Acute myeloid leukemia (AML) is a leading blood cancer subtype that can be caused by 27 gene mutations. Previous studies have explored potential vaccine and drug treatments against AML, but many were proven immunologically insignificant. Here, we targeted this issue and applied various clinical filters to improve immune response. KIT is an oncogenic gene that can cause AML when mutated and is predicted to be a promising vaccine target because of its immunogenic responses when activated. We designed a multi-epitope vaccine targeting mutations in the KIT oncogene using CD8+ and CD4+ epitopes. We selected the most viable vaccine epitopes based on thresholds for percentile rank, immunogenicity, antigenicity, half-life, toxicity, IFNγ release, allergenicity, and stability. The efficacy of data was observed through world and regional population coverage of our vaccine design. Then, we obtained epitopes for optimized population coverage from PCOptim-CD, a modified version of our original Java-based program code PCOptim. Using 24 mutations on the KIT gene, 12 CD8+ epitopes and 21 CD4+ epitopes were obtained. The CD8+ dataset had a 98.55% world population coverage, while the CD4+ dataset had a 65.14% world population coverage. There were five CD4+ epitopes that overlapped with the top CD8+ epitopes. Strong binding to murine MHC molecules was found in four CD8+ and six CD4+ epitopes, demonstrating the feasibility of our results in preclinical murine vaccine trials. We then created three-dimensional (3D) models to visualize epitope–MHC complexes and TCR interactions. The final candidate is a non-toxic and non-allergenic multi-epitope vaccine against KIT mutations that cause AML. Further research would involve murine trials of the vaccine candidates on tumor cells causing AML. Acute myeloid leukemia (AML) is a blood cancer subtype where an overproduction of abnormal myeloid cells causes improper development of platelets, red blood cells, white blood cells, and bone marrow failure . AML is NPM1), FMS-like tyrosine kinase 3 (FLT3), Runt-related transcription factor (RUNX1), and KIT, all of which are critical for hematopoiesis. The KIT gene encodes for a type III receptor tyrosine kinase that is critical for pathways involved in cell proliferation, survival, and differentiation of hematopoietic progenitor cells [Common mutations that cause AML are in the genes Nucleophosmin 1 , and gastrointestinal stromal tumors ,6. CBF-AThe KIT gene comprises six domains: Ig-like C2-type 1, Ig-like C2-type 2, Ig-like C2-type 3, Ig-like C2-type 4, Ig-like C2-type 5, and protein kinase. The protein kinase domain is intracellular, while the five other domains work on extracellular regions . KIT mutWT1), proteinase 3 (PR3), hyaluronic acid-mediated motility receptor (RHAMM), and mucinone1 protein (MUC1). However, these vaccines have had limited success in phase II clinical trials.AML is currently targeted with chemotherapy, drugs, and stem cell transplants . Less agThe KIT gene has not been targeted with vaccines to treat AML, but trials on the gene’s effectiveness in drugs and vaccines for other conditions exist. KIT gene mutations are common targets for the treatment of various cancers because of the gene’s role in cellular functions such as hematopoiesis, carcinogenesis, and melanogenesis ,15. FurtThis study relied on inducing an immunogenic response to point mutations on the KIT oncogene that have been found in AML patients. Single amino acid changes in genes can change the chemical properties of peptide sequences and have been associated with several cancers . The KITOur design was a multi-epitope AML vaccine predicted to induce effective CD8+ and CD4+ T-cell responses by targeting the intracellular and extracellular domains of the KIT oncogene. We used epitopes derived from common AML-inducing point mutations in the KIT gene and overlapping CD8+ and CD4+ epitopes. Our epitope vaccine design elicits T-cell immune responses by releasing epitopes cleared through the following clinically relevant variables: HLA binding affinity, immunogenicity, antigenicity, half-life, instability, toxicity, IFNγ release, allergenicity, population coverage, and murine MHC binding affinity. CD8+ T cells respond to endogenous antigens and participate in a cytotoxic activity, while CD4+ T cells respond to exogenous antigens and are helper T cells that induce a more refined immune response. Implementing CD8+ and CD4+ epitopes into cancer vaccines is predicted to safely induce an immune response that removes tumor cells expressing the same epitopes. This study is the first to target AML with a vaccine against KIT gene mutations.We targeted AML-inducing KIT mutations because of the gene’s critical roles in hematopoietic cell survival, proliferation, and differentiation  19,20,2,22. WhenThe final CD8+ dataset included 12 ninemer epitopes filtered for rank (<10), immunogenicity (>0), antigenicity (>0.4), half-life (>1 h), toxicity (non-toxin), allergenicity , and instability (<40). CD8+ epitopes were not filtered for IFNγ release. IFNγ release was not prioritized for the CD8+ epitopes because CD8+ T cells are cytotoxic and less involved in releasing IFNγ than CD4+ helper T cells. Next, we determined that the world population coverage for MHC Class I binding CD8+ epitopes was 98.55% . RegionsWe used the default thresholds provided by NetMHCpan-4.0 to determine strong- and weak-binding epitopes to murine MHC molecules. Strong-binding epitopes had a threshold of 0.5% and weak-binding epitopes had a threshold of 2%. There were four strong binders and six weak binders. Top CD8+ epitopes had strong binding to the murine MHC alleles H-2-Db, H-2-Dd, H-2-Kb, and H-2-Qa2. INAAI) from PCOptim-CD was used on CD8+ epitopes filtered for rank, immunogenicity, antigenicity, half-life, toxicity, allergenicity, and stability. The resulting dataset with optimal population coverage included four CD8+ epitopes . One optRDF, ARNILLTHGRITKIRDF, ARNILLTHGRITKIRDFG, ILLTHGRITKIRDFGLAR) overlapping with the CD8+ epitope GRITKIRDF from the same mutation. The K550N mutation resulted in the CD4+ epitope TYKYLQNPMYEVQWK overlapping with the CD8+ epitope NPMYEVQWK from the same mutation.The final CD4+ dataset included 21 epitopes filtered for rank (<10), immunogenicity (<50), antigenicity (>0.4), half-life (>1 h), toxicity (non-toxin), IFNγ (positive), allergenicity , and instability (<40). Two epitopes were 15-mers, two were 16-mers, five were 17-mers, and 12 were 18-mers. Thus, longer length epitopes had higher potency for MHC class II binding in our vaccine design. We determined that the world population coverage for MHC Class II-binding CD4+ epitopes was 65.14% . RegionsWe used the default thresholds provided by NetMHCIIpan-4.0 to determine strong- and weak-binding epitopes to murine MHC molecules. Strong-binding epitopes had a threshold of 1% and weak-binding epitopes had a threshold of 5%. There were six strong-binding epitopes and eight weak-binding epitopes. The top CD4+ epitopes had strong binding to the murine MHC alleles H-2-IEd and H-2-IEk. PCOptim-CD was used on CD4+ epitopes filtered for rank, immunogenicity, and antigenicity. The resulting dataset included six CD4+ epitopes  with a wWe combined the final filtered dataset for Class I and Class II MHC binding epitopes and used the IEDB population coverage tool to obtain 99.49% world population coverage. Population coverage rates for specific regions are listed in We modeled four top epitope–MHC complexes using MDockPep, CABS-dock, and PyMOL. We created 3D models for SDINAAIAF binding to HLA-A*01:01, GKSDLIVHV binding to HLA-A*02:06, GLARYIKNDSNYVVKGN binding to HLA-DRB1*04:01, and FGLARYIKNDSNYVVK binding to HLA-DRB3*01:01 . The 3D We selected a total of 33 CD8+ and CD4+ epitopes based on the filters: binding affinity/percentile rank, immunogenicity, antigenicity, half-life, toxicity, IFNγ release, allergenicity, and stability (12 CD8+ epitopes and 21 CD4+ epitopes). The KIT gene’s protein kinase domain, which affects intracellular signaling pathways, holds 26 of our top epitopes. There is limited research on treatments for AML that target the KIT gene. Instead, peptide vaccines and dendritic cell vaccines have targeted other tumor-associated antigens (TAAs), including WT1, PR3, RHAMM, and MUC1. Limited MHC allele interactions with epitopes have been tested in WT1 vaccines, indicating potentially low population coverage. Additionally, a WT1 vaccine restricted in HLA-A*02 had no immunological significance in its phase II clinical trial owing to minimal vaccine benefits and low sample size. A vaccine targeting OCV-501 with MHC class II molecules resulted in insignificant immunological improvements in its phase II clinical trial. TAAs are less effective than TSAs in eliciting safe immune responses to cancer cells. TSAs are only present in cancer cells and have a higher affinity to MHC molecules and TCRs, making them better candidates for anticancer vaccines. TAAs are more widely studied, but their potential toxicity and lack of specificity for tumors indicate that targeting TSAs may be an improved approach. Clinical trials with TSA-based anticancer vaccines have also been successful . FurtherClinical trials for CD8+ and CD4+ epitope vaccines against AML exist, but with limited success. One such vaccine targeting the WT1 gene reached phase II of clinical trials but did not develop strong immunological memory. We addressed this issue in our vaccine design by only selecting epitopes with high antigenicity scores. However, another vaccine targeting mutated WT1 peptides resulted in improved survival. Future trials for AML vaccines must prioritize targeting TSAs instead of TAAs to ensure proper and safe immune responses . In thisCurrent studies on treatments for AML that target the KIT gene emphasize drug therapy, such as combined treatment with nilotinib and chemotherapy  and midoOur vaccine design follows in silico methods predicted to safely induce CD8+ and CD4+ immunogenic responses. We demonstrated predicted vaccine efficacy by filtering epitopes through clinically relevant variables such as immunogenicity, antigenicity, toxicity, and allergenicity, to obtain top epitopes. Designing vaccines through bioinformatics offers a quick and cost-effective method of developing anti-cancer treatments before murine or pre-clinical trials. We identified four CD8+ and six CD4+ epitopes that were strong binders to murine MHC molecules, demonstrating potential use of our vaccine design in further research including murine trials.We filtered out many potential CD8+ and CD4+ epitopes because of low immunogenicity scores. In the CD8+ dataset, 50% of epitopes that passed the percentile rank filter also passed the immunogenicity filter. All top CD8+ epitopes failed to pass the IFNγ filter because IFNepitope was only developed for CD4+ epitopes. Thus, the IFNγ filter was disregarded for MHC I binding molecules. For the CD4+ dataset, IFNγ and allergenicity filtered out most of the epitopes in addition to immunogenicity.Our vaccine design was strengthened by the five CD4+ epitopes overlapping with the top CD8+ epitopes. Overlapping epitopes emphasizes their strength and our vaccine’s potential to elicit high immunogenic responses involving both cytotoxic and helper T cells. Population coverage for overlapping epitopes alone remains low, but the two potential immunogenic pathways that may be induced by the overlapping CD8+ and CD4+ epitopes indicate high potency for attacking cancerous cells. Further research on increasing population coverage of overlapping epitopes can help improve the vaccine’s effectiveness. Additionally, combined usage of CD8+ and CD4+ epitopes increase the likelihood of stability despite the short peptide lengths of CD8+ epitopes. CD8+ epitopes were limited to 9-mers, but CD4+ data included epitopes of up to 18-mers. Still, previous studies indicated that CD8+ and CD4+ immunogenic responses are inducible with vaccines using 9- or 10-mer peptides in patients with solid tumors ., are mainly instigated in the protein kinase domain. The protein kinase’s critical role in hematopoietic cell growth, proliferation and development makes the domain an important location for our top epitopes. Cancerous activities caused by mutations in the protein kinase domain can be primarily targeted by having most of our target mutations in this domain. Our study was unique in targeting a proto-oncogene for which not many have studied AML vaccine therapies. The 33 combined CD8+ and CD4+ epitopes induced a population coverage of 99.49%, ensuring that our vaccine may effectively improve AML prognosis for a large population. For both CD8+ and CD4+ epitopes, we determined population coverage based on HLA alleles that the peptides could bind to and the frequency of those alleles among various regions worldwide. High population coverage was optimal because more patients could effectively be treated with the vaccine. However, regions including Central America had lower population coverage for CD8+ and CD4+ epitopes. Large differences in population coverage such as between Central America and Europe were due to varying frequencies of HLA alleles in different populations. Each population has a unique frequency of HLA alleles, so the potency of our epitope design varies by region. In Central America, frequent HLA alleles include A*02:06:01, A*02, DQA1*05:01, and A*02:02 [The protein kinase domain of the KIT gene held 26 out of 33 top CD8+ and CD4+ epitopes. Each epitope was critical for our vaccine design. Still, the intracellular signaling pathways that the KIT gene is involved in, such as those outlined in Figure 2 A*02:02 . HoweverOur vaccine design would be the most effective on AML patients within Asia, Europe, and North America, which included regions with the highest population coverage. HLA alleles that our top epitopes bind to were more prevalent in these regions. AML is most reported in North America, Western Europe, and South Asia, which validates our vaccine design, as our targeted population would be the most reactive to our vaccine .Our data were weakly validated for population coverage owing to the minimal overlap between final epitope datasets and optimized epitopes from PCOptim-CD. However, PCOptim-CD was not as effective in our vaccine design as compared to other datasets—when PCOptim-CD was used on epitope data for a vaccine design targeting the HRAS gene for squamous cell carcinoma, the optimized dataset contained six epitopes . PCOptimPeptide vaccine designs are a cost-effective method of developing treatments to target tumors and/or viruses. Computational methods also allow for large protein datasets to be quickly tested for vaccine efficacy. When compared to in vitro and in vivo studies, in silico methods are unable to reflect direct testing with living cells. To address these challenges, tools for in silico vaccine studies are constantly being developed to form optimal vaccine designs. IntegralVac is an example of this, where MHCSeqNet, DeepVacPred, and hemolytic/non-hemolytic peptide predictors were combined to improve vaccine design accuracy and safety . Our datCompared to the population coverage of the CD8+ epitopes, the CD4+ epitopes had low coverage. Additional mutations were filtered through to find more epitopes, including combination mutations with double missense, but population coverage remained low. One potential reason was that IEDB had limitations in their HLA allele dataset—a few alleles in the final CD4+ dataset were excluded from the population coverage calculation. For example, HLA-DRB3*02:02 was not included in the CD4+ population coverage, but the allele could bind to 16 of the final CD4+ epitopes. HLA-DRB3*01:01 was also excluded from the population coverage but could bind to 12 final CD4+ epitopes. Thus, the accuracy of CD4+ population coverage was limited owing to the IEDB database.CD4+ epitope population coverage may have also been low because studies show that the KIT gene does not often interact with CD4+ T cells . The KIThttps://cancer.sanger.ac.uk/cosmic, (accessed on 2 June 2022)) [Common mutations of the KIT gene that cause AML are located on exons 17 and 8, and D816V is the most prevalent . Mutatioe 2022))  were usehttps://www.uniprot.org/, (accessed on 31 July 2022) [The “mast/stem cell growth factor receptor Kit” peptide sequence was obtained in FASTA format using UniProt (ly 2022) . Mutatedhttp://tools.iedb.org/mhci/, (accessed on 7 June 2022) [50 values of each peptide in the protein sequence with the IC50 values of other peptides found in the SWISSPROT database [9-mer CD8+ epitopes for each point mutation were obtained using the IEDB T Cell Epitope Prediction Tool with MHC I Binding (ne 2022) . The predatabase . Lower pStrong and stable epitope candidates were determined based on a variety of clinically relevant variables, including percentile rank (binding affinity), immunogenicity, antigenicity, half-life, instability, isoelectric point, aliphatic index, GRAVY score, toxicity, IFNγ release, and allergenicity. Only epitopes that passed these filters (IFNγ was disregarded for CD8+) were presented as top epitopes for our vaccine design.http://tools.iedb.org/immunogenicity/, (accessed on 13 June 2022) [Each epitope that passed the percentile rank filter was tested for immunogenicity using the IEDB Class I Immunogenicity tool (ne 2022) . IEDB trne 2022) .http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html, (accessed on 13 June 2022) [Antigenicity for top immunogenic epitopes was determined by VaxiJen v2.0 (ne 2022) . VaxiJenne 2022) ,45. Vaxine 2022) .https://web.expasy.org/protparam/, (accessed on 14 June 2022). To calculate half-life, ProtParam analyzed each epitope’s N-terminal amino acid [Half-life, instability, isoelectric point, aliphatic index, and GRAVY score were determined through ProtParam  [Toxicity was obtained with ToxinPred (ne 2022) . Toxicitne 2022) .http://crdd.osdd.net/raghava/ifnepitope/, (accessed on 14 June 2022) [IFNγ release was tested using IFNepitope (ne 2022) . IFNepitne 2022) . The IFNhttps://www.ddg-pharmfac.net/AllerTOP/, (accessed on 14 June 2022) [Allergenicity was determined using AllerTOP v2.0 (ne 2022) . AllerTOne 2022) .http://tools.iedb.org/population/, (accessed on 21 June 2022)). We used population coverage to determine our vaccine’s effectiveness on the world population and on the populations of 16 regions: East Asia, Northeast Asia, South Asia, Southeast Asia, Southwest Asia, Europe, East Africa, West Africa, Central Africa, North Africa, South Africa, West Indies, North America, Central America, South America, and Oceania. We observed population because HLA type representation varies by population and ethnicity, and maximum coverage is ideal for a vaccine design [Population coverage was calculated using the IEDB epitope analysis tool “Population Coverage” ) [We used the same method for filtering through the CD4+ epitope dataset as we did for the CD8+ epitopes. However, percentile rank/binding affinity and immunogenicity were calculated with different tools. Percentile rank/binding affinity was obtained using the MHC II Binding Prediction tool on IEDB (y 2022)) . IEDB inhttp://tools.iedb.org/CD4episcore/, (accessed on 4 July 2022)) [Immunogenicity was determined using the IEDB CD4+ T cell immunogenicity prediction tool (y 2022)) . This toy 2022)) . We calcThe final epitopes had several overlapping amino acid sequences. We developed PCOptim-CD to find an optimized epitope dataset with maximal population coverage to reduce redundancy in epitope selection. The original program, PCOptim, was only designed for CD8+ datasets. The modified version, PCOptim-CD, was programmed to obtain the optimized epitopes for the CD4+ dataset as well. PCOptim-CD  was baseEpitopes and their MHC-restricted alleles for optimal population coverage were obtained using PCOptim-CD . We usedhttps://services.healthtech.dtu.dk/service.php?NetMHCpan-4.0, (accessed on 28 June 2022) [https://services.healthtech.dtu.dk/service.php?NetMHCIIpan-4.0, (accessed on 19 July 2022) [Strong- and weak-binding CD8+ epitopes to murine MHC molecules were identified using NetMHCpan-4.0 for peptide-MHC class I binding (ne 2022) . NetMHCply 2022) , which ghttps://www.rcsb.org/, (accessed on 21 July 2022) [https://zougrouptoolkit.missouri.edu/mdockpep/, (accessed on 21 July 2022) [https://tcrmodel.ibbr.umd.edu/rtcrex/TCRSDM6_180718_160348, (accessed on 31 July 2022) [We found PDB files for four HLA alleles that were restricted by several of our top epitopes on the RCSB Protein Data Bank (ly 2022) . HLA allly 2022) ,65,66 anly 2022) , select ly 2022)  was usedSeveral studies have investigated how to treat AML, including drug therapies, combination therapy (drugs and chemotherapy), stem cell transplants, and vaccines. However, many treatments, including AML vaccines that target the KIT gene, remain unexplored. The purpose of this study was to develop a vaccine design for AML using in silico methods that target missense mutations on the KIT oncogene. We applied several clinically relevant variables to our vaccine epitopes, including percentile rank, immunogenicity, antigenicity, half-life, toxicity, IFNγ release, allergenicity, and stability, to ensure the vaccine’s safety and effectiveness. Then, population coverage demonstrated the broadness of our vaccine design’s potential. Using this method, we found 12 CD8+ and 21 CD4+ epitopes from mutated KIT peptide sequences that can be implemented in a vaccine and potentially used in murine trials. The 12 CD8+ epitopes were immunogenic, antigenic, non-toxic, non-allergenic, and had long half-lives. In comparison, the 21 CD4+ epitopes were immunogenic, antigenic, non-toxic, non-allergenic, have long half-lives, and release IFNγ. The CD8+ epitopes had a high population coverage of 98.55%, while the CD4+ epitopes had a lower population coverage of 65.14% owing to limitations in our tools’ datasets and minimal interactions between the KIT gene and CD4+ T-cells. PCOptim was modified into PCOptim-CD to analyze both CD8+ and CD4+ datasets for optimized population coverage. There was minimal overlap between the final filtered epitopes and the optimized epitopes from PCOptim-CD, proving that further research is needed to develop a stronger dataset with greater validity. The four CD8+ and six CD4+ epitopes that were strong binders to murine MHC alleles indicated that our results can lead to preclinical studies with vaccine trials on murine models. We designed a vaccine predicated to be safe and effective through in silico methods to help improve treatments for AML and develop cost-effective methods for vaccine designs before pre-clinical trials. Our data may be used to facilitate future studies in investigating the use of our vaccine design in murine and clinical trials and improving immunoinformatic tools. Murine trials with the peptide vaccine design would be the next step for advancing research on this treatment for AML. Using the top epitopes with strong binding to murine MHC molecules, hematopoietic and stem and progenitor cells from mice would be modified with genome editing in vitro. The treatment group would receive these cells intravenously (IV) in addition to radiation treatment  and IV-a


To evaluate MRI diagnostic performance in detecting clinically significant prostate cancer (csPCa) in peripheral-zone PI-RADS 4 lesions, comparing those with clearly restricted diffusion (DWI-score 4), and those with equivocal diffusion pattern (DWI-score 3) and positive dynamic contrast-enhanced (DCE) MRI.This observational prospective study enrolled 389 men referred to MRI and, if positive , to MRI-directed biopsy. Lesions with DWI-score 3 and positive DCE were classified as “PI-RADS 3up,” instead of PI-RADS 4. Univariable and multivariable analyses were implemented to determine features correlated to csPCa detection.p < 0.001). MRI showed a sensitivity of 100.0%, specificity 40.9%, PPV 46.5%, NPV 100.0%, and accuracy 60.9% for csPCa detection. Modifying the threshold to consider MRI positive and to indicate biopsy , the sensitivity changed to 93.9%, specificity 57.2%, PPV 53.0%, NPV 94.8%, and accuracy 69.7%. Age (p < 0.001), PSAD (p < 0.001), positive DWI (p < 0.001), and PI-RADS score (p = 0.04) resulted in independent predictors of csPCa.Prevalence of csPCa was 14.5% and 53.3% in PI-RADS categories 3up and 4, respectively , but positive dynamic contrast-enhanced (DCE) MRI, the overall PI-RADS score should be upgraded to 4.•  The current PI-RADS recommendation of upgrading PI-RADS 3 lesions of the peripheral zone to PI-RADS 4 because of positive DCE decreased clinically significant prostate cancer detection rate in our series.•According to our results, the most accurate threshold for setting indication to prostate biopsy is PI-RADS 3 or PI-RADS 3 with positive DCE both associated with increased PSA density.• The online version contains supplementary material available at 10.1007/s00330-023-09605-0. Over the last few years, evidence has established magnetic resonance imaging (MRI) as the most accurate and cost-effective diagnostic imaging modality for the detection and staging of clinically significant disease (csPCa) –4. RecenCurrently, international guidelines recommend performing MRI, acquired and reported using the Prostate Imaging—Reporting and Data System (PI-RADS) v2.1 –14. PI-ROn this basis, there are two possible types of PI-RADS 4 assignments: those with clearly restricted diffusion (DWI score 4) and size lower than 1.5 cm, and those deriving from the upgrading of a peripheral zone PI-RADS 3 lesion showing positive DCE. As far as we know, no previous prospective works investigated the prevalence of csPCa in PI-RADS 4 lesions deriving from PI-RADS 3 upgrading. Based on our empirical experience in a tertiary referral high-volume center, many of those lesions result in negative or ciPCa on targeted biopsy.Hence, the primary aim of the study was to evaluate the diagnostic performance of MRI and PI-RADS score in detecting csPCa in PI-RADS 4 categories with clearly restricted diffusion (DWI score 4) or after upgrading from PI-RADS 3 category because of positive DCE and assess the impact of a revised PI-RADS categorization on biopsy decisions. As a secondary objective, we aimed at determining the clinical and radiological features associated with csPCa in those subcategories.This observational prospective single-center cohort study received formal Institutional Review Board and Ethical Committee approval. Written informed consent was obtained from each participant. A total cohort of 468 patients was consecutively enrolled from February 2020 to February 2022. According to the study design, men with clinical suspicion of PCa  were referred to MRI and, if positive to MRDB (see below for the definition of “positive”). Inclusion criteria also included an acceptable MRI quality to rule in and/or rule out csPCa; only MR images with a Prostate Imaging Quality score , best MRI sequence on which the finding is identifiable (DWI/ADC vs DCE) and the PI-RADS score.p < 0.05.Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) v28.0.1.1. Tests were two-sided, and statistical significance was set at p < 0.001). The clinical, radiologic, and pathologic characteristics of the final cohort are summarized in Table The 389 patients included in the final analysis presented a median age of 67.2 years (interquartile range [IQR] 43–85), median total PSA value of 8.42 ng/mL (IQR 0.08–86.20), and median PSA density of 0.19 ng/mL/mL (IQR 0.002–1.60), with a statistically significant difference in PSA density between patients without PCa or having ciPCa and csPCa  were diagnosed with PCa, including 132/389 cases of csPCa (33.9%) and 60/389 cases of ciPCa (15.4%). The remaining 197/389 men (50.6%) were found negative for PCa, with most of them showing inflammatory patterns at histological examination.p < 0.001) and csPCa prevalence (p < 0.001).The prevalence of csPCa, ciPCa, and inflammatory patterns in each category of PI-RADS scores is summarized in Table k = 0.829).Inter-reader agreement considering the assignment of PI-RADS categories was substantial  for outcome 1 and outcome 2 of 0.84 (95% CI: 0.80–0.88) and 0.83 (95% CI: 0.79–0.87) versus 0.82 (95% CI: 0.77–0.86), and 0.82 (95% CI: 0.78–0.86), respectively , PSA density (p = 0.03 and < 0.001 respectively), DWI as best sequence (both p < 0.001), conventional PI-RADS score (p = 0.007 and = 0.04 respectively) and adjusted PI-RADS score (p < 0.001 and 0.03).Results of the univariable and multivariable analysis are shown in Table In this study, we applied an adjusted version of the PI-RADS scoring system, considering prostate peripheral zone lesions. We differentiated lesions classified as PI-RADS 4 (with DWI score 4) from those upgraded from PI-RADS 3 (DWI score 3) because of positive DCE and identified as PI-RADS 3up. Moreover, we considered a different threshold for defining MRI positive and setting indication to prostate biopsy: both PI-RADS 3 and PI-RADS 3up are associated with increased PSA density. Our analysis resulted in a reduction of MRI false positives and a significant increase in csPCa prevalence among patients with PI-RADS 4 lesions. Furthermore, in our analysis, we witnessed a marked improvement of PPV when PI-RADS 3up lesions are directed to MRDB only in case of association with elevated PSA density. PPV is currently still low and high-variable among centers ; this waThese results strengthened the diagnostic power of PI-RADS 4 categorization in the detection of csPCa, with the potential effect of reducing the excess of false positives, already widely described in the literature . This anThe secondary objective was to define the clinical and radiological features independently correlating with the presence of PCa and of csPCa, to profile patients at higher risk. The logistic regression models demonstrated how age, PSA Density ≥ 0.15, a clearer definition of the lesion on DWI (compared to DCE), and the PI-RADS score show independent correlation with csPCa. In this sense, when considering PI-RADS 3up as PI-RADS 3 and no longer as PI-RADS 4, 2.6% of csPCa would have been missed if not considering additional data. These results may suggest that clinical-radiological data, if included in nomograms, could support a risk-based stratification of PI-RADS 3 and 3up lesions.Considering what we found, a new subcategorization of PI-RADS 3 lesions could be proposed: (i) PI-RADS 3B consisting of PI-RADS 3 and 3up findings requiring to be directed to biopsy according to clinical data; and (ii) PI-RADS 3FU, consisting of PI-RADS 3 and 3up findings that should be directed to a follow-up MRI.Expectedly, DCE did not independently correlate with csPCa detection, which reflects its impact on the number of false positive MRI findings. Indeed, as already proved by other studies, DWI plays a key role in the diagnostic workup of PCa and the applicability of non-contrast MRI in the clinical practice is becoming more and more appealing and necessary, given the requested high burden of MR , and alsLimitations need to be acknowledged: first, the single-center study was conducted in a high-volume referral center with both readers being experienced; second, low-quality exams were excluded from the analysis. These might hamper the reproducibility of the results. Finally, we acknowledge that proposing in a retrospective fashion a new threshold for defining an MRI finding as positive could cause a selection bias in future investigations, but we believe that this study could represent a first experience in this scenario, setting the base for future research and randomized trials.In conclusion, we can affirm that the upgrade of lesions with DWI Score 3 to PI-RADS 4 given a positive DCE MRI does not provide a positive impact on the overall diagnostic performance of MRI for the detection of clinically significant prostate cancer, leading to a reduced cancer prevalence yield. Moreover, these results might boost the applicability of non-contrast MRI for selected populations and centers. Finally, a new subcategorization of PI-RADS 3 scoring could be proposed, consisting of PI-RADS 3 and 3up findings, and divided into: PI-RADS 3B requiring to be directed to biopsy according to clinical data, and PI-RADS 3FU, to be followed-up with MRI.Supplementary file1 (PDF 93 KB)Below is the link to the electronic supplementary material.


Journal of General Microbiology was first published in 1947, two years after the founding of the Society for General Microbiology. Although both names have changed, to Microbiology and the Microbiology Society, respectively, the former names capture a key aspect of the journal, in that it is inclusive of all microbes, from viruses through to fungi, protozoa and algae. Except for animal and plant virology, which branched off in 1967 to create the Journal of General Virology, this ambition continues to this day. The word ‘general’ was a counterpoint to the many specific applied microbiology journals that existed in this period, in the diary and brewing industries, for example. Whilst the Society was founded to bring together diverse groups of researchers, doing more fundamental research, the journal encompasses both fundamental and applied research, which can be seen from articles published right from its first issue to the current day.The In this two-part celebratory article, the team of senior editors associated with the journal at the start of our anniversary year have reviewed highly cited and influential papers published in the journal's 75 year history. The 40 or so selected papers, which date from 1956 to 2014, highlight some favourite themes, under a number of broader topic areas. While not comprehensive, we hope it will highlight of areas where the journal has championed important work and will encourage continued submission of strong research as the journal as it continues beyond its 75th year.The ability to culture microbes in the laboratory in controlled and reproducible ways was integral to the work of the early pioneers of our subject. This enabled experiments to tease apart how they resource food, convert this to energy and then build new biomass. We start with a series of early fundamental papers, focused on microbial physiology and growth.Aerobacter cloacae  was popular for such studies. This work was followed shortly by two other important papers. The first, from Schaechter and colleagues in 1958, determined the influence of media composition and temperature on cell size [Enterococcus faecalis when grown on glucose as the sole energy source and only using substrate-level phosphorylation for energy production. Using this system, they established a direct relationship between the amount of energy source and biomass. Knowing how ATP was generated from glucose they derived a measure for growth yield, termed YATP [Our earliest highlighted paper from 1956 is a classic on the use of continuous culture for the growth of bacteria, from scientists at the Microbiological Research Department at Porton Down, Wiltshire, an important research centre for microbiology in the UK from this period [ell size . The secell size . The claed YATP . This asSaccharomyces cerevisiae [S. cerevisiae during anaerobic glucose-limited culture, another growth mode that is available to this metabolically flexible class of microbes [Yeast physiology and metabolism also featured in early classic papers. De Deken in 1966 elucidated the generality of a process called the ‘Crabtree effect’, which is when yeast growing on certain sugars in the presence of oxygen use fermentative metabolism rather aerobic respiration. De Deken determined that this was due to a regulatory system that resulted in repression of the respiratory system when growing on selected sugars, and that this occurred in many yeasts in addition to the model brewer’s yeast microbes . This unEscherichia coli has often served as the model system, as being a facultative anaerobe it can grow easily in the laboratory either with or without oxygen and can quickly transition between these modes. An elegant paper in the early days of molecular genetics described a mutant of E. coli that cannot grow in anaerobic conditions using fumarate as the sole electron acceptor for respiration [nirA. This was later found to be identical to fnr [fnr twice!This transition between aerobic and anaerobic growth has also been the topic of many other papers in the journal. Here piration . The mutl to fnr , so the in vivo, as metals that are essential at low concentrations can be toxic at higher concentrations, and bacteria have evolved many ways to control the intracellular levels of potentially toxic metals such as copper, for example using the CopAB system in the plant pathogen Xanthomonas axonopodis [Pseudomonas aeruginosa acquires iron from organic sources was described in an important paper from 2000, where an elegant genetic study revealed two distinct uptake routes for iron capture via haem [As well as the regulatory mechanisms involved in this aerobic–anaerobic transition, the biochemical and physiological changes in the presence and absence of oxygen also highlight the use of metals in enzymes, where the journal has also published important papers. The use of metals in respiratory enzymes, but also more widely in many other enzymes, has prompted important reviews by Hughes and Poole  and Gaddopodis . Iron nevia haem , both of2). The appreciation that ROS are generated constantly during aerobic growth, through electron leakage from respiratory enzymes and by host organisms to kill invading microbes, triggered many studies from the mid-1990s onwards. Particularly strong contributions to Microbiology have shed light on adaptive strategies that allow obligate anaerobes to tolerate O2 and to scavenge ROS. Anaerobes are ubiquitous and include bacteria of environmental, medical or biotechnological interest, as well as commensals and pathogens of the gut microbiota. Consequently, they have been well studied by microbiologists from the outset of both the Society and the journal.One consequence of high concentrations of metals such as iron is the generation of reactive oxygen species or ROS from molecular oxygen H-dependent O2 reductase to protect against oxidative stress [Clostridioides difficile [Most anaerobes, despite their name, can tolerate transient exposure to air, low-Orobiosis . A similricans . The O2  journal , and man purpose . In anotficile .2 directly, microbes have diverse ways to handle ROS in complex adaptive responses. While Clostridium perfringens has surprisingly classical (in non-obligate anaerobes) enzymes of ROS detoxification, such as superoxide dismutase, catalase and hydroxyperoxide reductase [2 as a by-product. These mechanisms are not restricted to anaerobic bacteria either, and the role of a copper- and zinc-containing superoxide dismutase in the pathogenic fungus Candida albicans was established in a paper published in the journal [C. difficile confers a high level of resistance against peroxide via the secretion of a glutamate dehydrogenase-like enzyme through an unknown mechanism [2, eliminate endogenously produced ROS in aerated cultures, adapt their metabolism and efficiently repair damaged molecules.As well as dealing with Oeductase , other a journal . An appaechanism  and confC. albicans. This tool was used mainly as a reporter of gene expression, although it later became invaluable for protein localization and/or morphology studies in many yeast species.The interesting behaviours in which microbes engage often involve processes at the cell envelope or within organelles, where microscopy with direct visualization of microbial cells has been a key experimental tool. An important paper published in the journal was the first optimization of green fluorescent protein (yEGFP) for use in yeast , initialE. coli. In essence, a capillary is loaded with a defined buffer containing an attractant chemostimulus. One end of the capillary is then placed into a suspension of E. coli cells. As the attractant diffuses out of the capillary, a gradient is generated and the bacteria move up that gradient and into the capillary. After a predetermined amount of time, the capillary contents  are assayed. This simple assay allowed Adler to define the best conditions for assaying bacterial chemotaxis and set the scene for almost all subsequent work in the field and consequently has been (and continues to be) cited many times.One fascinating cellular process, well studied in many different microbes, is chemotaxis, the understanding of which has led to in-depth understanding of sensing and signal transduction pathways. A significant early methodological paper was published in the journal in 1973 by Julius Adler, considered by many to be the pioneer in this field . Adler aE. coli has a complex cell envelope comprising both an outer and inner membrane. One controversial paper, which was discounted for many years by the community, was published by Bayer in 1968 [E. coli were examined by transmission electron microscopy. Plasmolysis occurs when bacteria are placed in hypo-osmotic medium, with shrinkage of the cytoplasm leading to separation of the cytoplasmic membrane from the cell wall and outer membrane. The micrographs revealed areas where, unexpectedly, the two membranes remained in contact despite cytoplasmic shrinkage. Bayer and others proposed that these contact sites functioned in the export of lipopolysaccharides. These sites of membrane contact became known as ‘Bayer junctions’ or ‘Bayer patches’, although for many years they were dismissed as an artefact arising from the chemical fixation process used to prepare the cells for electron microscopy. Recently structural and biochemical analysis of the lipopolysaccharide transport machinery (LPT) have shown that it forms a transenvelope machine that connects both membranes. Since the structure formed during this process is resistant to plasmolysis, it likely gives rise to the contact sites seen in Bayer’s original images.As a Gram-negative organism,  in 1968 . In thisBacillus subtilis and P. aeruginosa. The formation of biofilms requires some matrix or ‘glue molecules’ to be secreted to hold the structure together, which is often a secreted polysaccharide. However, an important paper in the journal discovered that in the pathogen Acinetobacter baumannii, there is an essential role for the cell surface pili in this process and that biofilm development is abolished when biosynthesis of the pili, via a novel chaperone-usher system, is disrupted [While we considered growth of bacteria in liquid culture in the previous section, microbes can also be grown on surfaces as colonies, the kinetic study of which was described by Pirt in a classic paper in 1967 . Much moisrupted .The implications of life in biofilms was the subject of another classic paper from Jan-Ulrich Kreft in 2004, where he used mathematical modelling to argue that bacteria within biofilms are behaving altruistically . Unlike In part 2 we will continue our journey and look a papers based around the broader themes of microbial communities and evolution as we complete our journey through 75 years of the journal’s most impactful papers.


Hand edema is a common sequalae of burn injuries involving the upper extremity (UE) and larger total body surface area (TBSA) burns requiring fluid resuscitation. Hand edema can be challenging to manage depending on the severity of the injury and, if not adequately addressed, can have long-term functional implications. Multiple edema management techniques are available for therapists treating burn injuries, but little is known about their efficacy. A quality improvement project examined the efficacy of 4 strategies used at our burn center.Edema management strategies evaluated included: elevation using a pre-cut foam wedge and pillow, elevation and self-adhesive wrap, elevation and cotton-elastic wrap, and elevation with a chip-bag. Figure-of-8 measurements were taken pre and post intervention. Outcomes included length of time intervention was in place, reason intervention selection, time required to apply intervention, and if intervention was intact prior to removal. Training was completed with staff to ensure consistent application of intervention. Only adult patients were included.A total of 23 interventions were measured. At follow up, 61% of the interventions were not intact. In each instance this was due to displacement of either the wedge or pillow. Self-adhesive wrap (3.25 cm) followed by cotton-elastic wrap (2.57 cm) in conjunction with elevation achieved the greatest reduction in edema. Elevation of the UE with a wedge and pillow are less effective in managing hand edema alone, with a mean difference of .26 centimeters (cm) decrease in edema. On average, it took therapists less time to apply cotton-elastic wrap (6.4 minutes) vs self-adhesive wrap (14.25 minutes).In this limited sample, elevation with a pre-cut foam wedge or a pillow was least effective in reducing edema in the hand. Including a secondary edema management technique in conjunction with elevation is more effective in managing hand edema. A larger sample size may benefit increased understanding of edema management techniques within a burn center.Interdisciplinary training on maintaining edema management strategies is important to promote maintenance of positioning. Further research is warranted to examine the efficacy of hand edema intervention strategies in the acute burn patient.


C∞ value of 18.9% with higher resistant starch (RS) content and lower glucose release. Our results indicate that AmyM-mediated hydrolysis is an efficient pathway for the preparation of porous starches with different functionalities which can be used for a range of applications.Porous starch is attracting considerable attention for its high surface area and shielding ability, properties which are useful in many food applications. In this study, native corn starch with 15, 25, and 45% degrees of hydrolysis  were prepared using a special raw starch-digesting amylase, AmyM, and their structural and functional properties were evaluated. DH-15, DH-25, and DH-45 exhibited porous surface morphologies, diverse pore size distributions and pore areas, and their adsorptive capacities were significantly enhanced by improved molecular interactions. Structural measures showed that the relative crystallinity decreased as the DH increased, while the depolymerization of starch double helix chains promoted interactions involving disordered chains, followed by chain rearrangement and the formation of sub-microcrystalline structures. In addition, DH-15, DH-25, and DH-45 displayed lower hydrolysis rates, and DH-45 showed a decreased  Starch is a complex branched-chain homopolymer of glucose that is widely used in the food, feed, fuel, chemical, materials, pharmaceutical, and environments industries due to its nontoxicity, utilizability, and biodegradability . The watAmong granule-based starch products, porous starch with abundant micro-sized pores has attracted considerable attention due to its high specific surface area and potential molecular interactions, which make it suitable for a wide variety of applications, e.g., as an absorbent, carrier, protector , or multNative starch exists naturally as insoluble semi-crystalline granules of different particle sizes and shapes which are assembled by amylose and amylopectin. The insoluble surface and hierarchical interior structures are able to prevent access-binding and the action of amylase . Hence, In a previous study, we identified a novel maltohexaose-forming α-amylase—AmyM—with high starch liquefaction and maltooligosaccharide (MOS) formation ability . A furthCorallococcus sp. EGB, was prepared in Pichia pastoris GS115 using a feed-batch strategy involving waste molasses and industrial glucose [The corn starch was purchased from Yuanye Biotechnology Co., Ltd. . The maltohexaose-forming α-amylase (AmyM), derived from  glucose . Commercv/w) was dispersed in water and then treated with the prepared AmyM (0.5 U/mg dosage) at 45 °C and 180 rpm with different reaction times. During the hydrolytic process, the reducing sugars from the reaction system were identified using the 3′5-dinitrosalicylic acid (DNS) method [The corn starch (10% ) method , and theTo observe the morphological features, the dried starch samples with DH values of 15, 25, and 45 were fixed in glutaraldehyde and a scanning electron microscope (SEM) analysis was performed using an SU8010 microscope ; representative images were obtained. Untreated native corn starch was used as a control. The samples were coated with gold prior to observation and then examined at an accelerating voltage of 5 kV and 2500× magnification .To determine the pore diameter of modified corn starch, the starch samples were investigated using the Brunauer–Emmett–Teller method  with liquid nitrogen adsorption at −196 °C and relative pressure ranging from 0.01 to 1.0. The adsorbent was porous starch, and the adsorbent mass was nitrogen. The BJH equation was used to determine the surface area, volume, and average pore diameter. To determine the water solubility and swelling power, starch suspensions (2% concentration) were heated in a water bath shaker at 85 °C for 30 min, and, after cooling to room temperature, centrifuged at 3000 rpm for 15 min. The precipitate was weighed, and the supernatant was dried at 105 °C until it reached a constant weight. The water solubility index (WSI) and swelling power index were calculated according to the method described in . w/v) was prepared at 80 °C for 30 min under constant stirring, and the transparency of each starch sample was determined by measuring its light transmittance at OD650.The gel consistency was determined according to the method outlined in   operating at 40 kV and 40 mA with Cu Kα radiation (λ = 0.154 nm). The samples were equilibrated over a saturated NaCl solution until the water content reached about 9%, at which point they were packed tightly in a glass cell and scanned from 5–35° (2θ) at a rate of 2°/min and a step size of 0.02°. The relative reflection areas in the crystalline and amorphous portions were distinguished via the straight line joining the points of intensity at the 35° and 5° diffraction angles, and the relative crystallinity  of the starch samples, i.e., the ratio of the crystalline area to the total area between 5° and 35° (2θ) , was cal−1 and with a resolution of 4 cm−1. The collected spectra were automatically baseline-corrected using OMNIC 8.0 , and the region ranging from 1200 to 900 cm−1 was deconvoluted with a half bandwidth of 20 cm−1 and an enhancement factor of 1.8. Bands at 1022, 1047, and 995 cm−1 were detected in the spectra, and the ratios of the peak intensity of absorption bands at 1047/1022 cm−1 and 1022/995 cm−1 were used to characterize the short-range molecular order of starch samples, as well as the number of double helices [The FTIR spectra of the dried starch samples was obtained using a Thermo Nicolet Nexus FTIR spectrometer  equipped with a single-reflection diamond attenuated total reflection (ATR) crystal and a mercury–cadmium–telluride (MCT) detector. The spectra were scanned within a range of 500–4000 cm helices .w/v). The sealed pans were kept at room temperature overnight for equilibration, following which they were scanned from 20 to 100 °C at a heating rate of 10 °C/min using a differential scanning calorimeter  equipped with a thermal analysis data station. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy (ΔH) were calculated from the DSC curves obtained using the data-recording software [To analyze the thermal properties of the porous starch, 3 mg samples were weighted in stainless steel pans and mixed with 9 μL water with a starch/water ratio of 1:3 (software . 2) with pancreatin   in a shaking water bath at 37 °C. During the enzymatic reaction, starch sample aliquots (0.2 mL) were taken at 10, 20, 30, 40, 60, 90, 120, 150, and 180 min, and these were mixed with 0.2 mL ethanol (96% v/v) to stop the reaction. The mixture was centrifuged at 10,000× g for 5 min, and the precipitate was washed three times with 50% ethanol (200 mL). All the produced supernatants were pooled together for the subsequent amyloglucosidase treatment. Mixtures containing 0.1 mL supernatant, 0.85 mL buffer , and 50 μL amyloglucosidase (10 U/mL) were incubated at 40 °C for 30 min in a shaking water bath. The amount of released glucose was measured using a D-glucose assay kit , and the final starch hydrolysis curve was plotted according to the starch hydrolysis (%) against time. The formula used to calculate the digestibility of the starch in vitro as well as the method used to produce the digestograms of starch hydrolysis are outlined in [C∞), kinetic constant (k), area under the hydrolysis curve after 180 min (AUC 180), hydrolysis index (HI), and expected glycemic index (eGI) were calculated. Additionally, the resistant starch (RS) content was quantified according to the established method [The in vitro digestion of the raw starch and the DH-15, DH-25, and DH-45 samples was analyzed using the method described by Gularte and Rosell  . Differences with The enzymolysis of the raw corn starch was performed at 45 °C to obtain AmyM-modified starch samples with different degrees of hydrolysis , and SEM observations were performed to obtain visualizations of their surface morphologies. As is shown in To further clarify the porosity characteristics of the AmyM-modified raw starch samples, their adsorption, swelling power, solubility, and gel consistency were analyzed. Two different liquids (water and oil) were used, and the adsorption properties of DH-15, DH-25, and DH-45 were significantly improved after the enzyme treatment c. DH-15 Granule integrity and starch chain interactions affect the swelling power and solubility of starch in both its amorphous and crystalline states. The increased swelling power and the solubility of porous corn starch after enzymolysis are attributed to the abundant pores extending from the surface to the center, and to the disruption of the amorphous region, which allows each granule swell more freely . The amyThe molecular chains of amylose and amylopectin assemble in starch granules to construct multi-scale structural systems which directly affect the physicochemical properties of starch . ConsideThe enzymatic hydrolysis of raw starch preferentially occurs in the amorphous region and leads to an increase in the relative proportion of the crystalline region , thereby−1 and 2934 cm−1, were significantly enhanced in the DH-15, DH-25, and DH-45 compared with the control, and the value of the peak at 1649 cm−1, which we attribute to the bending vibration of the O-H in the water that was absorbed in the amorphous regions of starch [−1 and the C-H and C-O-H bending at 1080 cm−1 and 1019 cm−1 [−1 was obviously decreased compared with that of the DH-25, indicating that the exposed hydroxyl groups of the DH-45 promote the formation of ordered molecular structures via hydrogen bond interactions.The FTIR spectra of the native corn starch, DH-15, DH-25, and DH-45 were analyzed. A is shown in f starch , was als019 cm−1 , respect019 cm−1 . The pea−1 regions associated with the crystalline and amorphous phases of the starch were analyzed (−1 was observed in the DH-15 (this was lower than in the native corn starch), indicating that the disordered structure of the porous starch was produced during the initial stage (consistent with our previous assumptions). A similar result was also observed in the porous starch prepared using glucoamylase [−1 and a lower absorbance ratio of 1022/995 cm−1 were observed in the DH-25, and these represent the moderate molecular order of the double helices of the starch granules. Interestingly, the DH-45 displayed increased absorbance ratios of 1047/1022 cm−1 and 1022/995 cm−1 compared with the DH-25  to the DH-15 and DH-25, whereas the ΔgH of the DH-45 was significantly higher than that of the native starch. The DH-25 was regarded as the product of an intermediate stage of AmyM hydrolysis, and this was consistent with the increased ΔgH values and the increased temperature range. The effect of annealing may have been to enhance the degree of molecular order in the chain rearrangements [cT-oT) than the control, and this may have been a result of the rearranging of part of the double helix due to the presence of excess water [gH due to the hydrolysis of double helices, and transglucosidase in combination with maltogenic α-amylase and β-amylase can lower transition temperatures due to the reduction of longer chains, which leads to a greater number of shorter double helices and thereby decreases thermal resistance [Considering the water migration properties of the porous corn starch obtained from the pasting analysis, the gelatinization properties of the porous corn starch were further analyzed by using DSC thermograms to detect possible changes in the physical states of the starch slurry. As is shown in ngements ; this woss water . Maltogesistance ,49. The C∞), the kinetic constant (k), the area under the hydrolysis curve after 180 min (AUC 180), the hydrolysis index (HI), and the estimated glycemic index (eGI). As is shown in k) end point values (C∞)  . An 18.9granules . Decreasgranules . The minHydrolytic enzymes such as amyloglucosidase AMG, fungal α-amylase AM, and cyclodextrin glycosyltransferase CGTase have been shown to affect the amorphous and crystalline domains of starch, and the presence of pores on the surface increases the exposure of the granule to subsequent enzymatic digestion . HoweverFrom the above results, we suggest that modification via AmyM can induce structural changes in raw starch granules. AmyM hydrolyzes the amylose located at the surface and in the crystalline and amorphous regions during the initial stage (DH-15). The micropores that are produced allow AmyM to display ‘inside-out’ behavior toward corn starch granules. AmyM preferentially hydrolyzes the amylopectin in the crystalline region during the later stage (DH-45) via intermediate state DH-25 and exposes the granules to subsequent enzymatic binding. The associations of dissociative amylose chains and the interactions of maltohexaose-like oligosaccharides with long chains promote the formation of sub-microcrystalline structures via chain rearrangement, and the resulting short-range ordered structures retain the characteristics of native starch granules.C∞ values, increased the RS content, and lowered the AUC, HI, and eGI values. This has important implications for the application of AmyM in the preparation of porous starch. From this study, we conclude that the amylose-mediated ordered structure created via uniform enzymolysis is also the rate-limiting factor for digestion . However, more details concerning the structural features of porous starch are needed. Overall, modification via maltooligosaccharide-producing amylase at a low temperature offers an attractive alternative for obtaining porous starch granules that can be used in a variety of foods applications.In the present study, solid granular corn starch was treated using a maltohexaose-forming α-amylase (AmyM) below the gelatinization temperature, and this produced porous starch samples with porous surface and internal morphology via the decomposition of the amorphous and crystalline regions. Lower relative crystallinity, unstable granules, and the formation of short-term ordered structures by the potential amylose chain interactions in the amorphous region were observed. AmyM-mediated structural regulation conferred the porous properties of the samples, decreased the


A particular challenge for labeling and imaging transcripts are complex tissues that are often opaque and/or pigmented, preventing easy visual inspection. Here, we introduce a versatile protocol that combines in situ hybridization chain reaction, immunohistochemistry, and proliferative cell labeling using 5-ethynyl-2′-deoxyuridine, and demonstrate its compatibility with tissue clearing. As a proof-of-concept, we show that our protocol allows for the parallel analysis of cell proliferation, gene expression, and protein localization in bristleworm heads and trunks.Access to newer, fast, and cheap sequencing techniques, particularly on the single-cell level, have made transcriptomic data of tissues or single cells accessible to many researchers. As a consequence, there is an increased need for  Advancements in sequencing techniques have significantly increased the number of organisms for which molecular research can be conducted in. Moreover, single-cell RNA sequencing allows such investigations on the level of individual cell types . In turnin situ hybridization (ISH) have been developed. One of them is in situ hybridization chain reaction (HCR). In situ HCR is faster to perform than traditional ISH, and works at lower temperatures, which aids maintaining tissue integrity. Moreover, in situ HCR can be multiplexed by using different fluorophores compatible with fluorescent imaging  would yield additional insights. For example, such codetection could help to benchmark gene expression in cells or tissues for which protein markers are already established, or to directly assess ratios of RNA and corresponding protein . Combiniin situ HCR with immunostainings. Those describing a combined approach either rely on the availability of commercial antibodies and proprietary technology  . This fuPlatynereis dumerilii. This invertebrate has gained importance as a model organism in various biological fields, including evolution, development, regeneration, neurobiology, reproduction, chronobiology, and ecology [reviewed in  adjusted for salinity (to 34–35 parts per thousand).This mix is referred to as ASW/NSW throughout the manuscript. Animals were fed twice a week, once with organic spinach leaves cut to roughly 3 × 3  mm pieces in a blender, and once with a mix of ground Tetramin fish food and Spirulina powder.2 mixed 1:1 with ASW/NSW, then removing all segments posterior to the 30th segment with a scalpel, cutting perpendicular to the animals’ body axis, similar to previous reports  22.The concept of EdU incorporation  was usedFor EdU labeling worm heads, animals were incubated with EdU at the onset of darkness (ZT16) for 6 hr. Worm heads were subsequently sampled at ZT22.For EdU labeling of worm blastemas, animals that underwent amputations (see above) were incubated for 1h.The “click” reaction is the detection step during which fluorescent dyes are attached to EdU to label those cells that previously incorporated EdU during DNA synthesis. The reaction was performed according to the manufacturer’s protocol using the Click-iT™ EdU Cell Proliferation Kit for Imaging, with Alexa Fluor™ 488 dye. In short, following HCR probe binding and washes, samples were incubated for 30 min with Alexa Dye azides to label EdU in a copper-catalyzed, enzyme-free stable reaction. For more details, refer to the manufacturer’s protocol or the SHINE protocol, day 3, Step Case “HCR and EDU” or “HCR, EDU, and IHC,” Step 12.Images were taken on a Zeiss LSM 700 inverse confocal microscope using Plan-Apochromat 20x/0.8, WD 0.55 mm and LD LCI Plan-Apochromat 25x/0.8 mm (oil immersion) lenses. Upon screening, the acquisition parameters were set according to the specimen with the strongest fluorescence and applied on one entire set of samples, thereby avoiding possible overexposure and ensuring comparability. Using FIJI , regionsHCR probes were designed against the listed target genes  using a Platynereis hox3, 49 probe pairs for Platynereis pdp1, and 27 probe pairs for Platynereis per.The number of probe pairs depended on the length of each target sequence. As detailed in Details on the used antibodies are listen in Platynereis heads to correlate the expression of the circadian clock gene period with the photoreceptor protein L-Cryptochrome (L-Cry) and the proliferation marker EdU  is a key gene of the circadian clock . Visualization of EdU using click chemistry revealed positive cells in the anterior part of the head, in a dispersed, salt-and-pepper pattern with few cells also localizing to the posterior of the head  and D. APlatynereis L-Cry. L-Cry is a photoreceptor that shares regions of expression with period and is well described also on the sub-cellular level for P.dumerilii . These cells are found in a region of high proliferation and expression of genes belonging to the germline multipotency program, called the segment addition zone  carrying the same amplifier —that is expressed in a similar pattern—using a spectrally different HCR amplifier (B1 amplifier coupled to Alexa 546), tissue clearing did not improve HCR signal intensity to the same extent than it did for the B2 amplifier coupled to Alexa 647)  see . For bla647) see –D’.In summary, we find that SHInE is compatible with tissue clearing using DEEP-Clear, which, depending on the type of tissue and the fluorophore used for labeling, can improve the HCR signal quality and reduce background signal.in situ HCR with IHC and EdU labeling of proliferating cells. The simultaneous visualization of RNA and proteins in the same tissue, as well as the high sensitivity of HCR-mediated RNA visualization, make the protocol suitable for detailed studies on the level of organisms, tissues, and cells.Here we present a novel protocol that combines As we demonstrate, it is possible to successfully apply SHInE using home-made reagents, with HCR probes designed with a free webtool, home-made buffers and custom antibodies. This serves to keep the protocol affordable and flexible to many biological questions. The combination of multiple labeling methods within the same steps also greatly reduces the time required compared to similar protocols, further increasing the accessibility of these kinds of experiments, or allowing researchers to sample more replicates or experimental conditions.Additionally, SHInE gives the researcher full control over reagents. This opens the protocol up for future improvement or adaptation to other models and tissues. For example, dextran sulfate, which is part of the probe amplification buffer, has been reported to negatively impact some antibodies during IHC , so chanin situ HCR, but furthermore show an improvement in the signal-to-noise ratio of acquired images. However, the extent of this improvement may vary depending on tissue type and spectral range of the used fluorophores.As we show, compatibility of SHInE with tissue clearing allows for improved HCR signal intensity in originally opaque and pigmented tissue. Not only do we provide evidence for the compatibility of DEEP-Clear with Platynereis as a model species, the broad applicability of in situ HCR and DEEP-Clear for various invertebrate and vertebrate species suggests that the combined protocol we present here can easily be adapted for other model systems, and thereby help to complement the powerful possibilities opened by scRNAseq in a variety of model species.We believe that this protocol, with its flexibility and ease of use, combined with low cost and customization opportunities, will be a valuable resource. Whereas, we have focused on icad007_Supplemental_FilesClick here for additional data file.


Rice flour (RF) has become a promising food material. In the present study, RF with higher protein content was prepared using a granular starch hydrolyzing enzyme (GSHE). Particle size, morphology, crystallinity, and molecular structures of RF and rice starch (RS) were characterized to establish a hydrolytic mechanism; thermal, pasting, and rheological properties were determined to evaluate processability using differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and rheometer, respectively. The GSHE treatment resulted in pinholes, pits, and surface erosion through sequential hydrolysis of crystalline and amorphous areas on the starch granule surface. The amylose content decreased with hydrolysis time, while the very short chains (DP < 6) increased rapidly at 3 h but decreased slightly later. After hydrolysis for 24 h, the protein content in RF increased from 8.52% to 13.17%. However, the processability of RF was properly maintained. Specifically, the data from DSC showed that the conclusion temperature and endothermic enthalpy of RS barely changed. The result of rapid RVA and rheological measurement indicated that RF paste viscosity and viscoelastic properties dropped rapidly after 1 h hydrolysis and thereafter recovered slightly. This study provided a new RF raw material useful for improving and developing RF-based foods. Rice is one of the most widely grown and consumed cereal crops in the world . In addiAccording to the origin of the protein, the reinforcement can be divided into either exogenous reconstitution or endogenous enrichment. At present, exogenous reconstitution is the main way, and protein concentrates/isolates, such as rice protein , whey prHowever, with starch-degrading enzymes developing, hydrolysis of raw starch at moderate temperatures could avoid the starch gelatinization step. The key enzymes in this heterogeneous hydrolysis are called granular starch hydrolyzing enzymes (GSHE) or raw starch degrading enzymes (RSDE), including α-amylase, β-amylase, and amyloglucosidase ,22. TherIn RF, conceivably, the presence of non-starch components (protein and fat) will restrict the efficiency of starch hydrolysis by the GSHE, the key factor determining the extent of elevation of the endogenous rice protein content. Therefore, it is important to understand the mechanism of GSHE to improve the efficiency of enzymatic hydrolysis. Rice starch (RS) is the major component in RF and the substrate of GSHE. In RF, RS displays a multi-scale structure mainly including six levels: individual branches (level 1), amylopectin and amylose (level 2), semi-crystalline lamellas (level 3), growth rings (level 4), starch granules (level 5), and whole-grain structure (level 6) . EnzymatThe DH, total starch, and total protein content of native and partially hydrolyzed RF and RS  showed t50 (the median diameter), D (De Broncker mean diameter), D (Sauter mean diameter), and span factor are summarized in 50, D, and D decreased significantly. After 24 h of hydrolysis, the porous appearance was reduced or even disappeared from the surface of the starch granules (RF-24), but different sorts of pits and craters arose and changed the surface from smooth to rough. This trend of surface variation was similar for the RF and RS groups, but the change in the RS group was more dramatic. However, for the RS group, d50 and D only decreased slightly, while D and the span factor did not change significantly, indicating that the hydrolysis only occurred on the surface of the starch granules. In addition, a unique phenomenon of massive hydrolysis was observed in the RS group, occurring simultaneously on a single starch granule, leading to the formation of numerous pore structures or hollow shells  [995/1022  [To further understand the erosion patterns of GSHE on the surface of starch granules, the crystalline structure and molecular order of the RF and RS groups were analyzed by wide-angle X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) . The rel starch) , and R99regions)  were calregions)  and a weregions) . After tregions) . Consideregions) ,41.1047/1022 slightly increased in the RF group but decreased in the RS group with increasing hydrolysis time compared with the native counterparts, although no significant difference was observed except for RF-24. The reduction in RS-1, RS-6, and RS-24 in the RS group possibly stemmed from the erosion effect exerted by the GSHE, leading to more disordered starch granules’ surfaces. For the RF group, the dissociation of some starch granules on the agglomerate surface exposed the starch granules encased inside, which were not hydrolyzed by GSHE and showed a higher short-range order, while hydrolyzed starch granules demonstrated a decreased order. Therefore, the changes in molecular order for the RF group were more complex.Information on the molecular order obtained from the deconvoluted FTIR curves B indicatAmylose and amylopectin constitute the fine molecular structure of starch that determines the basis of the multi-scale structure and influences the physicochemical properties of RF and RS . TherefoFurthermore, while the chain length distribution was not affected only by annealing treatment for 24 h , it disto1 and Tp1 compared with RS-N, indicating that the complete gelatinization of RF required a higher temperature.Thermal treatment is one of the most common RF-based food processing methods used for rice paste, rice bread, and rice noodles, and the thermal properties of native and partially hydrolyzed RF and RS during gelatinization were determined by differential scanning calorimetry (DSC) . Intereso1, and Tp1 of RS samples increased significantly, and Tc1-To1 decreased. The ΔH in DSC curves showed the amount of heat energy required to melt the crystalline region of starch, reflecting the ratio of crystalline to amorphous regions [1 of RS-1 and RS-6 slightly decreased but showed no significant difference compared with RS-N. However, RS-24 had the highest ΔH1, which was consistent with the highest relative crystallinity in the RS group  . CompareSetback viscosity (SV) could reflect the degree of short-term retrogradation of paste during cooling after thermal processing due to molecular rearrangement . The SV The frequency sweep of pastes formed by native and partially hydrolyzed RF and RS  can reflIn this study, RF and isolated RS were hydrolyzed at 55 °C by GSHE (containing α-amylase and amyloglucosidase) aiming to improve the protein content in RF by partially removing starch. During hydrolysis, GSHE exhibited unique erosion patterns on starch granules, which significantly changed the granular and molecular structure and affected starch agglomerates in RF, which was related to the mode of action of α-amylase and amyloglucosidase.The relative hydrolytic activity of α-amylase and amyloglucosidase during hydrolysis of RS by GSHE was compared by measuring the composition and relative content of oligosaccharides released, based on the fact that exo-acting amyloglucosidase only releases glucose. The results in w/v) RS suspension as substrate in 50 mM sodium acetate, pH 5.0, using the 3,5-dinitrosalicylic acid (DNS) method to quantify released reducing sugars [Dry-milled Indica RF, made in Taizhou, Jiangsu Province (purchased online) was passed through a 100-mesh sieve for later use. The experimental enzyme mixture (Novozyme 5009) was generously provided by Novozymes Co. Ltd. , including fungal α-amylase  and amyloglucosidase  and named GSHE in this work. The activity (8111 U/mL) was assayed at 55 °C with 1.0% (g sugars  and glucw/v) NaOH 1:4 (w/v), stirred continuously at room temperature for 4 h, and centrifuged . The yellow protein layer on top of the precipitate was discarded, and the residue was washed 3 times using 0.2% (w/v) NaOH until the yellow layer disappeared. The slurry was neutralized to pH 7 by 1 M HCl and washed 3 times with deionized water. The final starch sediment was dried at 45 °C for 48 h, ground in a mortar, and passed through a 100-mesh sieve.RS was isolated following the method of Liu et al.  with sliRS  and RF  were suspended in 100 mL 50 mM sodium acetate, pH 5.0, and preheated in a water bath shaker . GSHE (50 U/g dry starch) was added to the RF and RS slurries and incubated for 1, 6, and 24 h . The reaction was terminated by placing the slurries in an ice bath to cool instantly and rapidly adding 3 mL 1 M NaOH. After 15 min with intermittent shaking, pH was adjusted back to 5.0 by adding 3 mL 1 M HCl, and the slurry was centrifugated . The supernatant was collected to analyze the degree of hydrolysis (DH), and the sediment was washed 3 times using abundant deionized water. Finally, the hydrolyzed RS and RF were dried , ground in a mortar, and passed through 100-mesh sieves. The samples were named RS-1, RS-6, RS-24, RF-1, RF-6, and RF-24. Native samples without any treatment were named RS-N and RF-N.Furthermore, to investigate the hydrolytic mechanism of GSHE in detail, RS was also hydrolyzed by GSHE under the above process for 3 and 9 h, and the obtained samples were named RS-3 and RS-9. RS subjected to the above process was used as the control, named RS-C, but without the addition of GSHE .The total starch and amylose content were determined using a total starch assay kit (K-TSTA) and an amylose/amylopectin assay kit (K-AMYL), respectively. The total protein content was determined by an automatic Kjeldahl nitrogen analyzer  using the factor 5.95 to convert nitrogen content to crude protein content.DH was determined by the amount of reducing sugar in the supernatant measured by the DNS method  using thg, 30 min). Acetonitrile solution  was used as the mobile phase with 0.8 mL/min flow rate, the column temperature was 30 °C, and the loading volume was 50 μL. Glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltospentaose (G5), maltohexaose (G6), and maltoheptaose (G7) were used as the standard.The relative composition and content of released oligosaccharides were determined by high-performance liquid chromatography  equipped with X-Bridge BEH Amide column (250 mm × 4.6 mm). The supernatant of hydrolyzed starch was diluted with ultrapure water, and the same volume of pure acetonitrile was added and centrifugated . Deionized water was used as a dispersant, and the sample flour was added gradually to the sample pool. The sample was completely dispersed by ultrasound 3 times for 3 s each time. The measurement was carried out under the conditions of a pump speed of 1600 rpm and a shielding range of 10–15%. The refractive indexes of water and samples were 1.52 and 1.33, respectively . The meaThe micromorphology was captured by scanning electron microscope . Flour adhered to the conductive tape, and gold was sprayed onto the flour in a vacuum. The final observation was conducted at ×3000 magnification operating at a low accelerating voltage of 15 kV and irradiation voltage of 1 kV.XRD was conducted by using a diffractometer  equipped with Cu-Kα radiation and operated with scanning from 4–40° (4.79 °/min) at 0.04° with a count time of 0.5 s. RC was calculated by using Jade 6.0 software  as previously described .−1 with a resolution of 4 cm−1 by 64 scans. The air background spectrum was deducted from each tested sample. The region of 1200−800 cm−1 was chosen to analyze the short-range order of samples with OMNIC 8.0, the half-bandwidth, and enhancement factor were set as 19 cm−1 and 1.9, respectively. The ratios between intensities at 1047 and 1022 cm−1 (R1047/1022) and 995 and 1022 cm−1 (R995/1015) over deconvoluted spectra were calculated. FTIR spectra were obtained by an FTIR spectrometer  equipped with an ATR accessory in the region of 400–4000 cmg, 10 min), the supernatant was filtered (0.22 μm membrane filter), and 20 μL was injected onto a CarboPac PA200 column at 30 °C, eluted at a flow rate of 0.4 mL/min using isocratic 150 mM NaOH and a linear gradient of 0–400 mM sodium acetate as mobile phase [The branch chain length distribution of RS samples was determined by high-performance anion exchange chromatography with pulsed amperometric detection . Starch (10 mg) was suspended in 5 mL 50 mM sodium acetate, pH 4.5, and boiled for 30 min to be completely gelatinized. The gelatinized starch was fully debranched by 2 U isoamylase . After inactivation by boiling for 10 min and centrifugation . First, 3 mg of sample powder and 6 mg of deionized water were added to an aluminum pan. After a slight shaking, sample pans were sealed and equilibrated overnight at 4 °C. The temperature was linearly increased from 30 to 100 °C° at 10 °C/min. An empty aluminum pan was used as a reference. To, Tp, Tc, and ΔH were reported for each endothermic peak.The pasting properties of samples were determined using a rapid viscosity analyzer . Deionized water was added to each sample (3.0 g on a 14% moisture basis) to a total weight of 28 g, and the analysis was conducted as previously described . ViscosiThe rheological characterization was done using a rheometer  with a parallel-plate system (40 mm diameter) and a working gap of 1000 mm. After the gelatinization of samples by RVA . A By controlling the reaction time of GSHE to RF, significant endogenous enrichment of the rice protein was achieved, while appropriate processing properties of RF were maintained. After the 24 h hydrolysis, the relative rice protein content improved from 8.52% to 13.17%. The results of the microstructure, crystal structure, and molecular structure of the RF indicated that the GSHE treatment rapidly increased the very short chains of amylopectin through the early-stage pinhole erosion, which then changed to pit erosion, further hydrolyzing the very short chains and increasing the relative content of the middle and long chains. In the whole process, the crystalline and amorphous regions were hydrolyzed, causing a slight increase in relative crystallinity. This hydrolytic mechanism led to a sharp decrease in viscosity during pasting after 1 h of hydrolysis followed by a slow increase, similar to the change in the viscoelastic properties of the pastes, but without significantly changing the thermal properties, which confirms that the physicochemical properties of RF can be properly preserved. The study brings more insights into structure–function relationships during the GSHE hydrolysis and creates a new RF raw material with high protein content by endogenous enrichment for application in various RF-based food, such as rice noodles and reconstituted rice, especially for gluten-free food, such as muffins, cakes, and bread.