We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aβ antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle’s binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer’s disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (A The pathologic cascade of neuronal dysfunction, synaptic loss, and neuronal cell death that leads to cognitive decline in AD has been linked to the formation of neurotoxic Aβ40–42 oligomers and aggregated Aβ fibrils in plaques .2. At 85% confluency, the cells were harvested using trypsin/EDTA (0.25%) and phenol red  and seeded into T25 flasks, 24-well plates, or 96-well plates, according to the experiment being performed.U-251 cells were obtained from the American Type Culture Collection (ATCC), sub-cultured, and then used to determine cytotoxicity after exposure to iron nanoparticles and NP-Ab. The cells were cultured in Gibco Roswell Park Memorial Institute (RPMI) 1640 media (Thermo Fisher Scientific), supplemented with 10% foetal bovine serum (FBS), 1% L-glutamine , and 1% antibiotic/antimycotic  at 37 °C under a humidified atmosphere of 5% COU-251 cells were used to evaluate the in vitro toxicity of iron nanoparticles and NP-Ab. Cells were seeded as described above into a flat-bottom 24-well plate and incubated at 37 °C. Iron nanoparticles were vortexed for 20 s and sonicated in a bath sonicator for 5 × 3 s with 5 s breaks in between to minimize agglomeration of the particles prior to cell treatment. NP-Ab were resuspended by gently pipetting. Different concentrations of both nanoparticles were added to the cells (0.1 µM−500 µM) and incubated for 24 h. Untreated cells were used as a negative blank control. All samples were assayed in triplicate. Following exposure, the cell supernatant was collected, and the cells were washed three times with 1x PBS. Then, 200 µL of 1x PBS was added to each well and a probe sonicator was used for 30 s in each well until the cells were completely homogenized.Homogenized cells and supernatants were kept at −20 °C until analysis. A 200 µL aliquot of supernatant was used to perform a lactate dehydrogenase cytotoxicity assay (Thermo Fisher Scientific), normalized to protein level obtained with a protein BCA assay kit per the manufacturer’s instructions. Each sample was assayed in triplicate. One-way ANOVA with Tukey’s post hoc test was used to evaluate differences between treatment groups .Salmonella typhimurium (TA100) carrying mutations in the operon coding for histidine biosynthesis and expressing the CYPP 1A1 enzyme. This test was carried out according to the manufacturer’s instructions. The growing Salmonella typhimurium TA100 strain culture was added to 96-well flat-bottomed plates, each containing a fixed concentration of iron nanoparticles. The plates were incubated at 37 °C for 3 days. Sodium azide was used as the positive control in this assay. The scores for the blank plate, the background control plates, and the positive control plates obtained were within the manufacturer’s specifications, indicating the validity of the results obtained for the aqueous extract.Bacterial mutagenicity of iron nanoparticles was determined using the Muta-chromoplate™ Kit , which is based on the widely used ‘Ames test’ . This te2 incubator. The cells were treated with 3 concentrations of iron nanoparticles and incubated for 24 h. Each treatment had 6 replicates. The ROS inducer pyocyanin (200 µM) and 50 µL of MilliQ water were used as positive and negative controls, respectively. Following treatment, the solution was removed from each well and 100 µL of ROS/superoxide detection reagent was added. The plate was incubated for 30 min at 37 °C and measured in a fluorescence reader for fluorescein  and for rhodamine .The production of reactive oxygen species (ROS) and superoxide in cells in response to the iron nanoparticles was determined using a total ROS/Superoxide detection kit, according to the manufacturer’s instructions . Cells were seeded onto black-walled 96-well plates and incubated at 37 °C overnight in a COU-251 cells were incubated for 24 h with iron nanoparticles and NP-Ab with a total concentration of 5 µg of iron. Following incubation, supernatants were collected, and cells were washed three times with 1x PBS. Washes were pooled with supernatant to capture any nanoparticles that had detached from the cell surface. Wells were filled with 200 µL of 1x PBS and homogenized using a probe sonicator for 30 s. The iron concentration in the intracellular and extracellular compartments was evaluated using ICP-MS.4 in 0.1 M sodium phosphate buffer for 60 min at 4 °C and dehydrated in increasing concentrations of ethanol. The samples were infiltrated with Procure 812 resin and polymerised at 60 °C for 48 h. Then, ultrathin (70 nm) sections were cut on a Leica EM UC6 Ultramicrotome and placed on carbon-coated copper mesh grids that were post-stained with uranyl acetate and lead citrate. Images were obtained using a Jeol JEM 1400 electron microscope operating at an accelerating voltage of 100 kV.U-251 cells were grown on coverslips to 70% confluence and treated with nanoparticles for different incubation times , followed by fixation with 2% glutaraldehyde and 2% paraformaldehyde for 24 h at 4 °C. After fixation, the coverslips were washed in 0.1 M sodium phosphate buffer (pH 7.4), post-fixed in 2% OsO® 488 rabbit monoclonal anti-LAMP1 antibody . Finally, cell nuclei were counterstained with 0.5 mg/mL DAPI for 40 min and following washing, coverslips were mounted onto slides. Stained cells were imaged using an Aperio XT slide scanner. Fluorescence and brightfield images were taken to capture the nanoparticles and antibody staining. Phalloidin was used as an actin skeleton marker for fluorescence quantification using CellProfiler .U-251 cells were grown on coverslips and incubated with NP-Ab and iron nanoparticles as for TEM imaging. Control cells were treated with 1x PBS at each time point. Following incubation, cell media was removed, and cells were fixed in 4% paraformaldehyde for 20 min at ambient temperature. The cells were washed three times with 1x PBS between each step. Antigen retrieval was performed in TE buffer (pH 9) at 90 °C for 10 min, followed by permeabilization with 0.25% Triton X-100 for 10 min. The cells were then incubated with a blocking buffer  for 40 min. Subsequently, the cells were incubated for 2 h at ambient temperature with a recombinant Alexa Fluorg at ambient temperature; subsequently, the media was removed, and the cell pellet was resuspended in 1x PBS to wash. This was repeated 3 times. Finally, cells were resuspended in 200 mL of 1x PBS and frozen at −80 °C until further analysis.Once the cells had reached 80–90% confluency in the T25 flasks, a 100 µM concentration of either iron nanoparticles or NP-Ab was added to the cell media and incubated for 24 h under normal cell growth conditions. Untreated flasks were used as controls. For proteome analysis, the media was removed and fresh unsupplemented media was added prior to harvesting the cells using a cell scraper. Cells were centrifuged for 5 min at 500× Cells were resuspended in RIPA lysis buffer  containing protease inhibitor, 0.1 mM phenylmethylsulfonyl fluoride . Cells were lysed using 5 × 5 to 10 s bursts of the probe sonicator, with chilling on ice conducted in between the sonication steps.Cysteine disulphides in proteins were reduced in 2 mM tris (2-carboxyethyl)phosphine (TCEP) at 60 °C for 60 min, followed by cysteine alkylation in iodoacetamide for 10 min at ambient temperature. To remove detergents, reducing and alkylating agents and other non-protein components, and 1 mL of cold acetone containing 1% HCl, were added to each sample and placed in a −20 °C freezer for 12–14 h to precipitate proteins. The protein precipitate was centrifuged , and the supernatant was decanted and discarded. The pellets were air dried for 2–3 min and resuspended in lysis buffer . Proteins were captured and desalted using S-Trap Mini spin traps , following the manufacturer instructions. On-trap digestion was performed by adding 1 µg of sequencing-grade trypsin  in 50 mM ammonium bicarbonate (100 µL) to each sample, overnight at 37 °C. Peptides were recovered from the S-trap using sequential elutions with 100 µL, each containing 0.2% formic acid and 50% acetonitrile, followed by a 2 min centrifugation  at each step, and pooling all eluents. The final pooled elutions were dried under vacuum centrifugation  and the desiccated pellet was resuspended in 50 µL of 1% formic acid containing 0.2% heptafluorobutyric acid. Prior to LC-MS/MS analysis, protein concentration was estimated using a micro-spectrophotometer  measuring absorbance at a wavelength of 280 nm, and quantification of mg/mL protein was determined using the extinction coefficient of albumin (BSA).2O/CH3CN of 98:2, containing 0.1% formic acid) and buffer B . The mass spectrometer settings included the following parameters: ion spray voltage 2000 V, capillary temperature 300 °C, positive ion mode, survey scan acquired (m/z 375–1750), and up to 10 multiply charged ions (charge state ≥ 2+) isolated for MS/MS fragmentation.Biological triplicates of all samples were analysed using a Q-Exactive Plus mass spectrometer  connected to a nano-LC, Dionex UltiMate 3000 high-performance liquid chromatography (HPLC) system , equipped with an autosampler . Mass spectrometer parameters for data-dependent acquisition (DDA) analysis, as well as nano-LC conditions, have previously been described . Brieflyhttps://www.maxquant.org (accessed on 9 August 2023)). The Uniprot database was used for searching and quantification, employing the label-free quantification (LFQ) method with a minimum ratio count of 2 and a minimum number of neighbours of 3. Default settings were used for all other identification and quantification parameters. One replication of the iron nanoparticles was excluded from the analysis due to low protein counts. Perseus computational platform version 2.0.6.0 (downloaded from https://www.maxquant.org/perseus (accessed on 9 August 2023)) was used for subsequent statistical processing [p-value threshold of <0.05, and the Student’s t-test was used for calculating the p-value. Gene ontology (GO) classification was performed with the aid of the DAVID website (https://david.ncifcrf.gov (accessed on 9 August 2023)), and the results were visualized using GraphPad PRISM 9. Canonical pathway analysis was performed with the Ingenuity pathway analysis (IPA) software . Lastly, the significantly upregulated and downregulated proteins were analysed using the STRING database (https://string-db.org/ (accessed on 9 August 2023)) to determine the protein networks.The raw data files obtained from the mass spectrometer were subjected to analysis using MaxQuant version 2.1.3.0 ). Their brains were extracted and fixed in 4% paraformaldehyde solution for 3 days, followed by paraffin embedding for sectioning on a microtome. Sagittal sections were cut at a 5 μm thickness.Brain sections used for staining were obtained from the brains of APP/PS1 and wild type (WT) mice under procedures approved by the University of New South Wales Animal Ethics Committee (Ethics number: 19/56b) and which were performed in accordance with the Australian National Health and Medical Research Council guidelines for the care and use of animals for scientific purposes. Mice were housed at a constant temperature of 21 °C with a 12 h light/12 h dark cycle with ad libitum access to standard chow and water. Animals were euthanized with a lethal dose of pentobarbitone , as per the manufacturers’ instructions. Adjacent sections on the same slide were incubated with either NP-Ab, rabbit polyclonal anti-Aβ antibody (1:600) as a positive control, or 1x PBS as a negative control for 20 min, followed 3,3′diaminobenzidine  for 5 min. Following three washes in 1x PBS, the slides were mounted with mounting medium and visualized using fluorescence microscopy .To determine the ex vivo Aβ plaques in ex vivo brain sections of mice were successfully immunostained using NP-Ab in the place of a primary antibody, indicating the presence of active binding sites on the nanoparticle-conjugated antibodies (β(1–42) peptide and rule out possible effects of the conjugation on antibody binding ability (β(1–42) peptides were used as they are fibrillogenic, the main constituent of Aβ plaques deposited in the brain [β(1–40) form of the peptide [β(1–42) peptide to approximate the NP-Ab-binding affinity for Aβ. The association and dissociation curves for the interaction between ProG-NP-Ab and Aβ are shown in β antibody functionalization.Spherical  iron cortibodies . BLItz/O ability . Aβ (p < 0.05). Interestingly, the highest LAMP1 intensity was observed at 1 h following incubation in both the iron nanoparticle- and NP-Ab-treated cells. The exception observed at the 1 h time point may suggest a potential temporal effect of the treatment on LAMP1 expression, resulting in increased lysosomal activity. Meanwhile, at the later timepoints, the iron deposits, which can be observed in the brightfield images, may quench the fluorescent signal in the images.Compared to the control cells treated with 1x PBS, cells treated with iron nanoparticles showed a significantly higher LAMP1 signal intensity at all time points examined ( < 0.05) E. In con, with the relaxivity values of the iron nanoparticles and NP-Ab being 129.29 mM−1s−1 and 55.99 mM−1s−1, respectively. R2 measurements of the commercially available contrast agent Resovist were also completed (2 =r 183.88 mM−1s−1).To evaluate the contrast-enhancing ability of iron nanoparticles and conjugated NP-Ab, the transverse T2 relaxation times at 9.4 T were evaluated using in vitro MRI. Increasing the concentrations of both the iron nanoparticles and NP-Ab led to the concentration-dependent darkening of the MR images . FunctioIn this study, label-free quantification (LFQ) was employed to measure the abundance of proteins. Following this, a total of 1398 proteins were quantified, with a false discovery rate of less than 1%, and were subsequently subjected to statistical sorting . The volTo determine the characteristics of the DEPs, we employed the GO enrichment analysis, which is categorized into three groups: biological process (BP), molecular function (MF), and cellular component (CC). The outcomes were sorted based on their enrichment score (EASE score), and we have presented the top 10 results for each category. In cases where there were limited outcomes to exhibit, we have included all significant enrichments .Between the iron nanoparticle and control groups, 93 DEPs showed most significant GO terms that were related to ‘sister chromatid cohesion’, ‘cytoplasmic translation’, ‘protein import into nucleus’, ‘apoptotic process’, ‘translation’, ‘establishment of meiotic sister chromatid cohesion’, ‘positive regulation of vascular smooth muscle cell proliferation’, ‘positive regulation of cell migration’, ‘mRNA export from nucleus’, and ‘regulation of cell shape’ in the BP category, ‘RNA binding’, ‘protein binding’, ‘chromatin binding’, ATPase activity’, ‘cadherin binding’, ‘protein C-terminus binding’, ‘syndecan binding’, ‘heparan sulfate binding’, ‘chondroitin sulfate binding’, and ‘endopeptidase activator activity’, in the MF category, and ‘membrane’, ‘extracellular exosome’, ‘cytosol’, ‘focal adhesion’, ‘cytoplasm’, ‘postsynapse’, ‘nucleoplasm’, ‘cytosolic ribosome’, ‘ribosome’, and ‘mitochondrial intermembrane space’ in the CC category A.GO analysis of the DEPs between the NP-Ab and control groups yielded the top 10 most significant results for ‘translation’, ‘cytoplasmic translation’, ‘mitochondrial translation’, ‘tricarboxylic acid cycle’, ‘2-oxoglutarate metabolic process’, ‘acetyl-CoA biosynthetic process from pyruvate’, ‘regulation of translational initiation’, ‘ER to Golgi vesicle-mediated transport’, ‘protein stabilization’, and ‘purine nucleotide biosynthetic process’ in the BP, ‘RNA binding’, ‘structural constituent of ribosome’, ‘protein binding’, ‘cadherin binding’, ‘pyruvate dehydrogenase (NAD+) activity’, ‘ribosome binding’, ‘ubiquitin protein ligase binding’, ‘mRNA binding’, ‘protein transmembrane transporter activity’, and ‘formate-tetrahydrofolate ligase activity’ in the MF, and ‘membrane’, ‘extracellular exosome’, ‘focal adhesion’, ‘cytosol’, ‘mitochondrial inner membrane’, ‘mitochondrion’, ‘ribosome’, ‘cytosolic ribosome’, ‘autolysosome’, and ‘mitochondrial small ribosomal subunit’ in the CC categories, as presented in p-value < 0.05). The affected canonical pathways are shown in p-value and z-score, which is an algorithm that is used to predict more active functions. Additionally, we conducted an analysis of the overlapping canonical pathways between the iron nanoparticles vs. control groups and NP-Ab vs. control groups, which were categorized as ‘Mitochondrial Dysfunction’, as shown in We employed ingenuity pathway analysis (IPA) to analyse the canonical pathways, which were evaluated based on the Fisher’s exact test .To better understand the interaction between these DEPs, we employed a protein–protein interaction (PPI) network analysis using the STRING database . The resAβ plaques in the brain. This would permit patients access to these disease-modifying treatments or, on the other hand, prevent misdiagnosis and provide more certainty around the prescription of these and future medications. The iron nanoparticles in our study are tailored for neurodiagnostic assessments, as they are able to achieve high saturation magnetization while keeping the core size small, a property essential for promoting blood–brain barrier permeation [Aβ-targeting ligand, while maintaining their stability and biocompatibility.A timely diagnosis of AD is necessary to ensure that patients benefit the most from therapeutic interventions earlier in the disease progression. Our project aimed to conduct a preliminary investigation of a nanoformulation developed to enhance MRI contrast between the amyloid plaques and the parenchyma in AD. This would enable an earlier imaging diagnosis of AD as an alternative to PET, which is less widely accessible than MRI and requires the use of a radioactive tracer. The recent FDA approval of the AD drugs aducanumab and lecanemab has amplified the urgent need for an accessible method for confirming the presence of rmeation . The addr2 relaxivity of our samples. Briefly, the expected signal change depends on the product of signal change per particle (as determined by its relaxivity) and the local particle density. The measured r2 relaxitivity of our particle without functionalization was 129.29 mM−1s−1, which is slightly lower than the r2 of Resovist (~180 mM−1s−1), which was used as a commercial reference. One possible reason for this is the currently thicker coating of our particles. This is something which will be addressed in future research; however, the focus of this study was to synthesize particles which can be directly functionalized—a feature not available on the commercial counterpart. Expectedly, the addition of antibodies to the surface decreased the r2 values in magnetic nanoparticles by increasing the distance between the water molecules and the metal centre, among other mechanisms. In our nanoparticles, relaxivity reduces from r2 = 129.29 mM−1s−1 to r2 = 55.99 mM−1s−1 when the antibodies were conjugated. Binding studies demonstrated the NP-Ab binding to the Aβ(1–42) peptide; although, their binding affinities were lower than that of the antibodies alone. Further developments will be necessary to improve the sensitivity of the contrast agents to require lower doses while maintaining an effective MRI signal.As a major direct indicator for expected MRI performance, we measured the β antibodies on cell survival has been primarily explored as a response to toxicity induced by Aβ [β, protects cortical neurons against Aβ-induced cell death by downregulating the c-Jun NH2 terminal kinase (JNK) pathway, which is activated during times of cellular stress, and the NF-κB signalling pathway [To ensure the clinical success of future nanoformulations, it is essential to comprehensively evaluate and describe the in vitro and in vivo behaviour of nanoparticles and possible mechanisms of toxicity and elimination routes. In our study, following 24 h of treatment, cell viability was initially evaluated using the lactate dehydrogenase assay. Cell viability remained high, even at the highest concentrations of iron nanoparticles and NP-Ab. Interestingly, functionalization with antibodies resulted in a decreased relative lactate dehydrogenase level at the highest concentration of 500 µM. However, the protective effect of anti-Aed by Aβ ,53. IgG ed by Aβ . Intrave pathway . AlthougElectron microscopy results demonstrated that both the iron nanoparticles and conjugated NP-Ab were readily endocytosed by glioblastoma cells within 5 min of incubation . MembranProteomics is a high-throughput technology which allows further insight into nanoparticle interactions with the cellular environment. The aspects of the proteome which are affected depend on their material composition, size, route of administration, and the cells or tissues used for the analysis. Previous studies looking at the proteomic changes induced by magnetic nanoparticles have looked at NRK-52E cell lines , SH-SY5Y3 [+2 into Fe+3 and prevents the production of ROS. Magnetic nanoparticles localizing in the lysosomes as part of the degradation pathway decompose to free iron ions [The existence of endogenous iron processing and recycling mechanisms within cells lends itself to the biocompatibility of iron nanoparticles. In our study, multiple DEPs following both treatments  corresponded to proteins that have been previously shown to be altered in neuroblastoma cells treated with FeCl3 , suggest3 ,69, whil3 . Downregron ions ,71, whicron ions . CorrespProtein changes related to cytoskeletal organisation and cell migration and motility correspond to the potential internalization mechanisms observed with TEM. Downregulation of the src substrate cortactin (CTTN), pleiotrophin (PTN), and upregulation of the hyaluronic acid receptor (CD44) and RDX was common to treatments with both iron nanoparticles and NP-Ab. Cytoskeletal organization and cell shape are regulated by PTN, which exerts its effects through the ligation of the cell surface-binding protein N syndecan, resulting in the phosphorylation of src and CTTN . In addiFollowing endocytosis, nanoparticles may be trafficked towards lysosomes via endosomes, either for degradation or lysosomal exocytosis . FollowiThe endoplasmic reticulum of cells is very sensitive to extracellular stimuli, and certain events, such as nutrient deprivation, oxidative stress, or saturation of protein folding, can result in endoplasmic reticulum stress. Exposure to nanoparticles, such as iron oxide nanoparticles, gadolinium-based contrast agents, and manganese oxide nanoparticles, has been shown to induce endoplasmic reticulum stress in mice . SimilarThis study provided an initial in vitro bioevaluation of novel iron nanoparticles and targeted nanoparticles for the diagnosis of AD. The cellular data support the biocompatibility of these conjugates; however, the cell lines used were not representative of in vivo conditions; moreover, only one cell type was evaluated in this study. It is essential to complete in vivo assessments of nanoparticle safety and efficacy, as well as their blood circulation time and stability. Proteomic investigations demonstrated the pathways implicated in cellular internalization and response to nanoparticle treatment. While the predominant proteomic changes involved energy metabolism, protein turnover, and cytoskeletal reorganization, these results also present the potential pathways of cytotoxicity and cellular stress. To ensure the successful clinical translation of the described nanoparticles, these pathways must be investigated further, and the dosing optimized to improve biocompatibility. Furthermore, a major challenge for neurodiagnostic and neurotherapeutic materials is achieving a sufficient crossing of the blood–brain barrier. As the blood–brain barrier is composed of endothelial cells, pericytes, and astrocytes , the endβ for their use as T2-weighted MRI contrast agents. The DMSA-coated iron nanoparticles and Aβ-targeted NP-Ab demonstrated in vitro biocompatibility, as determined using cellular assays and discovery proteomics, as well as favourable MRI relaxivity . TEM and ICP-MS studies demonstrated that the nanoparticles were readily internalized by glioblastoma cells and localized within cellular organelles. Proteomics results indicated that cells initiate compensatory mechanisms to respond to nanoparticle internalization, with enrichment observed in proteins implicated in energy metabolism and translation processes, and in the case of the NP-Ab, in the iron storage machinery. The NP-Ab were able to effectively stain ex vivo Aβ plaques and demonstrated their binding affinity for the Aβ(1–42) peptide. However, the addition of the targeting ligand increased the hydrodynamic size (369.5 nm) and reduced the MRI contrast-enhancing properties of the nanoparticles. Future work should focus on improving the sensitivity of the particles, while maintaining their stability in biological media, as well as assessing their efficacy and biocompatibility in an animal model. Early diagnosis is imperative for the successful treatment of neurodegenerative diseases, and contrast-enhanced MRI is a promising and powerful early detection tool. The results presented here demonstrate the excellent potential of biocompatible iron nanoparticles targeted at amyloid pathology for use in AD diagnosis.In this work, we propose an approach to improve the sensitivity of current neuroimaging methods for the early diagnosis of AD. We present novel iron core-iron oxide core shell nanoparticles targeted to A


Longitudinal epidemiological data are scarce examining the relationship between dietary patterns and respiratory outcomes in childhood. We investigated whether three distinct dietary patterns in mid-childhood were associated with lung function and incident asthma in adolescence.1), forced vital capacity (FVC), and forced expiratory flow at 25–75% of FVC (FEF25–75) were measured at 15.5 years and were transformed to z-scores based on the Global Lung Function Initiative curves. Incident asthma was defined by new cases of doctor-diagnosed asthma at age 11 or 14 years.In the Avon Longitudinal Study of Parents and Children, ‘processed’, ‘traditional’, and ‘health-conscious’ dietary patterns were identified using principal components analysis from food frequency questionnaires at 7 years of age. Post-bronchodilator forced expiratory volume in 1 s  and FVC , while the ‘processed’ pattern was negatively associated with FVC . Associations between the ‘health-conscious’ and ‘processed’ patterns and lung function were modified by SCGB1A1 and GPX4 gene polymorphisms. We found no evidence of an association between the ‘traditional’ pattern and lung function, nor between any pattern and FEF25–75 or incident asthma.In multivariable-adjusted models, the ‘health-conscious’ pattern was positively associated with FEVA ‘health-conscious’ diet in mid-childhood was associated with higher subsequent lung function, while a diet high in processed food was associated with lower lung function.The online version contains supplementary material available at 10.1186/s12931-023-02383-9. Whatisalreadyknownonthistopic: Evidence on the potential role of dietary patterns in the aetiology of childhood asthma and impaired lung function is very limited, and longitudinal studies are scarce.Whatthisstudyadds: We found that children with a higher score on the ‘health-conscious’ pattern had higher lung function in adolescence, whereas children with a higher score on the ‘processed’ pattern had lower lung function. These relationships were not explained by potential mediators such as adiposity, dietary fibre, vitamin D, long-chain omega-3, and antioxidants, but there was evidence that they may partly depend on genetic make-up. We did not find any evidence of association with incident asthma.How this study might affect research, practice or policy: Beyond possible effects of individual foods and nutrients, dietary patterns may have an additional influence on lung function growth. If our findings are replicated, this might inform potential dietary interventional studies to improve lung health.Accumulating evidence has shown the importance of maximal lung function attainment as lung function in early adulthood is linked to subsequent comorbidities and early mortality , 2. ChilPrevious studies on the link between various dietary patterns and asthma or lung function in childhood were mostly cross-sectional or case–control, with few exceptions that were either in children with asthma  , in veryIn this study, we have explored the relations of three distinct empirical dietary patterns at 7 years of age, previously derived using principal component analysis  , with luwww.alspac.bris.ac.uk, which contains details of all available data .We used the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based birth cohort that recruited pregnant women resident in Avon, UK  with due dates between April 1991 and December 1992. Participants have been followed up since birth with questionnaires and objective measures in research clinics. The study protocol has been described previously  and furtWe used three previously defined dietary patterns, ‘health-conscious’, ‘traditional’, and ‘processed’, which were derived using PCA on 57 standardised food items  collecteThe daily intake of each food group was estimated using standard portion sizes based on typical consumption patterns in Britain  adapted 1), forced vital capacity (FVC), and forced expiratory flow at 25–75% of FVC (FEF25–75), before and 15 min after administration of 400 mg of salbutamol. We transformed these lung function measurements to z-scores based on the Global Lung Function Initiative (GLI) curves, adjusting for age, height, and ethnicity, and separately by sex [https://github.com/thlytras/rspiro]  . ‘Post-bIncident asthma was our second primary outcome of interest. We defined current doctor-diagnosed asthma at ~ 7.5 years (91 months), ~ 11 years (128 months), and ~ 14 years (166 months) if mothers responded positively to the question “Has a doctor ever actually said that your study child has asthma?” and to at least one of the questions which asked if the child had had wheezing, wheezing and whistling in the chest, asthma, or asthma medication in the last 12 months. There was a good agreement between parental reports of a doctor’s diagnosis of asthma in ALSPAC and GP-recorded diagnosis  . We defiGSTT1 and GSTM1, and a missense substitution in GSTP1  [GPX4) gene (rs713041) [AHR) gene (rs2066853) [SCGB1A1) gene (rs3741240), which encodes the club cell secretory protein (CC16). The latter single nucleotide polymorphism (SNP) was shown to have the strongest correlation with serum levels of CC16 protein in a genome-wide association study [As the ‘health-conscious’ and ‘traditional’ dietary patterns are likely to be antioxidant-rich (and the ‘processed’ pattern antioxidant-poor) , we hypo rs1695) , 33; alss713041) , the ary2066853)  which is2066853) , and the) gene rs41240, whon study  and is pon study  , data on post-bronchodilator lung function measures at 15.5 years and incident asthma were complete for 2,950 − 3,085 (depending on the specific measure) and 4,491 participants, respectively , as well as body mass index (BMI) and fat mass index (FMI) at 9 years of age.10, tertiles) at age 7 years, and by antioxidant gene variants listed above  and outdoor particulate matter up to 10 µm in size  after a mean (± SD) 8.7 (± 0.3) years of follow-up, but no evidence of association with FEV1/FVC or FEF25-75 (Table 1 (P for non-linearity 0.03), but no evidence of association with FEV1/FVC or FEF25-75. The ‘traditional’ dietary pattern was not associated with any lung function measure.After adjusting for potential confounders, including several indicators of SES, we found a linear positive association between the ‘health-conscious’ pattern score and FEV1 and FVC were robust to further adjustment for the ‘processed’ and ‘traditional’ dietary patterns, maternal ‘health-conscious’ dietary pattern in pregnancy, and any supplement use, or exclusion of children of non-white mothers (~ 2.6%), and those with extreme energy intakes  and trace elements (zinc and selenium), vitamin D, preformed vitamin A, long-chain omega-3 fatty acids from fish, or fibre, nor by BMI or FMI at 9 years of age when we further adjusted for these factors separately  and FVC , but not in children without asthma  . However, the associations between the ‘health-conscious’ pattern and lung function were modified by GPX4 rs713041 genotype; positive associations were suggested in carriers of the T allele, but not in those who were homozygous for the C allele ; similarly, the ‘processed’ pattern was negatively associated with these outcomes only in the GG group . The only exception was a weak interaction between the ‘traditional’ dietary pattern and the GSTP1 rs1695 SNP .We did not find evidence of any association between the three dietary pattern scores and incident asthma Table , and thi1, whereas a higher ‘processed’ pattern score was associated with a lower FVC and FEV1, independent of multiple SES indicators. We did not find evidence for an association between the ‘traditional’ pattern score and lung function, or between any dietary pattern and incident asthma. To our knowledge, these are novel findings, robust to various sensitivity analyses.In ALSPAC children, we found that a higher ‘health-conscious’ dietary pattern score in mid-childhood was associated with a higher subsequent FVC and FEV1 between the top and bottom quartiles of ‘health-conscious’ and ‘processed’ dietary pattern scores were comparable to the mean difference in z-scores of pre-bronchodilator FEV1 comparing children with and without asthma  [1 and FVC (hence no association with their ratio), suggesting a proportional influence on airway and alveolar development. Furthermore, the stronger associations with post- than pre-bronchodilator FEV1 suggest that dietary patterns may be more related to growth and calibre, rather than tone, of large and small airways. Given that dietary patterns [The differences in FEV1, 0.37) ; thus wepatterns  and lungpatterns  track du1 [GSTM1, GSTT1, GSTP1, and AHR) or by air pollution . These findings suggest that the potential benefits of the ‘health-conscious’ pattern on lung function may not be explained by higher antioxidant intake. On the other hand, we found evidence of interactions between the ‘health-conscious’ pattern and maternal smoking and another antioxidant gene variant on lung function. The positive associations between the ‘health conscious’ pattern and lung function were only seen in carriers of the T allele of the rs713041 polymorphism of the GPX4 gene, who have an impaired antioxidant defence because of weaker GPX enzyme function, particularly when selenium intake is suboptimal [Dietary antioxidants have received attention for decades because of their anti-inflammatory effects and the transition from a traditional to a modern diet . Despite1 . In contboptimal . Thus, tboptimal , and in boptimal .SCGB1A1 rs3741240 SNP which regulates circulating levels of CC16 protein [1 and FEF25-75 in childhood, with a similar interaction with the SCGB1A1 polymorphism indicating a greater potential for up-regulation in the GG group [SCGB1A1 polymorphism.We also found evidence for interactions between both ‘health-conscious’ and ‘processed’ patterns and the  protein , 47, incGG group . HoweverThe ‘health-conscious’ and ‘processed’ patterns generally describe a nutrient-rich, versus an energy-dense, nutrient-poor diet, respectively , 31. In Of note, as the ‘health-conscious’ and ‘processed’ patterns scores were derived not to be correlated, their opposite associations with lung function may reflect different mechanisms. We speculate that the link between diet and lung function may be mediated by altered microbiota  or alterThe post-bronchodilator assessment of lung function, provided by the ALSPAC cohort, enabled us to better assess airway growth by eliminating reversible airflow limitation. Also, the availability of genotype data enabled us to test gene-diet interactions. We controlled for numerous potential confounders in the analyses; however, the possibility of unmeasured or residual confounding cannot be ruled out. Regarding the multiple analyses carried out, given the a priori nature of the hypotheses and the correlation between lung function measures, it did not seem appropriate to correct for multiple testing ; howeverA healthier diet in mid-childhood was associated with higher subsequent lung function, while a diet high in processed food was associated with lower lung function. Future studies are needed to replicate our findings and to elucidate underlying mechanisms.Additionalfile1:FigureS1. Study profile. FigureS2. Directed acyclic graph to study covariates and potential structural confounding bias for the association between child’s dietary patterns and lung function. TableS1. Summary of the factor loadings of foods in the dietary patterns extracted using principal component analysis from the FFQ at 7 years (loadings above 0.3 are shown in bold). TableS2. Details of selected polymorphisms. TableS3. Participant characteristics according to quartiles of ‘health-conscious’ dietary pattern score at 7 years of age. TableS4. Participant characteristics according to quartiles of ‘traditional’ dietary pattern score at 7 years of age. TableS5. Participant characteristics according to quartiles of ‘processed’ dietary pattern score at 7 years of age. TableS6. Linear regression coefficients  for pre-bronchodilator lung function measures (z scores) at 15.5 years according to quartiles of dietary pattern scores at 7 years, adjusted for potential confounders. TableS7. Linear regression coefficients  for post-bronchodilator lung function measures (z scores) at 15.5 years according to quartiles of dietary pattern scores at 7 years, adjusted for additional potential confounders as well as exclusions . TableS8. Linear regression coefficients  for pre-bronchodilator lung function measures (z scores) at 8 years according to quartiles of dietary pattern scores at 7 years, adjusted for potential confounders. TableS9. Linear regression coefficients  for post-bronchodilator lung function measures (z scores) at 15.5 years according to quartiles of dietary pattern score at 7 years, further adjusted for potential mediators. TableS10. according to quartiles of dietary pattern score at 7 years, stratified by asthma status at 7.5 years. TableS11. According to quartiles of dietary pattern score at 7 years, stratified by maternal smoking in childhood. TableS12. according to quartiles of the health-conscious dietary pattern score at 7 years, stratified by GPX4 genotype (rs713041). TableS13. Odds ratio  for incident asthma at 11 or 14 years according to quartiles of dietary pattern score at 7 years, adjusted for additional factors as well as exclusions .


Vigna angularis), V. riukiuensis “Tojinbaka” and V. nakashimae “Ukushima” were shown to have much higher levels of salt tolerance than azuki beans. To identify the genomic regions responsible for salt tolerance in “Tojinbaka” and “Ukushima”, three interspecific hybrids were developed: (A) azuki bean cultivar “Kyoto Dainagon” × “Tojinbaka”, (B) “Kyoto Dainagon” × “Ukushima” and (C) “Ukushima” × “Tojinbaka”. Linkage maps were developed using SSR or restriction-site-associated DNA markers. There were three QTLs for “percentage of wilt leaves” in populations A, B and C, while populations A and B had three QTLs and population C had two QTLs for “days to wilt”. In population C, four QTLs were detected for Na+ concentration in the primary leaf. Among the F2 individuals in population C, 24% showed higher salt tolerance than both wild parents, suggesting that the salt tolerance of azuki beans can be further improved by combining the QTL alleles of the two wild relatives. The marker information would facilitate the transfer of salt tolerance alleles from “Tojinbaka” and “Ukushima” to azuki beans.Wild relatives of crops have the potential to improve food crops, especially in terms of improving abiotic stress tolerance. Two closely related wild species of the traditional East Asian legume crops, Azuki bean ( Vigna angularis) is one of the most important legumes in East Asia, particularly in Japan and China [The azuki bean , while “Ukushima” prevented Na+ accumulation in leaves by filtering Na+ in the roots and stems (Na+ excluder) [To improve the salt tolerance in azuki beans, identifying promising genes is necessary. In our study, we screened 69 azuki bean landraces together with cross-compatible wild relatives consisting of 150 accessions in 8 species . The prixcluder) ,4. In adxcluder) . atpB-rbcL by Takahashi et al. [V. riukiuensis and V. nakashimae are different species but are genetically closely related. V. riukiuensis is adapted to the coastal saline environment in the Ryukyu islands, Japan, whereas V. nakashimae is adapted to the northeastern region of East Asia and is mainly found in and around the Korean Peninsula. Among the V. nakashimae populations, some populations such as “Ukushima” adapted to the coastal saline environment and acquired salt tolerance [V. riukiuensis and V. nakashimae are considered to have evolved from the common ancestral species V. minima [V. minima originated in mainland Southeast Asia, and V. riukiuensis and V. nakashimae might have evolved from V. minima in the course of the expanding geographical distribution to East Asia. The time of divergence between V. riukiuensis and V. nakashimae was estimated to be only 60,000 years ago [From DNA barcoding sequence analysis of rDNA-ITS and i et al. , it was olerance . V. riuk. minima . V. miniears ago . It is iVigna species, including azduki bean, has been reported [Genetic mapping is a powerful tool for detecting the loci involved in various biological processes . The idereported . Recentlreported ,15,16. Vigna species have been constructed, including azuki bean (V. angularis) [Vigna species V. vexillata (L.) A. Rich., V. angularis and V. unguiculata (L.) Walp [Vigna genomes of 10 species including the azuki genome are available in the following database (VigGS: http://viggs.dna.affrc.go.jp/ (accessed on 1 March 2023)) [The advancement of next-generation sequencing (NGS) technologies has accelerated whole-genome sequencing and genotyping methods. Whole-genome sequences for 14 gularis) ,21,22,23gularis) , has beeL.) Walp ,25,26,27h 2023)) . BecauseThe objectives of this study were to (1) confirm the differences in salt tolerance mechanisms between “Tojinbaka” and “Ukushima”, (2) detect the genetic loci of salt tolerance in “Tojinbaka” and “Ukushima”, which could function on the azuki bean genetic background, and (3) investigate the possibility of achieving higher salt tolerance in azuki beans by pyramiding non-allelic QTLs from “Tojinbaka” and “Ukushima”.Among the 377 SSR markers screened in each population, 370 (99.2%) and 374 (98.1%) SSR markers amplified a fragment in populations A and B, respectively . The perIn total, 193 (for population A) and 313 (for population B) clear polymorphic SSR products were mapped to linkage maps . Markers2 individuals were mapped onto the “Ukushima” genome. The average number of reads per F2 individual was 1,662,703. These mapped reads were used in SNP detection. In total, 74.1% (“Ukushima”), 62.0% (“Tojinbaka”) and 55.4% (F2) of the total number of reads were mapped onto the custom “Ukushima” genome. The mean coverage depths were 36.8 (“Ukushima”), 32.4 (“Tojinbaka”) and 17.4 (F2). The total number of detected SNPs was 25,350. Among them, 15,438 sites supported by Ukushima reads were reference-type alleles in Ukushima, and 5,469 of them were polymorphic in Tojinbaka. These sites were selected as bi-allelic RAD markers and were used in subsequent analysis. In F2, 2485 sites in Chr. 1 to 11 and 19 sites in scaffolds were available  , which c+ accumulation patterns among “Kyoto Dainagon”, “Tojinbaka” and “Ukushima”, the Na+ concentration in each of the following parts of the plants was measured: the 1st leaf to 5th (newest) leaf, the 1st to 3rd (newest) stem node and the roots. Yoshida et al. reported that Na+ concentration was measured, including the wilted leaves, and “Tojinbaka” was characterized as a “Na+ includer” and “Ukushima” as a “Na+ excluder” under 200 mM and 100 mM NaCl stress [+ allocation was measured in plants before wilting using radio-isotope 22Na+ with 100 mM NaCl treatment under artificial environmental conditions [+ concentrations.To confirm the differences in Nanditions . To conf+ includer”, while “Ukushima” showed a “Na+ excluder” type of Na+ accumulation pattern  had no wilting leaves like “Tojinbaka”  and three QTLs for percentage of wilt leaves  in population A  was considered to be the same allele because of the close mapped positions and the same direction of the allelic effect  survived for one month under 100 mM NaCl conditions A, but onjinbaka” D. No F2 lation A . The QTLc effect . “Tojinb2 plants (19.5%) could survive longer than “Ukushima” (2 plants (6.9%) had no wilting leaves  and three QTLs for percentage of wilt leaves  were detected in population B  could survive longer than “Tojinbaka”. Strong transgressive segregation was also observed for PWL. One hundred F2 plants (32.8%) showed lower PWL compared to “Tojinbaka” (2 plants (12.1%) showed no wilting leaves (PWL = 0).To clarify the combined effects of salt tolerance QTLs from “Tojinbaka” and “Ukushima”, F A and B . Therefo A and B C. In addjinbaka” F. Of theqDtW4-2) and PWL (qPWL4-2) were positioned close to one another and were considered to be the same locus  and two QTLs for PWL  were detected in population C . At all me locus . QTLs on+ excluder” and “Tojinbaka” is a “Na+ includer” . The Na+ concentration in the primary leaf of “Tojinbaka” was 2166 ppm, and this accession could survive up to 55 days under 250 mM saline conditions  in the F2 population  requires markers that can be analyzed individually, such as SSR markers. Therefore, it is necessary to develop a small number of SNP markers that can be used in the future at a low cost for the MAS to introduce salt-tolerant QTLs in azuki beans.In this research, three interspecific hybrids were developed, three linkage maps based on SSR or RAD markers were constructed using Fe groups , which ce groups . Althoug+ allocation patterns in three accessions used in this study were previously reported by Yoshida et al. [+ concentrations in leaves, stems and roots under 200 mM salt concentrations for 16 days after salt treatment, in which “Kyoto Dainagon” completely died [+ concentrations from the roots to the leaves, while “Tojinbaka” was an includer with a high Na+ concentration from the root to the leaf. However, since 200 mM NaCl is fairly strong salt stress for azuki and related salt-tolerant species, Na+ distributions were observed in dying plants. Noda et al. investigated the radioisotope 22Na+ distribution under 100 mM salt stress conditions [22Na+, they exposed whole plants to a storage phosphor screen for 24 h to visualize the 22Na+ distribution patterns in plants and then separated the root, stem and leaves and dried the samples to measure the 22Na+ radioactivity. At 3 days under 100 mM salt treatment, “Ukushima” and “Tojinbaka” mainly accumulated 22Na+ in the stem, while “Ukushima” maintained lower 22Na+ in the leaves compared with “Tojinbaka” [22Na+ filtering ability by the roots, resulting in the highest 22Na+ concentration in the roots and lowest 22Na+ concentration in the leaves. These results indicate that the Na+ allocation varies with salt concentration and salt stress period. In the present study, we cultivated these three accessions for 6 days under 100 mM salt treatment after 3 days under 50 mM salt treatment, dried them immediately and measured the Na+ concentration in each plant part based on the dry weight. Our results showed that the Na+ concentration was high and almost the same from the root to the first leaf in “Kyoto Dainagon”. This result indicates that “Kyoto Dainagon” does not have any Na+ filtering ability under 9 days of salt treatment (50 mM 3 days and 100 mM 6 days). “Ukushima” and “Tojinbaka” still retain the ability to filter Na+ from the stems and leaves upon 9 days of salt treatment, while the filtering ability was higher in “Ukushima”. This suggests that QTL analysis using populations A and B could detect salt-tolerant QTLs related to Na+ filtering ability from the stem and the leaf. Noda et al. [+ concentration in leaves might rely on Na+/K+ homeostasis in “Ukushima” and not in “Tojinbaka”. The different Naa et al.  and Nodaa et al. . Yoshidaely died . The autnditions . Three o+ accumulation in the leaves. The uppermost leaves of all accessions were the 3rd leaf at the beginning of salt treatment but only “Tojinbaka” could develop the 5th leaf when the Na+ concentration in each plant tissue was measured  treatment with 5 kBq 22Na+ [22Na+ was co-localized around starch granules developed in chloroplasts in the leaves of “Tojinbaka” and was not detected in other parts in the leaf cell. Therefore, they speculated that “Tojinbaka” has a Na+ trapping system using Na+-binding starch granules, which may be related to salt tolerance [Phragmites australis), a well-known salt-tolerant plant, also produced a large number of Na+-binding starch granules at the shoot base when salt stressed, and it was proposed as a novel salt tolerance mechanism not reported before [+-binding starch granules in the chloroplasts of the leaf cells, which could be part of the mechanism for salt tolerance in “Tojinbaka”. Interestingly, Noda et al. reported that “Ukushima” also contained starch granules in the chloroplasts [22Na+ with starch granules in the chloroplasts of “Ukushima”. Therefore, they proposed the possibility that the starch granules in “Ukushima” may lack Na+ binding ability. Further studies are needed to confirm this hypothesis.Generally, the detoxification mechanism is considered to be related to the vacuolar membrane transport systems, which lead to Navacuoles ,39,40,41Bq 22Na+ . The onlolerance . Common d before . The QTLroplasts . However+ filtering were detected based on the Na+ concentration in the primary leaf in population C (qNa8-1 and qNa11-1) increased the Na+ concentration. In population B, no salt-tolerance-related QTL was detected in these two regions. In population A, salt-tolerance-related QTLs were detected near the qNa8-1 region as qDtW8-1 and qPWL8-1, and no polymorphic markers were present in the qNa11-1 region  decreased the Na+ concentrations in the primary leaf. This indicated that “Tojinbaka”, which was characterized as a “Na+ includer” in previous studies [+ accumulation in the leaves. Surprisingly, these two “Tojinbaka” alleles at these two QTLs with Na+ filtering effects (qNa2-1 and qNa8-2) showed higher LOD (8.1 and 7.1) and PVE (11.1% and 9.7%) values  for Na+ filtering from leaves.Four QTLs related to Nalation C . Among t1 region . Thus, t studies ,4, had Q) values . In contqNa8-1) increased the Na+ concentration in population C and decreased the salt tolerance (qDtW8-1 and qPWL8-1) in population A , which is effective on increased Na+ accumulation in leaves, DtW dramatically decreased when the genotype of qDtW1-1/qPWL1-1 has the “Kyoto Dainagon” allele (AA). However, even when the genotype of qDtW8-1/qPWL8-1 has the “Tojinbaka” allele (BB), which is effective on increased Na+ accumulation in leaves, DtW does not decrease when the genotype of qDtW1-1/qPWL1-1 carries the “Tojinbaka” allele (BB). This phenomenon suggests that the “Tojinbaka” allele of qDtW1-1/qPWL1-1 might be involved in the detoxification of Na+ in the leaves. It is possible that these QTLs were involved in the Na+ trapping system using starch granules [qNa8-1 had no significant effect on DtW and PWL but an increased Na+ concentration in leaves , V. riukiuensis  and V. nakashimae . “Tojinbaka” was collected from Ishigakijima island in Okinawa prefecture, Japan (https://www.gene.affrc.go.jp/databases-plant_search_detail_en.php?jp=235833 (accessed on 1 March 2023)). “Ukushima” was collected from Ukushima island in Nagasaki prefecture, Japan (https://www.gene.affrc.go.jp/databases-plant_search_detail_en.php?jp=107879 (accessed on 1 March 2023)). The habitats of these wild accessions are close to the seashore.Three accessions from three V. angularis “Kyoto Dainagon” × V. riukiuensis “Tojinbaka”, (B) V. angularis “Kyoto Dainagon” × V. nakashimae “Ukushima” and (C) V. nakashimae “Ukushima” × V. riukiuensis “Tojinbaka”. In all cross combinations, an accession with higher salt tolerance was used as the male parent. For population A, the F2 and F2:3 population consisting of 190 individuals was used to develop a genetic linkage map and to phenotype salt tolerance. For population B, the F2 population consisting of 211 individuals was used to develop a genetic linkage map, and 87 F2:3 lines (with 1 to 10 plants per line) were used to phenotype salt tolerance. For population C, the F2 population consisting of 348 individuals was used to develop a genetic linkage map and to phenotype salt tolerance.To develop genetic linkage maps and to conduct QTL analysis, three interspecific hybrid populations were developed: (A) 2 plants and F2:3 lines of hybrid populations A and B were used for salt tolerance phenotyping, respectively. Seeds were sown in trays with Seramis clay granules (SERAMIS®) and were supported and kept in a growth chamber at 25 °C for 10 days. Each plant was transplanted to a hydroponic culture in a greenhouse of the Genetic Resources Center, Tsukuba, Japan , under short-day conditions (10 h light/14 h dark). A piece of each plant’s leaves was sampled for DNA extraction. The hydroponic culture solution contained a diluted nutrient solution with a 1:1 ratio of Otsuka house No. 1 (1.5 g/L): Otsuka house No. 2 (1 g/L) . The final solution was adjusted to an EC of 100 mS/m with water. Three weeks after transplanting, NaCl treatment was initiated. NaCl concentration was 50 mM for the first four days and was increased by 50 mM at four-day intervals until a final concentration of 200 mM was reached. After that, the NaCl concentration was maintained at 200 mM for 16 days . Accessions that survived the salt treatment were grown under lower salt conditions after the experiment and were used for F4 seed production.The F2 seeds were sown and transplanted as described above. Three weeks after transplanting, NaCl treatment was initiated. NaCl concentration was 50 mM for the first three days and was increased by 50 mM at three-day intervals until a concentration of 200 mM was reached. After that, the NaCl concentration was maintained at 200 mM for 16 days . The primary leaves were sampled 17 days after salt treatment began, and Na+ concentration in the primary leaves was measured by the method later described in “+ concentration measurement in parental linesNa”. Potentially because both wild parents have high levels of salt tolerance, many F2 individuals were still healthy 25 days after salt treatment began. Therefore, NaCl concentration was further increased to 250 mM and was maintained at 250 mM until 76 days after commencement of salt treatment.In the case of hybrid population C, the F+ accumulation in different plant parts, three seeds each from “Kyoto Dainagon”, “Tojinbaka” and Ukushima” were sown and transplanted to hydroponic solution, as described above. Three weeks after transplanting, NaCl treatment was initiated. NaCl concentration was 50 mM for the first four days and was increased to a final concentration of 100 mM. Six days after the NaCl concentration reached 100 mM , the leaves, stems and roots were harvested separately. The leaves were further divided according to the stem nodes on which they grew, from the primary node up to the fifth node. The stems were also divided into three parts: (1) from the base of the stem to the node of the primary leaf, (2) from the first node to second node and (3) the remaining part of the stem above the second node. To compare the pattern of Na+ concentration was conducted using the ion meter method [g) for 5 min, 200 µL of supernatant was analyzed with a LAQUA twin Na+ meter . Before measurements, the LAQUA twin Na+ meter was calibrated using the corresponding calibration solution. All measurement procedures were conducted at room temperature (approx. 20 °C). Measurement of Nar method , with slDtW), recorded the number of days from salt treatment until the plant wilted. The second trait, known as ‘Percentage of Wilt Leaves’ (PWL), measured the proportion of leaves that had wilted, a symptom of the plant dying. PWL was visually scored using eight categories: 0% = normal healthy leaves, 15% = 1–15% of leaves wilted, 30% = 16–30% of leaves wilted, 45% = 31–45% of leaves wilted, 60% = 46–60% of leaves wilted, 75% = 61–75% of leaves wilted, 90% = 76–90% of leaves wilted and 100% ≥ 90% of leaves wilted or plant completely dead. PWL scores were recorded 19 days after salt treatment began. Two salt-tolerance-related traits were recorded for each plant or line in populations A and B, respectively. The first trait, called ‘Days to Wilt’ . The method of Na+ measurement was the same as described above.In addition to DNA was extracted from young leaf samples using the EZ1 DNA Tissue kit . The extracted DNA samples were used for PCR amplification using 330 azuki bean SSR primer sets , 40 commPCR amplification was performed with a 5 μL reaction mixture containing 5 ng DNA template, 10x PCR buffer, 2 mM dNTP, 25 ng of each primer and 0.125 U Ex Taq polymerase enzyme. The thermal cycler protocol consisted of denaturation at 94 °C for 30 s, 40 cycles of 94 °C for 30 s, 55 °C for 60 s and 72 °C for 60 s, followed by 72 °C for 10 min cooling at 10 °C. PCR was performed in multiplex PCR reactions. Subsequently, PCR product size was analyzed on an ABI Prism 3100 genetic analyzer using GENEMAPPER ver. 3.0 (Applied Biosystems). The two linkage maps were constructed using AntMap .V. nakashimae “Ukushima” genome was constructed. The raw RAD reads of “Ukushima” were first processed via the Trimmomatic-0.30 to remove the reads with sequencing adaptors and of low quality  [http://viggs.dna.affrc.go.jp/ (accessed on 4 August 2015)) using Burrows–Wheeler Aligner (BWA) v.0.7.9a [V. nakashimae using GATK with FastaAlternateReferenceMarker, and the result was used as a custom “Ukushima” pseudo-reference genome.To improve the mapping quality of reads from wild species to the azuki genome, a pseudo-custom ity < 5) . Then, tv.0.7.9a . For locv.0.7.9a  were app2 plants from population C (V. riukiuensis × V. nakashimae) using the CTAB method [2 individuals was simultaneously digested with BamHI-HF and NlaIII and purified. Adaptor-1 and adaptor-2 were ligated to the digested DNA samples and purified. Adaptor-ligated biotinylated DNA samples were collected using streptavidin-coated magnetic beads . Adaptor-ligated DNA on the beads was amplified by PCR using Phusion High-Fidelity DNA polymerase  and the adapter primers. The size of the PCR-amplified fragments (smear between 200 and 1000 bp) was checked using electrophoresis in 1% agarose gel. The 96 purified PCR products were pooled and sequenced using the Illumina HiSeq2000 system. Sequence reads were mapped to custom pseudo-reference “Ukushima” genome, and local realignment and SNP detection were carried out as mentioned above. Error correction was performed based on the reference genome sequence. Genomic DNA was extracted from parents and 348 FB method . Each ge2 individuals and dropped segregation distortion markers . Suitable values for recombination fractions and LOD scores were estimated using the “est.rf” command. After these steps, initial linkage group construction was carried out using the “Form Linkage Groups” command, with a maximum recombination fraction of 0.25 and a LOD threshold of 15. Robustness of linkage groups was checked using the “plot.rf” command. Marker order within linkage groups was estimated using a program called TMAP [The genetic linkage group was constructed using R/qtl ver. 1.36.6 software . For potled TMAP . Some RASingle QTL analysis was run using the standard interval mapping method, as described in R/qtl . The sigV. angularis × V. riukiuensis and V. angularis × V. nakashimae populations of the azuki reference genome sequence (Vangularis_v1: cultivar Shumari in VigGS: https://viggs.dna.affrc.go.jp/ (accessed on 18 June 2015) [To compare QTL positions across the three populations, the physical positions of SSR markers mapped on the linkage maps of the ne 2015) ) were deV. angularis “Kyoto Dainagon” lacked Na+ filtering ability, while the salt-tolerant wild relatives V. nakashimae “Ukushima” and V. riukiuensis “Tojinbaka” were able to maintain lower Na+ concentrations in their leaves and had Na+ filtering ability. V. nakashimae had particularly strong Na+ filtering ability, but growth was stopped after salt treatment. In contrast, although V. riukiuensis accumulated Na+ in its leaves , growth was sustained. We developed three interspecific hybrid populations and identified 21 QTLs for salt tolerance. In population A (cross between azuki bean and V. riukiuensis), three QTLs were detected for DtW and PWL . In population B (cross between azuki bean and V. nakashimae), five QTLs were detected . In population C (cross between V. nakashimae and V. riukiuensis), four QTLs were detected . Additionally, four QTLs  that were detected in the leaf Na+ concentration analysis performed only in population C did not match the location of QTLs for DtW and PWL in population C. However, qDtW8-1/qPWL8-1 with the effect of the V. riukiuensis allele on reduced salt tolerance, which was detected in population A, was located on the same region as qNa8-1. This suggests that qDtW8-1/qPWL8-1/qNa8-1 has an effect on reduced salt tolerance by promoting Na+ accumulation in the leaves. Additionally, a genetic interaction was observed between qDtW8-1/qPWL8-1/qNa8-1 and qDtW1-1/qPWL1-1. This suggests that qDtW8-1/qPWL8-1/qNa8-1 is associated with Na+ accumulation ability in the leaves in V. riukiuensis, and qDtW1-1/qPWL1-1 is involved in the detoxification mechanism of Na+ that allows for growth under the presence of Na+ in the leaves. Furthermore, in population C, transgressive segregation was observed, indicating that the salt tolerance mechanisms in the two wild species might be different and that stronger salt tolerance could be acquired by combining genes conferring these mechanisms. Finally, this study not only provides useful marker information for subsequent fine mapping of candidate genes involved in salt tolerance and elucidating the genetic basis of Na+ filtering and detoxification mechanisms in the future but also contribute to developing super-salt-tolerant azuki beans by combining the salt-tolerant mechanisms of V. riukiuensis and V. nakashimae.This study demonstrated that the mechanisms underlying salt tolerance vary among azuki beans and their wild relatives. The salt-sensitive azuki bean


Vitis rotundifolia) is resistant to many of the pathogens that negatively impact the production of common grape (V. vinifera), including the bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf), which causes Pierce’s Disease (PD). Previous studies in common grape have indicated Xfsf delays host immune response with a complex O-chain antigen produced by the wzy gene. Muscadine cultivars range from tolerant to completely resistant to Xfsf, but the mechanism is unknown.Muscadine grape  signaling as well as seven R genes. Leveraging public data from previous experiments of common grape inoculated with Xfsf, we determined that most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome.We assembled and annotated a new, long-read genome assembly for ‘Carlos’, a cultivar of muscadine that exhibits tolerance, to build upon the existing genetic resources available for muscadine. We used these resources to construct an initial pan-genome for three cultivars of muscadine and one cultivar of common grape. This pan-genome contains a total of 34,970 synteny-constrained entries containing genes of similar structure. Comparison of resistance gene content between the ‘Carlos’ and common grape genomes indicates an expansion of resistance (R) genes in ‘Carlos.’ We further identified genes involved in Xfsf, within and between muscadine and common grape.Our results support the utility of a pan-genome approach to identify candidate genes for traits of interest, particularly disease resistance to The online version contains supplementary material available at 10.1186/s12864-023-09514-y. Vitis rotundifolia Michx., syn. Muscadinia rotundifolia 2n = 2x = 40) is a species of grape native to the southeastern United States . In comparison, the subgenus Euvitis (bunch grapes) exhibits greater diversity and includes about 60 species. Compared to the common table grape , muscadine grapes are a small-market crop . GE. GE31]. We ran RNAmmer, version 1.2 , on the We identified and classified predicted plant disease resistance-related genes (R-genes) in ‘Carlos’, ‘Trayshed’, ‘Noble’, and common grape to compare between tolerant, resistant, and susceptible individuals/species Fig. . We firsWe then identified orthogroups from GENESPACE that contained one or more R-gene candidates from ‘Carlos’. The 3,522 candidate R-genes were placed into 2,061 synteny-constrained orthogroups. Of these orthogroups, 250 were predicted to function as RLKs, 303 as RLPs, 84 as TNLs, 132 as CNLs, and 89 as other NLs. In comparing the number of genes in these orthogroups from ‘Carlos’ to those of common grape, we found that 518 of these orthogroups contain genes novel to ‘Carlos’, and 392 contain more genes in ‘Carlos’ compared to grape. The orthogroups unique to ‘Carlos’ contained 64 genes predicted to function as RLKs, 109 as RLPs, 50 as TNLs, 54 as CNLs, and 37 as other NLs. Of the 392 orthogroups with more ‘Carlos’ genes, 65 are predicted to function as RLKs, 166 as RLPs, 36 as TNLs, 87 as CNLs, and 57 as other NLs. For the remaining orthogroups, 244 contained more genes in grape than in ‘Carlos’, and 907 contained an equal number of genes between ‘Carlos’ and grape. These numbers support that a number of R-genes have either undergone expansions in muscadine or are unique to muscadine.IV.Differential expression in response to Pierce’s Disease.Xfsf in ‘Carlos’ cultivar plants, RNA sequencing of leaf tissue revealed 234 differentially expressed (DE) genes compared to mock-inoculated control plants  . To. ToXfsf III.Genome scaffolding and assembly.CLR reads were initially assembled into contigs using MECAT2 version 2019.03.04 , which iFollowing initial contig-joining, RagTag version 1.0.1  was usedIV.Gene annotation.Gene Model Maker (GeMoMa) version 1.8 was used to predict the ‘Carlos’ gene annotation using the existing annotation for the ‘Trayshed’ haplotype 1 genome assembly . GeMoMa V.Comparative genomics of ‘Carlos’ to other genomes.Vitis vinifera), version 2.1, annotation was included in the GENESPACE analysis [The ‘Carlos’ chromosomes were aligned to the ‘Trayshed’ haplotype 1 chromosomes and the ‘Noble’ chromosomes using MiniMap2 version 2.24 with default settings . The resanalysis . A custoanalysis . Plant danalysis .Due to grape and muscadine having a different total number of chromosomes, SyRI could not be run to compare assembly structure. Instead, Orthofinder version 2.3.12 was used to identify orthologs between ‘Carlos’ muscadine and common grape . ProteinVI.Differential expression analysis.p-value of 0.05. DRAGO2 was rerun on the differentially expressed genes to identify candidate R-genes.The ‘Carlos’ RNASeq data was trimmed using Skewer, version 0.2.2, with identical parameters to those used in the gene annotation Sect. . STAR, vwzy knockout in Xfsf, strain Temecula1, on host response (NCBI accession: PRJNA345471) [Wzy between the strains. To match the conditions of our ‘Carlos’ RNASeq data, samples were only included if collected 4 weeks after inoculation from petioles. A total of 9 samples were analyzed: 3 representing control, 3 representing infection with wild-type Temecula1 strain Xfsf, and 3 representing infection with mutant wzy knockout Temecula1 strain Xfsf. For both sets of samples, Skewer, version 0.2.2, was run using identical parameters to those used in the gene annotation section. The same version and parameters of STAR were run to align the reads with the V. vinifera genome, version 2.1. For the common grape data, expression was compared between the control and inoculated samples, the inoculated and mutant samples, and the control and mutant samples  using the same adjusted p-value to call differentially expressed genes.Common grapevine RNASeq data were obtained from a previous study that confirmed the effect of A345471) . Due to To compare the expression of genes in ‘Carlos’ muscadine to common grape, the results of the earlier OrthoFinder run were used. Orthogroups containing significantly differentially expressed genes from analyses were identified. Functional gene annotation of ‘Carlos’ was determined using EnTAP version 0.10.8 , which aBelow is the link to the electronic supplementary material.Supplemental Figure S1: Foliage and pollinated flowers of Vitis rotundifolia cv. ‘Carlos’ vines. Picture taken in Poplarville, MSSupplemental Figure S2: Pierce’s Disease symptoms in Vitis vinifera vines. Picture taken in Poplarville, MSSupplemental Figure S3: Riparian plot of synteny between the 4 gene sets of the Vitis pangenome. Riparian plot generated using GENESPACE version 0.9.4Supplemental Tables S1-S13: Summary of Early Forms of the Vitis rotundifolia ‘Carlos’ genome assembly. “Reference-Guided Scaffolds” refers to assembly before gap-closing


Under 405 and 442 nm Ramanexcitations, all of the marker bands of Chl a/c are observed and the isotope-based assignments providenew information concerning the structure of Chls a/c in the FCPs and their interactions with the proteinenvironment. Therefore, the Raman spectrum at 405 nm originates fromthe π–π* transitions of Chl a/c and not from a different, non π–π*electronic transition, as previously reported . Based on the 15N isotopeshifts of the Ca-N and in conjunction with other markerbands, two distinct conformations of five- and six-coordinated Chl a and Chl c are observed. In addition,two keto carbonyls were observed at 1679 (strong H-bonded) and 1691cm–1 (weak H-bonded) in both the 405 and 442 nmRaman spectra, respectively. Collectively, the results provide solidevidence of the nature of the vibrational modes of the active Chl a/c photosynthetic pigments in the FCPs.We report resonance Raman spectra of the light-harvestingfucoxanthin-chlorophyll  The correct assignment of the marker bandsof Chl a/c is of pivotal importancein understanding the molecular basis of their function since theydisplay a wide range of frequencies depending on the protein properties.In order to fully utilize the potential of resonance Raman scattering,it is essential to assign the normal modes of Chl a/c in the FCPs. Raman spectroscopy has been appliedin isolated FCPs from the centric diatom Cyclotellameneghiniana cells under frozen conditions(77K) with variable excitations from 406.7 to 476.5 nm.15 It was reported that under 441, 457.9, and 476.5nm excitation, two distinct Chl c2 C131 keto carbonyls were observed at 1675 (strong H-bonded) and at 1690cm–1 (weak H-bonded) associated with the presenceof two Ca-N breathing modes of Chl c2at 1355 and 1362 cm–1, indicating the presence oftwo conformers. Furthermore, it was reported that under 406 and 413nm excitation, there is no evidence for the presence of the Ca-N stretching modes and the C131 keto carbonylmodes of Chl c2 and the Ca-N breathingmodes of Chl a are absent. It was suggested thatthe 406 and 413 nm excitation Raman spectra originate from a different,non π–π* electronic transition. This suggestionis overly simplistic but suggests that the origin of the Raman modesobserved under 406 and 413 nm excitation is still unknown and raisesthe possibility that other vibrations despite the fact that they arenot conjugated into the π-system of the macrocycle could gainRaman activity. Vibrational assignments of Chl a/c based on isotopically enriched 15N-FCPs arenot available, yet. Resonance Raman spectra of 15N-enrichedChl a/c under 406 and 441 nm excitationare needed to establish a set of vibrational assignments of Chl a/c in the FCPs that will clarify theirseparate contributions in this energetic blue region.Resonance Raman spectroscopy has been applied extensivelyto characterizethe structure of chlorophyll-containing proteins.a/c pigment content in the FCPs from themarine diatom Fragilariopsis sp.19 The changes in 15N-containing Chl a/c bands in the FCP provide the first spectroscopicevidence for the characterization of the structural differences inChl a versus Chl c that lead toaltered photophysical and redox properties between the different classesof macrocycles, which are crucial for light harvesting and energytransfer processes. Based on the criteria that the Raman bands associatedwith large shifts upon 15N substitution are related tothe Ca-N stretching, where those that do not are relatedto the carbon–oxygen and carbon–carbon and/or the carbon–hydrogen,we have made the assignments of all Chl a/c modes. The 1361(−5) and 1354 (−5) cm–1 modes observed in the 405 and 442 nm excitation Ramanspectra originate from the pyrrole breathing Ca-N modesof two conformations of Chl c and those at 1377 (−5)and 1346 (−4) cm–1 to the Ca-Nof Chl a. There are also additional vibrational modesaffected by the 14N to 15N substitution in themacrocycles of Chls a/c. Two ketocarbonyls were observed at 1679 (strong H-bonded) and 1691 cm–1 (weak H-bonded) in both the 405 and 442 nm Ramanspectra.In thework presented here, we have extended our work in diatomsand employed 406 and 441 nm Raman excitation to probe the Fx-Chl Fragilariopsis sp. (CCAP 1029/24)was obtained from CCAP (Culture Collection of Algae and Protozoa).Cultures were grown in f/2-Si medium containing either Na14NO3 (NA) or 15NH4NO3 asnitrogen source, at 19 °C in a dark–light cycle (12h:12h)under white LED light. The light intensity used for the growth ofthe cell cultures was 150 μmol of photons m–2 s–1.The culture of Fragilariopsis sp. diatom wereharvested by centrifugation , and thesupernatant medium was removed. The cells were resuspended in 20 mMTris and sonicated in an ice bath for 20 min. Thylakoids were thensolubilized with the addition of β-1,4-dodecyl maltoside  in the resuspended cells while they were shaken for 20min on ice. Separation of solubilized proteins was carried out onan ion exchange column  in 25 mM Tris, 2mM KCl, and 0.4 mM β-DDM at pΗ 7.4 using a Shimadzu LC-20ADwith a SPD-20A detector with two-wavelength detection. The sampleswere loaded in a column, and fractions were eluted using a gradientfrom 0 to 750 mM KCl in a buffer made of 25 mM Tris, 750 mM KCl, and0.4 mM β-DDM, pΗ 7.4 at a flow rate of 3 mL/min. Fractionswere pooled and concentrated using Amicon filtration devices witha cutoff of 10 kDa and characterized, spectroscopically and biochemically.The rest of the fractions were stored at −80 °C untilfurther use. A 2:1:1 Chla:Chlc:Fxstoichiometry was determined.Cells from the –1. The 405 nm excitation wasprovided by a Coherent Laser, and the laser power incident on thesample was 10 mW. The 442 nm excitation was provided by a KIMMON He–CdLaser, and the laser power incident on the sample was 10–15mW. For the Raman measurements, the temperature of the samples waskept at −70 °C by using a Linkam cell with a liquid Nitrogencryostat.Absorption spectra were recordedwith a Cary 60 UV–vis spectrometer. Raman data were collected by a confocalLabRAM  equipped with a CCD detector,1800 grooves/mm grating, and an Olympus BX41 microscope. The spectralresolution was 5 cmFragilariopsis sp. at room temperature, at pH 8. The transitions at 440 (Soret),621 (Qx), and 673 (Qy) nm are attributed to Chl a, and at 457 (Soret),588 (Qx), and 637(Qy) nm to Chl c.19 The broadtransition in the 500–560 nm range has been attributed to redFxs, whereas blue Fx absorb in the 420–470 nm region. The pigmentanalysis  of the present FCP containsChl c2 possessing a vinyl group at the 8-positionconjugated to its porphyrin π-system.a-N inCh a/c by isotopic labeling of Ch a/c nitrogens (N). This way, the structure/functionand coordination state of Chl a/c in the FCPs will be determined. In the Chl a/c macrocycles, rings I and II are aligned along the y-axis and modes associated with these rings should be enhancedusing lines such as 405 nm excitation. Rings II and IV are alignedalong the x-axis, and modes associated with theserings should be enhanced using lines such as 441 and/or 458 nm closeto the Bx axis. Excitation within theBy absorption band using the 406.7 nmline produces a spectrum dominated by totally symmetric Franck–Condon-activemodes aligned along the y-axis of the macrocycle.The assignment of the Chl a vibrational modes isstill controversial, whereas that of Chl c is stilllimited. The 406.7 nm RR spectra of Chl a in solutionobtained by the group of Bocian13 and thoserecorded by 457 nm excitation by the group of Koyama18 in conjunction with 15N isotopes and normalcoordinate analysis have been reported. Upon 15N substitutionand normal coordinate analysis by the group of Koyama,18 the modes with the largest shift were νCaN(II)/νCaNCa(I) at 1138 (−11),νCbH(IV) at 1188 (−8), γCbH(IV) at 1220 (−5), νCaN at 1284(−7), νCaN(IV) at 1342 (−6), and νCaN(IV) at 1376 (−5) cm–1. In addition,1488 (νCaCm), 1534 (νCbCb)), 1550 (νCbCb + CaCm(III)), 1604 (νCaCm + CbCb)), and 1698 (νC9 = O) cm–1 have been identified.One objective ofthis work is to identify the C14N-containing(trace a) and 15N-containing FCP (trace b) at pH 8, 25°C. The assıgnments of the peaks in trace a are securedvıa the 15N shifts shown ın trace b. The modeswith a larger isotopic shift were associated with the Ca-N stretching modes. In trace a, the peaks at 918, 1146, 1138, 1115,1288, 1361, and 1377 cm–1 have contributions fromCaN of Chl a/c and 1275and 1346 cm–1 from CmH and CaN. The clearest indicator of Chl c is the Ca-N mode at 1361 cm–1. Its frequency is similarto that observed for the oxidation state marker band in heme-containingprotein with the same porphyrin ring structure.21 The five-coordinate Chl a complexes are characterizedby modes in the frequency ranges 1605–1612, 1551–1555,and 1527–1529 cm–1 whereas those of six-coordinateare downshifted to 1596–1600, 1545, 1548, and 1518–1521cm–1. We assign the peaks at 1609 and 1550 cm–1 to five-coordinated Chl a and thepeaks at 1619 cm–1 to the vinyl of Chl c. For the assignments of 1550, 1656, and 1679 cm–1, see below. The peaks at 965, 1005, 1046, 1160, 1185, 1195, 1490,and 1530 cm–1 originate from Fx, in agreement withprevious works.2314N-containing(trace a) and 15N-containing FCP (trace b) at pH 8, 25°C. The assıgnments of the peaks ın trace a are securedvıa the 15N shıfts shown ın trace b andvia the spectra obtained with 405 nm excitation. The most significantdifferences in the data presented in c) and decrease of 1346 (Chl a) modes and (2) the presence of the 1550 cm–1 mode observed in the 406 nm excitation and at 1556 cm–1 in the 442 nm excitation spectra. The latter indicates that 1556cm–1 has contributions from the ν(CaCm+ Cb-Cb) of Chls c whereas the peak at 1550 cm–1 originates mostlyfrom the ν(CaCm+ Cb-Cb) of Chl a. The distinct isotopic shift of the Ca-N modes of Chl a/c in the 15N-labeled FCP confirm in conjunction with the presence ofthe 1608 and 1586/1593 cm–1 modes the presence offive- and six-coordinate Chl a. The magnitude ofthe isotopic shift agrees well with the calculated shift based onempirical normal coordinate analysis using the isotopes of Chl a.–1 regions of the data presented in –1 that arise from five- and six-coordinated Chl a and two from Chl c at 1361 (−6)and 1354 (−6) cm–1 that originate from thetwo conformations of Chl c, as previously reported.15 However, the two conformations were observedonly at 441, 457, and 476 nm excitations but not at 406 or 413 nmexcitation. Interestingly, the same authors assigned the 1620 cm–1 observed in their 413 nm excitation spectra to thevinyl C31=C32 of Chl c butnot the more intense CaN modes of Chl c. In addition, the CaN vibrations of Chl a were not reported. It should be mentioned that the Raman spectrain ref  and at692 (−2), 717 (−2), 736 (−6), 746 (−4),756 (−2), 764 (−20), and 800 (−3). In 15N isotope shifts of a number of bands at 520 (−3), 574 (−3),700 (−4), 717 (−2), 733 (−3), 745 (−3),787 (−5), and 800 (−3) are depicted. The crystal structureof FCP has demonstrated that Chl a/c are coordinated by His, Tyr, and H2O axial ligands. Basedon the vibrational analysis of heme- and chlorin-based assignments,we tentatively assign the 214 cm–1 mode presentedin c) ofthe five-coordinated Chl c and the 338 cm–1 mode to the asymmetrical stretch of the six-coordinated Chl c. The following tentative analysis of the 15N-sensitive bands is based on the normal coordinate analysis of Ni-OEP.24 The 263 (ν9) and 351 (ν8) cm–1 modes are pyrrole-substituent bending vibrations.In the 351–400 cm–1 region, there are uniquebending vibrations related to the C–C–C of the acrylatesubstituents of Chl c. There are candidate RR bandsfor the acrylate moiety in Chl c, at 351–370and 387–400, modulated by different orientations relative tothe porphyrin plane of Chl c. These bands are insensitiveto isotope labeling on the ring. In the 471 to 607 cm–1 region, there are three 15N-sensitive bands at 520 (−3),564 (−3), and 574 (−3) cm–1 that mayrepresent Mg-ligand vibrations. In the 690 to 800 cm–1 range, there are RR bands sensitive to 15N that may havecontributions from both Chl c and Chl a. The vibrations are assigned to ν7 (700 cm–1), γ11(717 cm–1), γ5 (733 cm–1), ν15 (745 cm–1), and ν6 (800 cm–1).The high-frequency skeletal modes are understoodand have beendiscussed several times with respect to their function as marker bandsfor oxidation/coordination/spin state sensitivity. Below the ν4bands in the 1300–1400 cma/c molecules in the lowest excited singlet and triplet statesplaykey roles in the primary processes of photosynthesis. The studiesreported herein for Chl a/c in theFCP provide insight in the key structural element that distinguishesChl a from Chl c. Comparison ofthe FCP of Fragilariopsis sp. to those from othermarine diatoms with known crystal structures may provide the meansto identify conserved structural features that can be assumed to beinvolved in basic functions to different classes of FCPs. In contrast,dissimilarities between these FCPs are likely to be involved in thefine-tuning to specific needs demanded by differences in the localprotein environment of the FCPs. Furthermore, with the identificationof the major coordination marker bands of Chl a/c, the changes in the electronic and molecular structuresof the macrocycles upon singlet and triplet excitation can also beobtained with more certainty.The structures of the Chl