Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder forming hamartomas throughout the body. Facial angiofibromas (FAs) occur in 75% of TSC patients, which are often enlarged, impairing the appearance of the face, and reducing the patient’s quality of life (QOL). The aim of this study was to characterize the impact of topical sirolimus treatment on the health-related QOL in patients with FA associated with TSC.We investigated a total of 33 patients who received sirolimus gel treatment for FA associated with TSC and assessed the changes in the health-related QOL using the Medical Outcomes Study 36-Item Short Form (SF-36) Health Survey. SF-36 surveys were performed before and after 3 months of treatment. The conditions of the patients after using the sirolimus gel were categorized into the following three categories: “improved,” “unchanged,” and “aggravated.” Adverse events were investigated using the CTCAE v5.0-JCOG.The median age of the patients was 25 (range 14–55) years. After 3 months of sirolimus gel treatment, three scale scores of the SF-36, vitality (VT), social function (SF), and mental health (MH), were significantly improved compared to before the treatment. The VT and SF in patients who had improved FA were significantly better than those in the other patients. There were no significant differences in any scale scores between patients with and without adverse events at 3 months after the initiation of sirolimus gel treatment.This is the first report regarding improved health-related quality of life in patients treated with sirolimus gel for FA associated with TSC by using the SF-36. The three scale scores associated with mental health were significantly improved compared to before the treatment. The health-related QOL in patients receiving sirolimus gel treatment is more strongly affected by the treatment efficacy than adverse events.Sirolimus gel treatment improves the health-related QOL in patients with FA associated with TSC. TSC1 gene on chromosome 9 or the TSC2 gene on chromosome 16, which encode hamartin and tuberin, respectively [Tuberous sclerosis complex (TSC) is a rare autosomal dominant inherited disorder characterized by systemic hamartomas and hypomelanotic macules . It is cectively . Dysfuncectively . Most syWataya-Kaneda et al. reported phase 2 and 3 randomized clinical trials of topical sirolimus treatment with a sirolimus gel formulation in TSC patients and showed significant reductions in the size and color of the FAs associated with TSC , 6. In JIn the present study, we evaluated the changes in the health-related QOL of patients receiving sirolimus gel treatment for FA associated with TSC, using the Medical Outcomes Study 36-Item Short Form (SF-36) Health Survey, one of the most widely used surveys for health-related QOL .Ⓡ gel; Nobelpharma, Tokyo, Japan). The sirolimus gel contained 0.2% sirolimus and additives including alcohol. Each patient was instructed to spread 400 mg of the sirolimus gel evenly on the FA site twice a day. The severity of FA was assessed using the Facial Angiofibroma Severity Index [FA associated with TSC was diagnosed based on the International Tuberous Sclerosis Complex Consensus Conference (ITSCCC) diagnostic criteria after consultations with an internist and dermatologist. A total of 33 patients met the diagnostic criteria and received topical sirolimus  were significantly improved compared to before treatment Fig. . For theWe then assessed the influences of the therapeutic efficacy and adverse events of sirolimus gel treatment on the health-related QOL. Figure TSC induces the development of various hamartomas throughout the body, including angiomyolipomas and angiofibromas. The mTOR signaling pathway has been reported to be activated in these tumors . SirolimTo date, several studies have reported the influence of topical mTOR inhibitors treatment on patients’ QOL. Wataya-Kaneda et al. assessed the QOL improvement of patients with sirolimus gel treatment for FA associated with TSC using the Dermatology Life Quality Index (DLQI) and Children’s DLQI (CDLQI). They reported that the total scores on the DLQI and CDLQI after 12 weeks of treatment did not differ markedly between the sirolimus gel group and the placebo group . SimilarIn this study, we used the SF-36 health survey to prospectively assess the changes in the physical and mental health of Japanese patients who had FA associated with TSC and were treated with sirolimus gel. The three scale scores associated with mental health, VT, SF, and MH, were significantly improved compared to before the treatment.FAs influence the mental status of TSC patients and their families . Since FWe further assessed the treatment efficacy and adverse events. It has been recognized that these two major factors are directly associated with patient satisfaction and health-related QOL during treatment. Patients in whom FA improved had significantly higher mental health scale scores for VT and SF than those whose condition did not improve. Furthermore, there was no marked influence of adverse events related to sirolimus gel treatment on the health-related QOL. These findings suggested that mental health became better in patients whose FA improved after administration of sirolimus gel. In this study, the adverse events with sirolimus gel treatment were tolerable, with only low-grade severity.However, our study had several limitations. First, several surveys for the evaluation of health-related QOL have been developed, including as the Sickness Impact Profile (SIP) and the Functional Assessment of Cancer Therapy-General (FACT-G) , 16. In We evaluated the changes in the health-related QOL in patients with sirolimus gel treatment for FA associated with TSC by using the SF-36. Three scale scores associated with mental health, VT, SF, and MH, were significantly improved compared to before the treatment. Furthermore, improvements of the VT and SF scores were observed in the improved group compared with the unchanged group. However, further investigations are necessary to achieve a better understanding of the efficacy and safety of sirolimus gel treatment for FA associated with TSC.


Background: The newly identified coronavirus known as 2019-nCoV has posed a serious global health threat. According to the latest report (18-February-2020), it has infected more than 72,000 people globally and led to deaths of more than 1,016 people in China.Methods: The 2019 novel coronavirus proteome was aligned to a curated database of viral immunogenic peptides. The immunogenicity of detected peptides and their binding potential to HLA alleles was predicted by immunogenicity predictive models and NetMHCpan 4.0.Results: We reportin silico identification of a comprehensive list of immunogenic peptides that can be used as potential targets for 2019 novel coronavirus (2019-nCoV) vaccine development. First, we found 28 nCoV peptides identical to Severe acute respiratory syndrome-related coronavirus (SARS CoV) that have previously been characterized immunogenic by T cell assays. Second, we identified 48 nCoV peptides having a high degree of similarity with immunogenic peptides deposited in The Immune Epitope Database (IEDB). Lastly, we conducted ade novo search of 2019-nCoV 9-mer peptides that i) bind to common HLA alleles in Chinese and European population and ii) have T Cell Receptor (TCR) recognition potential by positional weight matrices and a recently developed immunogenicity algorithm, iPred, and identified in total 63 peptides with a high immunogenicity potential.Conclusions: Given the limited time and resources to develop vaccine and treatments for 2019-nCoV, our work provides a shortlist of candidates for experimental validation and thus can accelerate development pipeline. It has further challenged the scientific and industrial community for quick control practices, and equally importantly to develop effective vaccines to prevent its recurrence. In facing a rapid epidemical outbreak to a novel and unknown pathogen, a key bottleneck for a proper and deep investigation, which is fundamental for vaccine development, is the limited -- to almost no -- access of the scientific community to samples from infected subjects. As such,in silico predictions of targets for vaccines are of high importance and can serve as a guidance to medical and experimental experts for the best and timely use of the limited resources.The emergence and rapid spread of the recent novel coronavirus known as 2019-nCoV has posed a serious global health threatde novo prediction from 2019-nCoV 9-mer peptides. Here, we found i) 28 SARS-derived peptides having exact matches in 2019-nCoV proteome previously characterized to be immunogenic byin vitro T cell assays, ii) 22 nCoV peptides having a high sequence similarity with immunogenic peptides but with a greater predicted immunogenicity score, and iii) 44 + 19 nCoV peptides predicted to be immunogenic by the iPred algorithm and 1G4 TCR positional weight matrices respectively.In this regard, we report our recent effort to computationally identify immunogenic and/or cross-reactive peptides from 2019-nCoV. We provide a detailed screen of candidate peptides based on comparison with immunogenic peptides deposited in the Immune Epitope Database and Analysis Resource (IEDB) database including those derived from Severe acute respiratory syndrome-related coronavirus (SARS CoV) along with4). These peptides have been shown to bind various HLA alleles, although with higher tendency towards HLA-A:02:01, from both class I and class II, and can be target for CD8+ and CD4+ T cells respectively.We collected all peptides in IEDB .In order to investigate the extent to which the difference between the source (2019-nCoV) and target (IEDB) peptides influences the immunogenicity of the source peptides we used a recently published immunogenicity modelWe could see a similar  immunogenicity scores for a number of IEDB and 2019-nCov peptides especially for those with high immunogenicity scores . While aIt is worth noting that in general predicting immunogenicity of given a peptide is challenging and not a fully solved problem, and therefore current models for predicting immunogenicity are suboptimal. iPred is also not an exception. In fact, we could see that a substantial number of IEDB immunogenic peptides were scored < 0.5 (the threshold score used to classify immunogenic vs non-immunogenic). This led us to ask whether we can gather any other evidence of either immunogenicity or cross-reactivity.de novo search of immunogenic peptides against the 2019-nCov proteome sequence. We scanned 9-mers from 2019-nCoV proteome with a window of 9 amino acids and step length of 1 amino acid . The immunogenicity of 9-mer peptides were predicted using iPred and MHC presentation scores were gauged using NetMHCpan 4.06 for various HLA types. In this task, we focused on haplotypes common in Chinese and European populations, which include HLA-A*02:01, HLA-A*01:01, HLA-B*07:02, HLA-B*40:01 and HLA-C*07:02 alleles (Data Table 34).As a complementary reciprocal approach, we conducted aBased on MHC presentation and immunogenicity prediction, we detected 5 peptides predicted to bind 4 different HLA alleles of which 2 had strong immunogenicity scores . For thode novo candidates are appealing shortlisted targets for experimental validation, it does not provide information about target T cell receptors (TCRs). We therefore set out to interrogate the possibility of cross reactivity with one well-studied TCR.While our7 and exploit the possibility of cross reactivity of this TCR to any 2019-nCoV peptide.T cell cross-reactivity has been instrumental for the T cell immunity against both tumor antigens and external pathogens. In that regard, a number of T cells have been extensively characterized including 1G4 CD8+ TCR, which is known to recognize the ‘SLLMWITQC’ peptide presented by HLA-A*02:01. We therefore set out to leverage the data from a recently published study4). The distributions of binding, activation and killing scores along with their multiplicative score and geometric mean are illustrated inHere, we scanned all 9-mers from the 2019-nCoV proteome (9613 peptides) with Binding, Activating and Killing Position Weight Matrices  and associated each peptide with the geometric mean of these three assays as a measure of immunogenicity Data Table 44. The d8. Looking at the LV peptide, we identified 44 2019-nCoV peptides of which 2 peptides had immunogenicity score > 0.7 and 12 peptides > 0.6  and valine (V) in anchor positions 2 (P2) and 9 (P9) respectively. Previous studies have shown that for MHC-I HLA-A02:01 specific peptides, 9-mer peptides with leucine at P2 and valine at P9 are preferably presented on the surface of HLA-A02:01s > 0.6 . Thus, hIn this study we provide a profile of computationally predicted immunogenic peptides from 2019-nCoV for functional validation and potential vaccine developments. We are fully aware that an effective vaccine development will require a very thorough investigation of immune correlates to 2019-nCoV. However, due to the emergency and severity of the outbreak as well as the lack of access to samples from infected subjects, such approaches would not serve the urgency. Therefore, computational prediction is instrumental for guiding biologists towards a quick and cost-effective solution to prevent the spread and ultimately help eliminate the infection from the individuals.9. Ourin silico approach takes the search beyond presenting only common immunogenic peptide between SARS and 2019-nCoV and provides the experimental community with a more comprehensive list includingde novo and cross reactive candidates. On the other hand, considering the fact that two studies have been accomplished independently with distinct approaches, this serves to demonstrate a high level of confidence in reproducing the results. Reproducibility of computational prediction is always of high importance and becomes even more significant under urgent scenarios as of this outbreak.With a rising global concern of novel coronavirus outbreak, numerous research groups have started to investigate and publish their findings. At the time of preparing this manuscript, we became aware of a similar study conducted in comparing 2019-nCoV proteome with SARS CoV immunogenic peptidesOur study also suggests the need for further efforts to develop accurate predictive models and algorithms for the characterization of immunogenic peptides.In this study, we provide potential immunogenic peptides from 2019-nCoV for vaccine targets that i) have been characterized immunogenic by previous studies on SARS CoV, ii) have high degree of similarity with immunogenic SARS CoV peptides and iii) are predicted immunogenic by combination of NetMHCpan and iPred/1G4 TCR positional weight matrices. Given the limited time and resources, our work serves as a guide to save time and cost for further experimental validation.MN908947.3). All sequences subjected for analysis are deposited in GitHub repository.2019-nCoV open reading frame sequences were downloaded from NCBI .We then scanned 2019-nCoV virus protein sequence with each of B, A and K PWMs and associated each of 9613 9-mers with a cross reactivity score. At the same we utilized NetMHCpan and associated each 9-mer with its presentation score. Our final list of cross-reactive candidate peptides were those with a cross-reactivity sore >= 0.8 and reported as strong binders from NetMHCpan and have ‘L’ and ‘V’ amino acids at anchor positions. The custom R codes are accessible from GitHub repository .2019-nCoV open reading frame sequences were downloaded from NCBI (In silico identification of vaccine targets for 2019-nCoV (Data tables).http://doi.org/10.5281/zenodo.367688610Zenodo:– Table1 nCoV peptides having exact match with immunogenic SARS CoV peptides.xlsx (Table of nCoV peptides having exact match with immunogenic SARS CoV peptides)– Table2 nCoV peptides with high sequence similarity with immunogenic IEDB peptides.csv (Table of peptides with high sequence similarity with immunogenic IEDB peptides)– de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and iPred.csv (Table of results ofde novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and iPred)Table3– de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and PWM.xlsx (Table of results ofde novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and PWM)Table4This project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the in silico prediction approach. Overall, they present 28 2019-nCoV-derived peptides that exactly match epitopes of SARS-CoV, and additional 48 2019-nCoV peptides that overlap with already known immunogenic epitopes reported in IEDB. The latter have been evaluated using immunogenicity prediction resulting in 22 2019-nCoV peptides with higher immunogenicity scores than their IEDB counterpart. Additional analysis of encoded 2019-nCoV peptides using HLA binding likelihood and immunogenicity scores predicted by NetMHCpan 4.0 and iPred, respectively, showed 13 additional vaccine candidates regarding prevalent European and Asian HLA-types. Finally, the authors modeled possible cross-reactivity of a well described HLA-A*02:01-NY-ESO-1-peptide-reactive TCR to 2019-nCoV peptides with predicted HLA binding affinity and found 19 hits. With this, authors provide a shortlist of potential candidates for development of a 2019-nCoV vaccine applicable to a broad population.The authors present a list of potential 2019-nCoV vaccine candidates based on an The paper would benefit from clearer labelling of the figures and tables, and more extensive description of shown results in the figure legends. Column titles should be consistent among tables and figures, and ideally not consist of the internally used abbreviations but be self-explanatory. In Figure 1A, the axis labeling (Counts and Alignment Score) seems to be switched. 1B: Adding a note that these are non-exact peptides with alignment score >=4 would be helpful. In Figure 2, 45 peptides are shown, but 48 peptides are mentioned in the text. Please clarify. General remarks: It would be better to use “encoded proteins” than the term “proteome”, as the data derives from 10 ORF sequences. The authors should indicate in the methods the employed thresholds of the used predictors to label a peptide a binder or immunogenic. These thresholds could also be shown in the figures where appropriate. As iPred only uses MHC binding likelihood and amino acid qualities to predict peptide presentation which is not very different from general NetMHCpan approach , the authors should discuss why they used iPred instead of well described tools such as NetTepi or NetCTL and NetCTLpan. It does also not come across why an anti-NY-ESO-1 TCR should be cross-reactive to any 2019-nCoV peptide?Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Therapeutic cancer vaccination; T cell epitope prediction,  identification and validation; ImmunopeptidomicsWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. in silico approach to identify antigenic peptides derived from 2019-nCoV. First, they screened ten open reading frame of 2019-nCoV sequence used the IEDB database, finding a series of peptides potentially immunogenic. The first piece of data relies on the identification of 28 peptides that were exactly matching SARS CoV peptides. Moreover, using combinatory approaches, modelling HLA and TCR binding, they identified 13 peptides potentially able to bind a given TCR in HLA A2 restriction fashion, and cutting a list of peptides able to bind several HLA alleles and characterizing several peptides that may be de novo candidates or crossreactive peptides to be used either to study immune response to 2019-nCoV or to set a vaccine.The Authors use anIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:T cell responses in Health and Diseases.  Modulation of Immune Responses. Clinical Immunology.  VaccinesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is an important manuscript identifying potential vaccine targets for 2019-nCoV, using computational prediction. In the absence of patient samples, such approaches are instrumental to guide towards quick and efficient identification of vaccine candidates. The authors used 10 open reading frame sequences of 2019-nCoV deposited at NCBI and conducted sequence alignment against immunogenic peptides deposited in the Immune Epitope Database and Analysis Resource (IEDB) database. They identified 28 peptides with sequences matching exactly to severe acute respiratory syndrome-related coronavirus (SARS CoV), that have previously been characterised as immunogenic by T cell assays. These findings are very promising and have the added benefit of potentially developing a vaccine against both SARS and COVID-19. Additional peptides were identified to most likely bind common Chinese and European HLA alleles and have high immunogenicity potential. The authors provide a shortlist of peptides as potential vaccine candidates. While this manuscript presents a good model for identifying such targets, a comment should be included in the discussion about the necessity of expanding the analysis to include wider HLA allele types, considering that the virus is likely to spread worldwide.‘Towards target peptide length > 3’.‘Normalized alignment scores’ .The importance of leucine (L) and valine (V) in anchor positions for MHC binding (for data presented in Table 5). Minor comment: Please explain the methods and analysis in greater details, including the following terms:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:T cell immunologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Prof Wilkinson, Thank you so much for your time and valuable suggestions. In our new and revised version we have taken your suggestions into consideration. Best regards Hashem Koohy


Mesenchymal stem cells (MSCs) are derived from multiple tissues, including amniotic fluid (AF-MSCs) and the umbilical cord (UC-MSCs). Although the therapeutic effect of MSCs on sepsis is already known, researchers have not determined whether the cells from different sources require different therapeutic schedules or exert different curative effects. We assessed the biofunction of the administration of AF-MSCs and UC-MSCs in rats with caecal ligation and puncture (CLP)-induced sepsis.CLP was used to establish a disease model of sepsis in rats, and intravenous tail vein administration of AF-MSCs and UC-MSCs was performed to treat sepsis at 6 h after CLP. Two phases of animal experiments were implemented using MSCs harvested in saline with or without filtration. The curative effect was measured by determining the survival rate. Further effects were assessed by measuring proinflammatory cytokine levels, the plasma coagulation index, tissue histology and the pathology of the lung, liver and kidney.We generated rats with medium-grade sepsis with a 30–40% survival rate to study the curative effects of AF-MSCs and UC-MSCs. MSCs reversed CLP-induced changes in proinflammatory cytokine levels and coagulation activation. MSCs ameliorated CLP-induced histological and pathological changes in the lung, liver and kidney. AF-MSCs and UC-MSCs functioned differently in different tissues; UC-MSCs performed well in reducing the upregulation of inflammatory cytokine levels in the lungs and inhibiting the inflammatory cell infiltration into the liver capsule, while AF-MSCs performed well in inhibiting cell death in the kidneys and reducing the plasma blood urea nitrogen (BUN) level, an indicator of renal function.Our studies suggest the safety and efficacy of AF-MSCs and UC-MSCs in the treatment of CLP-induced sepsis in rats and show that the cells potentially exert different curative effects on the main sepsis-affected tissues. Sepsis is defined as a systemic inflammatory response syndrome (SIRS) that is induced by an infection, leading to an imbalance in the adaptive and innate immune systems in an individual. Sepsis is a heterogeneous syndrome caused by different pathogens and different effect sites that collectively result in sustained excessive inflammation and immune suppression within individuals . In the clinicaltrials.gov. The safety and efficacy of MSCs should be explicitly determined before their large-scale clinical application, and the study of MSC biofunction in sepsis pathology would benefit from acquiring an advanced application scheme for the clinical setting.Mesenchymal stem cells (MSCs) are adult stem cells, so named because they were first derived from bone marrow . Over thIn this study, we provide evidence that intravenous tail vein injections of human amniotic fluid MSCs (AF-MSCs) and UC-MSCs are both therapeutic programmes that reduce mortality in rat models of caecal ligation and puncture (CLP)-induced sepsis. Notably, we documented that the curative effects of these MSCs included a reduction in the infection-induced increase in inflammatory cytokine levels, reductions in pathological tissue damage and cell apoptosis and the prevention of the functional deterioration of the tissues. The cellular therapeutic effects of AF-MSCs and UC-MSCs on rats with sepsis reported here provide a new perspective for the study of the biological effects of these MSCs on different tissues in a sepsis model.6 cells in 150 μl.The study was approved by the Academic Committee of the Third Affiliated Hospital of Guangzhou Medical University. Animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The rat CLP model was used as the disease model of sepsis, and intravenous tail vein administration of AF-MSCs and UC-MSCs was performed to treat sepsis at 6 h after CLP. We established two phases of animal experiments to determine the effects of MSCs with and without filtering through a 70-μm filter on a rat model of CLP-induced sepsis. The amounts of AF-MSCs and UC-MSCs administered were 1 × 10n = 3), sepsis control (n = 7), sepsis + AF-MSCs (n = 7) and sepsis + UC-MSCs (n = 7) groups. A statistical analysis of the mortality of the rats in each group over a 72-h period was performed.MSCs were harvested in saline without filtration. The following four groups were established in this phase: normal saline sham . Six surviving rats from each group were sacrificed at 24 h after CLP for physiological and biochemical analyses, and the remaining rats in each group were used for the survival analysis at 72 h. The levels of oedema in the lungs were analysed in 3 rats, leaving 3 rats for further lung analyses. The liver and kidney tissues of 5 rats were analysed.2. The cells were harvested and passaged with trypsin-EDTA  upon reaching 70–80% confluence.The derivation, maintenance and expansion of MSCs were performed using previously published protocols . BrieflyAdult (8–10 weeks) male Sprague-Dawley rats  were randomly assigned to the 4 study groups. Rats with medium-grade sepsis were produced by CLP using a protocol , with soSix rats from each group were sacrificed at 24 h, and three were randomly selected for an analysis of the wet and dry weights of the lung. The wet weight was determined by weighing the lung immediately following dissection, while the dry weight was determined by weighing the lung after drying in a 65 °C incubator for 2 days.Histological study and staining of organs from each study group were conducted, including the right lung, liver and kidney. The organs were fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin. Specimens were cut into paraffin sections for further assays. Haematoxylin–eosin (HE) staining  was performed to identify general histology. Masson staining  was carried out to label collagen in the connective tissue. Periodic acid–Schiff (PAS) staining  was utilized to identify glycogen accumulation within liver tissue. TdT-mediated dUTP Nick-End Labelling (TUNEL)  Biotech Co., Beijing, China) was used to label dead cells, according to the manufacturer’s instructions. All pictures were recorded by a microscope .The concentration of immune cytokines in the serum and bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA) kits purchased from CUSABIO BIOTECH  according to the manufacturer’s instructions, including IL-1β (Cat# CSB-E08055r), IL-6 (Cat# CSB-E04640r), IL-10 (Cat# CSB-E04595r) and TNF-α (Cat# CSB-E11987r). Coagulation-related biochemistry was assessed using commercial kits from the Instrumentation Laboratory  on an ACLTOP700 analyser  according to the manufacturer’s instructions; the reported parameters included prothrombin time (PT) , activated partial thromboplastin time (APTT)  and plasma fibrinogen (FIB) . Tissue function-associated biochemistry was assessed using commercial kits from Roche  on a COBAS C702 instrument according to the manufacturer’s instructions and included aspartate aminotransferase (AST) (Cat# 05850819190), alanine aminotransferase (ALT) (Cat# 05850797190), blood urea nitrogen (BUN) (Cat# 05171873190) and creatinine (Cat# 05168589190).P values < 0.05 were considered statistically significant.Quantitative results of experiments were expressed in a scatter plot, unpaired Mann-Whitney test were applied to compare the difference between two groups and Tukey’s test was used for comparisons among multiple groups. The statistical log-rank test (Mantel-Cox) was used to compare the survival data of the different groups. Adult Sprague-Dawley rats that underwent CLP were established as a sepsis model and treated with different MSCs resuspended in saline or saline alone 6 h after CLP surgery. Six surviving individuals from each group were sacrificed for histological and biochemical analyses at 24 h, and the survival rate of the remaining rats was determined at 72 h and a statistical analysis was performed Fig. a. We genSix rats from each group in the second round of the animal experiment were sacrificed for further biological analyses. For blood pathology, we assessed changes in the serum levels of proinflammatory cytokines and coagulation activation parameters. The serum concentrations of four typical inflammatory cytokines were measured, namely, IL-1β, IL-10, IL-6 and TNF-α Fig. a–d. CompCLP-induced sepsis involving the lung causes histological changes, induces oedema, increases cell death and upregulates the levels of proinflammatory cytokines. The administration of both types of MSCs attenuated these changes, and UC-MSCs were much more successful in reducing inflammatory cytokine expression than AF-MSCs. Sepsis-induced histological changes and the ability of MSCs to inhibit these changes in the lungs were assessed using HE Fig. a and MasCLP-induced sepsis causes histological and biochemical changes in the liver, which were equally attenuated by AF-MSC and UC-MSC administration. In the sepsis group, a few necrotic hepatocytes with an eosinophilic cytoplasm and a pyknotic nucleus, degenerating cells with considerably intact cell morphology and a degenerative area of cytoplasm weakly stained with HE were observed, while the histology of both of the MSC groups was more homogeneous and similar to the sham group, without any understained areas Fig. a. RegardCLP-induced sepsis causes histological changes, cell death and biochemical changes within the kidney. The administration of MSCs attenuated the sepsis-associated phenotype, and AF-MSCs were more effective than UC-MSCs. Histology of the healthy kidney revealed normal glomerular and tubular structures in the sham group. In the sepsis model, pathological changes in the glomeruli were not typically observed, but some histological changes in tubular structures were evident, including epithelial swelling, loss of the brush border of luminal cells and the appearance of vacuolated areas. Prominent improvements induced by MSC therapy included a reversal of the tubular swelling and the observation of a normal morphology of the endothelial cells Fig. a. Both MThe CLP rodent model is the most commonly used animal model in sepsis studies . After CSafe administration of MSCs can be determined by using certain therapeutic schedules. For the clinical usage of cytotherapy, the first requirement is safety, and some concerns have been noted regarding the administration of MSCs. Reports of brachial vein  and pulmBased on criteria from the ISCT , MSCs caThe curative effects of AF-MSCs and UC-MSCs on rats with CLP-induced sepsis differed. UC-MSCs are one of the most widely used cell types in cytotherapy, and their bioeffect on curing sepsis has been described in several studies , 30–32, The beneficial effect of AF-MSCs was probably related to the paracrine action of secreted growth factors . A WJ-MSIn summary, in addition to highlighting that different cell production processes may lead to diverse physicochemical properties, biological characters and therapeutic effects of MSCs, our studies have identified the safety and efficacy of AF-MSCs and UC-MSCs in the healing of rats with CLP-induced sepsis and their potential differences in ameliorating the effects of sepsis on different tissues. Further preclinical studies are needed to define the molecular variations in MSCs derived from different sources via different production schemes, to establish the relationship between the cell phenotype in vitro and the cytotherapeutic biofunctions of MSCs in vivo, to improve the curative effects of MSCs and to obtain a better understanding of the differences in the curative effects of MSCs from different tissue sources.


Aspergillus spp., is a concern for immunocompromised populations. Despite substantial research efforts, conventional treatments of PA using antifungal agents are associated with limitations such as excessive systemic exposure, serious side effects and limited availability of the therapeutics in the lungs for an adequate duration. To overcome the limitations associated with the conventional regimens, pulmonary delivery of antifungal agents has become a focal point of research because of the superiority of local and targeted drug delivery. Dry powder inhalers and nebulized formulations of antifungal agents have been developed and evaluated for their capability to effectively deliver antifungal agents to the lungs. Moreover, progress in nanotechnology and the utilization of nanocarriers in the development of pulmonary delivery formulations has allowed further augmentation of treatment capability and efficiency. Thus, the following review provides an insight into the advantages and therapeutic potential of the utilization of nanocarriers in pulmonary delivery of antifungal agents for the treatment of PA. In addition, discussions on formulation aspects and safety concerns together with the clinical and regulatory aspects of the formulations are presented, which suggest the possibility and desirability of utilization of nanocarriers in the treatment of PA.Pulmonary aspergillosis (PA), a pulmonary fungal infection caused by  Mycoplasma pneumoniae and Staphylococcus aureus, while chronic infectious bronchiolitis is mostly caused by both bacteria and mycobacteria  (ammonium slat) (DSPE-mPEG2000). The developed formulations (PEG-LN AmB) had smaller mean particle size (< 100 nm) and high zeta potential (−41.3 ± 5 mV to −50.4 ± 5 mV) which indicated good long-term stability. The smaller particle size and high zeta potential were due to the presence of anionic lipid, DPPA and DSPE, which induced strong repulsive forces between the LNs. Besides, DPPC and DPPA had strong hydrophobic interaction with AmB, which resulted in the high EE of the PEG-LN AmB formulation in addition to the strong interactions of AmB with CHOL. Pharmacokinetics study in Sprague-Dawley rats suggested prolonged circulation half-life of PEG-LN AmB with half-life of 5.591 h as compared with Fungizone® (3.825 h) and AmBisome® (3.673 h). PEG-LN AmB demonstrated an almost 2-fold higher AUC0–24h (19.448 µg.h/mL) than AmBisome® (10.204 µg.h/mL) and 19 times higher AUC0–24h than Fungizone® (1.084 µg.h/mL). This prolonged half-life in the bloodstream was due to the formation of hydrodynamic layer on the LNs by DSPE-mPEG2000. which might inhibit RES uptake and opsonization of the LNs. The in vitro antifungal activity of the PEG-LN AmB against C. albicans and A. fumigatus was better than Fungizone® and AmBisome® where the MIC of PEG-LN AmB against C. albicans and A. fumigatus were 0.125 µg/mL and 0.25 µg/mL when compared to Fungizone  and AmBisome® . This might be due to the higher affinity of AmB incorporated into the LNs to ergosterol in fungal cell membrane than to CHOL in LNs. The in vivo antifungal efficacy of PEG-LN AmB tested in ICR mice was superior than AmBisome® with higher mean survival times which might be attributed to the effect of PEG molecules on pharmacokinetic and toxicological behaviour of AmB [A study by Jung and team developed an AmB-entrapping lipid nanoparticles (LN) IV formulation with 1,2-dipalmitoyl-r of AmB .l-a-phosphatidylcholine (eggPC) and DSPE-mPEG2000. The developed NLC IV formulations have particle size in nanometer range (192.7 ± 14.4 nm to 241.9 ± 13.5 nm) and high incorporation efficiency (83.0 ± 1.3% to 95.2 ± 1.7%). The high solid lipid concentration serves as immobilization capacity for incorporation of drug into the lipid core while the liquid lipid weakened the crystallinity of the lipid core during lyophilization. The in vitro release study suggested that the release rate of ITZ from the NLC formulations could be controlled by modulation of the amount of liquid lipid in lipid core. This is due to the less mobility of lipid core as compared to liquid-solid LN. As proportion of liquid lipid increases in the formulations, the cumulative drug release of the formulations increased which could be due to faster drug release induced by the weak crystallinity of liquid-solid LN. The pharmacokinetics study in male Sprague-Dawley rats suggested sustained release of ITZ from the NLC formulation with less amount of liquid lipid as LN with high amount of liquid lipid will be degraded rapidly by enzymes in the blood circulation and were cleared in the blood faster. The sustained release of ITZ-NLC was due to higher solid lipid proportion in the formulation as well as the presence of steric stabilizer, DSPE-mPEG2000 used in the formulation. In addition to prolonging of the circulation time of NLC in bloodstream, reduction of gel formation during preparation and storage due to steric hindrance induced by DSPE-mPEG2000 also increased the stability of the formulation [2000 using hot high-pressure homogenization method has developed with a drug high payload. The ITZ-loaded NLC formulation has high incorporation efficiency (89.6%), loading capacity (18.3%) as well as particle size of 285.9±14.8 nm and PDI of 0.195 ± 0.015. The high DL capacity was due to the formation of disordered of crystal lattice by the addition of small amount of liquid lipid in lipid core, which created larger space for accommodation of ITZ. Besides, addition of liquid lipid into the lipid core also slows down the recrystallization of solid lipid. This reduced crystallinity in lipid core results in reduced drug expulsion during storage [® IV was evident in male Sprague-Dawley rats, whoch might be due to the presence of PEG as stearic stabilizer preventing the rapid clearance of NLC by RES through avoidance of protein adsorption and subsequent opsonization [To overcome the limitations associated with SLN, NLC, which is the second generation SLN was utilized in the delivery of antifungal agents. In a study, ITZ-loaded NLC IV formulations were developed with tristearin as solid lipid, triolein (TO) as liquid lipid, Tween 80, egg yolk mulation . To furt storage . The res storage . High ITnization .Other intravenous approaches against lung infectionsThe particle size of an IV formulation may influence its lung-targeted drug delivery. An IV formulation of AmB was prepared with sodium deoxycholate (DCH) (AmB-DCH), which had a mean particle size of 404.9 ± 1.7 nm. Based on the in vivo drug distribution study in Oncins France 1 male mice, the AmB-DCH formulation has significantly higher drug concentration in lungs as compared to liposomal AmB formulation (9.173 ± 0.498 µg/g versus 2.527 ± 0.386 µg/g) while the drug concentration in kidney of AmB-DCH was 15-fold lower than the liposomal AmB formulation. This suggested better lung-targeted delivery of drug and furthermore, less nephrotoxicity of the AmB-DCH, which might be due to the presence of AmB poly-aggregated form, which is a small size micellar system induced by the surfactant action of DCH and stabilized by the ionic interaction between DCH and AmB. The AmB poly-aggregated form with higher particle size (404.9 ± 1.7 nm) showed greater lung distribution as compared to commercial liposomal AmB (around 100 nm) which showed high renal distribution suggesting influence of particle size on the distribution of AmB to different organs. The higher particle size of AmB-DCH allow faster opsonization of the drug by alveolar macrophages, resulting in higher drug distribution in lungs which would be adequate to treat aspergillosis (MIC 0.5–8 µg/mL) Polymeric drug delivery system for intravenous treatment of lung infectionsAntifungals incorporated in polymeric nanocarriers for IV route administration have also been explored by researchers worldwide. Owing to the biocompatible and biodegradable properties in drug delivery, many researchers have developed polymeric nanoparticles by using a diblock copolymer, namely D-α-tocopheryl polyethylene glycol 1000 succinate-β-poly(ε-caprolactone-ran- glycolide) (PLGA-TPGS). Tang et al. had developed AmB loaded PLGA-TPGS nanoparticles (AmB-NP) using a modified nanoprecipitation method to overcome the limitations of AmB in the treatment of pulmonary fungal infections. The AmB-NP had a mean hydrodynamic diameter between 120 nm to 130 nm, which was claimed effective in cellular uptake and internalization. The PDI of AmB-NP was around 0.2 indicating uniform particles size distribution ,103. DL C. albicans which were treated with AmB-NP intravenously had lower fungal burden and higher survival rates compared to the C. albicans-infected BALB/c mice treated with free AmB. In addition, the authors found an improvement on the lung lesions of the C. albicans infected mice that treated with AmB-NP compared to the infected and untreated BALB/c mice as well as the C. albicans-infected BALB/c mice treated with free AmB  and PLGA-loaded AmB nanoparticles (PLGA-AmB) (107 nm to 117 nm) were comparable. In terms of DL and EE, AmB-NP were in accordance with the previous study. The DL and EE of PLGA-AmB was slightly lower than the AmB-NP reflecting higher binding affinity of lipid soluble drug to the PLGA-TPGS nanoparticles. However, PLGA-AmB had a slightly higher zeta potential of around −23.6 mV compared to the AmB-NP with zeta potential of around −19.5 mV. In vitro release studies showed that the AmB-NP had a biphasic release profile with a initial release of 47.38% of the drug in 5 days followed by a sustained released for 22 days. Both PLGA-AmB and AmB-NP had the similar MIC value against fections . In addi effects . Furtherof D-AmB .P. brasiliensis-infected BALB/c mice treated with 60 µg three times daily of Nano-D-ITZ over 30 days showed lesser lesions and fungal burden compared to the treatment with 1 mg daily of free ITZ indicating higher antifungal activity of Nano-D-ITZ. The developed formulation with lower dose and dose frequency provided higher efficacy than the free drug suggesting another alternative to replace the free drug [Since the focus had been directed towards triazoles as the treatment of pulmonary fungal infections, Cunha-Azevedo et al. recently developed another PLGA-DMSA nanoparticle formulation containing ITZ (Nano-D-ITZ). In comparison to oral free ITZ, IV Nano-D-ITZ showed a higher concentration of ITZ in the lungs, which might be due to the targeting effect of DMSA and entrapment of nanoparticles in the first encountered capillary bed of lungs after IV administration. In addition, ITZ was also found in the spleen and liver of the experimental mice but undetected in the plasma following administration of Nano-D-ITZ. ITZ in spleen was mainly due to the opsonization of nanoparticles, which was then identified and cleared by the phagocytes and tissue macrophage from the blood, whereas, metabolism of ITZ into a huge number of metabolites in the liver by CYP3A4 might be responsible for the concentration of ITZ in the liver. In evaluating the antifungal activity, ree drug . NanocarThe delivery of antifungal agents to treat pulmonary fungal infections through pulmonary route has been investigated for the advantages mentioned in this review. Additionally, the use of nanocarriers in pulmonary delivery of antifungal agents has shown impressive potential but the full potential of nanocarriers has yet to be exploited. From the available literature, micronized drugs show enhanced dissolution profiles and antifungal activity. This is beneficial for the delivery of poorly water-soluble antifungal agents as micronized drug formulations provide the possibility of using a reduced dose of antifungal, which subsequently reduces the systemic exposure of the drug, leading to reduced toxicity of the antifungal agents. Besides, the microstructured crystalline formulation of antifungal agents for pulmonary route of administration could provide prolonged retention of the entrapped drug in the lungs, allowing local action of the drug while avoiding extensive systemic exposure of the antifungal agents, thereby reducing associated toxicities.The formulations delivered through pulmonary routes focus on the aerosolization properties of the formulation to estimate the in vitro deposition of drug in the lungs. Good aerosolization properties of the formulations developed suggest deep lung penetration of the antifungal agents, which would be beneficial in the treatment of pulmonary fungal infections where most fungi colonize the deep lungs. With comparable aerosolization properties, DPI formulations with lipid-based nanocarriers possess advantages over the DPI formulations with only course carriers in that they offer the opportunity of modulated release of the drug. Depending on the quantity and type of the phospholipid used in the formulations, the DPI formulations with lipid-based nanocarriers can achieve burst release profiles or sustained release profiles. The different release profiles of drugs depend on the interaction of the antifungal agents with the lipid components of the lipid-based nanocarriers, which could result in rapid expulsion of the encapsulated drug or a slower release of free drug from the lipid barriers. From the literatures reviewed, reduced cytotoxicity of the antifungal agents could be achieved through incorporation of antifungal agents into lipid-based nanocarriers. Besides, the use of lipid-based nanocarriers also provided the possibility of enhanced targeting of the antifungal agents through incorporation of targeting ligand as compared to DPI formulations with only course carrier.In addition to lipid-based drug delivery systems, polymeric drug delivery systems in DPI formulation have also being studied widely to enable efficienct delivery of antifungal agents and to overcome the drawbacks of the conventional antifungal for the treatment of pulmonary fungal infections. Based on the reported research, it could be said that the polymeric nanoparticles containing antifungals in DPI formulation possess good aerosolization properties, which increases their efficacy in treating pulmonary fungal infections. Sustained release is a major advantage of the polymeric drug delivery systems which was shown in most of the polymeric formulation developed by researchers that may also enhance the efficacy of the therapy and patient compliance due to the reduction in the frequency of dosing when compared to conventional therapy. It can also be pointed that polymeric drug delivery can improve the solubility of hydrophobic antifungals as AmB and triazoles that have the limitations of low solubility that hinder their effectiveness. Overall, microcarriers and nanocarriers are promising approaches that aid in the delivery of antifungals to the lungs, which could overcome the limitation of conventional therapy in the treatment of pulmonary fungal infections.The suitability of pulmonary drug delivery using nanocarriers is demonstrated by their enhanced efficacy, stability, and most importantly, their improved safety profile. These improved safety profiles of pulmonary drug delivery formulations of antifungal agents using nanocarriers are illustrated through the use of ideal excipients as well as reduced cytotoxicity of the developed formulations. The use of ideal excipients to enhance the safety of the formulation developed was demonstrated in the study by Beinborn et al. where they judiciously used PVP K25 as the polymeric stabilizer in the formulation . The reaThe enhanced safety profile of the pulmonary formulations with nanocarriers was also demonstrated through reduced cytotoxicity against cultured cell lines. A study by Vyas et al. demonstrated lower toxicity of aerosolized liposomal AmB formulation to human RBC in the hemolysis assay. This reduced hematotoxicity of the liposomal formulation can be attributed to the more stable and compact configuration of bilayers and intercalation of AmB in the lipid bilayers . This fi® and AmBisome®. This was due to the slower release of drug from the stronger barrier provided by the lipid matrix of LN, which suggested that the LN formulation of AmB could reduce the nephrotoxicity more effectively. Lower hematotoxicity of the LN formulation of AmB towards Sprague-Dawley rat RBC suggested lowered direct contact of AmB to RBC due to lipid matrix of LN and slower release of drug due to interaction of AmB with phospholipids and cholesterol [Beside liposomes, NLCs, lipid micelles and SLNs have also demonstrated enhanced safety profiles through reduced cytotoxicity. For instance, the NLC formulation developed by Pardeike et al. showed no signs of cytotoxicity on A549 cells as evident through undetected effect on cell mitochondria and on the membrane integrity of A549 cells at therapeutically used concentrations. This indicated good tolerability of ITZ-loaded NLC, which could be due to the utilization of well tolerated lipids (Precirol ATO 5 and oleic acid) and an optimized concentration of surfactant (Eumulgin SML 20) in developing the NLC formulation . Anotherlesterol .® as the lytic and cytotoxic effects of deoxycholic acid of Fungizone® can be prevented [®, another commercial AmB product used in Brazil. Apart from that Nano-D-AmB did not show significant viability of peritoneal macrophages which also proves the safety of the formulation, whereas the viability of peritoneal macrophages was reduced after incubation with Anforicin B® [P. brasiliensis-infected BALB/c mice receiving free ITZ presented alopecia  is a dry powder inhaled formulation of ITZ currently under development by PULMATRIX , a clinical stage biopharmaceutical company. It is an investigational product which is intended for the treatment of ABPA to curb the limitation of oral antifungals and to potentially reduce the steroid burden [PUR1900 (Pulmazoled burden . The prod burden ,114.The increasing incidence of pulmonary fungal infections, particularly in immunocompromised patients, requires highly efficient delivery of antifungals into the lungs to prevent and eradicate the fungal cells. The development of novel approaches that target the lungs are needed to compensate the limitations of the conventional therapy of pulmonary fungal infections. Lipid- and polymer-based nanotechnology in cooperation with antifungal formulatiosn have shown positive results regarding enhanced pharmacokinetic and antifungal activity. Thus, targeted delivery by administering these nano-formulations via the pulmonary route is the promising option for the treatment of pulmonary fungal infections. However, future studies and more well-designed clinical trials are needed to ensure the suitability of the nano-formulations for effective and safe delivery of antifungals, as well as to elucidate their associated risks.


Background: Isolated tumor cells or small clusters of tumor cells observed in the vicinity of the main tumor mass in pathology sections, termed tumor budding, are common in cancers and have been associated with prognosis in some settings. This study examined the clinical associations and treatment efficacy implications of tumor budding in breast cancer patients receiving neo-adjuvant therapy. Methods: Breast cancer patients that received neo-adjuvant therapy before definitive surgical treatment in a single cancer center over a 7-year period were included, and their records were reviewed. Data extracted included patient demographics, tumor characteristics and pathologic response to treatment at surgery. The initial breast cancer biopsy before any therapy was reviewed by two pathologists, and a hot spot area was evaluated for tumor budding (defined as 1 to 5 cancer cells observed detached from the main tumor mass). Results: Seventy-five patients who received neo-adjuvant therapy  were included. Tumor budding was observed in two-thirds of the patients. There were no significant differences in patient (age and menopause status) and tumor  characteristics between the group that had tumor budding and the group that did not have tumor budding in the pre-treatment biopsy. Likewise, no statistically significant differences were observed in the frequency of complete or partial responses between the two groups. Conclusion: In this cohort of breast cancer patients receiving neo-adjuvant therapy, tumor budding was frequent, but it was not associated with tumor characteristics or pathologic responses to treatment. The value of tumor budding as a prognostic factor in the neo-adjuvant setting within the general breast cancer population could not be confirmed, but such a value in specific sub-groups deserves further investigation, given the pathophysiologic rationale and data from other settings. Breast cancer is a highly prevalent malignancy in women and a cause of significant morbidity and mortality . It is tNeo-adjuvant chemotherapy is increasingly used in locally advanced breast cancer with the aim of breast conservation and in cases with positive lymph nodes. In addition, neo-adjuvant therapy provides invaluable information regarding the chemosensitivity of cancers that may be incorporated into post-treatment algorithms. The molecular sub-type of breast cancer is a predictor of response to neo-adjuvant chemotherapy, with cancers of triple negative and HER2-positive sub-types more commonly obtaining complete pathologic responses than ER-positive cancers. However, beyond tumor sub-type, no other clinical or pathologic markers have been consistently proven to predict therapy response in the neo-adjuvant setting ,6. Thus,Tumor budding has been observed in various cancers. The exact definition of tumor budding varies among studies, but it generally refers to a small number of cancer cells, usually up to five cells, which are detached from the main bulk tumor mass and are observed as isolated cells or small clusters of cells in histologic sections . When tuThis study describes the association of tumor budding with clinicopathologic factors in breast cancer patients that received neo-adjuvant chemotherapy and explores its potential value as a predictor of response.Breast cancer patients who received neo-adjuvant therapy over a 7-year period were identified in the breast cancer section of the electronic patient database at our cancer center, and their records were retrieved and reviewed. Patient demographics and tumor characteristics were recorded in a database constructed for the study.2; low-grade tumor budding: less than 5 buds per 20× power field in a hotspot area of 0.785 mm2) were reviewed with the same microscope by two experienced general pathologists and recorded in the study database. We elected to use a 5-bud limit for the categorization of tumor budding grade based on the recommendations of the International Tumor Budding Consensus Conference for colorectal cancer [Tumor budding was examined in eosin and hematoxylin sections of the initial diagnostic biopsy specimens of patients. These specimens were obtained by an interventional radiologist through a core needle biopsy under ultrasound guidance before the initiation of treatment. The presence or absence of budding and the grade  was defined as no residual tumor in the breast or lymph nodes after neo-adjuvant chemotherapy, a partial response (PR) was defined as minimal or moderate residual disease and evidence of response to therapy, and no response (NR) was defined as extensive residual disease in the breast and/or the lymph nodes without evidence of therapy effect on tumor cells. This practical, three-tier, semiquantitative response evaluation was used based on the ease of evaluation and the fact that different degrees of partial response to therapy forecast an intermediate prognosis between complete response and no response.x2 test for comparison of ratios and the t-test for comparison of mean differences of continuous variables. Overall survival was evaluated with the Kaplan–Meier method. The Log Rank test was used to compare Kaplan–Meier survival curves. All statistical comparisons were considered significant if p < 0.05.Statistical analysis was performed with the Fisher exact test or the The protocol of the study obtained approval from the Ethics review board of the institution.Seventy-five breast cancer patients who received neo-adjuvant chemotherapy were included in the study . All patThe majority of patients in the series  received neo-adjuvant chemotherapy with the FEC-D regimen . Other regimens used included AC-paclitaxel (4 cycles of adriamycin with cyclophosphamide followed by 4 cycles of paclitaxel every 2 w) in 4 patients, docetaxel-cyclophosphamide for 4 cycles every 3 w in 2 patients, carboplatin-docetaxel for 6 cycles every 3 w in 3 patients and weekly paclitaxel monotherapy for 12 weeks in 1 patient . All HERAmong the 75 patients, 72 completed neo-adjuvant chemotherapy and underwent surgical resection of their breast cancer. Fifteen of the 72 patients (20.8%) had CR, 20 patients (27.8%) had PR and 37 patients (51.4%) had NR . CR rateAmong patients with tumor budding, 36 patients (72%) had intermediate-/high-grade tumor budding (five or more buds per hotspot high-power field) and 14 patients (28%) had low-grade tumor budding. The degree of tumor budding was not associated with response to therapy .p = 0.8, Overall survival was not different between patients with tumor budding and patients with absence of tumor budding in pre-operative biopsies (Log Rank test Tumor budding in cancer is defined as isolated cancer cells or small clusters of cancer cells separated from the main tumor mass, seen in histologic sections . From a As a putative depiction of the initial steps of the metastatic cascade, presence of tumor budding has been found to be of prognostic significance in various cancers, including carcinomas of gastrointestinal origin, lung cancer, head and neck carcinomas, and breast cancer ,25,26,27This study reports on the associations and predictive implications of tumor budding in a series of breast cancer patients that received neo-adjuvant therapy and had a diagnostic core needle biopsy before staring neo-adjuvant treatment. These biopsies yielded sufficient material for budding evaluation in all cases in this series. The feasibility of budding evaluation was consistent with the experiences in colorectal cancer literature, where endoscopic biopsies have been used for rectal cancer evaluation before neo-adjuvant chemoradiation . Two-thiThe current study is the first to explore tumor budding in the neo-adjuvant setting. It confirmed that tumor budding is a common pathologic occurrence in breast cancer and is associated with all molecular sub-types. There was no association of tumor budding with response to neo-adjuvant treatment in this series. However, due to the comparatively small size of the cohort, associations of tumor budding with response to therapy in specific breast cancer sub-types, such as triple negative cancers as observed in the post-operative setting by other investigators, were not excluded. As there were only 10 triple negative patients in the current series, the lack of association of tumor budding with response to therapy should be interpreted with caution in this group. Other limitations of this study include the retrospective design and the fact that it was performed at a single center, with evaluations performed by two experienced pathologists. As a result, whether findings can be replicated in other centers or by pathologists less experienced in breast pathology remains unknown.Overall, we have shown the common occurrence of tumor budding as a pathologic phenomenon at diagnosis before any treatment in breast cancer. Tumor budding may be identified in eosin and hematoxylin histologic sections without the need of additional stains or special slide preparation. The value of tumor budding as a prognostic marker in specific sub-sets of breast cancer remains an open question that will require more extensive studies. Whether further characterization of budding cells with immunohistochemistry stains for key proteins of the metastatic process may add value to their study as prognostic factors in breast cancer awaits future evaluations.


Statistics shows that Japan now has 28.4% of population aged over 65, which marks the highest in the world. Moreover, one in four over 65 is said to have ADRD or MCI. Traditional family caregiving derived from filial piety is giving way to moving to care facilities in order to reduce a burden on children or get professional care. This suggests that everyday communication for older adults involves younger conversational partners with varying degrees of shared knowledge and experiences. Such intergenerational communication can be challenging and stressful on both sides. To date, empirical studies that observe interactional strategies of both younger caregivers and older adults is unknown. Using 21 recordings of weekly conversational activities (45-60 minutes) led by recreation workers  taken at a nursing home in Japan, this qualitative study demonstrates how (1) sharing of the past and present becomes a learning opportunity for both recreation workers and older participants with ADRD (aged 87-95) , (2) reference to nursing-home living establishes and reaffirms their interpersonal relationship, and (3) intergenerational conversation becomes a tool to express life satisfaction for the older participants. This evidence-based study proposes mundane conversations during recreational activities as part of non-pharmacological person-centered care which serves to improve the quality of life and life satisfaction of older adults at care facilities. It is therefore important to study communicative strategies of professional caregivers since they affect social engagement as well as emotional and psychological well-being of those at the end-of-life stage.


Alzheimer’s disease is the 4th leading cause of death in North Carolina for people 65 and older. People with dementia are hospitalized more often and have prolonged stays, poorer outcomes, higher costs, and increased readmission rates. Hospital employees have expressed the desire to have specialized training to learn how to more effectively communicate with and provide better care to patients with dementia. To address identified patient and hospital employee needs, the University of North Carolina (UNC) Center for Aging and Health is disseminating hospital-specific dementia-friendly training at five hospitals within the UNC Health System. The training is being delivered via online modules and follow-up didactic sessions over a three-year period to clinical and non-clinical staff who interact with patients. To date, 1,948 employees at three of the five hospitals have launched the online training; 1,102 have completed the training. The pilot training took place at the UNC Hospitals--Hillsborough Campus  in 2019. Hillsborough Hospital staff (n=195) who participated in the dementia friendly training completed a survey to assess their ability to recognize symptoms and provide appropriate care to dementia patients pre- and post-training. Clinical staff answered 23 Likert scale self-efficacy questions; non-clinical staff answered the first 12 of these questions. Positive change in self-efficacy ratings from pre- to post-training was significant for every question (p < .0001). Additional results will be included in the poster. The dementia-friendly hospital initiative is preparing employees to provide better care for people with dementia and is effective in increasing employee self-efficacy.


Primary cutaneous lymphomas (PCLs) are a heterogeneous group of extranodal non-Hodgkin lymphomas, limited to the skin, with no evidence of extracutaneous disease at the time of diagnosis. PCLs are the most frequent extra-nodal lymphomas, with an incidence around 10 cases per million inhabitants per year. Among all PCLs, primary cutaneous B-cell lymphomas (PCBCLs) account for ~25–35%. According to the 2018 World Health Organization (WHO)-European Organization for Research and Treatment of Cancer (EORTC) classification for primary cutaneous lymphomas, PCBCLs are classified into three most common entities: primary cutaneous marginal zone B-cell lymphoma (PCMZL), primary cutaneous follicle-center lymphoma (PCFCL), and primary cutaneous diffuse large B-cell lymphoma-leg type (PCLCL-LT). PCMZL and PCFCL are considered clinically indolent and have an excellent prognosis, with 5-year survival rates higher than 90%. PCLCL-LT is less common and more aggressive, with 5-years survival rate lower than 60%   to TNM was proposed to better stage patients with cutaneous T-cell lymphomas presenting as mycosis fungoides (MF) or Sézary syndrome (SS), which together account for the 75–85% of all PCLs. Nevertheless, TNM+B classification system is not applicable for non-MF/SS primary cutaneous lymphomas due to their different clinical behavior . For thin (FISH) . In partn (FISH) . The chrBorrelia infection by antibody test using enzyme linked immunosorbent assay (ELISA) and by polymerase chain reaction (PCR) of Borrelia DNA in tumor tissue. To this regard, there is some evidence that Borrelia infection in North American and European endemic areas  may play a role in the pathogenesis of indolent type of PCBCLs because of chronic inflammation (Helicobacter pylori infection in gastric MALT lymphoma) might be useful in treating these types of lymphomas. On the other hand, the largest available study aimed to evaluate the prevalence of Borrelia infection in patients with cutaneous lymphomas failed to demonstrate an association between PCBCL and this microorganism in non-endemic area  the identification of ammation –11. Thismic area . ConsequAn appropriate imaging study should be performed in all patients with PCBCLs and includes either a chest, abdominal and pelvic computerized tomography (CT) scan with contrast or a whole body positron emission tomography (PET) technique together with CT scan, PET/CT scan , 13. In Next step in staging PCBCLs is the bone marrow biopsy, but the role of this diagnostic tool is controversial . AccordiIn summary, an appropriate diagnostic process has important implications for the management of patients with PCBCLs. The clinician starts with the diagnosis issued by the pathologist and may ask for additional molecular and/or microbiological investigations. The subsequent diagnostic work-up of PCBCLs should determine the extent of disease and can be achieved through a careful staging assessment which should be based on the biological behavior of the different subtype of PCBCLs, notably indolent or aggressive.GT, MF, and MA have made substantial contributions to conception and design of the article and have all been involved in drafting the manuscript and revising it critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nuclear Medicine has been at the heart of theranostics even before the term was coined. It provides effective tools for precision medicine particularly with positron emission tomography (PET), and radioligand therapy. It allows the investigation of phenotypes and functions in all areas of medicine and provides innovative tools to kill cancer cells. This Research Topic for Frontiers in Medicine focuses on the role of Nuclear Medicine in the context of Personalized Medicine.Jamet et al. have demonstrated the interest of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) PET in the management of patients with multiple myeloma (MM). [18F]FDG-PET is highly sensitive and specific for bone lesions detection at baseline. The presence of extra-medullary disease, the number of bone focal lesions and the maximum standardized uptake value [SUVmax] have independent pejorative prognostic value on progression-free survival and overall survival. For therapy response assessment, [18F]FDG-PET is considered as the reference imaging technique. [18F]FDG-PET and bone marrow flow cytometry are complementary for detection of minimal residual disease before maintenance therapy. New PET tracers such as [11C]methionine, choline or acetate,[68Ga]Ga-pentixafor, which targets CXCR4, and immuno-PET targeting CD138 and CD38, also show promising results.Three reviews reported the growing interest of PET in onco-hematological diseases. Bailly et al., [18F]FDG-PET changed response assessment and therapy strategy in diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma. [18F]FDG-PET might also have a significant impact in the management of mantle cell lymphoma (MCL). [18F]FDG-PET at baseline in MCL patients has good sensitivity for staging and SUVmax provides prognostic information. They conclude that [18F]FDG-PET results should be integrated in the definition of MCL treatment strategy to identify patients who might benefit from more intensive therapy.As discussed by 18F]FDG uptake has been questioned. Thus, new tracers such as [18F]Fludarabine, a nucleoside analog, have been developed. Early results with [18F]Fludarabine have been reviewed by Barré et al.. Favorable preclinical results in murine models  have prompted a “first in man” study. In DLBCL patients, increased uptake was observed in sites considered abnormal by CT and [18F]FDG. In chronic lymphocytic leukemia patients, increased uptake coincided with lymph-nodal sites expected to be involved by the disease. [18F]Fludarabine uptake was also high in the spleen and bone marrow. No uptake was observed in the cardiac muscle and brain. They conclude that [18F]Fludarabine might correctly quantify the disease burden, in the presence of confounding inflammatory processes.The specificity of [Bodet-Milin et al., focusing on medullary thyroid carcinoma. They conclude that pretargeted PET imaging has a high potential for antibody-based diagnostics and theranostics.Monoclonal antibody (mAb)-based therapies and immunotherapy have experienced considerable growth in cancer management. Labeled mAbs also show promise for theranostics. While PET can be performed using directly radiolabeled mAbs, pretargeting improves imaging contrast. The development of pretargeted immuno-PET in tumors expressing carcinoembryonic antigen  has been reviewed by in situ activation or inhibition of immune cells or on the administration of immune cells selected, activated, or transformed ex vivo. It is most important to delineate, by in vivo imaging, the distribution, activation and migration of immunologically active cells. Methods designed to monitor the fate of these cells and to define their immunological status have been reviewed by Perrin et al., focusing on cell tracking in cancer immunotherapy, particularly CAR (Chimeric Antigen Receptor) T-cell therapy, and on its potential impact on these new therapeutic modalities.Immunotherapy relies on Gérard et al. Preliminary in silico studies investigate the conversion of hypoxia maps into dose-distribution objectives for EBRT dose painting in view of future clinical trials.Severe hypoxia, frequent in glioblastoma multiforme, is associated with resistance to ionizing radiation. It contributes to treatment failures after external-beam radiation therapy (EBRT). It would be logical to deliver higher radiation doses to hypoxic tumor regions. This calls for the delineation of hypoxic zones as examined by 188Re) is an attractive candidate for therapy and has a favorable gamma emission for imaging purposes. It is readily extracted from 188W/188Re generators and exhibits chemical properties similar to those of technetium-99m, which might constitute an additional, purely diagnostic companion radionuclide. The development of radiopharmaceuticals based on 188Re, including peptides, mAbs, and particles has been reviewed by Lepareur et al., demonstrating that 188Re is a cost-effective alternative for routine clinical use.The ability to monitor the distribution of radioactivity inside the body is a major advantage of nuclear medicine procedures. Rhenium-188  proves useful in the follow-up and treatment assessment of brain diseases. Carbon-11 and fluorine-18 labeled tracers have been derived from the chemical structure of cocaine. The review by Lonceint et al. as a major concern for health organizations. In hospitals, medical personnel may be exposed to ionizing radiation and the highest doses (up to a few mSv) are recorded in nuclear medicine departments. The review aims at understanding the attitude of health professionals toward the risks of exposure and how they combine patient care with self-protection. The coexistence of care and radiation protection logics was shown to be a source of contradictions for nuclear medicine professionals and of differences in risk regulation strategies according to occupational groups.To complete this overview, risk management has been discussed by JB wrote the editorial, which was revised, proofed, and accepted by all authors.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.


With the tremendous advancements in genome sequencing technology in the field of pharmacogenomics, data have to be made accessible to be more efficiently utilized by broader clinical disciplines. Physicians who require the drug–genome interactome information, have been challenged by the complicated pharmacogenomic star-based classification system. We present here an end-to-end web-based pharmacogenomics tool, PharmaKU, which has a comprehensive easy-to-use interface. PharmaKU can help to overcome several hurdles posed by previous pharmacogenomics tools, including input in hg38 format only, while hg19/GRCh37 is now the most popular reference genome assembly among clinicians and geneticists, as well as the lack of clinical recommendations and other pertinent dosage-related information. This tool extracts genetic variants from nine well-annotated pharmacogenes (for which diplotype to phenotype information is available) from whole genome variant files and uses Stargazer software to assign diplotypes and apply prescribing recommendations from pharmacogenomic resources. The tool is wrapped with a user-friendly web interface, which allows for choosing hg19 or hg38 as the reference genome version and reports results as a comprehensive PDF document. PharmaKU is anticipated to enable bench to bedside implementation of pharmacogenomics knowledge by bringing precision medicine closer to a clinical reality. Pharmacogenomics (PGx) is the field that studies how genetic makeup affects a person’s response to drugs. Even though the concept of pharmacogenomics has been around since the 1950s . By way Pharmacogenomics plays an instrumental role in drug safety and efficacy. Studies indicate that the most commonly prescribed pharmaceuticals are effective in only 25% to 60% of patients . FurtherGenetic variants together with environmental factors play an important role in an individual’s response to drug treatment. As sequencing has become more affordable, many health centers can now easily get patient genomes sequenced. Genetic markers in pharmacogenomics are identified by means of numbers and letters and separated from gene names by a star known as star allele nomenclature. For example, CYP2B6*2 identifies the genetic variant in gene CYP2B6 at genomic position g.5071C > T, leading to amino acid substitution R22C . Star alCustom-designed pharmacogenomic arrays were the technology of choice for their ability to provide faster, cost-effective solutions, particularly for large sample sizes as part of research studies. The Affymetrix-developed Drug Metabolizing Enzymes and Transporters (DMET) Plus array  was one Next-generation sequencing (NGS) approaches are gaining more popularity in PGx, involving either pharmacogene-targeted/whole exome sequencing (WES)  or wholeThe next challenge is the translation of genetic test results into clinical action. PharmGKB has published PGx-based drug dosing guidelines by several consortia, including the Clinical Pharmacogenetics Implementation Consortium (CPIC) , the DutFinally, the success of PGx implementation relies heavily on its acceptance among patients and clinical healthcare professionals. The major stumbling block to its widespread implementation among general physicians and clinical geneticists appears to be the lack of knowledge of genetics and an unfamiliarity with PGx data and tools. CDS delivered through electronic health records (EHRs) has proved indispensable in facilitating gene-based drug prescription for patient care ,40.Considering the current state of clinical pharmacogenomics together with the availability of pharmacogenomic resources, we have created a web-based tool that facilitates the easy transition of a person’s whole genome variant data into clinical recommendations. Through this, we aim to enable clinicians and geneticists to more broadly implement pharmacogenomics in patient care. The initial version of this software covers nine well-annotated pharmacogenes that cover the activity of 37 drugs.Gene-specific information tables provided jointly by PharmGKB and CPIC  were useGenetic markers in pharmacogenomics are indicated using star-allele nomenclature—numbers and letters and separated from the gene name by a star. Several bioinformatics software tools that aid in the conversion of a genome variants to star-allele nomenclature are available including Astrolabe , PharmCAhttp://www.pgrn.org) (accessed on 7 September 2020). Drug dosage information was retrieved from the Pharmacogenomics Knowledge Base  (accessed on 15 December 2020).We used the diplotype-phenotype table, from the gene-specific information tables, to map the sample diplotype assigned in the previous step to its phenotype. Allele functionality data was also obtained from the diplotype-phenotype table. Medication/drug name information for the corresponding gene was obtained from the Clinical Pharmacogenetics Implementation Consortium (CPIC) of the National Institutes of Health’s Pharmacogenomics Research Network ; phenotype status corresponding to allele functionality  and clinical recommendations suggesting usual dose  or adjust dose (for other phenotypes). There is an exception in the nomenclature of phenotype status in the SLCO1B1 gene according to the diplotype-phenotype file, where instead of the above, the conventions used are indeterminate/possibly decreased/decreased/normal/possibly increased/increased/possibly poor/poor function. The second section provides a detailed interpretation of the findings (consult note). For each gene listed in the first section, this will detail the consequence of the genotype on the allele functionality and phenotype. Wherever possible, dosage suggestions and changes are also recommended , cancer drugs (11%) and cardiovascular/lipid-modifying drugs (3%), will shed more light on the necessity for more studies in this field.Limitation: Although the cost of WGS continues to decline, it remains prohibitively expensive for widespread clinical use. However, its use is justified by the fact that a one-time genomic test to determine a person’s pharmacogenomic profile would inform clinicians about dosing and effectiveness for a multitude of drugs. Inclusion of this information into the EHR would be invaluable to patients throughout their lifetime.


Influenza virus containing eight ribonucleoprotein (RNP) complexes. Each RNP incorporates multiple copies of nucleoprotein (NP), a fragment of the viral genome (vRNA), and a viral RNA-dependent RNA polymerase (POL), and is responsible for packaging the viral genome and performing critical functions including replication and transcription. A complete model of an Influenza RNP in atomic detail can elucidate the structural basis for viral genome functions, and identify potential targets for viral therapeutics. In this work we construct a model of a complete Influenza A RNP complex in atomic detail using multiple sources of structural and sequence information and a series of homology-modeling techniques, including a motif-matching fragment assembly method. Our final model provides a rationale for experimentally-observed changes to viral polymerase activity in numerous mutational assays. Further, our model reveals specific interactions between the three primary structural components of the RNP, including potential targets for blocking POL-binding to the NP-vRNA complex. The methods developed in this work open the possibility of elucidating other functionally-relevant atomic-scale interactions in additional RNP structures and other biomolecular complexes.Seasonal flu is an acute respiratory disease that exacts a massive toll on human populations, healthcare systems and economies. The disease is caused by an enveloped  Influenza viruses from the Orthomyxoviridae family, which are in continuous circulation around the world /3/1967) ,25,26,27luenza A  to the R/3/1967) luenza A ,30,31,32luenza A . Lastly,With infectious diseases exacting an increasingly ruinous toll on human populations and economies, the value of being able to identify the structure-function dependencies of biomolecular complexes in pathogens, and identify where novel therapeutics might best affect their pathogenic function, is greater than ever . Over 15i represents the nucleotide of interest. In order to provide a general representation of the range of the known structure space of RNA in the PDB we use the crystal structure of T. thermophilus 30S rRNA (PDB accession code: 1J5E). This allows for a correlation between We defined each nucleotide along the length of the vRNA by two torsional angles: Influenza. This approach allows the full RNP complex to be assembled using partial structures of proteins and RNA at different resolutions from both X-ray crystallography and cryo-electron microscopy (cryo-EM) data sources.The MMFA described in our earlier work  is used Structural assembly of the RNP hairpin is accomplished by superposition of individual NPs from circular and helical suprastructures. The MatchMaking option in the Structural Comparison Tool of Chimera  allows fUsing 62 high-resolution (1 Å or better) non-homologous protein structures from PDB as structural references, we identified short loops with the same sequence length as our targets, and match the loop ends to the ends of the template protein. A target threshold of 0.1 Å RMSD is used to find matching main-chain atoms of the templates and targets. Out of our set of potential candidates, we further eliminate those which have bad contacts with the neighboring residues of the loop.To resolve any bad contacts in our RNP model we employ a pairwise-distance calculator to check contacts between individual proteins (NP or POL) with one another, and between individual proteins and the vRNA. Resolving close contacts and the most extreme van der Waals overlaps  is done by regenerating side-chains of clashing residues in different orientations that do not clash.Lastly, we perform energy minimization and microcanonical ensemble (NVE) simulations using the GROMACS software package (ver. 4.5.5)  with theThe goodness-of-fit for select components of our model is calculated using the Fit-to-Map utility in Chimera .Molecular graphics images in this work are produced using the UCSF Chimera package  from theOur modeling approach utilizes multiple types of sequence and structural information, and we model each RNP component using different information sources . As we gWe determine initial models for vRNA and nucleoprotein (NP), and orient them relative to one another using electron microscopy envelopes described by experimental data.Influenza genome using only the phosphorous atom trace to guide placement [Influenza carries a large sum of negative charges in its phosphate backbone. Packaging of this molecule is only possible due to the many positively-charged residues in the RNA-binding groove on the surface of NP. This encapsidation is critical to transcription, replication and viral packaging [The backbone of the single-stranded vRNA provides the primary point of contact with the RNA-binding grooves of each NP. This allows us to insert a genome segment of the lacement ,21. Usinackaging .i represents the nucleotide of interest. We calculate T. thermophilus (PDB accession code: 1J5E) and observe a linear correlation between both angles  are able to connect the vRNA bound to NP-12 in the helical suprastructure to the vRNA bound to NP-13 in the circular suprastructure  aligning the terminal sugar moieties of the loop to the terminal sugar moieties in the vRNA already bound to the NP, and (ii) making sure that the modeled RNA loop does not encroach on the excluded volumes of the NPs, or the NP-bound vRNA. We identify RNA loops that satisfy both of these constraints by drawing structures from the PDB . As an etructure  and formInfluenza A/Albany/3/1967  we find that 33 NP molecules can be arranged into the full hairpin structure, providing a comparison to the sequence and structure of the 8th RNP of Influenza A [According to experimentally-determined structures of RNP (EMDB accession code: emd_2205 and PDB accession code: 4BBL) each NP molecule in the helical suprastructure incorporates an average of 26 phosphorous atoms over its surface and through the space to its nearest neighbor NP. Thus, approximately 26 RNA residues coil around each NP molecule in the helical suprastructure. In the circular suprastructure (EMDB accession code: emd_1603 and PDB accession code: 2WFS) each NP molecule has an average of 20 associated phosphorous atoms. At the junction between the helical and circular suprastructures, the NP molecules show an average of 22 phosphorous atoms associated with each NP molecule. By fitting a segment of the vRNA NS1, the smallest gene, from luenza A . Our arrInfluenza A genome.By assembling multiple known protein and RNA structures we thus generate an initial NP—vRNA hairpin structure that compactifies a full gene from the Binding of polymerase (POL) to the NP—vRNA complex is critical for the accurate assembly of the complete RNP macromolecule ,21,24,25Loop-binding modes for POL are known , but the located ,17,44.Influenza A genome have identified two secondary structures that describe the 5Sequence-based studies of the rkscrew”  and a “prkscrew” ,47. For Influenza polymerase complex have been determined, including one from bat Influenza A (PDB accession code: 4WSB), which includes the bound RNA promoter region [Influenza A/Albany/3/1967 virus and anchors the POL to the rest of the NP—vRNA complex. Using the Chimera Fit-to-Map utility, a goodness-of-fit calculation identifies 78.4% of all heavy atoms within the outer EM contour in the final model.Several new crystallographic structures of the whole r region . In thisInfluenza A/Albany/3/1967 viral sequence can be threaded directly into the promoter model. The connecting RNA loops  can then be modeled based on the phosphorous atom coordinates ; incorporating some of the same data used to model the RNP hairpin . Using t termini b. The “hates see .The final assembly of the NP—vRNA—POL complex incorporates a single NP oligomer with 33 molecules, a single vRNA chain (NS1 gene) with 855 residues, and a viral polymerase that encloses the “half-corkscrew” promoter at the 5Using an all-atom force field with previous applications to RNA and protein-RNA compounds ,39,40 weormation  was prodAs we have described before, our model is able to describe the atomic coordinates of all protein and nucleic acid components of the complete RNP complex while simultaneously recapitulating the global structures defined by cryo-EM experiments. Thus we deliver unprecedented precision in terms of molecular geometry at scales where drug-binding and sequence mutations will have a demonstrable effect.i) to the binding pocket of its neighbor NP-(i) and E339 of NP- even provides us with an explanation for the functional efficacy of the viral polymerase.Two regions of PA  form direct interactions with the vRNA b. Most aFor PB1, residues 177–214 form a residues b. This hthe vRNA , and in s of PB1 .One other salt bridge between PB1 and vRNA has functional implications for polymerase activity: the R198 side-chain of PB1 and the phosphate backbone at residue C842 on vRNA. We find that the arginine side-chain has the requisite length to bring its guanidino group near enough to interact with the vRNA backbone, and is stabilized in our simulations. However, when the residue is mutated to lysine (R198K) the interaction is lost due to K198 not having enough bond lengths necessary to bridge the space between PB1 and C842 of vRNA c. The poWhile solving structures of biomolecular complexes in an experiment is critical, computational methods can also provide useful new insights into the mechanistic dependencies of large-scale biomolecular functionality. Our motif-matching fragment assembly (MMFA) method is able to leverage the vast amount of structural information from the PDB and EMDB as part of our structure determination process. Additional sequence information from GenBank  or the IInfluenza A virus: the RNP complex. To the best of our knowledge, no other work has described the complete NP—vRNA—POL complex at this level of detail. It should be noted that there is no cryo-EM map of reasonable resolution for the full NP—vRNA—POL complex, so the structural validation done in this work has been conducted on certain components  . Due to The second big challenge was assembling the two higher-order NP suprastructures  into the hairpin conformation of RNP. Using our knowledge of oligomeric structures of NPs, the requisite orientations of the folded domains (residues 402–421) relative to the binding pockets of neighboring NP monomers, and our knowledge of the flexibility of biopolymer loops, we modeled the loops responsible for orienting the folded domains throughout the entire RNP complex. Thus we formed one continuous NP oligomer with helical and circular supramolecular structures that describes the hairpin conformation of RNP and is consistent with observed cryo-EM structures.Influenza polymerase (POL) binds to both 5Influenza A polymerase-vRNA complex as a template we modeled a “half-corkscrew” structure of vRNA and successfully docked POL complex to the NP—vRNA complex using a low-resolution (22.1 Å) cryo-EM map of a POL-NP complex as a guide. Our loop modeling techniques then fill in the remaining gaps in the vRNA structure between the POL and NP.tructure ,46,47. UFollowing simple energy minimizations and an NVE simulation to refine our model, we identified multiple salt-bridge interactions involving side-chain and nucleobase groups that are stabilized in our final RNP model. Importantly, these interactions provide valuable insight into the functional behaviors of the RNP that are only apparent after our final assembly is complete.At atomic-scale we find that NP-NP oligomerization is stabilized by a salt bridge between NP-NP-(i+1)E9, as exp studies . Our modNP-(i+1)E9, as expWe find that the vRNA contributes further to RNP stability with salt bridges between PB1(R198) and vRNA(-POactivity . The intactivity , howeveractivity . These rInfluenza. These results could open the door to future studies in viral protein mutagenesis or viral sequence analyses to assess the efficacy of POL-binding and activity.In addition to providing structural evidence that supports the results of various experiments involving protein mutations, our findings also call into question the effects of the vRNA sequence on POL-binding. Such specific interactions as we find have never been observed or discussed before, meaning that our methods could be used to seek out additional protein/nucleic-acid interactions for other viral genome segments, or offer the possibility of identifying novel drug targets for inhibition of One of the biggest challenges for structural modeling in the coming decades will be solving macromolecular structures of large biomolecular complexes, such as the genomic segments from living systems.Influenza virus has eight fragmented genome pieces, and each fragment is a stand-alone molecular machine that carries out functions related to the viral life cycle. Our progress towards understanding how the virus functions through its life cycle is hindered by the lack of a detailed structure of an intact Influenza NP-vRNA-POL complex. Through multiple efforts by many laboratories and scientists, sequence and structural information of this complex at different levels of detail, resolution and completeness have become available, but have never before been consolidated to model a complete RNP complex in atomic detail.The Influenza RNP complex and its associated polymerase in atomic detail—a crucial advance to complement many years and multiple studies of RNP structure determination through cryo-EM [Influenza. This work is an important step toward modeling RNP complexes at various stages in the viral life cycle [By combining these data with in silico MMFA modeling approaches we have developed a model of a single  cryo-EM ,17,18, a cryo-EM ,28,42. T