MSI high, p53 wild, and aberrant types, in contrast to moderate levels of agreement reported for the classical histomorphological parameters . In this way, molecular classification preoperatively applied seems to provide earlier and more reliable prognostic information to guide clinical management . The bioThe integration of the TCGA molecular groups in the ESGO/ESTRO/ESP guidelines of endometrial carcinoma have offered the possibility of limiting the current under- and overtreatment of women with endometrial carcinoma. However, there are still several limitations that should be overcome, especially regarding the management of MMRd and NSMP carcinomas. Major improvements that might be hypothesized are (i) lumping together MMRd carcinomas, regardless of grade and histotype (with the exception of SWI/SNF-deficient UEC/DEC) and (ii) substratifying the risk in NSMP EECs based on further prognostic factors . Moreover, methods and criteria to apply the molecular classifier are not without pitfalls and limitations and are expected to evolve over time. Further improvements in the prognostic definition of endometrial carcinomas will be achieved only integrating clinical, histomorphological, immunohistochemical, and molecular data in a multidisciplinary approach.