Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins’ primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.In this investigation, we reported the production of prototype breads from the processed flours of three specific  The wheat varietal selection undertaken by breeders in the last decades was tailored mainly to improve its technological and productivity-related traits; however, the latter resulted in a considerable impoverishment of the genetic diversity of wheat-based products available on the market. A recent debate supported the idea that such phenotype-based selection might have led to a greater immunogenicity of modern varieties, causing the increasing prevalence of celiac disease (CD) and other gluten-related disorders . In lineConsequently, the comprehensive proteomic characterization of wheat genotypes played a key role in providing new insight into gluten protein expression, not only supporting the drive to scout genotypes combining lower toxicity with satisfactory technological properties, but also posing convenient bases for the development of new detoxification strategies ,14. All The current detoxification technologies mainly rely on enzymatic-based protein hydrolysis treatment  or modifTriticum turgidum wheat collection through a multidisciplinary approach  > 270 ppm). The assay was repeated for this sample by applying an increasing dilution factor of the gliadin extract in order to reach the proper levels for the gluten quantification. The averaged results are reported in By comparing the CTRL and HYD samples for each wheat genotype, a reduced amount of gluten was assessed to be present in all of the three prototype breads from the hydrolysed flours; the reduction was directly ascribed to the proteolytic activity of the enzymatic/microbial mixture designed for the current investigation. However, the fermentation process—applied under comparable conditions to the three selected wheat flours—provided a variable efficacy in gluten degradation depending on the specific genotype. In particular, the sample prepared by the sourdough fermentation of Colosseo flour (HYD-1) resulted in a final prototype bread which can be labelled as GF, due to residual gluten content below the 20 ppm threshold limit (11.3 ± 1.3 ppm). The prototype bread HYD-3, prepared with PI 56263 flour, presented a residual gluten concentration of 36 ± 7 ppm, which is referred to as ‘very low gluten content’ (20 ppm < [gluten] < 100 ppm). In both cases, the gluten degradation efficiency was assessed to be very high (≥99.5%), and the final prototype breads could potentially be included in the diet of CD patients. On the contrary, the HYD-2 sample prepared using Neolatino flour maintained a high level of residual gluten (7600 ± 700 ppm), and was thus not acceptable for direct consumption by CD patients. Notably, in this investigation, it was proven for the first time that the efficiency of gluten detoxification strategies are strictly related to the specific protein profile of the wheat flour. Interestingly, the genotype-depending efficiency reported here poses specific challenges to food technologists because it constrains the validity of all of the previous investigations dealing with gluten hydrolysis by the enzymatic treatment and/or sourdough fermentation of the specific flours on which they were developed and validated. In order to increase the understanding of this experimental evidence, we carried out a detailed proteomic characterization using high resolution tandem mass spectrometry (HR-MS/MS).For the proteomic characterization of prototype samples, a comprehensive protein extraction was carried out under previously optimized conditions . A stronIn order to characterize the low-molecular weight (LMW) fraction resulting from flour hydrolysis, the ultrafiltration of the total protein extracts on cut-off membranes was applied, with a size limit of 3 kDa. The permeate fraction (LMW) was directly analysed by untargeted HR-MS/MS for the sequence identification. For comparison, we applied the same protocol and analysis to both the hydrolysed and control breads, notwithstanding the absence in the latter of the fermentation/hydrolysis step.Triticum taxonomy. Notably, such a database was significantly extended compared to our previous investigation [Triticum turgidum subsp. durum (taxonomy ID 4567), which was made publicly available after the full sequencing of its genome [T. turgidum subsp. durum reference proteome. Given the high complexity of the proteolysis accounted for by the simultaneous microbial and fungal activities, a non-specific cleavage was set for the database indexing. As was expected, very few peptides were detected in the LMW fraction of the three control samples, confirming that all of the sequences identified in samples HYD-1, HYD-2, and HYD-3 were directly ascribed to the detoxification strategy devised and carried out on these samples. In particular, 312, 242 and 384 peptides were detected in samples HYD-1, HYD-2, and HYD-3, respectively, as is reported in The HR-MS/MS analysis was carried out in data dependent acquisition mode, and the fragmentation spectra were processed via commercial software for sequence identification against a customized database containing all of the protein accessions currently assigned to the tigation , becauses genome . TherefoThese latter two classes of proteins were not taken into consideration for further discussion because they were not relevant for the CD immunoreactivity perspective. Interestingly, all of the storage proteins, namely gliadins  and glutenin  were affected, to a certain extent, by the microbial/enzymatic degradation. Of particular interest was the ability to hydrolyze α- and γ- gliadins, especially in the sample HYD-3, as they are mainly responsible for the toxicity level of durum wheat flours towards CD patients. In addition, the protein accessions referred to as ‘AAI domain-containing proteins’ were counted as an independent category because they featured partial sequence homology with γ-gliadin, α-gliadin, and LMW-glutenin accessions, and thus deserved attention for the toxicity risk evaluation.http://www.allergenonline.org/celiacfasta.shtml). All of the peptides detected in the LMW fractions were searched for an exact match with the immunostimulatory sequences, and the positive matches were counted in Such an evaluation was accounted for by an in-silico assessment of the sequence identity with known T-cell epitopes  by meansTriticum taxonomies were identified in all of the samples, especially in the protein extract from the CTRL prototype breads  fraction of the protein extracts was also characterized by untargeted HR-MS/MS analysis. A typical workflow for a bottom-up proteomic approach, with chymotrypsin as specific enzyme, was applied to both the HYD and CTRL samples, keeping the latter as the internal reference to trace back to the susceptible and resistant sequences. Hundreds of sequences ascribed to eads see . Intereseads see . Notablyeads see . On the http://www.allergenonline.org/celiachome.shtml) and some further information about toxicity and the HLA-DQ antigen, as well as the number of peptides per sample encrypting the specific epitope. As was already mentioned, the possibility to identify the peptide sequences by searching against the reference proteome of durum wheat provided an undeniable advance for the current investigation. Browsing the list of the identified epitopes  digestion experiments. The aim was to evaluate the digestibility and toxicity risk for CD patients of the hydrolyzed gluten proteins in such processed samples, in experimental conditions, which simulate the human GD digestion process. The standardized static protocol applied  providedtriticum-belonging proteins was quite short; only 291 peptides for the GD digest of bread HYD-1, and 227 peptides for the GD digest of bread HYD-3. This proved that the extensive hydrolysis affected the wheat proteins after the combined effect of the detoxifying strategy and the GD digestion process. Moreover, an in-depth analysis of the data acquired showed that, in both cases, most of the peptides detected were below the 9 AA length cut-off, and thus do not pose a risk to elicit any immune-response in CD patients. In particular, only 79 peptides out of the 291 detected for the HYD-1 bread were greater than or equal to 9 AA in length. Similarly, only 71 peptides out of the 227 detected were greater than or equal to 9 AA in length for the HYD-3 bread.Notably, the final lists of peptide sequences assigned to These short lists of sequences were screened against the CELIAC Database, as previously described, in order to disclose the presence of epitopic sequences that were resistant to the detoxification process applied and survived the GD digestion, as well. Notably, no epitope was detected in the GD digests of both the HYD-1 and HYD-3 breads. This experimental evidence confirmed that the detoxification strategy applied to these two prototype samples was successful in hydrolyzing the toxic/immunogenic sequences expressed in the relevant monovarietal flours down to concentration levels thar become not detectable in the in-vitro simulated human GD digests.Triticum turgidum wheat genotypes, which were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains and fungal endoproteases. The immunoassay-based characterization suggested a differential efficiency in the gluten degradation according to the specific monovarietal flour, which was investigated in-depth by HR mass spectrometry and in-silico epitope mapping. The in-vitro simulated human GD experiments also proved the absence of toxic/immunogenic epitopes that are relevant for CD patients in the prototype breads produced, confirming the relevance of this investigation for the improvement of the dietary habits of vulnerable individuals. Notably, the advanced proteomic analysis provided new insight for the development of detoxification strategies assessing a genotype-depending efficiency of the proteolytic activity strictly related to the punctual differences of the primary protein structure. Taking advantage of the full sequencing of the durum wheat genome, a detailed list of the susceptible and resistant epitopic sequences was achieved in the current investigation, suggesting the need to constrain the validity of any detoxification strategy to the specific flours on which they are developed.In this investigation, we reported the production of prototype GF breads from processed flours of specific


Gliosarcoma is a rare variant of IDH- wild type glioblastoma with both glial and mesenchymal differentiation. It accounts for approximately 2% of glioblastomas and has a poor prognosis similar to that of classic glioblastoma. It is seen mostly between 40 and 60 years of age with a mean age over 50 years. Pediatric gliosarcoma is even rarer than gliosarcoma in adults. We describe the clinicopathological features of gliosarcoma in patients under 20 years of age and determine whether there are significant differences from gliosarcoma in adults. We also present detailed review of published literature on pediatric gliosarcoma.Slides of gliosarcomas in patients under 20 years of age were reviewed. Clinicopathological features were noted in detail and follow up was obtained.Eleven cases of gliosarcoma were reported in patients under 20 years of age. Ages ranged from three to 19 years (mean age 13 years). Frontal, parietal and temporal lobes were the commonest locations. Mean and median tumor size was six and five cm respectively. All 11 cases demonstrated the classic biphasic pattern. In 10 cases, glial component was astrocytic and was highlighted on GFAP. Sarcomatous component in most cases resembled fibrosarcoma and was high grade in 72.7%. Glial areas were reticulin poor while sarcomatous areas were reticulin rich. In over 45% cases, bizarre tumor giant cells were seen in the sarcomatous areas. In 1 case, sarcomatous areas showed extensive bone and cartilage formation. Other histologic features included hyalinized blood vessels, hemorrhage, infarction, gemistocytic cells, rhabdoid cells etc. Follow up was available in nine patients, five received chemoradiation post resection while three received radiotherapy only. Prognosis was dismal and eight patients died within one to 14 months following resection.Gliosarcomas in patients under 20 comprised 13% of all gliosarcomas reported during the study period. Frequency and mean age were higher compared to other published reports. Pathological features were similar to those described in literature. Clinicopathological features and prognosis of pediatric gliosarcomas were similar to adult gliosarcomas. Gliosarcoma, World Health Organization (WHO) grade IV is a rare variant of Isocitrate Dehydrogenase (IDH)- wild type glioblastoma with both glial and mesenchymal differentiation and accounts for approximately 2% of glioblastomas. It has a poor prognosis similar to that of classic glioblastoma. It is seen mostly between 40 and 60 years of age with a mean age above 50 years, is more common in males and occurs mainly in the cerebral hemispheres with the temporal and frontal lobes being the commonest locations. It is characterized histologically by a biphasic pattern composed of alternating glial and mesenchymal (sarcomatous) areas. Both glial and mesenchymal components represent monoclonal proliferations. The clinical profile, imaging, spread and macroscopic appearance of this variant are similar to classic glioblastoma. It is often superficial and deceptively circumscribed. Pediatric gliosarcoma is even rarer than gliosarcoma in adults , 2.Histologically, the glial component is usually astrocytic (like the classic astrocytic glioblastoma) with anaplastic features. Sarcomatous component usually manifests as a spindle cell sarcoma with nuclear atypia, mitoses and necrosis. The glial areas are reticulin poor while the sarcomatous areas are reticulin rich (highlighted on reticulin stain). On immunohistochemical (IHC) staining, glial areas express glial fibrillary acidic protein (GFAP) while the sarcomatous areas are negative .Pediatric gliosarcoma, as stated above, is even rarer. To the best of our knowledge, 45 cases have been reported in literature –36.Herein, we present a series of 11 cases of gliosarcoma reported in patients under 20 years of age. The aim of this study is to describe the clinicopathological features of gliosarcoma reported in patients under 20 and to determine whether there are significant differences from gliosarcomas occurring in adults. We also present a detailed review of published literature on these extremely rare tumors.The Surgical Pathology files of the Section of Histopathology, Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, were searched for gliosarcomas reported between July 1, 2011 and June 30, 2019. Cases reported in patients under 20 years were identified. Slides of these cases were retrieved and were reviewed by the senior authors. The diagnosis was confirmed. Clinical and pathological features were described in detail. Follow up was obtained through verbal telephonic communication with the parents. Ethical exemption was obtained from the Aga Khan University Ethical Review Committee (ERC). All procedures performed on patient tumor samples in this study were in accordance with the ethical standards of the Institutional ERC and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Detailed review of published literature was conducted by the authors.We systematically searched PubMed, Google Scholar and Web of Science detailed for articles on pediatric gliosarcoma with restriction of ‘original article’ and ‘case reports’. We only included articles with English abstracts. We searched using the Medical Subject Heading (MESH) terms and key words ‘pediatric gliosarcoma’. A total of 34 articles were included. Review articles were not included. We collected demographics, clinicopathological and follow up information. All selected articles were assessed for eligibility. All were found to be eligible. No duplicates were found. All 34 were included in qualitative synthesis.. Glial component was astrocytic in 10 cases (90.9%) and oligodendroglial in 1 case , 84 cases of gliosarcoma were reported. Of these, 11 31.1%) were reported in patients younger than 20 years of age. Clinicopathological features are summarized in Table 1–2019, 81.1% werePediatric gliosarcomas are even rarer than adult gliosarcomas. Clinical and morphological features are similar to adult gliosarcomas. However, they may mimic more common tumors on radiological and histological examination. The clinicopathological features and differential diagnostic consideration are discussed. Published literature is reviewed.On radiological examination, the relative discreteness of these tumors may mimic meningioma , 38. SimAt the molecular level, gliosarcomas including those in the pediatric age group demonstrate Phosphatase and Tensin homolog (PTEN) and TP53 mutations and Cyclin-dependent kinase inhibitor 2A (CDKN2A) deletions. Epidermal growth factor receptor (EGFR) amplification is infrequent. Except for the last, their genetic profile is similar to that of IDH-wild type glioblastoma. Gains on chromosome 7 are seen in 75% cases while losses on chromosome 10 are seen in 35% . In a stOccasional case reports documenting pediatric gliosarcomas in locations other than the cerebral hemispheres have been published. Neelima et al. reported a case occurring in the thalamus . A case Various studies have emphasized the importance of gross total resection in achieving relatively better prognosis , 32. StuMallick et al. published a series of five cases of pediatric gliosarcoma and investigated the value of concurrent and adjuvant temozolamide in the treatment of these tumors. They showed that temozolamide is well tolerated by pediatric patients and survival data with temozolamide therapy was encouraging. The two-year progression free and overall survival rates were 44.2 and 62.9%, respectively .Follow up was available in only 9 out of 11 casesMolecular workup was not performed.Pediatric gliosarcomas are extremely rare. Clinicopathological features of pediatric gliosarcoma are similar to adult gliosarcoma. However, pediatric gliosarcomas may mimic more common tumors on radiological and histological examination. On histological examination, gliosarcomas may sometimes mimic sarcoma and carcinoma if specific mesenchymal and glandular differentiation is present. In pediatric age group, osteosarcoma, fibrosarcoma, teratoma, and atypical teratoid/rhabdoid tumor should be excluded. Like their adult counterparts, pediatric gliosarcomas have a dismal prognosis in spite of aggressive chemoradiation. Slightly better survival times have been demonstrated in some studies with gross total resection although other studies have shown extremely poor survival even with apparent gross total resection.


NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo mosaic virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.Host factors play a pivotal role in regulating virus infection. Uncovering the mechanism of how host factors are involved in virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated  Bamboo mosaic virus (BaMV), a flexuous rod-shaped virus, has a single-stranded positive-sense RNA genome and is classified in the Potexvirus genus of Alphaflexsiviridae ..N. benthNbLTP1 is involved in BaMV infection, we used Tobacco rattle virus (TRV)-based virus-induced gene silencing to knock down the expression of NbLTP1 in N. benthamiana plants. The phenotype of NbLTP1-knockdown plants did not differ from that of the negative control (using the luciferase gene fragment in the knockdown vector) plants (NbLTP1 in knockdown plants was reduced to 36% of that of the control (NbLTP1-knockdown leaves was significantly reduced to 53% of that of the control at 5 dpi (To examine how ) plants . The exp control A, and that 5 dpi B.NbLTP1-knockdown protoplasts were inoculated with BaMV RNA. The accumulation of BaMV CP in NbLTP1-knockdown protoplasts was decreased to 70% and 61% of that of control protoplasts at 24 and 48 h postinoculation (hpi), respectively , -/P(+), and -/CaM(-) were associated with chloroplasts . Becausea leaves A, BaMV wa leaves B. By conTo successfully invade its host, a viral pathogen needs to overcome the host defense mechanisms and access the host protein efficiently for assisting in viral multiplication. Revealing strategies against the viral pathogens requires identifying these host proteins with a role against or assisting the viral pathogens. In this study, we identified a downregulated gene, NbLTP1, that was demonstrated to assist in BaMV accumulation by knockdown experiments (loss of function)  and tran2+-binding CaM [2+ via the conserved contact residue Tyr102  for viral RNA accumulation. However, the detailed mechanism still needs to be revealed.In conclusion, herein, we have identified a gene from


During tapping experiment of 10,000 cycles, the TENG could stably harvest electrical energy. The harvested energy from the proposed TENG is sufficient to illuminate more than 55 LEDs and drive small electronic devices, and the TENGs exhibit excellent performance as a wearable energy harvester.Surface modification of polyimides (PIs) using electrospinning would significantly improve the performance of TENGs because of the larger surface area of the electrospun friction layer. However, PIs generally have high solvent resistance, so it is complicated to convert them into nanofibers using electrospinning process. This study aims to fabricate PI nanofibers via simple, one-step electrospinning and utilize them as a friction layer of TENGs for better performance. PI nanofibers were directly electrospun from PI ink made of polyimide powder without any additional process. The effect of PI concentration on spinnability was investigated. Uniform and continuous nanofibrous structures were successfully produced at concentrations of 15 wt% and 20 wt%. Electrospun PI nanofibers were then utilized as a friction layer for TENGs. A TENG with 20 wt% produced an open circuit voltage of 753 V and a short circuit current of 10.79 μA and showed a power density of 2.61 W m Among various types of nanogenerators such as triboelectric, pyroelectric, thermoelectric, and piezoelectric nanogenerators, triboelectric nanogenerators (TENGs) have gained enormous attention over past few years owing to their simple configuration, low weight, and cost-effective fabrication process. TENG’s electrical performance is usually assessed by power density (W m−2), including voltage and current. TENG’s power density is relatively small compared to other types of nanogenerators because the current generated by TENGs is insufficient.The rapid development of flexible electronics has promoted the wide application of various low power consumption electronic devices. Thus, an effective power source of these devices has gained increasing attention. Nanogenerators are good candidates due to their ability to harvest electrical energy sustainably from environmental sources11. In addition, PI exhibits excellent stability as a friction layer under repetitive external pressure or deformation due to its outstanding mechanical properties.In order to enhance the performance of TENGs, various approaches have been conducted. Selecting materials of the friction layers according to the triboelectric series is an easy, reliable, and straightforward way. The farther two materials on the triboelectric series table, the higher the amount of charges generated. Many synthetic polymers have the nature of being negatively charged while nylon, cotton, and aluminum are generally used as positive friction layers. Polyimide (PI) has been widely used as a negative friction layer for TENGs due to its highly negatively charged nature14. They achieved relatively high performance, but either the complicated, expensive photolithography process or the E-beam evaporation method was used to fabricate the surface microstructures. Others developed porous structures inside the friction layer using pore foaming technology, such as particle templating methods18. This approach was cheap and easy to handle, but fabricated TENGs showed insufficient results. Thus, a simple and efficient way to enhance the surface area of the friction layer and hence improve the performance of TENGs has recently been required.Some researchers have tried to further improve the performance of TENGs by enlarging the surface area of the friction layer with enhanced surface morphology23. In electrospinning, a solution is ejected from a nozzle by applying a high electric potential difference between a nozzle and a collector. After ejection, the jet is elongated by whipping instability before deposition due to the interactions between the charges existing on the jet, resulting in continuous fiber structures. The electrospun nanofibers show a remarkably large surface area per unit volume and a rough surface as well. It has been found that electrospun nanofibers can improve the triboelectric effect, and they allow the generation of more charges26.Electrospinning could be one of the best approaches for manipulating the friction layer because this technique is capable of producing long continuous nanofibers directly28. In other words, it is challenging to fabricate electrospun PI nanofibers. To overcome this problem, PI nanofibers are commonly made through the following two-step approach: (1) electrospinning of polyamic acid (PAA) precursor via a polymerization process to form PAA nanofibers, (2) conversion of the PAA nanofibers into the PI nanofibers through thermal/chemical imidization process under several hundred degrees of Celsius for a few hours31. As expected, this two-step approach is complicated and expensive. Furthermore, since TENGs generally use flexible plastic substrates, high process temperature over a few hundred degrees of Celsius should be avoided. As a result, one-step, simple, direct electrospinning of PI nanofibers is crucial to fabricate more enhanced TENGs with higher performances.Therefore, electrospinning of PI nanofiber membranes would lead to the enhanced performance of TENGs because of both PI’s triboelectric nature and its enlarged surface area. However, PI has high solvent resistance, and they are typically insolubleIn this study, we first developed a simple, one-step process for fabricating electrospun PI nanofibers from PI powder without any additional process. PI powders were dissolved into dimethylacetamide (DMAc), and the effect of PI concentration on the nanofiber structure was investigated. Then, TENGs were fabricated using electrospun PI nanofiber membrane. We investigated the influence of PI concentration on the performance of TENGs with contact-separation mode. The fabricated TENGs were also tested in various applications such as capacitor charging and LED illumination. It is expected that electrospun PI-nanofiber based TENGs could be utilized as an energy-harvesting device.As mentioned earlier, the two-step process is generally used to fabricate electrospun PI nanofibers due to PI’s high solvent resistance. PAA monomers are first electrospun to fabricate the nanofibrous structure, and they are converted into PI through imidization process. In contrast, PI powder is soluble in DMAc, which is one of an organic polar solvent, so it is worthwhile to investigate whether direct electrospinning of PI solution is applicable. Since direct electrospinning does not require thermal/chemical post-processing, it has an advantage in the fabrication of TENGs with a flexible plastic substrate. The PI friction layer with electrospun nanofiber structures has a much larger surface area than film structures, so TENGs with nanofiber membrane would produce more enhanced electrical performance.33. The solution properties such as polymer concentration and molecular weight of the ejected polymer significantly affect fiber formation in comparison to other governing parameters like surface tension and conductivity. For low molecular weight polymers, a high polymer concentration is necessary to fabricate uniform nanofiber structures. In other words, once the polymer and the solvent capable of electrospinning are determined, the characteristics of the electrospun nanofibers are mainly influenced by the polymer concentration in ink.In order to fabricate nanofibrous membranes using electrospinning technique, we first investigated the solubility and spinnability of the PI powders with various solvents  between the electrodes, the amount of transferred charge (Q) in between, and the separation distance (x) between the two triboelectric charged layers . The output voltage of a typical TENG operated in contact-separation mode is determined as follows6:ρA, εr, and S are the charge density on the surface of the contact materials, the relative permittivity of friction layer, and the surface area, respectively. In addition, d and x(t) denote the thickness of the friction layer and the distance variation between the contact materials, respectively. In the open circuit condition, the output voltage is linearly proportional to the product of ρA and x(t) because Q is zero. When we define the effective thickness constant d0 as d/εr, the output current in the short circuit is highly proportional to the S and ρA, as follows:For evaluating the performance of TENGs, power generation is defined by three parameters: the voltage  due to different d is too small to have an influence on output voltage. Therefore, the thickness of the friction layers has little impact on the electrical performance of TENGs. The low performance at 5 wt% was due to the structures of the layer. When the 5 wt% ink was used, it was sprayed instead of spun, and the PI was in the form of granules rather than nanofibers. The film was severely damaged during the measurement since the adhesion among the granules was very weak. The damaged PI film was represented in Supplementary Information Fig. OC values for the TENGs with 10 wt% and 15 wt% were 300.8 V and 366 V, respectively. The ISC values were 6.15 μA and 6.52 μA, respectively. However, we were not able to measure the corresponding values of the TENG with 20 wt% because the layer was detached entirely from the ITO-PET at the very beginning of the measurement  was dissolved in dimethylacetamide  for 12 h using a magnetic stirrer at room temperature. The concentration of PI varied from 5 to 20 wt% with an interval of 5 wt%. An indium-tin-oxide coated polyethylene terephthalate (ITO-PET) was used as a collector. Aluminum was used as a top electrode of the TENGs.2) was ultrasonicated for 5 min in deionized water to remove contamination on the surface. ITO-PET was placed on the moving stage, and electrospun PI nanofibers were collected on the ITO-PET substrate. The distance from the nozzle tip to the surface of the collector was fixed at 150 mm. PI ink was fed into the nozzle using a syringe pump  with a fixed flow rate of 6 μl min−1. The input voltage was set at 10 kV, and PI electrospinning was performed for 1 h for all the experiments in this study. The surrounding humidity was kept below 35% by using a dehumidifier .Electrospinning was performed using an in-house system  and the short circuit current (ISC), were measured using an oscilloscope  and a preamplifier . The microstructure of the electrospun PI nanofibers was observed using a field-emission scanning electron microscope .To compare film-like structures and nanofibrous structures, a commercial PI film and a screen-printed PI film were also prepared as friction layers for the TENGs. To fabricate the screen-printed PI film, PI ink with a concentration of 15 wt% was directly screen-printed onto the ITO-PET film using a razor blade and was heated to 60 °C for 3 h. The pressing force was measured with a load cell  and a data acquisition board . The electrical performance parameters, including the open circuit voltage (VSupplementary Information.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


A photosensitizer was loaded in pre-formed [DS/PArg]4 hollow microcapsules with or without exposure to heat. Loading efficacy and drug release were quantitatively studied depending on the capsule concentration to emphasize the interactions between the DS/PArg multilayer network and Cholosens. The loading data were used to determine the dosage for heated and intact capsules to measure their PDT activity in vitro. The capsules were tested using human cervical adenocarcinoma (HeLa) and normal human dermal fibroblast (NHDF) cell lines, and two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results provide compelling evidence that encapsulated forms of Cholosens are efficient as PDT drugs for both eukaryotic cells and bacteria at specified capsule-to-cell ratios.Microencapsulation and targeted delivery of cytotoxic and antibacterial agents of photodynamic therapy (PDT) improve the treatment outcomes for infectious diseases and cancer. In many cases, the loss of activity, poor encapsulation efficiency, and inadequate drug dosing hamper the success of this strategy. Therefore, the development of novel and reliable microencapsulated drug formulations granting high efficacy is of paramount importance. Here we report the in vitro delivery of a water-soluble cationic PDT drug, zinc phthalocyanine choline derivative (Cholosens), by biodegradable microcapsules assembled from dextran sulfate (DS) and poly- Photodynamic therapy (PDT) is one of the modern methods for the treatment of cancer and infectious diseases that hold the promise of selective erasing of the pathologic area ,2,3. PDT3 vaterite sacrificial templates via alternate adsorption of biodegradable polyelectrolytes represent the system of choice for the delivery of PDT drugs, owing to their long-term structural stability and low toxicity 4) capsules loaded with Cholosens for the PDT treatment on eukaryotic cells and bacteria studied in vitro. Human adenocarcinoma (HeLa) cells and normal human dermal fibroblasts (NHDF) were used as corresponding models of cancer and normal eukaryotic cells. The antimicrobial PDT effect was studied on Gram-positive and Gram-negative strains, Staphylococcus aureus and Escherichia coli, respectively. In our work, we post-loaded the capsules with Cholosens with and without exposure to high temperature (80 °C for 60 min), while also varying the capsule concentration to get a better understanding of ionic interactions between the DS/PArg multilayer capsules and Cholosens. PMC loading was then followed by detailed research of the heat treatment’s influences on the potency of the encapsulated form of Cholosens, i.e., the drug release rate, internalization of capsules by cells, and their dark and light toxicities.Small-to-medium molecular size PDT drugs stable to heating up to 70–90 °C are excellent models with which to discover the potential of PMC as a delivery platform for therapeutics encapsulated via the post-loading method. In this work, we used the phthalocyanine-based dye, octakis{methylene}chloride zinc phthalocyanine, M = 1610 g/mol , which i2 . A recen2 . The curw/w water solution of poly , dextran sulfate, sodium salt , poly-l-arginine hydrochloride , α-chymotrypsin from bovine pancreas, calcium chloride dihydrate, anhydrous sodium carbonate, ethylenediaminetetraacetic acid trisodium salt (EDTA), rhodamine 6G (RhD6G), fluorescein 5-isothiocyanate (FITC), phosphate-buffered saline (PBS), Dulbecco’s minimum essential medium (DMEM), fetal bovine serum (FBS), Alamar blue, and calcein-AM were purchased from Sigma-Aldrich. Minimum essential medium (MEM), penicillin, streptomycin, trypsin, and trypan blue were purchased from Thermo Fisher Scientific. Hydrochloric acid was obtained from Merck. Zinc phthalocyanine (Cholosens) was kindly provided by the Institute of Organic Intermediates and Dyes . All chemicals were used as received without further purification.Poly(sodium 4-styrenesulfonate) , a 20% Staphylococcus aureus and Escherichia coli were from ATCC .Normal human dermal fibroblasts (NHDF) and HeLa cell cultures were obtained from the Department of Cell Engineering, Education and Research Institute of Nanostructures and Biosystems, Saratov State University, Russia. −1 from a three-stage Milli-Q Plus 185 purification system was used in the experiments.Deionized water with specific resistivity higher than 18.2 MΩ cmA single batch of hollow PMC was prepared and used in all experiments described in this manuscript.3 microparticle template was synthesized by mixing 2 mL of each of 1 M CaCl2 and Na2CO3 solutions under vigorous agitation for 30 s. The obtained CaCO3 spherical particles with (average diameter 4 μm) were collected by centrifugation and thoroughly washed with DI water. Multilayer capsules comprising four bi-layers of DS/PArg were then assembled on CaCO3 via the layer-by-layer (LbL) method. DS and PArg were alternatively adsorbed from 2 mg/mL and 1 mg/mL of respective aqueous solutions, also containing 0.5 M NaCl, starting from the DS layer. After each single layer formation, the particles were thoroughly washed with water to remove the uncoupled polymer. The obtained coated particles were treated with 5 mL of 0.2 M EDTA for 15 min to remove the inorganic phase resulting in the formation of hollow polymeric capsules. Capsule suspensions containing variable numbers of particles were then re-dispersed in 1 mL of an aqueous solution of Cholosens (0.05 mg/mL). After one hour of incubation needed for the infiltration of Cholosens, each suspension was divided into two specimens. Capsules in specimen 1 were immediately washed with DI water by centrifugation to remove the unloaded Cholosens, whereas capsules in specimen 2 were heated up to 80 °C and kept for 60 min at constant shaking (500 rpm) before cooling down for ten minutes and washing. The supernatants were collected to measure the concentration of Cholosens . Here and further in the manuscript, the concentration of Cholosens in supernatants was determined spectroscopically  by measuring the intensity of fluorescence at λex = 685 nm, λem = 715 nm. The fluorescence intensity data were then converted to concentrations using a calibration curve plotted for a series of dilutions with a known concentration of Cholosens, which exhibited linear character in the measured concentration range. Each calibration solution was prepared in 1× PBS to match the ionic strength of the tested samples.The capsules were assembled and loaded with Cholosens following the method previously described by our group . In brieThe PMC were post-loaded with RhD6G of FITC dyes via incubation of the microcapsule suspension in the respective solutions (0.1 mg/mL) for 60 min followed by three washing steps with DI water.Cholosens-loaded capsule suspensions were lyophilized in FreeZone 12 Labconco freeze drier. Each sample was then mixed with 1 mL of 1 mg/mL α-chymotrypsin dissolved in 1× PBS (pH 7.4) in 2 mL centrifuge tubes and kept at 37 °C for 24 h. Undissolved polymeric complexes were then extracted by centrifugation and resuspended again in the enzyme solution. The respective supernatants were analyzed with fluorescence spectroscopy to determine the amount of Cholosens released from the capsules after the complete digestion of the PMC.8 [DS/PArg]4 microcapsules in 1 mL of 1× PBS (pH 7.4) was measured with fluorescence spectroscopy over 48 h. The medium was fully refreshed at each timepoint.The cumulative release of Cholosens from 4.5 × 10ex = 491 nm, λem = 516 nm). The cells were seeded in 24-well cell culture plates at the density of 10000/cm2 in 0.5 mL DMEM supplemented with 10% FBS and 1% penicillin–streptomycin appropriate for the cell passage and incubated for 24 h. The medium was then replaced by the fresh DMEM containing FITC-labelled PMC (5 capsules/cell). After incubation for 1, 2, 4, and 24 h to allow the capsule uptake, the cells were rinsed with PBS (pH 7.4) three times, trypsinized, centrifuged, and kept in ice-cold PBS until study with a FACSCalibur flow cytometer. The cells were treated with trypan blue to quench the extracellular fluorescence of FITC and to detect the signal from internalized capsules solely 4 microcapsules were visualized at λex = 685 nm, λem = 715–780 nm. Photodynamically-induced damage of HeLa cells by different forms of Cholosens was visualized by irradiation of the respective samples with white laser  at 670 nm. A single cell in the treated area received approximately 0.1 mW irradiation dose.HeLa cells were plated into a Petri dish and incubated for 24 h before staining with a calcein-AM dye (λp < 0.05 and < 0.01).Statistical analysis of data was conducted using unpaired two-tailed Student’s t-test. Data are presented as the means ± standard deviations (SD). Two levels of significance were established .Cholosens loading was assessed by the quantitative analysis of the loading efficiency that was essential for administration dosage of heated and intact encapsulated drug forms for in vitro studies. Previously we showed that the amount of rhodamine B detected in DS/PArg capsules was lower for heated analogs in comparison with the untreated PMC. Moreover, the loading of heated capsules did not depend much on the capsule concentration that was varied in the range of 6.9 × 10e sample . The par4 microcapsules was measured by two complementary approaches, i.e., (1) by measuring the residual concentrations of the drug in the supernatants obtained after loading  loading . Spectro loading a reveale8 capsules/mL. The observed result contains no contradiction if we take into account the residual concentrations of Cholosens observed for the capsule concentration 4.5 × 108 capsules/mL, which were approximately 20% and 10% of the initial amount in suspension for intact and heated capsules, respectively. As such, at a higher capsule concentration, the drop in the loading per capsule was well-expected because almost the same amount of Cholosens was spread across a larger number of capsules.The loading data obtained through measurements of Cholocesns released from enzymatically-degraded capsules  and thro8 capsules/mL, intact PMC displayed better loading than their heated counterparts.The total amount of loaded Cholosens significantly increased when increasing the capsule concentration for both intact and heated capsules . Besides4 microcapsules in the cell culture medium. As such, we observed no changes for the capsules suspended in water or 1× phosphate-buffered saline (PBS). Capsule cellular internalization was then studied on HeLa cells by analyzing approximately 104 cells per sample by flow cytometry. The PMC were post-loaded with FITC for analytical purposes. In each sample, the cells were mixed with capsules in a concentration of 10 capsules/cell. The uptake level (the proportion of cells (%) with at least one capsule) was determined as the median fluorescence intensity in each sample related to the median fluorescence intensity of the control cells incubated without capsules. The results are reflected in Before engaging in the in vitro experiments, we studied the morphology of [DS/PArg]4 microcapsules by HeLa cells was a time-dependent process, wherein the number of cells with internalized capsules had gradually increased. Similarly to the data described elsewhere 4  of the difference between the toxicity of intact capsules after laser irradiation compared to non-irradiated samples was confirmed for all studied capsule/cell ratios in both cell lines  and lower compared to the dark toxicity of intact PMC over the whole studied density range. On the contrary, intact capsules gradually became more toxic upon increasing the capsule-to-cell ratio. Maximal light toxicity for E. coli achieved with heated PMC was the ~28% of cells remained viable. The light-induced toxicity of intact capsules was significantly higher than of their heated counterparts for all capsule/cell ratios  the loading efficacy exceeded 90%. Besides, unlike what was observed for rhodamine B, the loading per capsule was higher for heated capsules compared to intact ones all the way until the capsule concentration reached 9 × 108 capsules/mL (8 capsules/mL in case of Cholosens), the number of displaced dye molecules will overcome the amount of those entrapped in the capsules. The data in In contrast to a poor loading of rhodamine B in DS/PArg microcapsules upon heating , Cholosesules/mL . At thisWhile the heat treatment did not affect the loading, it had an impact on the release of Cholosens from PMC. The heated capsules released the drug slower than their intact counterparts, which was also a reason for their lower antimicrobial PDT activity in comparison to the untreated PMC (c.f. discussion later in the text).4 microcapsules and free drug were comparatively studied on eucaryotic cells  and bacteria (S. aureus and E. coli); we aimed to determine the cytotoxicities, bacterial toxicities, and anticancer and antimicrobial PDT efficacies of different drug forms.The developed Cholosens-loaded [DS/PArg]4 microcapsules, the drug toxicity was significantly mitigated, thereby providing distinctive evidence of the postulated need for Cholosens encapsulation.Non-encapsulated Cholosens inhibited the metabolic activity of both human cell lines, which we attributed to its electrostatic binding to the plasma cell membrane. Upon loading of Cholosens in [DS/PArg]We figured out that capsules mainly deliver the photosensitizer to NHDF and HeLa cells through cellular uptake. The internalization of capsules 4–6 µm in diameter by cancerous cells, including HeLa, has been well-described in literature ,58, suppNHDF cells poorly tolerated the encapsulated drug forms, showing viability below 80% already at five added capsules per cell. This result indicates the necessity for a more profound understanding of the mechanism of the capsule-cell interactions through in vitro and in vivo studies, while it highlights the importance of cancer-targeting by delivery systems. Besides, further research on the design of PMC for delivery of Cholosens or analogs needs to consider a short lifetime of singlet oxygen (about a few µs or less), so its oxidative effect cannot be extended past a proximate locale. Thus, the cell could either undergo apoptosis or necrosis depending on which crucial organelle or functional molecule  was affected by the delivered drug. For example, damage in DNA ,60,61 orS. aureus and E. coli without activation by light significantly higher than both encapsulated forms at most of the studied capsule densities and drug concentrations. . The current mechanism of action for many antibacterial drugs includes an initial attachment of the oppositely-charged ionic drug to the bacterial surface by electrostatic interaction. Aside from the size disparity, PMC-loaded Cholosens has a much more complex and better-balanced ionic interaction of Cholosens molecules and polymeric layers in the network and/or inside the capsules than the pure drug. Therefore, not only the size of the PMC-loaded Cholosens but the reduced ionic interaction of the loaded capsules with the bacterial surface makes the pure drug more prone to get attached to the bacteria, further providing a desired photodynamic efficacy. The results of our study are entirely consistent with the proposed mechanism of antimicrobial activity of the developed capsules. Both heated and intact capsules loaded with Cholosens displayed the antimicrobial PDT activity upon irradiation with 680 nm light against Our study introduces the DS/PArg PMC as an efficient delivery system for the water-soluble PDT drugs. Further development of the encapsulated drug forms would be aimed at the tailoring mechanisms for controlled delivery, e.g., capsule size reduction for efficient tumor targeting via the EPR effect. We believe that PMC produced via consecutive surface self-assembly of complementary biocompatible, naturally-derived polyions will outperform the vast majority of nanomaterials in the treatment of infectious diseases and cancer due to the stable structure and favorable safety profiles.


Antibody-mediated rejection (AMR) occupies a major position for chronic rejection after kidney transplantation. Regulatory B cell (Breg) has been reported to have an inhibitory immune function, which contributes to the resistance for AMR.A nested case–control study for nine healthy donors, 25 stable (ST) patients, and 18 AMR patients was performed to determine the type of Breg in maintaining immune tolerance and preventing AMR.+ Bregs, but not Bregs, significantly decreased. The receiver operating characteristic (ROC) curve analysis revealed that rather than the circulating Bregs, decreased circulating IL-10+ Breg levels were positively associated with AMR. However, kidney B cell and IL-10 infiltration was significantly increased in the AMR group with high expression of C-X-C motif chemokine 13 (CXCL13). In addition, circulating IL-10+ Bregs, rather than Bregs, remained higher than those at pre-operation, during the 90-day post-operation in immune homeostasis.Compared to the ST group, circulating interleukin (IL)-10+ Breg levels are more appropriate measures for assessing the resistance of AMR after kidney transplantation.The circulating IL-10 Kidney transplantation is the best choice for the end-stage treatment of chronic kidney disease. At present, antibody-mediated rejection (AMR), which is characterized by the presence of a donor-specific antibody (DSA) directed to human leukocyte antigen (HLA), has emerged as the leading cause of chronic damage to the kidney graft, which largely limits the long-term survival of the graft. , 2. HoweAccumulating evidence suggests that under inflammatory circumstances, B cells can differentiate into antibody-secreting plasmablasts and regulatory B cells (Bregs), which are a special B cell subset that secretes immunosuppressive cytokines, particularly interleukin (IL)-10. –7. Thus,+CD24+CD27+ (+CD24+CD38+ (+ mBregs) defined with CD19+CD24+CD27+IL-10+ (+ tBregs) defined with CD19+CD24+CD38+IL-10+ , and AMR groups. Then, the biodistribution of B cell, IL-10, and chemokines in the kidney grafts of healthy, ST, and AMR patients were determined. Furthermore, the dynamics of the four subpopulations of circulating Bregs in ST patients for 90 days after surgery were investigated. These present results, together those reported by previous studies . The clinical trial registration number is ChiCTR1900022501.These patients were divided into three groups: (1) healthy control group : healthy kidney donors within 18–55 years old (free of disease), consisting of the siblings and spouses of the recipients; (2) recipients with stable graft function group : patients within 18–55 years old, whose kidney function was stable (free of infection or rejection before and after kidney transplantation) during the follow-up period (≥6 months); 3) recipients with AMR group : patients within 18–55 years old, who were diagnosed with AMR. The kidney grafts of recipients with ST and AMR were obtained from healthy kidney donors. The differential diagnosis between the ST and AMR groups was confirmed by two kidney pathologists, according to the Banff 2017 consensus  recipien.The peripheral blood samples from the healthy group were collected at pre-operation, and at 1, 7 and 14 days post-operation. Samples obtained from the ST group were collected at pre-operation, and at 1, 7, 14, 30, and 90 days post-operation. Samples obtained from the AMR group were collected when rejection occurred (before the anti-rejection therapy for AMR). Peripheral blood mononuclear cells (PBMCs) were isolated from 5 ml of blood by Ficoll-Paque gradient centrifugation and preserved with 10% dimethyl sulfoxide  in liquid nitrogen.The core needle renal biopsy specimens obtained from the healthy group were collected before surgery for time-zero donor kidney biopsies. The core needle renal biopsy specimens obtained from the ST group were collected at 90 days post-operation after their kidney function stabilized due to the protocol biopsy. In light of the second puncture impairment of kidney function, all AMR patients refused the core needle renal biopsy after anti-rejection therapy for AMR. Thus, core needle renal biopsy specimens from the AMR group were only collected when rejection occurred (before the anti-rejection therapy for AMR). A total of three healthy donors, nine ST patients, and nine AMR patients provided a signed informed consent before the core needle renal biopsy. The remaining six healthy donors, 16 ST patients, and nine AMR patients refused the core needle renal biopsy due to fear of major bleeding in association with the biopsy. Single cells were isolated from the core needle renal biopsy specimens of three healthy donors, three ST patients, and three AMR patients, by combining mechanical dissociation with the enzymatic degradation of the extracellular matrix, which maintains the structural integrity of tissues . The kidney cells were preserved with 10% dimethyl sulfoxide  in liquid nitrogen, and the remaining core needle renal biopsy specimens were embedded in paraffin at 4°C.6 PBMCs were incubated with the Roswell Park Memorial Institute 1640 dilution-cell stimulation cocktail (500×), which comprised of the PMA (Phorbol 12-Myristate 13-Acetate), a calcium ionophore (Ionomycin), and protein transport inhibitors Brefeldin A and Monensin, , at 37°C with 5% CO2 for 5 h. Dead cells were excluded by staining with 7-Aminoactinomycin D , and the surface markers of Bregs were stained with fluorochrome-labeled antibodies /Cy7 anti-human CD24, Alexa FluorR647 anti-human CD27, Alexa FluorR488 anti-human CD38, and Brilliant Violet 510™ anti-human CD45; the appropriate isotype controls: Brilliant Violet 421™ Mouse IgG1 κ Isotype Control, PE/Cy7 Mouse IgG2a κ Isotype Control, Alexa FluorR647 Mouse IgG1 κ Isotype Control, Alexa FluorR488 Mouse IgG1 κ Isotype Control, and Brilliant Violet 510™ Mouse IgG1 κ Isotype Control; Biolegend, San Diego, CA, USA) for 30 min at 4°C, with protection from light. For the intracellular staining, cells were stained with antibodies targeting the intracellular cytokines of Bregs  for 30 minutes at room temperature, with protection from light. The labeled cells were analyzed using the BD Biosciences Canto II instrument . A total of 100,000 events were acquired in the lymphocyte gate. The data analysis was performed using FlowJo software .Cells stored in liquid nitrogen were quickly thawed in a water bath set at 37°C for 3 min. A total of 2–3 × 10Immunohistochemistry staining of CD19, IL-10, and C-X-C motif chemokine 13 (CXCL13) in kidney tissues was performed, respectively. Briefly, 4 μm sections obtained from formalin-fixed, paraffin-embedded kidney tissue samples were incubated with 1:500-diluted anti-CD19, 1:1000-diluted anti-CXCL13 , and 1:500-diluted anti-IL-10  primary antibodies overnight at 4°C, followed by Horseradish Peroxidase-conjugated 1:20,000-diluted goat anti-rabbit secondary antibody  for 1 h at room temperature and then 3,3′-diaminobenzidine for another 10 min. DAPI appears in blue. Immunohistochemistry images were acquired with an Aperio ScanScope AT Turbo . Numbers of CD19, IL-10, and CXCL13 positivities were scored as follows: 0 positivity, score = zero; 1–5 positivities, score = one; 6–10 positivities, score = two; 11–20 positivities, score = three; >20 positivities, score = four.2 test, a corrected χ2 test, or Fisher’s exact test, as needed. One-way repeated measures analysis of variance was used at various time points. P-values <0.05 were considered statistically significant.In the nested case–control study, receiver operating characteristic (ROC) curve analysis was performed by MedCalc v18.11.3. The remaining statistical analyses were performed using IBM SPSS 21.0. Normally distributed measurement data with a homogeneity of variance were expressed as mean ± standard deviation . However, the incidence of both anti-class I and anti-class II DSA-positivity in the AMR group was significantly higher, when compared to those in the ST group . Furthermore, compared with ST patients, AMR patients had significantly higher scores in tubulitis (t), interstitial inflammation (i), and peritubular capillaritis (ptc) , according to the Banff classification     in PBMCs were similar in the healthy, ST, and AMR groups (P = 0.095). The percentage of both mBregs and tBregs in B cells did not differ among groups   and healthy group  (As shown in + mBreg or IL-10+ tBreg ratios in B cells exhibited a good sensitivity and area under the ROC curve (AUC) in diagnosing patients with AMR. Meanwhile, compared to the mBreg or tBreg ratios, the IL-10+ mBreg or IL-10+ tBreg ratios in B cells exhibited a statistically significant superior ability to discriminate between ST and AMR patients . Furthermore, similar results appeared between the healthy (before surgery) and AMR (before the anti-rejection therapy) groups (The diagnostic capacity of four subpopulations of circulating Bregs was compared between the ST (at day 90 post-operation) and AMR (before the anti-rejection therapy) groups by ROC curve analysis , althoug) groups Table 3.P = 0.020). Then, it was found that the mean count scores in IL-10 positivity were significantly higher in the AMR group than in the ST group (P = 0.008). Additionally, compared with ST patients, AMR patients had significantly higher scores in CXCL13, which is a B cell specific chemokine (P = 0.007)  and healthy group  , the four subpopulations of circulating Bregs had no significant differences between pre-operation and post-operation (P > 0.05) (P > 0.05), the four subpopulations of circulating Bregs remained higher than those at pre-operation during the three months following transplantation (P < 0.05) . However, the percentage for both IL-10+ mBregs and IL-10+ tBregs in B cells were significantly higher at days 1, 7, and 14, post-operation, in the ST group, when compared to those in the healthy group  . The clinical trial registration number is ChiCTR1900022501. The patients/participants provided their written informed consent to participate in this study.YL, FL, and KZ performed the experiments, analyzed the data, and wrote the manuscript. SW and HZ participated in the study design, material support, and coordination. XY, WS, JW, ZW, XP, YF, LL, and HX carried out parts of the experiments. JL and GF conceived the study and performed critical revisions of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 82070771) and Foundation of Henan Provincial Health Bureau (SBGJ2018022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer CY declared a shared affiliation, though no other collaboration, with one of the authors FL to the handling editor.


Antimicrobial resistance (AMR) is one of the most complex global health challenges today: decades of overuse and misuse in human medicine, animal health, agriculture, and dispersion into the environment have produced the dire consequence of infections to become progressively untreatable. Infection control and prevention (IPC) procedures, the reduction of overuse, and the misuse of antimicrobials in human and veterinary medicine are the cornerstones required to prevent the spreading of resistant bacteria. Purified drinking water and strongly improved sanitation even in remote areas would prevent the pollution from inadequate treatment of industrial, residential, and farm waste, as all these situations are expanding the resistome in the environment. The One Health concept addresses the interconnected relationships between human, animal, and environmental health as a whole: several countries and international agencies have now included a One Health Approach within their action plans to address AMR. Improved antimicrobial usage, coupled with regulation and policy, as well as integrated surveillance, infection control and prevention, along with antimicrobial stewardship, sanitation, and animal husbandry should all be integrated parts of any new action plan targeted to tackle AMR on the Earth. Since AMR is found in bacteria from humans, animals, and in the environment, we briefly summarize herein the current concepts of One Health as a global challenge to enable the continued use of antibiotics. Antibiotics are the foundation of modern medicine: they are the reason for the survival of immunocompromised patients and transplant recipients, they support trauma care, as well as implants or prostheses; the whole medical world relies on the possibility to treat infections: without safe and effective antibiotics, indeed, the whole edifice of modern medicine itself will crumble because of the spread of antimicrobial resistance (AMR) .Several interconnected human, animal, and environmental habitats can contribute to the emergence, evolution, and spread of AMR, and it is essential to recognize that the health of people is tightly connected to the health of animals, that diseases are transmitted from humans to animals and vice versa, and that AMR must therefore be tackled in both. Such a perspective also encompasses the environment, which is clearly tied to humans and animals and likewise a potential source of new resistant microorganisms. In addition, antibiotics are often used for nonmedical uses, such as beekeeping, ethanol production, horticulture, in antifouling paints to prevent barnacle build-up on boat hulls, and food preservation . UnfortuThe defined “One Health” approach is essential for developing comprehensive and integrative measures to address AMR. This approach should include the surveillance of microbes in humans, animals, and environments to better understand AMR, and to develop effective preventive and control strategies . One HeaIn this review, we summarize the current knowledge about the three parts that make up our world from the globally expanding threat of AMR.AMR is one of the greatest health challenges of the 21st century ,11,12,13Besides, patient and public expectation and pressure to prescribe antibiotics, or any situation that allows for financial benefit from the supply of medicines, such as unregulated over-the-counter availability and use, can drive inappropriate antimicrobial prescribing. Indeed, another major threat that fosters the spread of AMR and endangers the patients’ safety is drug counterfeit. As a consequence of globalization, a criminal market of drug without standard manufacture requirements has flourished, and even antimicrobials were—and indeed are—not exempt from this phenomenon. Warnings and alerts have arisen in the last years all around the world ,15,16,17Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase (ESBL), and high-level AmpC producing Enterobacteriaceae and carbapenemase producing Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii [S. aureus and Gram-positive pathogens), it will be transmitted to the next generations. In the case of horizontal transfer , the resistance gene moves to neighboring bacteria, and therefore, the spread of resistance can be even greater [AMR is widespread in Gram-positive and Gram-negative bacteria, and the following resistance phenotypes have a significant clinical public health impact: methicillin-resistant aumannii . Generalaumannii . AMR mec greater ,25.E. coli, community-acquired-MRSA); the spread of MDR bacteria into the community is a crucial development, and it is associated with increased morbidity, mortality, healthcare costs, and once again, antibiotic overuse [Healthcare institutions such as hospitals, nursing homes, and rehabilitation facilities are hotbeds for MDR bacteria, but some MDR organisms have become quite prevalent causes of community-acquired infections , but also among Bacteroides species and residing Gram-positive bacteria [However, humans themselves may act as a potential reservoir and spread AMR: antibiotics in the early stages of life are capable of definitively altering the microbiome with the selection of resistance in some commensal strains, then triggering the spread of such mechanisms to others ,27; morebacteria .The threats posed by AMR are of increasing concern even in low- and middle-income countries (LMICs), as their rates of antibiotic use increase. An understanding of the burden of resistance is rather lacking in LMICs, particularly for MDR pathogens. AMR spread affects not only the national budgets or gross domestic product (GDP) of countries, but also the attributable mortality or related disabilities. Cassini et al. , throughOne important conclusion that can be drawn from this study is that most of the estimated burden was in hospitals or other healthcare settings, thus suggesting the urgent need to address AMR as a patient safety issue. Antimicrobial classes that are used in human and animal health encompass the same or very similar molecules to a large extent, and this can clearly drive the transmission of resistance between animals and people, either directly or via the environment. Antimicrobials used in animal farming are often administered in sub-therapeutic doses and for prolonged periods, thus framing ideal conditions for bacteria to fix genes that confer resistance, and contributing fundamentally to the global AMR crisis. These genes can subsequently be transferred to human-adapted pathogens or to human gut microbiota especially through contaminated food at origin, an unsafe food chain, or the environment. Moreover, they support ideal conditions for the amplification of genes that may have arisen in people or the environment . The relationship between health and AMR in the food chain encompasses both the pathogenic and non-pathogenic microorganisms, as both can have serious consequences . There amcr-1 positive E. coli isolates in Norway, have been recently reported, thus highlighting new potential pathways to transfer AMR genes to “low prevalence” countries [The first one is the potential unsafety of food linked to contaminated water, preparation, or poor hygiene ,36,37,38ountries ,41,42,43Antimicrobials have been and are still widely used for disease prevention and growth promotion in food animals. Expanding human population ever demands more animal-based protein, which in turn leads to more industrialized methods of food animal production, including sub-therapeutic antibiotics for growth promotion and disease prevention ,45,46,47The high proportion of poor-quality veterinary medicine for therapeutic use in livestock exacerbates the problem of antibiotic overuse or misuse, particularly in LMICs . As seenIntensive farming is also a stress factor for animals due to crowding and a lack of hygiene, causing illnesses and increased shedding. Antibiotics consumed by animals can be excreted in urine and feces into the surrounding environment, potentially inducing or selecting for the development and maintenance of antibiotic resistance genes (ARGs) into the environment ,66,67. Imcr-1 and mcr-2 through the facilitation of the capture and dissemination of potential colistin resistance genes from aquatic bacteria, such as Aeromonas and Shewanella, which can be naturally resistant to colistin [mcr-1 positive Escherichia coli through Whole-Genome Sequencing (WGS), concluding that these strains were highly prevalent in the aquaculture supply chain and resistant to most antibiotics, and that mcr-1 could be transferred to humans via the aquatic food chain [Bacillus cereus, and found 100% antibiotic resistance to rifampin and most β-lactams, thus highlighting the need for continuously monitoring AMR in B. cereus in aquatic products and controlling drug use in aquaculture [In 2014, China produced over 45 million metric tons of fish, crustaceans, and mollusks by aquaculture with more than 50% of this production exported . The heacolistin . This hyod chain . Likewisaculture .The higher the amount of antibiotics used in animals, whatever the reason, the higher the anticipated AMR rates . MoreoveEnterobacterales [The last aspect to be considered in the strict interplay between humans and animals about AMR is the relationship of humans with pets, companion animals, or animals living close to humans for work or recreational activities. Dogs are not only potential carriers of MRSA, but they can also be colonized by plasmid-mediated quinolone resistance genes or different ESBL-producing cterales ,74,75. Overall, these studies highlight also the importance of raising awareness in patients and healthcare workers: even if pets or companion animals may be very helpful for some “frail” patients for their social activities, nevertheless, they must be trained to optimal animal waste disposal and contact precautions, ideally by “trained trainers”; this would avoid patient colonization with MDR pathogens. AMR develops in, and is maintained and transmitted across humans, animals, and the natural environment. In the natural environment, antibiotics are produced by microorganisms to better compete against other microbial competitors, or just as molecular signaling between cells . TherefoStreptomyces sp. strain AM-2504 exerts a molecular mechanism to protect itself from the activity of dityromycin that can be reproduced in E. coli, which is one of the commonest commensals in humans [Antibiotic-producing microorganisms can protect themselves from the toxic effect of the drug using different strategies: one of the most common involves the modification of the antibiotic’s target site. The soil bacterium n humans . Antibiotic resistance genes (ARGs) are ubiquitous in nature : they caAcinetobacter comprised up to 29% of the 16S rRNA reads, and a strong correlation was found between the abundance of Acinetobacter and the OXA-58 carbapenemase gene [Moreover, these environments frequently contain very high levels of antibiotics and pharmaceuticals ,87: the ase gene . Other ease gene ,90,91.The counterpoint of any human activities potentially releasing whatever ARG in the environment is that MDR bacteria or even pathogens excreted or eliminated via effluents and sewage can re-contaminate humans and animals  even durSurprisingly, even remote environments or the sponge microbiota may host AMR genes or plasmids ,96, and Finally, it must be reminded that healthcare settings themselves may act as a potential reservoir for environmental pollution with MDR or even hospital-acquired outbreaks with MDR pathogens ,103.Local. Allow healthcare professionals to make better informed clinical decisions to ensure better patient outcomes.National. Guide policy and ensure appropriate and timely public health interventions.Global. Provide early warnings of emerging threats and data to identify and act on long-term trends.Therefore, integrated surveillance between human medicine, veterinary medicine, and the environment is of paramount importance to detect and track emerging threats over time ,104. AMRYet it has proven difficult to achieve these objectives on a global scale, and especially in LMICs, largely because current surveillance systems deliver data that are extremely variable in quality and quantity . NowadayHowever, an integration of different surveillance systems for animal and human health encompassing also environmental concerns is unfortunately still lacking among most of the countries: it has been clearly indicated that despite huge but often individual efforts, even the most relevant AMR action plans are partly deficient because they do not tackle all the potentially relevant pathways and drivers of AMR in the environment, and the science to inform policy is still lacking ; these gAMR is one of the most complex global health challenges today, and antibiotics are the foundation of modern medicine: not only the medical world, but also the planetary health rely on the possibility of treating infections: without safe and effective antibiotics, the “main building” of modern medicine itself will crumble, after decades of overuse and misuse in human medicine, animal health, agriculture, and dispersion into the environment . The One Health concept stresses the ecological relationships between human, animal, and environmental health : since A


Crown ethers are common building blocks in supramolecular chemistry and are frequently applied as cation sensors or as subunits in synthetic molecular machines. Developing switchable and specifically designed crown ethers enables the implementation of function into molecular assemblies. Seven tailor-made redox-active crown ethers incorporating tetrathiafulvalene (TTF) or naphthalene diimide (NDI) as redox-switchable building blocks are described with regard to their potential to form redox-switchable rotaxanes. A combination of isothermal titration calorimetry and voltammetric techniques reveals correlations between the binding energies and redox-switching properties of the corresponding pseudorotaxanes with secondary ammonium ions. For two different weakly coordinating anions, a surprising relation between the enthalpic and entropic binding contributions of the pseudorotaxanes was discovered. These findings were applied to the synthesis of an NDI-[2]rotaxane, which retains similar spectroelectrochemical properties compared to the corresponding free macrocycle. The detailed understanding of the thermodynamic and electrochemical properties of the tailor-made crown ethers lays the foundation for the construction of new types of molecular redox switches with emergent properties. Pedersen discovered crown ethers in 1967 while searching for multidentate ligands for the vanadyl group . He was 2+, and Ba2+ rotaxane formation  TTFC7 and exTTFC7 were designed in analogy to their crown-8 analogs  from BC7  rotaxane binding). Generally, the oxidation potentials are shifted to higher values, and the reduction potentials are shifted to lower values in pure DCE due to decreased charge stabilization.The electrochemical properties of the TTF and NDI-bearing macrocycles and pseudorotaxanes are summarized in E1/2ox1, TTF→TTF•+ and E1/2ox2, TTF•+→TTF2+). E1/2ox1 and E1/2ox2 of the free exTTFC8 and exTTFC7 crown ethers are anodically shifted compared to those of TTFC8 and TTFC7, in which the TTF units are directly incorporated into the crown ethers. This behavior is known also for the two redox-active TTF building blocks exTTF and tetramethylene-TTF, which are not part of a macrocycle rotaxanes and facilitates the synthesis of a [2]rotaxane. Due to the remote position of the NDI unit and the rigid linker, rotaxane formation does not have a significant impact on the optoelectronic properties of the NDI moiety. The electrostatic interaction observed for NDI2− and ammonium ions observed in the pseudo[2]rotaxane A1·PF6@NDIC8, which even results in dethreading of the pseudorotaxane, may give rise to new switching modes in more complex molecular structures.On the contrary, Overall, these findings demonstrate the benefit of detailed and systematic studies on the noncovalent interactions cohering the components of switchable MIMs for the construction of new architectures with emergent properties.File 1Experimental section, including synthetic procedures, copies of NMR spectra, ITC, electrochemical, mass spectrometric and spectroelectrochemical data.File 2exTTFC7, NDIC7, and NDIC8.Crystallographic data (cif) for


Duplication of the superficial femoral artery (SFA) is an extremely rare anatomic variation, with few case studies reported. We report one case of the duplicated SFA, discovered by both ultrasonography (US) and computed tomography angiography (CTA). We also reviewed literatures concerning 6 cases of the duplicated SFA (including our present case), and summarized the clinical and imaging features of the anatomic variation.A 55-year-old woman presented to our hospital with an intermittent cramp in the lateral aspect of the right leg. The patient underwent Doppler US examination on bilateral lower extremity arteries and veins to examine potential vascular abnormality. Incidentally, US discovered the duplicated left SFA and CTA of bilateral lower extremities revealed the anatomic orientation, course, length, diameter and distance of the duplicated left SFA. It was revealed to be divided into two trunks with similar luminal diameter and courses parallel. They reunited at distal thigh level. The findings of US and CTA examination did not correspond with the symptom of the patient, and the patient was discharged.We report a rare case of the duplicated SFA diagnosed with the combinations of US and CTA examination, which served as valuable imaging methods to detect and diagnose the vascular anatomic variation in lower extremities. The precise knowledge of vascular anatomy and its variations is crucial as the treatment with endovascular techniques has increased. Variations of the femoral artery are rarely reported, especially for the superficial femoral artery (SFA) –3. Few cA 55-year-old woman presented to our hospital with nonspecific knee pain in the lateral aspect of the right leg. She had no symptoms on the left leg. She had hypertension, diabetes mellitus, and dyslipidemia, and she was on a treatment for intervertebral disc herniation in another hospital. Her physical examination was nonspecific, and the right and left ankle-brachial pressure index was 1.06 and 0.88, respectively.The patient underwent Doppler US examination on bilateral lower extremity arteries and veins to examine potential vascular abnormality. There was no abnormality in vessels of the right side. The left SFA was revealed to be divided into two trunks with similar luminal diameter and courses parallel Fig. . They reFor further evaluation, CTA of bilateral lower extremities was performed. The left SFA appeared to originate from left common femoral artery at the same level of the contralateral side. It appeared to run as a single vessel, 4 cm long, then split into two branches, medial and lateral ones. The luminal diameter of the medial one of the SFA was 5.3 mm, whereas that of lateral one was 4.4 mm, measured in each proximal portion Fig. . Both thVariations of the femoral artery are rarely reported, including aplasia and hypoplasia of the SFA with associated persistent sciatic artery, duplication or hypoplasia of the deep femoral artery, trifurcation of femoral artery, and duplication of the SFA , 2. To dA literature search on PubMed database was performed to review the previous reports concerning the duplicated SFA. The detailed analysis is summarized on Table Lower extremity arterial embryology begins at 6-mm human embryo. The sciatic artery develops from the umbilical artery and provides the major blood supply to the lower limb bud , 8, 12.Between the 6–and 33-mm stage of embryo development, the iliac artery system is expanded to femoral artery system, which allows the development of lower limbs during sciatic artery regresses . On the In previous literature, only one report describes a case of the duplicated SFA diagnosed with US . AlthougIn summary, we report an extremely rare case of the duplicated SFA diagnosed with the combination of US and CTA examination.


Alzheimer’s disease (AD) is the most common form of dementia and results in progressive neurodegeneration. The incidence rate of AD is increasing, creating a major public health issue. AD is characterized by neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein and senile plaques composed of amyloid-β (Aβ). Currently, a definitive diagnosis of AD is accomplished post-mortem. Thus, the use of molecular probes that are able to selectively bind to NFTs or Aβ can be valuable tools for the accurate and early diagnosis of AD. The aim of this review is to summarize and highlight fluorinated molecular probes that can be used for molecular imaging to detect either NFTs or Aβ. Specifically, fluorinated molecular probes used in conjunction with  Alzheimer’s disease (AD) is the most common form of dementia and is characterized by a progressive loss of cognitive function. It accounts for an estimated 60% to 80% of all cases of dementia . As of nThe use of molecular probes to image NFTs and Aβ plaques has mainly focused on the use of positron emission tomography (PET) . This meMagnetic resonance imaging (MRI) is another medical imaging modality used for studying AD. MRI has been used to measure water diffusion changes in the brain, AD-associated changes in brain perfusions, structural changes, and brain atrophy and study the effects of AD on the functional connectivity network of the brain ,11,12. DAnother upcoming technique used for molecular imaging is fluorescence imaging with near-infrared light. The fluorescence emission of each probe used should be within 650 and 900 nm to achieve an adequate penetration depth and high sensitivity. The advantage of optical imaging includes the low cost, real-time imaging with the option of multitargets, wide availability, and no radioactivity . NotwithThe deposition of amyloid-β (Aβ) plaques within the brain has been considered the initiating event in the development of Alzheimer’s disease (AD) and occurs before clinical symptoms emerge ,17. ThusTrifluoromethoxy-benzylated compounds such as 2-(4’-dimethylaminostyryl)-6-(2-([3’-trifluoromethoxy](benzylamino)ethoxyethoxy)benzoxazole (TFMB-2Et) and 2-(4’-dimethylaminostyryl)-6-(2-(3-trifluoro-methoxy)benzylaminoethoxyethoxyethoxy)benzoxazole (TFMB-3Et) have been investigated as candidate probes for amyloid imaging in vivo and have stronger 19F signals than FSB ..27].p-(trifluoromethyl)phenyl)-1,3-benzothiazol for the detection of AD using ex vivo brain tissue at 3T MRI has been demonstrated -2-naphthyl)ethylidene)malononitrile ((18F)FDDNP). The probe crossed the blood-brain barrier in proportion to the blood flow and exhibited a greater accumulation and slower clearance from NFT and Aβ-dense brain regions . In vitrositions . Furtherositions . The majositions . This trSince then, newer 18F-labeled radiopharmaceuticals such as (18F)florbetapir , (18F)florbetaben , and (18F)flutemetamol  have been synthesized ,50,51,52Lastly, (18F)flutemetamol was FDA approved in 2013 and is an 18F derivative of PIB, thus demonstrating similar kinetic properties . PET scaAdditional compounds used for Aβ imaging include 11C-labeled 5-(6-([tert-butyl(dimethyl)silyl]oxy)-1,3-benzothiazol-2-yl)pyridin-2-amine (11C-AZD2184), which has a high affinity for amyloid fibrils in vitro (Kd = 8.4 ± 1.0 nM). In human studies, a high binding of 11C-AZD2184 was observed in the cerebral cortical regions of AD patients in comparison with the control subjects . All molN-methyl benzenamine  and lansoprazole for potential tau radiotracers . . 104]. Additional BODIPY-based fluorescent probes include BD-tau and have been used to visualize tau aggregates in live cells. It was found that BD-tau was permeable to tau-induced cells and showed a 2.2-fold fluorescent increase upon interaction. Additionally, it was able to visualize tau aggregates in live cells but not under fixed-cell conditions. BD-tau was also able to detect filamentous tau aggregates in brain tissues, suggesting its use as a fluorescent probe in vivo  105]. S. S105]. All fluorinated molecular probes used for fluorescence tau imaging are demonstrated in Molecular imaging of the Aβ deposition and NFTs is crucial for the early detection and treatment monitoring of AD. Although the number of proposed fluorinated molecular probes is large, most of them have not reached the clinical translation stage. To facilitate future transitions into the clinic, molecular probe parameter optimizations are necessary. 19F or 18F, respectively. In order to develop fluorescent molecular imaging probes, molecules have to contain an electron-donating group linked to an electron-withdrawing group by a highly polarized conjugated π-electron chain. Alternating donor and acceptor groups can be used to control the relative energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). This leads to a smaller HOMO-LUMO gap and the desired long-wavelength absorption/emission bands [Molecular probes designed to bind to Aβ and hyperphosphorylated tau protein are small molecules comprised of at least two aromatic rings and a linker that may also serve to modify the probe’s affinity. For example, the vast majority of fluorinated molecular probes developed for AD detection contain either a methylamine or dimethylamine group that binds to π-conjugated structures, yielding a nanomolar affinity ,106,107.on bands ,87. TurnThe particular challenge that should be considered before synthesizing a novel fluorinated molecular imaging probe is the potentially high lipophilicity of the desired probe, which can limit the administrating concentrations. Thus, conjugating a hydrophilic group to improve the blood solubility should be considered. 17–21 and Aβ30–40 allow for hydrophobic and aromatic interactions with potential molecular probes. Thus, the aromatic core plays an important role in binding to Aβ [Two significant hydrophobic-binding sites located in Aβng to Aβ . Since Ang to Aβ . 19F nuclei per molecule, which yields a low MRI signal and low image resolution. To eliminate this issue, increasing the amount of magnetically equivalent fluorine atoms per molecule is needed. This can be done either through attaching additional CF3 groups to the probe or through conjugating the probe to perfluorinated macromolecules, which are currently used to track the cell activity in vivo using MRI [Taking a close look at the affinity of all the studied fluorinated molecular probes, it can be noted that a vast majority have an association constant on the order of nM. A future development includes improving the probe’s association to the targeted protein structure. This approach involves precise biochemical calculations and modeling of the probe–target interactions. However, this approach is less beneficial for PET than MRI and fluorescence imaging, since the sensitivity of PET is extremely high, with a low spatial resolution. On the other hand, the MRI signal is directly proportional to the number of molecular probes at the disease site, and, therefore, a linear increase in the MRI signal with the probe’s affinity is expected. In addition, an increase of the probe affinity to their target decreases the background signal due to nonselective binding. Therefore, the signal-to-noise ratio of the MRI image is expected to increase even more. Currently, the major drawback of fluorinated MRI probes is the small number of sing MRI ,111. A h18F-labeled PET molecular probes since PET imaging agents are highly developed, and the low resolution of PET images is limited by the positron traveling range. At the current stage of development, molecular imaging probes with low nonspecific binding should be preferred over other imaging probes. MRI imaging of fluorinated molecular probes can be improved by developing a dedicated phased-array brain coil that can substantially increase the SNR of an MRI image [The other approach that potentially helps to improve the quality of the imaging of fluorinated AD molecular probes is the technical improvement of the signal detection systems. For example, the transition from whole-body PET to organ-specific PET can be beneficial by building a dedicated PET detector for brain imaging . In addiRI image . In addiRI image . These tSubstantial improvement in the field of AD fluorescence imaging can be achieved by developing a probe with a large Stokes shift and high quantum yield. A large Stokes shift means the emission of the photons with a longer wavelength yielding to a better tissue penetration and reducing the fluorescence background.19F atoms per molecule, in addition to a high affinity and low nonselective binding. To make a proper decision about choosing a molecular probe for fluorescence imaging, molecules with the highest quantum yield and Stokes shift should be suggested. In addition, the changing of the fluorescence properties of the probe once bonded to the target can be a useful ability. Considering the desired properties of the molecular imaging probes, we can suggest the following block scheme (Depending on the imaging modality used, the desired properties of the molecular probe can be determined. For example, PET imaging probes should have a high affinity to the targeted protein but have a low nonspecific binding to decrease the background signal. MRI probes should contain a high number of k scheme  of develOverall, the early detection of AD with fluorinated molecular probes is an extensively developing field that has the potential to be translated into clinical practice. The successful clinical translation of fluorinated molecular probes can help increase the understanding of AD development, Aβ deposition, and NFT formation. These molecular probes can potentially help monitor AD treatment and, therefore, might be useful tools for AD drug development. In addition, these probes can facilitate early-stage AD detection, which makes them a valuable AD diagnostics instrument.