The online version of this article (10.1007/s10853-020-04662-y) contains supplementary material, which is available to authorized users. Chemicadelivery  or the adelivery , 14.π–π conjugation, leading to the loss of mechanical strength, as well as reduced electrical and thermal conductivity [3 times lower than pristine graphene [Most existing covalent functionalisation of graphene family nanomaterials is based on grafting molecules through oxygen-containing functional groups of graphene oxide (GO), followed by chemical or thermal reduction to obtain reduced graphene oxide (rGO) , 16. Grauctivity , 19. Youuctivity , and evegraphene . GO-baseπ system which forms attractive hydrophobic and π–π interactions with aromatic molecules [Functionalisation of pristine graphene can be achieved by both covalent and non-covalent interactions . Pristinolecules , 22. Priolecules , 24, pyrolecules , triphenolecules , and corolecules , as wellolecules . Althougolecules  and condolecules  of the psp2 carbons on the basal plane using diazonium salts [π system and so degrades the product’s conductivity due to the conversion of sp2 carbons to sp3 carbons [Covalent modification of pristine graphene can be achieved through free radical addition to um salts –37 or beum salts  to form um salts –47. This carbons , 33, 48, carbons .3 is traditionally used. Benzoic acid derivatives with polyphosphoric acid and phosphorus pentoxide have been used for milder graphene acylations [Edge-selective covalent functionalisation minimises chemical reactions on graphene’s basal plane and the attendant deterioration of engineering properties. Ball milling graphite in the presence of gases or gas mixtures has been reported to produce edge-specific functionalisation . The tecylations , 56, 57.ylations –60. Otheylations . Furtherylations .The current work extends the top-down method of electrophilic aromatic substitution to create five distinct types of edge-modified graphene with no intermediate carbonyl moiety. Our modifications significantly modified the graphene nanoparticles’ dispersibility. The synthetic utility of the directly attached reactive moieties was demonstrated by creating a “glycographene” through radical addition of allyl mannoside to edge-thiolated graphene. Chemical modifications were confirmed by FT-IR and XPS. Edge localisation was visualised on modified CVD graphene samples by scanning electron microscopy of gold nanoparticles attached to thiol groups, epifluorescence microscopy of a fluorescently tagged lectin–glycographene bioconjugate, and quenched stochastic optical reconstruction microscopy (qSTORM) –66, a su2 g−1 and 300 m2 g−1, respectively, corresponding to 4–5 graphene layers for C750 and 8–9 layers for C300. The batches used in this work had BET specific surface areas of 794.9 ± 1.6 m2 g−1 and 268 m2 g−1. The M25 grade has a nominal lateral dimension of 25 µm. The specific surface area specified by the manufacturer is 120–150 m2 g−1, corresponding to about 17–22 graphene layers; the batch used in this work had a BET specific surface area of 55 m2 g−1. On receipt, the GNPs were portioned into 50-ml Falcon tubes and sealed with Parafilm.Graphene/graphite nanoplatelets  were purchased from XG Sciences . The C750 and C300 grades have nominal specific surface areas of 750 m2 sheets to a silicon wafer covered with 300 nm thermal oxide. Before use, CVD graphene was stored in a covered Petri dish under ambient conditions.Monolayer graphene produced by chemical vapour deposition (CVD) on copper was provided by 2-DTech  and transferred as ~ 1 cmN-methyl-2-pyrrolidone (NMP) at 37 Hz for 48 h in an ultrasonic bath (Elmasonic PH750EL). Remaining graphite was removed by cetrifugation (3 × 20 min at 4000 rpm). The supernatant was a graphene dispersion (ca. 0.4 g l−1). Graphene reaction dispersions were vacuum filtered through 0.02-µm Whatman Anodisc membrane filters to create graphene laminates. For analysis, samples were dried as laminates on the filter membrane or re-dispersed in water by sonication and freeze-dried . For long-term storage, the samples were kept in a desiccator at room temperature.Graphene dispersions were also prepared by ultrasonic exfoliation of natural graphite flakes . Following the method of Hernandez , 2 g graN,N-dimethylformamide  and methanol were purchased from Fisher Scientific, UK. Sodium borohydride (≥ 96%), iodine (≥ 99.8%) and triphenylphosphine (99%) were purchased from Sigma-Aldrich. Raney nickel catalyst (50% in water) and chlorosulphonic acid  were purchased from Merck Chemicals. All chemicals were used as supplied. Water was purified to a resistivity of 18.2 MΩ cm at 25 °C (Milli-Q).SnakeSkin dialysis tubing  was purchased from Thermo Scientific. Disposable folded capillary cells for Zetasizer Nano Series were purchased from Malvern.3−, Scheme Sulphonated graphene (G–SO3) in 30 ml of toluene and sonication under N2 atmosphere for 15 min. Then, the reaction flask was connected to a condenser with nitrogen flow. 2.5 g of triphenylphosphine and 200 mg iodine were added into the mixture that was left stirring at 80 °C for 21 h. The product was filtered using vacuum filtration and 0.45-μm HV membrane filter and washed with toluene, acetone, 0.1 M sodium thiosulphate solution, and deionised water, followed by freeze-drying.Thiolated graphene  were bound to thiolated CVD graphene. The nanoparticle solution was diluted tenfold in phosphate-buffered saline . A sample of modified CVD graphene was immersed in 10 ml of the diluted nanoparticle suspension overnight at ambient temperature. The sample was rinsed with water and acetone and dried in a nitrogen gas stream and stored at room temperature.Gold nanoparticles  in a glass vial wrapped in aluminium foil. The CVD glycographene sample was added to the vial and incubated at 37 °C for 2 h. The sample was rinsed with water and dried in a nitrogen gas stream.Glycographene Scheme  was made−1 using a Renishaw inVia, with a 633-nm excitation laser set to 10% (0.89 mW) power or a 514-nm laser (0.176 mW). The energy resolution was 0.3 cm−1. Raman shifts were calibrated by setting the G (E2g) peak from silicon to 1581 cm−1 [−1 every 0.5 µm . Data were processed using WiRE 4.2 software to zero the baseline and remove cosmic rays. Nonlinear fits (SI) were applied to the peaks following the methods of Puech et al. [D band (i.e. ID/IG) using the Tuinstra–Koenig relationship (D band (i.e. AD/AG) using the relationship from Cançado et al. (LD) was estimated using the expression from Lucchese et al. (Eq. S4) [Raman spectra were recorded from 100 to 3200 cm581 cm−1 , 69. The581 cm−1 . Raman mh et al. . Domain  Eq. S1) , the rel Eq. S2) , and the Eq. S3) . The ave(Eq. S4) .−8 mbar during measurements. Peaks were fit using CasaXPS with Shirley background correction. The C 1s energy was calibrated by fixing the binding energy for the sp2 component to 284.5 eV. The C 1s peak was fit as five components summarised in Table sp3:sp2 C ratio were from Monte Carlo simulations within CasaXPS. Fits to 2p1/2 and 2p3/2 peaks were constrained to have identical FWHM values and an area ratio of 1:2.X-ray photoelectron spectra (XPS) were acquired using a Kratos Axis Ultra with an Al Kα source 1486.6 eV) operated at 15 kV and 10 mA. The pressure of the vacuum chamber was below 5 × 10486.6 eV −1, 32 scans, 4 cm−1) were acquired using a Nicolet 5700 FT-IR spectrometer in air. Background spectra were recorded every 10 min.FT-IR samples were prepared by mixing approximately 0.2 mg modified GNPs with 300 mg KBr  using an agate mortar and pestle then pressed at 10 tons from a hydraulic press for 5 min to obtain a sample disc. Transmission spectra (4000–400 cmx–y stage (PRIOR HLD117) and a PRIOR ProScan III controller. The sample was illuminated by laser beams delivered to the back of the microscope using an optical fibre. A vibration motor was attached to the fibre to remove coherent artefacts in the final image due to laser speckle. Light from the sample was collected by the lens and incident on a Hamamatsu ORCA Flash v2 sCMOS camera. Up to 20000 individual images were acquired for each area with an exposure time of 10 ms. External and internal edges of thiolated CVD graphene were labelled with BODIPY FL L-cystine  and Alexa Fluor 647 maleimide . The graphene samples were immersed in 10 mL deionised water to which 10 mM dye stock in DMSO was added for 2 h at room temperature. After coupling, the samples were washed with water and acetone, dried under a nitrogen stream, and kept at 4 °C and away from light. Image data were processed using ImageJ with the ThunderSTORM plugin [STORM images were taken using a previously described custom-built STORM system , consistM plugin .Epifluorescence images were collected on an Olympus BX51 upright microscope using UPlanFLN objectives and captured using a Coolsnap camera (Photometrics) through MetaVue software (Molecular Devices). A specific bandpass filter set for FITC  was used.2/Si were mounted on 12.5-mm aluminium stubs. No surface coatings were applied.Scanning electron microscopy (SEM) images were acquired using an FEI Quanta 650 FEG-SEM operating at 1–5 kV. CVD graphene samples on SiOSpecific surface area was determined from the Brunauer–Emmett–Teller (BET) method using a Micromeritics Gemini V surface area and pore size analyser.−1 from 30 to 800 °C in a N2 atmosphere.Thermogravimetric analysis (TGA) was performed on a TA Instruments Q500 thermogravimetric analyser. 1–3 mg of graphene sample was heated at 10 °C min−1) in deionised water were prepared and placed in disposable foldable capillary cells for testing. The measurement was repeated 6 times for each type of functionalised graphene.Zeta potential was determined using a Malvern Zetasizer Nano series. Aqueous graphene suspensions (0.05 mg mlk0) to pristine and edge-modified CVD graphene samples were carried out in a two-electrode set-up described Valota et al. [Estimates of the interfacial electron transfer rate  at 550 °C. The micropipette was filled with 5 mM potassium ferricyanide  in 6 M LiCl aqueous solution . A high concentration of LiCl was used to prevent droplet evaporation . The fil64− adsorption on highly oriented pyrolytic graphic (HOPG) lowered electron transfer rates [k0 values [−6 cm2 s−1 was used based on previous measurements using the Randles–Ševčík equation [A camera with a microscope objective  and light source were positioned parallel to the sample surface . Droplet deposition was monitored and recorded using Infinity Analyze software (v.4.2) . A second camera above the sample provided low-resolution, live imaging of the sample to identify areas for deposition. After depositing a droplet, cyclic voltammetry was run using NOVA software (v. 1.11). An uncertainty of 24.4 mV was assigned according to the step size during the scan. Cyclic voltammograms (CVs) were conducted for seven scan rates in nine separate droplets. A set of measurements on a single droplet typically required ca. 30 min, an important consideration in light of the observations by Patel et al. that Fe(CN)er rates . Typical0 values . A diffuequation . Some elequation , 83.3−, Scheme Graphene/graphite nanoplatelets (GNPs) and monolayer CVD graphene were treated with chlorosulphonic acid and then hydrolysed with base to produce edge-modified graphene sulphonate  than the thiol S 2p peaks (165.1 ± 0.2 eV and 164.0 ± 0.2 eV). The S 2p spectrum for G–SH showed that the reduction of the sulphonate to the thiol was incomplete (–SO3−/–SH = 2.8 in the example shown). Quantification of the XPS survey spectra for G–SO3− and G–SH gave a S:C atomic ratio of about 0.3:100  –89, an etransfer .sp2 carbon in the GNPs after edge modification was deduced from deconvolution of the C 1s peak , caused by a one-phonon defect-assisted electron–defect scattering event from edges, grain boundaries, or internal defects [ID/IG ratios and equally prominent D′ features . Larger GNPs (XG C300 and M25) showed a decreasing ID/IG ratio with increasing GNP size , consistent with the Tuinstra–Koenig relationship [ID/IG caused by the edge modification. A Raman map of sulphonated CVD graphene  showed that the ID/IG ratio was lowered at the edges, demonstrating that the chemical modifications complicate this simple metric for graphene order. This effect has previously been discussed for graphene with its basal plane modified by diazonium salts [areas of the D and G peaks, namely the average domain diameter, LD, were more consistent. Sets of modifications to commercial GNPs and graphene produced by mechanically exfoliating graphite in NMP showed little changes in LD on modification relative to the source materials (Table S6). For CVD graphene, LD consistently dropped by about half and Raman analysis of the GNPs before and after edge modifications  of the edge-modified G–SO3− produced in this study, regardless of whether the GNPs or graphite exfoliated in NMP was used (Table S5). The Raman 2D peak shifts to lower energy with decreasing layer number when the number of layers is below ~ 10 [Sulphonated graphene (SGnP) has been produced by chemical exfoliation of graphite in chlorosulphonic acid. The work of Abdolmaleki et al.  reportedlow ~ 10 , but no low ~ 10 –96.D peak, and a ID2/IG ratio of over 2 and a FWHM of 24 cm−1, indicative of single-layer graphene [ID2/IG ratio falls below 1 for bilayer graphene and drops further as the layer number increases. The spectra taken from the edges of the graphene sheet had a measurable but low ID/IG and lower ID2/IG ratio, consistent with the literature [ID/IG ratio, consistent with the more ragged appearance of the flake by visible light microscopy  and STORM  of the samples over a range of 100–900 °C  between the unmodified samples and the dye-labelled G–SH  × 10−4 cm s−1 . The large uncertainty in the result is likely due to inhomogeneity of the CVD surface . The mean k0 for the drops sitting at the edges of CVD G–SO3− was (3.6 ± 1.7) × 10−4 cm s−1 . Electron transfer rates to the edges of single-layer graphene are expected to be two orders of magnitude greater than the basal plane [k0 reduced to (1.0 ± 0.5) × 10−5 cm s−1 , comparable to unmodified CVD graphene. Some of the decrease must be attributed to the bulky dye slowing edge electron transfer because the value of LD estimated from Raman spectra remained about half the value of unmodified CVD graphene, as it was for G–SH (Table S6). The electron transfer rates for the unfunctionalised CVD graphene and dye-labelled CVD graphene are comparable to those of the basal plane of HOPG exposed to air for > 60 min after exfoliation, based on comparable peak separations from scans at 100 mV s−1 [The al plane . The onl0 mV s−1 .The edge modifications produce marked changes in the graphenes’ dispersibility, consistent with the chemical changes . Sulphonation produces graphene that is water dispersible; reduction to the thiol creates a material dispersible only in toluene. Glycographene is moderately dispersible in water, making it a conceivable candidate for applications in biosensors or biomaterials. Contact angle measurements (Table S4) were consistent with these observations.The edge sulphonation of larger GNPs also produced an unexpected trend. These were presumed to have a smaller amount of edge length for a given mass. Elemental analysis by XPS  showed the opposite trend; however, the largest flakes had the highest S/C ratio. This observation could be an artefact residual, unreacted chlorosulphonic acid trapped between the layers of the GNPs. Consistent with this, graphite has previously been intercalated by tosylate  and meth2) was performed and reduced to aminographene (G–NH2). FT-IR  confirmed the presence of the nitrogen-containing functional groups. G–NO2 showed a distinct, sharp N–O vibration at 1385 cm−1 characteristic of nitrated aromatic systems. This signal disappeared when G–NO2 was reduced to form G–NH2, and distinctive N–H vibrations appeared from 3700 to 3300 cm−1 along with C–N stretches and N–H scissoring bands at lower energies.The nitration of GNPs . The relative amount of the nitro group (Eb = 406.9 ± 0.3 eV) decreases between the G–NO2 sample and its reduced G–NH2 analogue. The largest peak at 400.1 ± 0.2 eV can be assigned to organic nitrogen groups, including amines and nitrogen heterocycles [2 sample. A small contribution at 402.8 ± 0.4 eV was attributed to nitrogen in an intermediate oxidation state, such as a hydroxyl amine. Nitration produced products with a greater heteroatom content (N:C around 0.5:100). Analysis of the G–NH2 was complicated by the presence of by-products from the nitro reduction over Raney nickel.The N 1rocycles –105, butAs a nitration caused too damaging to CVD graphene, epifluorescence of FITC-labelled GNPs M25 aminographene was an alternative method to highlight the specific edge modification in this study . A faint green fluorescence on the edge of GNP  could be a possible dye attachment. Nevertheless, conventional fluorescence microscopy was not suitable to observe the edge nitration of GNPs due to a low fluorophore attachment.sp3:sp2 C ratio, and estimated distance between defects (LD) from Raman are evidence of chemical functionalisation predominantly at the edges and internal defects. This straightforward and scalable method of edge-specific functionalisation creates a versatile platform material for biotechnological and biomedical applications of graphene, rather than graphene oxide or its derivatives. Ongoing and future work on these materials includes measurements of their electronic conductivity and interfacial electron transfer coefficients, applying them in nanoscale theronostics devices, using them as platforms for tissue engineering and regenerative medicine , synthesising MRI contrast agents (e.g. with Gd-DOTA–based side groups), creating multiple reactive groups (e.g. both thiol and amine) for bio-orthogonal functionalisation, and assessing whether the mechanical and electrical properties are maintained upon edge functionalisation.Super-resolution microscopy of fluorophore-labelled edge-modified graphene, the s XPS spectra; elemental analysis based on XPS survey spectra; Raman spectra; schematic for microelectrochemistry and representative measurements; thermogravimetric analysis of pristine and edge-modified GNPs; dispersibility and contact angle measurements; Raman comparison of GNP source for edge sulphonation; Raman and XPS analysis of GNP size on edge sulphonation. In compliance with the University of Manchester’s requirements and those of the funding agencies, these data for this work are available to download from https://data.mendeley.com/datasets/mwrvrcj639/draft?a=20782f0e-4a57-4056-a4e7-c24057beded6Supporting information available: synthesis of nitrographene and aminographene; Raman spectrum analysis methods; representative survey and C 1 (PDF 3805 kb)Below is the link to the electronic supplementary material.


Blank  and experimental  groups of lotus seeds were stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for about 30 days, respectively. The mildew was observed and the changes of Cw, Aw, together with the production of aflatoxins were measured. Results showed that no mildew was found and aflatoxins were not detected in lotus seeds when they were stored for 30 days at 25 °C and relative humidity of 40%, 50% and 60% with Cw < 12% and Aw < 0.6. While, when the relative humidity was up to 70%, the Cw and Aw values rose quickly, and the Cw exceeded the officially-permitted level (14%). Although no mildew was observed, AFB1 was still detected, increasing the potential risk of lotus seeds regarding aflatoxins. For warranting the quality with economic and safe storage, lotus seeds are suggested to be stored at 25 °C and relative humidity lower than 60% with 12% and 0.6 as the safe Cw and Aw control points, respectively, to prevent medicinal and edible products from mildew and the contamination of aflatoxins.Affected by the inner properties and the external environmental conditions, medicinal and edible lotus seeds are susceptible to mildew with fungal infection under suitable temperature and humidity conditions, leading to the production and contamination of various mycotoxins, along with threats to its quality and safety. In this study, the changes of water content (C Foods including medicinal and edible products, feeds and medicinal plants are vulnerable to fungal conidia and mildew if improperly treated in the processes of growth, harvest, processing, storage and transportation , as a valuable and commonly-used edible and medicinal matrix, not only has the properties of tonifying spleen, stopping diarrhea, stopping bandage, tonifying kidney, nourishing heart and tranquilizing mind, but also is a common food or food additive with wide nutritional and edible functions. However, affected by the nature of their components and the external environmental conditions, lotus seeds are susceptible to mildew with fungal infection under suitable temperature and humidity conditions, especially during the plum rain season, leading to the production and residue of various mycotoxins, such as aflatoxins, ochratoxins, etc. Aflatoxins, mainly containing aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) with strong hepatotoxicity and carcinogenic, teratogenic and mutagenic effects , water activity (Aw) and environmental temperature and humidity, to ensure their quality and safety.Various mycotoxins have been found with residue in different kinds of matrices  system. In practice, foods are commonly dried to reduce the water content to prevent them from mildew. In the other hand, salt or sugar is added to decrease the Aw of matrices to inhibit the growth of toxigenic fungi. Therefore, in public view, effective control Cw and Aw could prevent foods from mildew and the production of mycotoxins to ensure their safety.It is generally believed that in the processing, storage and transportation processes, foods, medicinal plants and other matrices should be dried sufficiently to control their water content for inhibiting the growth of mildew or pests. In addition, Aw and Aw changes of lotus seeds under different storage conditions according to the experimental design in Fig. w and Aw and mildew for exploring the optimum water content and water activity control points for the safe storage. These findings will provide powerful supports and valuable references for screening for the reasonable, reliable and effective storage conditions to prevent medicinal and edible foods and other products from mildew and mycotoxins contamination for ensuring their quality and safety.Therefore, through the investigation of the C1 and AFG1, and 0.5 mg/mL of AFB2 and AFG2 was purchased from Pribolab Pte. Ltd. (Singapore). The ToxinFast®-Aflatoxins Test immunoaffinity columns were bought from Huaan Magnech Bio-Tech Co., Ltd . The aflatoxigenic  Aspergillus flavus (A. flavus) lyophilized powder (CGMCC 3.4410) were got from the China General Microbiological Culture Collection Center  and cultured to 1 × 107 conidia/mL suspension for use.The mixed aflatoxins standard solution containing 2.0 mg/mL of AFB2HPO4.12H2O, NaH2PO4, KH2PO4, and tween-20 were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd . Purified Wahaha water from Wahaha Group Co., Ltd  was used for all the experiments.HPLC-grade acetonitrile and methanol were obtained from Thermo Fisher Scientific Inc . NaCl, KCl, NaNelumbo nucifera Gaertn.) samples, produced in Hubei province,  were purchased from Beijing Tong Ren Tang Co., LTD. All the samples were divided into the blank and experimental groups. For the blank groups: 20 g lotus seeds were added into 16 petri dishes, respectively, followed by irradiation for 5 h under an ultraviolet lamp. For the experimental groups: 20 g lotus seeds were added into another 16 petri dishes, respectively. After irradiation for 5 h, 1 mL of A. flavus conidia solution was artificially added onto the surface of lotus seeds and were air-dried. Then, all the petri dishes containing the raw and treated samples were stored in an incubator at 25 °C and relative humidity conditions of 40%, 50%, 60% and 70%, respectively. On the 0, 10th, 20th and 30th day, all the samples were observed by naked eyes regarding mildew. In addition, the samples were treated for the measurement of Aw and Cw, along with the determination of potential aflatoxins by the optimized high performance liquid chromatography coupled with photochemical derivatization and fluorescence detection (HPLC-PCD-FLD) method on the 0, 3rd, 6th, 9th, 12th, 22nd and 32nd day.Lotus seed  of sample was accurately calculated based on the lost weight.The weight loss method recommended in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission w) by using a HD-6  intelligent moisture activity measuring instrument. 15 min later, data of Aw were recorded.Five gram lotus seeds out of the above samples stored at 25 °C and different relative humidity  conditions were taken out on the 0, 3rd, 6th, 9th, 12th, 22nd and 32nd day for the measurement of water activity (Av/v) and water was selected as the mobile phase with isocratic elution at a flow rate of 1.2 mL/min. The column temperature was set at 30 °C. The injection volume was 20 μL. The AURA INDUSTRIES PCD reactor  was consisted of a mercury lamp (λ = 254 nm) and a knitted reactor coil of 0.74 mL (15 m × 0.25 mm). The eluate was monitored by using a fluorescence detector at an excitation wavelength of 360 nm and an emission wavelength of 450 nm.Chromatographic separation of four aflatoxins was performed on a MG-III-C18 column  through a Shimadzu LC-20AT HPLC system  consisting of two LC-20 AT pumps, an SIL-20A autosampler, a CTO-20A column oven, a CMB-20A controller, the post-column photochemical derivatization (PCD) reactor and an RF-20AXL fluorescence detector (FLD). Methanol–acetonitrile  solution was obtained by dispersing 0.2599 g NaH2PO4, 6.4204 g Na2HPO4.12H2O and 40 mL of tween-20 in 1960 mL of purified water.Firstly, Phosphate buffer saline (PBS) solution was prepared by dissolving 0.2 g KCl, 8.0 g NaCl, 0.2 g KHv/v) solution. After blending by vortex and ultrasonication for 30 min, the mixture was followed by centrifugation for 5 min at 10,000 r/min and 5 mL of the supernatant was collected and transferred into a 50-mL EP tube containing 40 mL of 1% PBT solution. The solution was mixed with pH being adjusted to 6–8, followed by filtration through a 0.45-µm syringe nylon filter. 40 mL of the filtrate was precisely measured and elutriated through an immunoaffinity column at a flow rate of 1–2 drop(s) per second, then 10 mL of PBS and water were taken, respectively, to wash the column successively, and finally 800 µL of methanol was added to elute the target aflatoxins. 1 mL of the eluent was collected and filtered through a 0.22-µm syringe nylon filter for HPLC-PCD-FLD analysis.Then, 5 g homogenized lotus seed sample powders that have been stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for 30 days were accurately weighed and placed in a 50-mL centrifuge tube with the addition of 1 g NaCl and 25 mL of methanol–water  versus the injection concentration (x). Then, the contents of aflatoxins in the tested samples were quantified according to the standard curves. All the working solutions were freshly prepared just before being injected into the HPLC analytical system.The mixed standard solution of aflatoxins was diluted to 9 different concentrations of working solutions, and 20 µL of the dilution was injected successively from low concentration to high concentration into the HPLC-PCD-FLD system. The peak area of each aflatoxin was recorded and the standard curve was established by linear regression of the peak area (w and Aw were expressed as mean ± standard deviation (SD) and analyzed by using the Origin 2016 software.All data regarding Cw of lotus seeds stored at 25 °C and different relative humidity conditions of 40%, 50%, 60% and 70% within 32 days was measured and shown in Fig. w values exhibited a fluctuation under different relative humidity conditions within the first 10 days, and then were relatively stable during the next 22 days, which was increased successively from low humidity to high humidity. The higher the relative humidity was, the bigger the Cw value was. The samples stored at relative humidity 70% exhibited the highest Cw values. After storage for 12 days at 25 °C and relative humidity 70%, the Cw value exceeded the prescribed limit (14%) for lotus seeds in the Chinese Pharmacopoeia , and further to prevent this medicinal and edible food from mildew.The Cw) data of lotus seeds stored at 25 °C and different relative humidity conditions of 40%, 50%, 60% and 70% within 32 days were determined and listed in Fig. w values expressed a similar trend with Cw, which presented some fluctuation under different relative humidity conditions within the first 10 days, and then were stable during the next 22 days. After storage for 10 days at 25 °C, the Aw values were increased with increasing the relative humidity from 40% to 70%, which reached the highest values at relative humidity 70%. After storage for 12 days at 25 °C and relative humidity 70%, the Aw values were bigger than 0.7, which exceeded the relative humidity value of 70%. When the relative humidity condition was lower than 70%, the Aw values were all smaller than 0.6, which might be considered as the suitable water activity control point for safe storage of lotus seed in practice. These data illustrate that the increase of relative humidity of storage condition will lead to the enhancement of the water activity of lotus seeds, which may raise the incidence of mildew of this edible and medicinal food. While, from then on, the maximum limit standard, as well as some regulations on Aw for lotus seed and other matrices are lacking. To prevent lotus seeds from mildew and mycotoxins contamination, it is suggested that the relative humidity conditions for the storage environment of lotus seed should be effectively controlled to lower the Aw in an acceptable range (not more than 0.6).The water activity  and experimental samples (inoculated with A. flavus conidia that could produce aflatoxins) stored at 25 °C and relative humidity 40%, 50%, 60% and 70% for 30 days was observed at an interval of 10 days. It could be seen in Fig. The above results have shown that the CyB1, yB2, yG1, yG2) of four aflatoxins at different concentrations (x) were determined and the linear regression equations with their correlation coefficient (R2), as well as the limit of detection (LOD) and quantitation (LOQ) were obtained and listed in Table Under the optimized chromatographic conditions, the HPLC-PCD-FLD chromatogram of four aflatoxins was recorded and shown in Fig. 1 was detected in both the blank and experimental lotus seed samples after they were stored at 25 °C for 30 days, and the concentration in the samples of experimental group reached 6.6 μg/kg, which has exceeded the officially-recommended MRL (5.0 μg/kg).Although no visible mildew was observed on the surface of lotus seeds by naked eyes within 30 days, it was not definite if toxic aflatoxins were produced in the samples. Herein, the lotus samples stored at 25 °C and relative humidity of 40%, 50%, 60% and 70% for 0, 10, 20 and 30 days were collected and prepared for aflatoxins determination using the developed HPLC-PCD-FLD method. The results in Table 1 could still be detected in lotus seeds stored for long time (about 30 days) at 25 °C and high relative humidity (around 70%), especially in the samples without incubation with toxigenic fungi, which would increase the risk incidence of potential safety threatens of lotus seeds, and should be given more attention. Therefore, in practice, the relative humidity of storage environment for lotus seeds should be effectively controlled in less than 70% with the optimum water content and activity control points at 12% and 0.6, respectively, for safe storage to ensure the quality and safety of this edible and medicinal food related products, as well as the health of consumers.These results indicated that lotus seed was a susceptible matrix to mildew and aflatoxins contamination. Although no visible mildew was observed, AFBw and Aw of the matrices for fungal growth, further may result in the occurrence of mildew and mycotoxins contamination . If storage conditions are poorly managed, some fungi species can infect these matrices, leading to mycotoxin contamination. Environmental temperature and humidity conditions are the two key factors for the growth of fungi, which should be in effective control to prevent foods, feeds and medicinal plants from mildew. In common, most fungi will grow and multiply quickly at 20–35 °C and relative humidity more than 70%  and experimental  samples of lotus seeds were stored at 25 °C and different relative humidity conditions to measure the Cw and Aw changes. Our results have shown that both the Cw and Aw values tended to be stable after storage for 10 days, and no obvious mildew was observed on the surface of all tested samples for 30 days. When the relative humidity was up to 70%, the Cw and Aw values were increased quickly, and the Cw value exceeded the official-permitted level (14%), which was in agreement with the reports  starch (Zhang et al. w and Aw conditions were seldom paid special attention and failed to control. Changes of weather and environmental conditions will lead to large humidity fluctuations. When the environmental humidity is above 70% for a long time, the water content and activity of foods will be high, which will easily result in the mildew of TCMs, along with mycotoxins contamination. Our results have exhibited that when the relative humidity was no more than 60%, the Cw and Aw values approximately equal to about 12% and 0.6, respectively, which might be recommended as the optimum water content and water activity control points for safe storage of lotus seeds in practice at 25 °C with the environmental relative humidity less than 60%, to ensure the quality, safety and effectiveness of medicinal and edible products, as well as the health of the consumers.In the practical medicinal and edible foods and TCMs storage process, the experimental temperature and humidity, as well as the C


TOMM40). The highly variant ‘523’ poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer’s disease, but whether it plays a role in modifying the risk or clinical course of PD it yet to be elucidated. The TOMM40 ‘523’ allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson’s Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 ‘523’ and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 ‘523’ allele groups were not associated with disease risk, while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 ‘523’ polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for age of symptom onset warrants further investigation in other PD populations.Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson’s disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of outer mitochondrial membrane 40 homologue gene ( The pivotal role of mitochondrial dysfunction in PD is supported by several parkinsonism-causing toxins, genetic mutations and retrotransposon insertions4, which specifically impair mitochondrial function. Insights from these toxins and mutations imply that mitochondrial dysfunction in the pathogenesis of PD can arise from a wide array of biological processes, such as bioenergetic disturbances, nuclear and mitochondrial DNA mutations, impaired fusion and fission, defective mitophagy, and abnormal morphology and size5. For example, a significant cause of mitochondrial dysfunction in PD is the inhibition of mitochondrial complex I, an ETC defect that leads to severe oxidative stress and ROS- and caspase- mediated dopaminergic cell death5. Notably, the latter impairment is a major pathological feature of PD induced by familial PTEN-induced kinase 1 (PINK1), alpha-synuclein (SNCA) and Daisuke-Junko-1 (DJ-1) gene mutations or the toxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)3. Thus, mitochondrial dysfunction is thought to cause neuronal stress and degeneration, eventuating in this neurodegenerative disease, PD.Parkinson’s disease (PD) is increasingly known as a multifaceted neurodegenerative disorder with a heterogeneous and burdensome symptom presentation and progression. Abnormal or deficient mitochondrial functioning is widely implicated as a key process in the selective neuronal death and pathogenesis of PD. Mitochondrial dysfunction, resulting in a loss of electron transport chain (ETC) efficiency or decline in ATP-synthesising capacity, appears to elicit dopaminergic cell death via a number of mechanisms, including reactive oxygen species(ROS)—generation, impaired ATP production and disrupted calcium homeostasisTOMM40) gene encodes the pore-forming subunit (TOM40) of the protein-transport channels in the mitochondrial outer membrane, thus playing a fundamental role in mitochondrial functioning6. The TOM40 protein facilitates the import of approximately 1500 externally synthesised proteins and peptides into the mitochondria, and plays a key role in the mitophagy degradation pathway8. Altered or abnormally functioning TOM40, mediated by genetic changes or atypical protein expression, is thought to contribute to mitochondrial dysfunction and protein accumulation in Alzheimer’s disease (AD) and PD8. This may occur through different mechanisms: (1) mitochondrial import impairment, which may prevent essential proteins and peptides from reaching their designated mitochondrial targets, or allow unwanted and mutant proteins to aggregate in the mitochondria; or (2) mitophagy disruption, which may enable damaged and malfunctioning mitochondria to accumulate. Given evidence for mitochondrial dysfunction in PD, genetic variants affecting TOMM40 expression may have risk and disease-modifying effects.The translocase of outer mitochondrial membrane 40 homologue (TOMM40 is apolipoprotein E (APOE), which is in a region of strong linkage disequilibrium (LD), or non-random association, with TOMM40 variation11. Though the extent to which common variants in TOMM40 can have APOE-independent effects on disease risk or disease-modifying effects remains uncertain, polymorphisms in TOMM40 have been independently associated with a range of primarily cognition-based neurodegenerative diseases, including AD, frontotemporal dementia and dementia with Lewy bodies, as well as non-pathological ageing15. One of these variants is situated in intron 6 of TOMM40 and is a homopolymer of repeating thymine (T) nucleotides known as rs10524523 or ‘523’16. The ‘523’ variant is polymorphic, with lengths that vary from 11 to 39 T residues in Caucasians17. To better categorise and describe this diverse structural variant, three allelic groups for the homopolymer have been established previously, according to the number of constituent T residues: ‘Short’ , ‘Long’  and ‘Very Long’ (T ≥ 30)18. Although the functional effects of the ‘523’ variation are mostly unknown, increasing length of the poly-T repeat is thought to increase TOMM40 expression20. Therefore, the effect of the TOMM40 ‘523’ variant and altered TOM40 protein levels may contribute to mitochondrial dysfunction in PD21. While a vast number of published studies report conflicting roles of the various TOMM40 ‘523’ variant lengths in AD and ageing populations26, there have only been two previous studies within PD cohorts with differing findings. The first was a study of a relatively large Polish PD cohort which found no significant associations between TOMM40 ‘523’ and PD risk or age of onset27. Subsequently, a significant overrepresentation of the L/VL ‘523’ genotype was reported in a Swedish PD cohort compared to healthy controls, though it should be noted that these findings are yet to be published in full28. Thus, with conflicting findings in these two European cohorts, further investigation in other populations is required.Downstream of TOMM40 in mitochondrial import and mitophagy, both plausible sources of PD-causing mitochondrial dysfunction, the ‘523’ variant in TOMM40 is therefore a potential risk factor and disease modifier in PD. This study investigated the distribution of ‘523’ alleles and their association with disease risk and age of symptom onset in two independent PD cohorts, one Australian and one international , using PCR-based and whole genome sequencing approaches.Due to the essential role of 29. Clinical and demographic data, including the age of symptom onset, were recorded in the database. All PwP were examined by a movement disorder neurologist prior to inclusion in the study for verification of the diagnosis in accordance with the UK Brain Bank criteria for idiopathic PD and reported no family history of PD30, while healthy controls were confirmed to have no history of any neurological disorders. This study was approved by the Sir Charles Gairdner Hospital Human Research and Ethics Committee . Written informed consent was obtained from all participants, in accordance with the Australian National Health and Medical Research Council research guidelines.The first cohort comprised of 235 home-based people with PD (PwP) and 231 healthy controls of European heritage from the Perron Institute for Neurological and Translational Science PD Database, as previously reportedhttp://www.ppmi-info.org/data). This cohort comprised of 368 PwP and 172 healthy controls, and only individuals of European heritage were included in order to reflect the composition of the Australian cohort.The second cohort was derived from the international Parkinson’s Progression Markers Initiative (PPMI) database  and stored until DNA extraction. Alternatively, blood was collected from the medial cubital vein. DNA was extracted and purified from these samples using QIAamp DNA mini kits , according to the manufacturer’s protocol. DNA concentration was determined using absorbance readings calculated by a NanoDrop One Microvolume UV–Vis spectrophotometer .‘523’ variant in the Australian cohort was completed using fluorescently labelled primers, as previously described19. The forward primer sequence was 5′-/6-FAM/-TGCTGACCTCAAGCTGTCCTC-3′ and the reverse primer was 5′-GAGGCTGAGAAGGGAGGATT-3′, synthesized by Integrated DNA Technologies Pty Ltd . Endpoint PCR was performed using 5 μL of 5 × MyFi Reaction Buffer , 1 μL of MyFi DNA Polymerase (Bioline), 0.2 μL of each forward and reverse primer (20 μM) (IDT), 0.25 μL of 1% dimethylsulfoxide , 50 ng of genomic DNA, and 11.35 μL of dH2O ; to a final volume of 25 µL. Optimised PCR conditions were as follows: 1 cycle at 94 °C for 3 min, 27 cycles at 94 °C for 15 s, annealing at 65 °C for 20 s and extension at 70 °C for 30 s, and 1 cycle at 70 °C for 5 min. Applied Biosystems SimpliAmp Thermal Cyclers  were used for endpoint PCR cycling. Post PCR products were stored at 4 °C until capillary fragment separation, which was conducted by the Australian Genome Research Facility . Electropherograms were analysed using Peak Scanner Software . ‘523’ allele lengths were determined according to a previously established method by Linnertz et al.19. Briefly, the highest intensity peak(s) in each peak cluster between 160–190 bp were identified and sized, and 150 bp (accounting for flanking regions and primers) was subtracted from the peak sizes to determine poly-T allele lengths. Alleles were grouped using the convention established by Roses et al.9: Short , Long  and Very Long .PCR-amplification of the http://www.ppmi-info.org/data) in binary alignment map (BAM) format that had been aligned to the human reference genome GRCh38 using the Burrows-Wheeler transform alignment algorithm31. Resultant BAM files were analysed using the Integrative Genomics Viewer32 in order to calculate the length of the poly-T repeat, as previously demonstrated33. Alleles were grouped using the convention established by Roses et al.9: Short , Long  and Very Long .Whole genome sequencing (WGS) data were obtained from the PPMI database (available at APOE ε were determined using the single nucleotide polymorphism (SNP) and PCR-restriction fragment length polymorphism (PCR–RFLP) analyses. APOE ε2/ε3/ε4 genotypes were determined by sequencing two SNPs (rs429358 and rs7412) using the MassARRAY system  at the Australian Genome Research Facility 34. For PCR–RFLP analyses, endpoint PCR reactions were prepared to a final volume of 10 µl using primer sequences previously described35. Reactions contained 7.2 µl dH2O , 2 µl MyFi reaction buffer , 0.05 µl MyFi DNA polymerase , 0.375 µl forward and, 0.375 µl reverse primer  at 200 ng/µl, and 25 ng DNA. The PCR amplification protocol followed, being an initial hold temperature of 95 °C for 4 min 30 s and 35 cycles of denaturation at 95 °C for 30 s, annealing 60 °C for 30 s and extension at 72 °C for 1 min 30 s. Restriction enzyme reactions were prepared to a final volume of 20 µl, containing 6.3 µl dH2O , 0.2 µl acetylated BSA , 2 µl C buffer , 0.5 µl Hhal  and 10 µl PCR product. Reactions were incubated for 4 h at 37 °C prior to polyacrylamide gel fractionation. Restriction enzyme digested products were fractionated on 12% (w/v) 29:1 polyacrylamide gel  in 1 × TBE. Electrophoresis fragment separation was performed at 100 V for 3 h on the DCode Universal Mutation Detection System . Gels were stained in 1 × TBE containing SYBR Gold nucleic acid gel stain  for 4 min before visualization using a BioRad Chemidoc MP Imaging System. APOE ε genotype data relating to the PPMI cohort was obtained from the online database (available at www.ppmi-info.org/data).Genotyping of p-value of < 0.05 was employed for all statistical tests. Variables were described using mean and standard deviation , or frequency and percent , as appropriate. Normality was assessed and subsequent clinical characteristics were analysed using Independent Samples T-Test, Mann–Whitney U, or Chi-square, as appropriate. For cross-sectional analysis, Chi-square, stratified Mantel–Haenszel tests and binary logistic regression models were used to evaluate the association between TOMM40 ‘523’ genotypes and risk of PD in the Australian and PPMI cohorts. Binary logistical regression models were run both with and without correction for the APOE ε4 status ) and patient sex. Analysis was also run considering all combinations of ‘523’ length category and APOE ε genotype, to examine for interactive effects without a priori assumptions. The aforementioned analyses were performed separately in the PPMI and Australian cohorts, and after combination of the cohorts. Following this, binary logistic models were carried out when considering populations of APOE ε3/ε3 carriers only, as previously examined in this fashion and stated as a requirement for replication studies13.The Australian and PPMI cohorts were analysed separately, and together, using IBM-SPSS software . A significant nominal TOMM40 on age of disease onset, correcting for APOE ε allele status and sex. Again, GLMs were carried out when considering populations of APOE ε3/ε3 carriers only. Residual plots were examined for all models and no violations were noted. Correction for multiple comparisons was conducted using Bonferroni pairwise comparisons, where appropriate.Generalised linear models (GLMs) were also constructed in order to study the interaction of TOMM40 ‘523’ S allele carrier status  and APOE ε4 status  were combined to produce 5 groups. Mean comparisons were then analysed using the Kruskal–Wallis one-way analysis of variance and univariate GLMs. This analysis was repeated in the combinations of TOMM40 ‘523’ S allele carrier status and APOE ε2 status, and TOMM40 ‘523’ VL allele carrier status and APOE ε4 status.Subsequently, the load combination of TOMM40 ‘523’ genotype as well as both the TOMM40 ‘523’ and APOE genotype. To compare the survival curves, the log rank test was applied, placing weight on longer survival periods37. Allelic stratification by TOMM40 ‘523’ was also run using Kaplan–Meier analysis. Additionally, all distributions of ages at onset adjusting for sex were compared via Cox proportional hazard regression models.Finally, Kaplan–Meier curves for age at PD symptom onset were estimated, stratified by A summary of the cohorts included in this study is presented in Table TOMM40 ‘523’ genotyping was obtained following optimisation of a PCR-based assay, with representative electropherograms presented in Supplementary Fig. 1. Genotyping of the TOMM40 ‘523’ variant in the PPMI cohort was completed using a WGS method, previously reported as an alternative method to fragment analysis and Sanger sequencing33. The distribution of the ‘523’ alleles in PwP compared to healthy controls for both cohorts are presented in Fig. 9 (Supplementary Table 1). APOE ε genotypes of the Australian cohort were further validated using PCR–RFLP, with representative samples ε2/ε3, ε2/ε4, ε3/ε3 and ε3/ε4 presented in Supplementary Fig. 2. APOE ε genotype and allele groupings for both cohorts exhibited similar distributions, however there was an absence of individuals with the ε2/ε2 genotype in the Australian cohort (see Supplementary Table 2).In the Australian cohort, ‘523’ distribution and no significant difference was observable . Each distribution was also compared to distributions on Webstr database, similarities were seen between the current study cohorts and both the Gtex (predominantly European self-reported ancestry) and 1000 genomes European distributions. Thus, the current cohort were considered reflective of a population of European descent and the two cohorts could be combined to increase the sample size and power to detect ‘523’ effect.Both groups consisted of individuals with self-reported of European heritage, though country of origin information was not available for all cases. To determine whether cohorts could be appropriately combined, Levene's Test for Equality of Variances was used to compare the TOMM40 allele distributions between cohorts, case–control analyses were completed in both individual cohorts and in a combined cohort. When examined genotypically, the TOMM40 ‘523’ variant was not significantly associated with the risk of PD in either the Australian cohort , or the PPMI cohort , or when cohorts were combined . Moreover, when combined, allelic distribution of the S , L  and VL  binned groups was not significantly different between PD and controls . Analysis considering all combinations of ‘523’ genotypes and APOE ε genotypes also failed to show any significant differences between PD and control groups. As with prior findings, analysis utilising stratified Mantel–Haenszel tests did not find significant differences.As there were no significant differences in APOE ε4 status and patient sex into account, which again exhibited no significant association between TOMM40 ‘523’ genotype and PD risk. Models considering all combinations of ‘523’ and APOE ε genotypes also returned non-significant results. Binary logistic regression models were used subsequently to assess this association allelically. Neither naïve nor covariate corrected models, in either cohort or the combined cohort, exhibited a significant allelic association between TOMM40 ‘523’ and disease risk (Table APOE ε3/ε3 carriers (Supplementary Table 3), and again failed to show any significant associations between TOMM40 ‘523’ alleles or genotypes and risk of PD.Binary logistic regression models were analysed comparing each genotype or allele to all other genotypes or alleles Table . GenotypTOMM40 ‘523’ on age of disease onset, GLMs were constructed, with data from naïve and corrected models reported in Table TOMM40 ‘523’ alleles or genotypes and age of PD symptom onset was observed. However, in the PPMI cohort, carriage of the TOMM40 ‘523’ S/S genotype was associated with a 2.58 year later age of symptom onset (p = 0.040). This difference was not significant after controlling for APOE ε4 load and sex in multivariable GLMs, although a trend for later age of onset was still observable . It is important to note, however, that there was a lack of co-carriage of the S/S genotype with the APOE ε4/ε4 genotype in this cohort (see Supplementary Table 4) and that APOE ε4/ε4 homozygotes had a mean age of symptom onset almost 4 years earlier than other genotypes, although this was not statistically significant (p = 0.314). Thus, significance may have been lost due to opposing directionalities of these two genotypes on age of symptom onset. Carriage of the S/S genotype was also associated with a 2.69 year later age of PD symptom onset in the PPMI cohort (p = 0.032) after correcting for APOE ε2 load and sex. However, when adjusting for multiple comparisons, model-derived estimated means revealed the S/S genotype did not result in a significantly different age of PD symptom onset when compared to individual genotypes (p = 0.326).To investigate the influence of TOMM40 ‘523’ and age of PD symptom onset (Table APOE ε3/ε3 homozygotes (Supplementary Table 5), none of which revealed a significant association between TOMM40 ‘523’ and age of symptom onset. Finally, when considering the load combination of the TOMM40 ‘523’ S allele and APOE ε4 or APOE ε2, no significant differences were found in the mean age of symptom onset between groups in either cohort or in the combined cohort according to the copy number of these alleles.Subsequent analysis of the PPMI cohort exhibited no other statistically significant associations (at the 0.05 level of significance), either allelically or genotypically. Furthermore, when the cohorts were analysed as a combined cohort, no significant associations were exhibited between TOMM40 ‘523’ genotype, in the Australian cohort , respectively. Subsequently, Kaplan–Meier curves for the age at which symptoms of PD began were analysed, stratified by TOMM40 ‘523’ and APOE genotype as in Roses et al.13. Again, neither the Australian cohort (p = 0.550, Fig. p = 0.867, Fig. p = 0.660, Fig. TOMM40‘523’ allelic stratification also failed to produce significant results. Finally, all analyses completed using the Kaplan–Meier method were re-run using Cox proportional hazard regression models, which adjusted for sex, and no significant results were noted.Kaplan–Meier curves for the age at which symptoms of PD were first reported were stratified by ort Fig. A, the PPort Fig. B and theort Fig. C. No or TOMM40 ‘523’ structural variant as a potential PD risk factor and modifier of the age at symptom onset. The distribution of ‘523’ poly-T alleles did not vary between PwP and healthy controls, or in the two independent cohorts examined. The similarity in ‘523’ allelic distribution between the PPMI and Australian cohorts is worth noting as this study utilised two different approaches in the calling of the TOMM40 ‘523’ variant, establishing a PCR-based and a WGS-based assay for use in the Australian and PPMI cohorts, respectively. Assay development for the TOMM40 ‘523’ variant is generally considered to be difficult, as poly-T variants are challenging to sequence19. Despite optimisation within this study, PCR stuttering was observed in the PCR-based assay similar to previous reports19, which is a standard complication when amplifying repetitive genomic sequences, and highlights the need for further optimisation of the assay. By comparison, calling from WGS files revealed a similar distribution as reported previously33. While it has been presented as having fewer challenges in optimisation and sequencing, calling of ‘523’ variants was an arduous process and the aforementioned study has stated that the correlation between ‘523’ calling by WGS and PCR-based methods decreases with increasing size of the ‘523’ allele33.The current study aimed to investigate the TOMM40 ‘523’ variant is implicated in PD risk and found no association between carriage of allelic variants and the risk of developing PD. When binned, genotypic and allelic frequencies appeared to be similar between controls and PwP in both independent cohorts, and when the cohorts were combined. This agrees with previous work in a Polish PD cohort which showed no association of ‘523’ alleles, genotypes or haplotypes with the risk of PD or age of symptom onset27. In contrast, in a Swedish population, a higher frequency of the L/VL genotype was observed in PD patients compared to controls28, though these findings are yet to be published in full. Given that these studies were conducted in different ethnic groups, and that ‘523’ allele frequencies are ethnic-specific18, the contradictory findings in the studies to date may indicate that ‘523’ is a risk factor in some populations, but not in others. As this is only the third study to investigate the role of TOMM40 ‘523’ in PD risk, further studies should be performed in other ethnically diverse populations.The present study then examined whether the 39. Previous studies have implicated TOMM40 ‘523’ length in the age of onset of AD, particularly in carriers of the APOE ε3 allele22, but mixed findings exist. For instance, although Roses et al.9 reported that longer ‘523’ allele lengths were associated with risk of AD and earlier age of onset, the initial findings were not replicated in other populations40. Such varied findings may be a result of varied methodology and varied consideration of the influence of the APOE ε locus13, which is well-established as the strongest genetic predictor of AD. Most studies to date report no association of APOE ε variation with susceptibility to PD42, as was also the case in the current study. While the present findings did not show any positive evidence of interactive effects between TOMM40 ‘523’ and APOE ε genotypes in relation to PD risk, they do suggest that the two loci may have small independent and opposing effects on determining the age of onset of the symptoms. While initial regression analyses in the PPMI cohort showed that age of onset was significantly delayed by carriage of the S/S ‘523’ genotype, this was not replicated after correction for covariates, or in the Australian cohort. However, it is possible that this difference is caused by a country- or geographical-specific effect, as seen in other genetic studies43. It is worth noting that only one prior study has examined this association27, though it is not clear whether this study completed rigorous analysis of the interactive effects of TOMM40 and APOE, as was conducted in the current study. As such, further in-depth analysis in larger PD cohorts is required to determine the significance of the present findings.As mitochondrial involvement in PD is thought to be a key contributor to neuronal dysfunction and degeneration, it is plausible that a genetic variant that modulates mitochondrial function could modify age of symptom onset. Furthermore, AD and PD share a number of clinical, pathological and molecular features including toxic protein accumulation and mitochondrial dysfunction in the form of respiratory chain defects, oxidative stress, mitochondrial DNA damage and morphological abnormalities‘523’ allele length are poorly understood due to a scarcity of research in this area. While several studies have suggested that the VL allele increases TOMM40 mRNA expression and the S allele represses expression44, others have found no significant differences in mRNA levels between S and VL variants45. A recent study demonstrated that overexpression of TOMM40 in vitro was associated with greater mitochondrial membrane potentials, respiratory rates, spare respiratory capacities, ATP levels, amyloid-beta resistance, and protein uptake16. On the other hand, another study observed a correlation between TOM40 protein deficits and enhanced oxidative stress, reduced ATP production and abnormal complex I protein concentrations in the brains of PD patients and in alpha-synuclein overexpressing murine models21. As the literature currently stands, further elucidation of the biological consequences of up- or down-regulated TOMM40 expression is required to give insight into its potential role in PD risk and disease modification.Currently, the functional effects of variation in TOMM40 ‘523’ variant in two independent PD cohorts. TOMM40 plays an essential role in mitochondrial import and mitophagy, and the ‘523’ polymorphism has been associated with age of onset of AD and with age-related cognitive decline. While this study clarifies that TOMM40 ‘523’ is not in itself a predictor of PD risk, it raises the possibility that, as in AD, it may be a genetic marker for the age of symptom onset in PD. Whilst not conclusive, our findings in the large international PPMI cohort suggest that carriage of the S/S ‘523’ genotype may be protective in terms of delaying the age of symptom onset, and may warrant further investigation in other populations. Importantly, the effect of co-carriage of the APOE ε4 allele, which appears to have an opposing effect on age of onset, must be considered in future studies. Though not significant in modulating risk of PD, future studies should consider the possible role of TOMM40 ‘523’as a determinant of age of symptom onset, and symptom trajectory. Given a phase 3 clinical trial of a therapeutic for the prevention and delay of onset of AD was recently conducted involving participants stratified by TOMM40 ‘523’46, the findings reported herein are noteworthy, and may allow the design of symptom-focused studies in PD for much-needed improvements in patient outcomes and care.Overall, this study aimed to investigate the risk and disease-modifying role of the Supplementary Information


Diversity in metacommunities is traditionally viewed to consist of the diversity within communities ( Diversity in metacommunities is traditionally viewed to consist of the diversity within communities that is complemented by the differences between communities so as to result in the total diversity of the metacommunity  nor environmental diversity alone but rather the combination of both.The effect of this combination on (joint) diversity becomes apparent through the associations between the two components “trait”  and “community affiliation” . Associations rank between complete association  and the absence of association . Particularly complete association underlines the role played by the “flanking” or “marginal” distributions, since in this case, the joint diversity equals the overall  trait diversity (when differentiation of communities for types is complete) or the diversity of communities . The criterion is also known as the principle of transfers  of their effective numbers into components relating to the marginal variables “trait” and “community affiliation.” Herewith, one component (the leading component) is distinguished by realizing a minimization criterion. As applied to community affiliation as leading component, for example, the criterion determines the minimum number of communities required to realize the observed joint diversity given the observed trait distribution. The criterion does not rely on the concept of effective numbers at the outset, but it is amenable to an interpretation in terms of such numbers. The minimally required number lays the basis for a unique factorization of the joint diversity into the leading  and the associated marginal component (trait). The relation of the factorization to a more recently proposed method of partitioning diversity is demonstrated that avoids the arbitrariness inherent in averaging diversities within communities , the joint diversity equals the overall  type diversity  equals the product Consequently, equating the product with the observed joint diversity effective number C as a leading component, for which the number community-centered perspective of the factorization. One then arrives at the multiplicative decomposition (or factorization) of joint diversity in the formThe latter fact asks for distinction between a leading and an associated component of the factorization, where the leading component reflects the desirable properties. The above demonstrations suggest For Rényi-diversities , one would then aim at finding the minimum number of types required to obtain the observed joint diversity in the first place. In this way, the contribution of types to the ecological diversity of a metacommunity is assessed on the basis of the observed partition of the metacommunity into its constituent communities. This done, the associated effective number of communities is to be specified. Since community determinants are frequently considered to be less dynamic than the traits of the community members existing on or adapting to these determinants, this may be the more relevant perspective to be taken in many ecological studies.The apparent asymmetry in the above treatment of the community and type component T and C. All of the conclusions obtained so far apply analogously. Thus, one first determines the minimum number In essence, this perspective simply marks the dual analogue of the previous community-centered approach to the decomposition of joint diversity into community and type components. It therefore results from just reverting the roles of For Rényi-diversity, indicator of structural characteristics under the community-centered perspective when considering the deviation of The minimum number The latter suggests consideration of varies from 1 towhile structural characteristics vary from complete differentiation to complete absence of differentiation among communities.When compared with the observed number of communities, N again be the largest natural number with N, one infers that generally whilevaries from 1 tothe marginal type frequencies start with monomorphism and end with maximum polymorphism .Characterization of the structural aspects encoded in This result is obvious for Rényi-diversities, since there always The structural characteristics inherent in the factorization of joint diversity illustrate more vividly the conceptual advantages of the present approach, which focuses on the numbers of communities and types that essentially determine metacommunity diversity. By “essential,” two issues are addressed, factors and their interaction. The factors are specified by the minimally required number of communities (which depends on their differences in type composition) and the associated number of types . Their interaction is “orthogonal” in the sense of an independent operation in producing the joint diversity.The factorization reminds of the common approaches to partitioning diversity in such a way that the total (metacommunity) type diversity Chiu et al.  destabilize polymorphisms and by this imply tendencies toward monomorphism.Since in self-incompatibility systems, heterotypic combinations are preferred irrespective of community affiliations, potential differences in community affiliation between types tend to be equalized. This situation is characterized by small It remains to demonstrate how the two perspectives are connected. Since both perspectives rest on the same joint diversity entclass1pt{minimaThe latter observation becomes evident when considering the fact that Often, the spatial distribution of communities does not show a sufficient degree of fragmentation to allow for determination of a unique subdivision into subcommunities. As a consequence, more than one subdivision may become relevant, each of which may show a different partitioning of the same underlying overall type diversity. Similarly, different criteria for subdivision, such as the spatial distribution of environmental variables, may be relevant in comparative causal analyses of observed genetic or species variation. In this case, the states of each variable give rise to a subdivision into “communities,” which in turn enables comparison of the effects of the various variables on the observed type variation.structure , which is established especially in population genetics and implemented in methods such as An assignment of the above kind specifies a joint distribution of types and community affiliations and therefore allows computation of the joint diversity together with its factorization into In most cases, the models underlying an analysis of latent factors are based on probability laws that specify likelihoods or posterior probabilities of the assignments. These probabilities serve as the primary qualification criterion that is to be maximized . This allows consideration of the most likely structural characteristics indicated by


General knowledge questions are used across a variety of research and clinical settings to measure cognitive processes such as metacognition, knowledge acquisition, retrieval processes, and intelligence. Existing norms only report performance in younger adults, rendering them of limited utility for cognitive aging research because of well-documented differences in semantic memory and knowledge as a function of age. Specifically, older adults typically outperform younger adults in tasks assessing retrieval of information from the knowledge base. Here we present older adult performance on 421 general knowledge questions across a range of difficulty levels. Cued recall data, including data on the phenomenology of retrieval failures, and multiple-choice data are available. These norms will allow researchers to identify questions that are not likely to be known by older adult participants to examine learning or acquisition processes, or to select questions within a range of marginal accessibility, for example. Comparisons with young adult data from prior databases confirms previous findings of greater knowledge in older adults and indicates there is preservation of knowledge from early adulthood into older adulthood.The online version contains supplementary material available at 10.3758/s13428-020-01493-2. A standard categorization of long-term, declarative memory is as either episodic or semantic Tulving, . Many, iIn contrast, the nature of semantic memory or the knowledge base is such that assessing the contents of this system can be challenging. The assumption is that the contents include pre-experimentally acquired information that is relatively stable over the lifespan and can be accessed across contexts Tulving, , 1985. IInterestingly, GK has been found to predict recent and current event knowledge , so having an accurate assessment of what someone knows is important, to control for differential effects of prior knowledge. For example, researchers examining marginal knowledge , to item selection effects , to changes in reading habits among younger cohorts  and multiple-choice questions. The former typically require more effortful search strategies in memory, whereas the latter, because the answer is provided, are more sensitive to discrimination among related foils. Older adults, in episodic tasks, generally show more marked deficits in tests that offer less environmental support, such as cued-recall, than tests such as recognition , were normed in a cued-recall test. The questions were divided into four sets ranging from 70 to 148 questions each. For each question, participants had the option of providing an answer, indicating they could not remember (DR), or indicating they did not know (DK) the answer. Specific guidelines on when and how to use DR and DK were not provided  platform . Fifty-seven participants completed the first set of questions  consisted of 55 older adults recruited on MTurk (see Table n = 32) and at Claremont McKenna College . Five participants reported English was not their first language (see Table Participants tested in the lab were community-dwelling older adults (ages 60+). For the Set C questions, the participants were 67 older adults recruited from the Waterville, Maine, community who participated in two experimental studies examining the phenomenology of retrieval failures .As mentioned above, four different sets of questions were used. Two sets (A and B) were developed for the purposes of gathering the present normative data; the other two (C and D) were originally used in experimental tasks in our labs. The encoding phase of the experimental tasks was similar to the norming task, in that participants provided responses to open-ended questions about a variety of topics or responded DR or DK. The questions in all sets covered a variety of topics, ranging from literature to sports, geography, history, science and technology, pop culture, and music (see the https://github.com/el-cms/Open-trivia-database) and the online version of the Encyclopedia Britannica, which includes an online quiz platform (www.britannica.com/quiz). Set B included 134 questions from Burke et al. . If you do not remember the answer, please type DR ("Don't Remember"). Please do not leave any questions blank.”Importantly, no additional instructions were provided on when and how to use these options. They were further told some of the general knowledge questions were quite difficult and were informed that the purpose of the study was simply to assess what people know. Because MTurk participants are paid by the number of jobs they complete online, we were reasonably confident participants would not spend additional time searching for correct answers online.Participants who were tested in the lab were tested individually. The study was programmed using E-Prime . Errors were coded as errors of omission  or errors of commission . DR and DK responses were coded as such. Table Analyses are reported at the item level. As noted above, for each item between 54 and 67 participants responded . Responses were hand scored by a research assistant. Responses were scored as correct if the participant provided the correct answer or a clear misspelling or minor morphological/wording variation , indicating a wide range of question difficulty was successfully obtained. Errors ranged from 0 to .82 . The proportion of DR responses was overall low ; however, the range of 0 to .56 does indicate some questions in the pool might be reflective of marginal knowledge. DK response rates ranged from 0 to .93 . Very few questions were left blank overall , indicating participants were generally following instructions. Rank was calculated in order of increasing difficulty; ties were left as such in the database.To examine whether the rate of responses varied as a function of item difficulty, we binned all questions into quartiles, using accuracy as a proxy for difficulty. By binning the items into quartiles, we provide a basic stimulus selection framework for researchers, who might be interested in identifying items with a specific level of difficulty or items that elicit a high rate of DR or DK items. Because the data are necessarily ipsative and not independent , we focus on the descriptive statistics rather than providing any inferential statistics. Thus, the 421 questions were divided into quartiles ranging in overall accuracy from most difficult , to moderately difficult , to moderately easy , to easiest . As can be seen in Figure The variable rates of DR and DK responses suggest that questions that are in a moderate difficulty range elicit the highest proportion of DR responses whereas questions that are too difficult elicit the highest rate of DK responses. This reflects the fact that general knowledge varies in accessibility as well as in availability .Mean response times (RT) in milliseconds as a function of response  were calculated for each participant. DK responses were faster  than all other response types, which were similar . The rank order in the present study was correlated to the orders in previous studies: Compared to Tauber et al.  and to Nelson and Narens  there appears to be both generational and longitudinal stability overall.As was reported by Tauber et al. , we compd Narens  norms weM = .24, SEM = .02), intermediate in the Nelson and Narens’ norms , and lowest in the Tauber et al. norms , F = 82.98, MSE = .014, p < .001, ηp2 = .58 . This suggests that there is greater overlap in the knowledge base between today’s older adults and college students from the late 70s than between older adults and current young adults. Furthermore, although the knowledge base does increase over the lifespan (as evidenced by the higher accuracy among our older adults), the information that was known by young adults at the time of Nelson and Narens’ study appears to be preserved in older adults but very obscure to today’s younger adults. In concert with Tauber et al.’s conclusions about the need to regularly update general knowledge norms for younger adults due to fluctuations in the relevance of information, these findings point to the need to have age-appropriate norms for older participants as well. We acknowledge that these analyses are predicated on the strong assumption that age (or cohort) is the main factor that distinguishes our sample from the college students tested in the Nelson and Narens and Tauber et al. studies. Clearly, other factors, such as overall educational achievement, individual and group differences in information-seeking behaviors, and even personality characteristics such as conscientiousness, might contribute to the age-related differences. We return to these points in the General Discussion.In addition to examining overall ranking of difficulty as Tauber et al.  did, we Because general knowledge questions are used across a variety of tasks, in addition to providing cued-recall performance, we administered a multiple-choice version of the task to a set of new participants, all from online sources. Multiple-choice questions provide additional environmental support and, on tests that do so, such as item recognition, older adults often show reduced performance deficits relative to younger adults Craik, . Thus, pA total of 201 participants were recruited. All participants were recruited on MTurk, using the same restrictions described above. Data from five participants were omitted because they self-reported their age under 55 (three from Set A and one each from Sets C and D), leaving 49 participants in each set. All but four participants (two in Set B and two in Set C) reported being native speakers of English . We also confirmed that these participants were not pressing the same key on every trial nor were they responding quickly.What is the last name of the criminal who was killed by FBI agents outside of a Chicago movie theater”, to which the correct answer is Dillinger, names of criminals who were called “Public Enemies” by the FBI, just as Dillinger was, were listed as foil choices. The The same sets of questions were used. For each question, three alternative, incorrect responses were added. For set A, the foils were developed in the lab or were selected from the online quizzes where provided. For the questions in Set B, the Burke et al.  foils weThe procedure was very similar to that used in Experiment ρ(419) = .81, p < .001, although not perfect, indicating some variability in performance as a function of the demands on the retrieval process. To examine whether item difficulty based on the cued-recall task also predicted accuracy in multiple-choice, we used the quartile bins based on cued recall accuracy from Experiment SEM = .02) for the most difficult questions to .51 (SEM = .02), to .69 (SEM = .02), to .86 (SEM = .02) for the easiest questions, F = 212.00, MSE = .03, p < .001, ηp2 = .60. At all levels of difficulty, performance was above chance, estimated at .25 given four alternatives . Thus, the questions that were difficult to answer in one format were similarly difficult in the other format.Item-level information is presented in Table ρ49 = .81, F = 8.84, MSE = 16433694, p < .001, ηp2 = .06. Correct responses  were faster than errors , F = 31.34, MSE = 12122700, p < .001, ηp2 = .07. The interaction was significant, F = 18.93, MSE = 12122700, p < .001, ηp2 = .12. For easy and moderately easy questions, correct responses were faster than errors for the easier items ; however, as difficulty increased, the pattern reversed, such that errors were slightly faster than correct responses for the most difficult items t(105) = 2.0, p = .047, d = 20. RTs for moderately difficult items did not differ as a function of accuracy, t(104) = 1.44, p = .15, d = .14.RTs to correct and incorrect multiple-choice questions were examined in a one-way ANOVA as a function of question difficulty using the cued-recall rank as a between-items factor Fig. . This anF = 1018.79, MSE = .014, p < .001, ηp2 = .71  = 19.41, MSE = .014, p < .001, ηp2 = .12. The interaction  x 4  mixed ANOVA. Unsurprisingly, accuracy was higher in the multiple-choice format  than in the cued-recall format , n = 23). Thus, there appear to be some GK items that are more affected by incorrect foil presentation than others; although we acknowledge a very small set in our database fit this description. Overall, items that are more accessible in a cued-recall task also seem to be more accessible in a multiple-choice task. One caveat of this conclusion is that different participants completed the two tasks; future studies aimed at more directly assessing the relative accessibility or availability of specific units of knowledge might use alternative methods, such as directly comparing free recall, cued-recall, and recognition in a within-participants design. Alternatively, methods such as Buschke’s . Although significant, the effect was not particularly large, accounting for less 6% of the variance. This pattern held in all sets , except for Set A , which included the most difficult questions, suggesting that education predicts general knowledge level across a range of difficulty, but not when items are largely inaccessible. There was no systematic relation between rate of DR responses and education (r = .01). Years of education were negatively correlated with DK responses for all sets , in which DK was the modal response. This suggests that some items are simply not known and do not depend on educational attainment. Note that the items in Set D were specifically selected based on young adult norms to be especially difficult. In the multiple-choice task, there was an overall modest positive correlation between education and accuracy . Within each set of questions, the correlation was only significant for participants in Set C , which had the highest rate of correct answers overall. Thus, education, not surprisingly, does affect accuracy and presumably the knowledge base of participants.One important way in which the samples tested here and those included in the previous studies differ concerns student status and overall educational attainment. Whereas participants in Tauber et al.  and NelsAccording to several theories of intelligence


Substituent effects were interpreted in terms of the Hammett equation, showing a good correlation for carbons para- to the substituent group, not for the meta- ones. Similarly, the Lynch correlation shows the additivity of the substituent chemical shifts in the case of both H and C nuclei, again with the exception of the carbons in the meta- position. Density Functional Theory (DFT)-predicted 1H and 13C chemical shifts correspond closely with experimentally observed values, with some exceptions for C NMR data; however, the correlation is valid only for the aromatic moiety and cannot be extended to the heterocyclic ring of the chroman-4-one scaffold.The attribution of  Some time ago, pioneering studies demonstrated that Hammett parameters can be correlated, not only with reaction rates and equilibrium constants, but also with ultraviolet absorption spectra, infrared frequencies, and NMR chemical shifts [P Hammett parameters were successfully correlated with the chemical shifts of 1H and 13C NMR spectra in solution of a series of tetrabutylammonium iron(III) porphyrin complexes, in turn revealing how the electronic effect of the substituents affected the electronic configuration [F and σR) were employed as a molecular descriptor to characterize the substituent effects [Linear free-energy relationships (LFER) establish a direct correlation between thermodynamic parameters and kinetic data, offering insights into reaction mechanisms. LFERs create a connection between several measurable quantities and well-known parameters, Hammett substituent constants  of a given substituent on the electron density at the portion of molecule under study. Accordingly, there should be a direct correlation with any property affected by the electron density at the considered site. Therefore, the rationalization of substituent effects propagation in a molecule leads to a deep understanding of structure-property relationships and shines light on reaction mechanisms, two essential concepts in chemistry ,9,10.1H and 13C chemical shift values [0 is the chemical shift value of the unsubstituted compound [Frequently, NMR spectroscopic parameters are used to understand the transmission of substituents electronic effects within organic molecules, with a specific focus on routinely used t values ,12,13. TWhen good linear correlations are obtained applying Equation (1), the substituent effect on the chemical shift values can be considered electronic in origin, and the electron density around the nucleus under study is mostly affected by the electron-donating or -withdrawing ability of the substituent itself.In recent years, computational chemistry, in particular Density Functional Theory (DFT), has provided strong support to experimental chemists, giving insight into the properties and reactivity of different molecular entities. This is particularly apparent in the realm of organic chemistry, where the behavior of a given molecular group  can be tuned by the nature of the attached substituents, if any. On one hand, it has been demonstrated that DFT can reproduce nicely the experimental data and, on the other one, it may have a predictive value as well, e.g., in those cases where experimental data are not available ,15,16,17Herein, we report the application of these methods to 2,2-dimethylchroman-4-one derivatives, for which substituent effects have not been previously assessed. The chroman-4-one  ring system occupies an important position among oxygen heterocycles, as it is featured in a wide variety of compounds endowed with biological, medicinal, and synthetic interest ,22,23,24With the objective of understanding the factors underlying the transmission of substituents effect within the chromanone system, a library of substituted 2,2-dimethylchroman-4-one derivatives  has been studied and their chemical structures are shown in 1H and 13C NMR spectroscopic data of 2,2-dimethylchroman-4-one derivatives from Series 1–3 [Lynch correlations to evaluate the proportionality relationships with respect to the appropriate substituent chemical shift (SCS) in monosubstituted benzene; and (ii) Hammett correlations to show the resonance and inductive transmission of substituent electronic effects. Finally, prediction of the 1H and 13C nuclei chemical shifts by using DFT calculations has been also undertaken, followed by an analysis of the existing correlation among theoretical and experimental data.Based on our previous work and after an accurate survey of the literature, we decided to collect the ries 1–3 ,31,32. I1H chemical shifts of chroman-4-one derivatives belonging to Series 1, 2 and 3  displays a deshielding effect for all the studied compounds, as can be inferred from the data collected in the tables. Thus, this signal is located at ca. 2.70 ppm in the 1H-NMR spectra, as a result of the effect of both the carbonyl and the alkoxy groups belonging to the chroman-4-one scaffold. However, the substituents attached to the phenyl moiety  of the 6-substituted derivatives (Series 1) are those expected for this substitution pattern, with values of J5,7 from 1.6 to 2.4 Hz and J7,8 from 8.0 to 8.6 Hz. The J constants of compounds belonging to Series 2 (substituted in the 7-position) show values of J5,6 from 8.0 to 8.8 Hz and J6,8 from 1.0 to 2.4 Hz, while those of Series 3 (substituted in the 5-position) show values of J6,7 from 8.0 to 8.8 Hz and J6,8 from 1.0 to 2.4 Hz.The experimental nd 3 see  are repond 3 see  —SM—. Thety R in  do not p13C chemical shifts of the 2,2-dimethylchroman-4-one derivatives from Series 1, 2 and 3 are reported in 13C chemical shifts of the methylene group in the 3-position and the methyl groups in 2-position were observed, as apparent from the data collected in the tables. A “normal” substituent effect on the 13C chemical shift of the carbonyl group was found for the compounds in Series 2, as can be judged from the deshielding effect on moving from electron-releasing to electron-withdrawing substituents, viz. 190.9 ppm for -NMe2 group vs. 195.0 and 201.3 ppm for -NO2 and -Cl substituents, respectively. On the other hand, no substituent effect on the same group was observed for derivatives from Series 1 and 3, due to a noticeable spread of the data. In addition, the substituent effect is nicely observed on ortho- and para- carbons for all the 2,2-dimethylchroman-4-one derivatives studied, while the same effect on the meta- carbons showed no linear trends due to the spread of the data (see below).The 1H and 13C chemical shifts of the investigated 2,2-dimethylchroman-4-ones according to the Lynch correlation was carried out [EXP is the substituent chemical shifts (SCS) of 1H or 13C nuclei observed in 2,2-dimethylchroman-4-one derivatives, while Δδ(Benzene) is the SCS of 1H or 13C nuclei observed in monosubstituted benzene; a is the slope that compares the substituent effect on both aromatic moieties and b is the intercept. As an example, 1H nuclei of chroman-4-one derivatives belonging to Series 1, while the same analysis for the 13C nuclei of the aromatic ring is shown in Analysis of substituents effect on the ried out . This ana values < 1) than in the monosubstituted benzene ring and this is particularly apparent for compounds belonging to Series 1.The obtained Lynch correlations are collected in 13C NMR data depicts a different scenario, since the correlation cannot be extended to all the carbon atoms. In particular, the carbon atoms meta- with respect to the substituent R show a spread along the vertical axis for all the series and, accordingly, were not further considered in the data set  − 0.04, with a R-square value of 0.90, while for 13C nuclei chemical shifts (carbons meta- to the substituent R were not considered), the Lynch correlation afforded the following relationship: Δδexp = 1.0 Δδ(Benzene) − 0.1, with a R-square value of 0.90. These results reinforce the conclusions for each series previously described.Notably, cumulative Lynch correlations of ries see  in SM co13C chemical shifts as the probe to analyze this type of LFER. This choice was based on the advantages of carbon atoms over their hydrogen counterparts because 13C nuclei span over a larger chemical shift range and smaller dependence on medium effects is commonly observed. Thus, looking for a correlation involving 13C chemical shifts of chroman-4-one derivatives grouped in Series 1–3, different Hammett parameters have been screened, including σp+, σm and σI values [p+ Hammett parameters, when carbon atoms in the para- position with respect to the substituent R  were considered. The choice of the σp+ Hammett parameter is related to the specific position of the analyzed carbons, as well as to the presence in the chromanone scaffold of both the carbonyl and alkoxy groups attached at positions C-4a and C-8a, respectively. Accordingly, the attached R substituent can interact directly with the alkoxy or carbonyl groups through resonance effect [p+ Hammett parameter [0 around 160 ppm) Series 2 and 3 show very similar δ0 values (around 120 ppm), as a consequence of the different arrangement of the R substituent with respect to the carbonyl and the alkoxy groups of the chroman-4-one skeleton.Linear Hammett correlations for 2,2-dimethylchroman-4-one derivatives have been also investigated, adopting solely e effect ,35,36. Tarameter . Notably13C chemical shifts of carbon atoms in the meta- position , indicating that the chemical shifts are sensitive to the inductive effect of the substituent attached to the aromatic ring. A similar behavior was observed for the C-8a of compounds in Series 2 with both Hammett parameters. On the other hand, no correlation (R-square consistently < 0.4) with either σm or σI Hammett parameters was observed for C-8 of Series 1, C-5 of Series 2 and for both C-7 and C-8a of Series 3. This behavior can be attributed to the fact that the electronic effect of the substituent is poorly transmitted to meta-carbons, resulting in modest chemical shift changes (<3 ppm range). Accordingly, subtle non-electronic effects on the chemical shift may have a role, preventing to obtain a correlation with Hammett parameters.Next, we analyzed Hammett correlations of tion see , by choo1H and 13C chemical shift values have been collected in 1H chemical shift values (δTHEOR) and the experimental ones (δEXP), the corresponding plots being shown in The results of DFT calculations for all the 2,2-dimethylchroman-4-one derivatives belonging to Series 1, 2, and 3 in terms of predicted ortho-, meta- or para-) with respect to the substituent group R, nice linear correlations between the predicted and experimental H-NMR chemical shifts have been obtained with slopes near unity (between 0.96 and 1.16) and R-square ≥ 0.95 . Thus, the nitro group is markedly tilted with respect to the aromatic plane in the modeled conformer of 3f , to Series 3  and, finally, to Series 2 , increasingly higher chemical shifts values can be expected. Moving to para-hydrogens  reported in 1e and 1j, which clearly deviate from the set of the other chroman-4-ones, albeit no specific geometric features can be related to such anomalous behavior. Furthermore, it is important to point out that the reported data have been calculated by considering the average chemical shift of the non-equivalent hydrogens , as obtained from the calculation carried out on the most stable conformer of each compound.On the other hand, methylene groups of chroman-4-one derivatives give a worse linear correlation d than th13C chemical shifts, and the plots are shown in ipso-, meta-, and para- with respect to the substituent R for all the examined Series, with some exceptions in the case of ortho-carbons. The R-square values of the linear correlations are shown in ipso-carbons in Series 1–3 and the obtained slope value (0.88) is lower than unity. Indeed, the presence of a certain spread of data was found, resulting in a quite poor linear correlation (R-square = 0.75). On contrast, good linear correlations were obtained for the ortho-carbons in Series 2 and 3 (not in Series 1), as well as meta- and para-carbons in Series 1–3  of Series 1–3 and the plots are given in It is worth noting, however, that in the case of bons see c. FinallThe synthesis and full characterization of the 2,2-dimethylchroman-4-one derivatives belonging to Series 1–3 have been reported in previous studies ,31,32.3)4Si) calculated at the B3LYP/6-311+G level of theory as reference.DFT calculations were carried out with the Gaussian09 (rev. D.01) software  . All the1H and 13C NMR Lynch correlations, an estimate of the substituent effect transmission on the nuclei of 2,2-dimethylchroman-4-ones compared to the case of monosubstituted benzenes can be inferred. The data reported for the first time in this work clearly reveal that in all cases good linear correlations were found. Indeed, 1H NMR Lynch correlations showed slope values of 0.90 (for Series 1), while Series 2 and 3 consistently gave a slope around 0.95. Likewise, application of Lynch equation to 13C NMR data showed good linear correlations and the slopes were found to be between 0.94 and 1.05, demonstrating again that the substituent effect is well transmitted, similarly to the case of monosubstituted benzenes. However, the carbons meta- to the substituent R showed no correlation.From the analysis of the p+ Hammett parameters and the carbon atoms in the para-position with respect to the substituent R belonging to Series 1–3 and, in all cases, the ρ values were positive indicating that the chemical shifts are sensitive to the electronic effects of the substituents attached to the aromatic ring. Indeed, the resonance effect of electron-donating groups causes upfield chemical shifts, while that of electron-withdrawing groups causes downfield chemical shifts. However, for the case of the 13C nuclei in meta- position with respect to the substituent R in Series 1–3, the correlation with σm and σI Hammett parameters demonstrated that these carbon atoms are not sufficiently sensitive to the substituent inductive effect. This behavior can be attributed to the fact that the chemical shift changes on meta- substitution are in every case of comparable magnitude (<3 ppm range), therefore subtle non-electronic effects on the chemical shift may have to be postulated.On the other hand, good linear correlation fittings were observed between the σortho- to the substituent). These results demonstrate that the prediction of the chemical shifts of the aromatic hydrogen and carbon atoms using the adopted theoretical approach is accurate, in turn leading to a good estimation of the desired values.Finally, nice linear dependences between the theoretically predicted and experimental hydrogen and carbon NMR chemical shifts were obtained with slopes near unity, with only a few exceptions in the case of C-NMR (notably for carbon atoms