Pseudourostyla cristata by high-throughput sequencing and qRT-PCR. A total of 853 differentially expressed lncRNAs were identified. Compared to vegetative cells, 439 and 414 lncRNAs were upregulated and downregulated, respectively, while 47 lncRNAs were specifically expressed in dormant cysts. A lncRNA-mRNA coexpression network was constructed, and the possible roles of lncRNAs were screened. Three of the identified lncRNAs, DN12058, DN20924 and DN30855, were found to play roles in fostering encystment via their coexpressed mRNAs. These lncRNAs can regulate a variety of physiological activities that are essential for encystment, including autophagy, protein degradation, the intracellular calcium concentration, microtubule-associated dynein and microtubule interactions, and cell proliferation inhibition. These findings provide the first insight into the potentially functional lncRNAs and their coexpressed mRNAs involved in the dormancy of ciliated protozoa and contribute new evidence for understanding the molecular mechanisms regulating encystment.Ciliated protozoans form dormant cysts for survival under adverse conditions. The molecular mechanisms regulating this process are critical for understanding how single-celled eukaryotes adapt to the environment. Despite the accumulated data on morphology and gene coding sequences, the molecular mechanism by which lncRNAs regulate ciliate encystment remains unknown. Here, we first detected and analyzed the lncRNA expression profile and coexpressed mRNAs in dormant cysts versus vegetative cells in the hypotrich ciliate  They are found at various trophic levels of the food chain, which makes them important for the proper functioning of the ecosystem2. In addition, ciliates are single-celled organisms with highly specialized cell structures as well as a dual nature as “cells” and “animals”, which make them important models in many biological research fields, such as cytology, genetics, developmental biology and evolutionary biology1. Ciliates have great adaptability and various survival capabilities, and transforming into dormant cysts under adverse conditions is the one that has attracted extensive attention8. Studying the cystic state of ciliates has historically been an important aspect for exploring the relationship of structure and function and the formation of and mechanisms controlling cell patterns in eukaryotes9. To date, a great deal of morphological data has been accumulated on ciliate encystment at the microscopic and submicroscopic levels, and these data have shown the basic dedifferentiation processes of cellular patterns and offered new insight into ciliate phylogeny12. With the intensification of research, increasing studies have been performed on the gene/protein expression and biochemical changes related to cyst formation in ciliates1. For instance, Grisvard et al. measured differentially expressed (DE) genes during the encystment–excystment cycle of the ciliate Sterkiella histriomuscorum by DNA microarray analysis. The 16 most commonly observed DE genes were upregulated in cysts and excysting cells7. Chen et al. identified 26 DE proteins in dormant cysts of the ciliate Euplotes encysticus by comparative proteomic analysis. Among these DE proteins, 12 were specifically expressed proteins, and 14 were DE proteins5. Gao et al. found 6 specific proteins in dormant cysts of Pseudourostyla cristata (P. cristata) by using a shotgun LC–MS/MS approach6. More recently, two studies identified mRNA expression profiles related to the encystment of Colpoda aspera and P. cristata8. These two studies not only analyzed DE genes related to cyst formation but also identified the signaling pathways that may facilitate encystment, such as the cAMP, mTOR, PI3K/AKT, calcium and AMPK signaling pathways. The data from these molecular studies offer an in-depth view of the regulatory mechanism of cellular patterns during encystment and the function of organelles.Ciliated protozoa are a diverse group of eukaryotes that are commonly found in different ecosystems in Earth’s biosphere8. Increasing evidence has demonstrated that lncRNAs play notable roles in a broad variety of biological processes, such as the modulation of epigenetic, transcriptional or posttranscriptional gene expression and the stress response15. However, to the best of our knowledge, the role of lncRNAs in ciliate encystment has not been studied.LncRNAs are no less than 200 nucleotides in length and lack substantial protein-coding capability. In recent years, important regulatory functions have been established and extended to study various cellular processesP. cristata, which can form massive cysts in culture, was used as the experimental model to study the potential function of lncRNAs in ciliate encystment. High-throughput sequencing (HiSeq), quantitative real-time PCR (qRT-PCR) and bioinformatic techniques were used to determine the expression profiles of lncRNAs in dormant cysts versus vegetative cells, and DE lncRNAs in the encystment process were analyzed. In addition, the roles of DE lncRNAs and their coexpressed genes in the encystment mechanism were addressed. The current research work shows, for the first time, lncRNAs that regulate ciliate encystment, contributing to a better understanding of the complicated molecular mechanisms regulating encystment stress resistance in ciliates.In the present study, The complete experimental design is shown in Supplementary Fig. To differentiate lncRNAs from protein-coding transcript units, three filtering processes mentioned in the system were used. We identified 11,959 accurately expressed lncRNA transcripts Fig. . In addip < 0.05 and |log2 FC|> 1. The IDs of lncRNAs are shown in Supplementary Table A total of 853 DE lncRNAs were identified between the vegetative cell and dormant cyst libraries based on the criteria of P. cristata, we constructed a lncRNA-mRNA coexpression network and investigated the potential interactions between the lncRNA transcripts and mRNA transcripts. We constructed a coexpression network with the top 500 transcripts according to the p-value from low to high. There were more than 140 nodes in the network. Among these nodes, seven nodes with the highest correlation (COR) values were selected, and a coexpression network was constructed using R software  were not annotated and had an unknown function. We conducted correlation analysis between the ncRNAs and protein-coding mRNAs based on the expression of DE transcripts to predict the functions of the ncRNAs. To detect key lncRNAs and their potential functions in the encystment of P. cristata through bioinformatic analysis and comparative analysis of data from our previously published articles8. Among these three lncRNAs, the upregulated lncRNA DN12058_c0_g1_i1_2 (DN12058) had 184 coexpressed mRNAs, and the top 30 coexpressed mRNAs were identified as upregulated after screening of the COR values. The top 30 coexpressed mRNAs included ATP1, ND4, EEF1A, ND5, hptG (HSP90 α), ATP6, COX3, UBC, RPS13, petF, acpP, PABP, RPL7, etc.  and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses for mRNAs in the lncRNA‐associated network. We focused on three lncRNAs that were likely to be associated with cysts of ftnA, AGL, OSBPL5_8, ZIP9, TOP3, VAMP7, EIF1, UBE1, etc.  and downregulated lncRNA DN30855_c0_g1_i1_1 (DN30855). the lncRNA DN20924 had 376 coexpressed mRNAs, and the top 30 coexpressed mRNAs were identified as upregulated after COR value screening. The top 30 coexpressed mRNAs included GGH, TTLL11, MELK, amyA, ICK, ATP2B, etc. , further confirming the high reliability of the HiSeq results for the DE lncRNAs.To validate the HiSeq results, 16 DE lncRNAs were randomly selected for qRT-PCR analysis. As shown in Fig. 16. Studies on the molecular biological functions of lncRNAs in cyst formation in ciliates have not yet been reported. LncRNAs are considered to be widely distributed in the genome, to be significant regulators of gene expression, and to participate in a wide range of important cellular processes. The regulatory roles of lncRNAs have been a focal point of research in the life sciences15. In this study, for the first time, we identified DE lncRNAs between dormant cysts and vegetative cells. As discussed below, these up- or downregulated lncRNAs can engage in various encystment-related activities through their coexpressed mRNAs. Although the results are preliminary, the large number of DE lncRNAs can form the basis of further studies on the molecular mechanisms of lncRNAs in ciliate encystment.Research on the mechanism underlying cyst formation in ciliates has gradually advanced from the level of morphology to the level of molecular biology, but the research is still mainly focused on coding genesi.e., the ubiquitin–proteasome pathway and the autophagy pathway, were active in the degradation of proteins and organelles during cyst formation. These findings are consistent with the finding of Pan et al.8 that the proteasome system was activated to degrade excess proteins during P. cristata encystment. Furthermore, these molecular events corresponded well with dramatic changes in cell morphology, including rapid cell volume contraction, autophagy and autophagosome formation during the cyst formation process17.Among the 853 identified DE lncRNAs and their coexpressed mRNAs, we mainly focused on analyzing three DE lncRNAs and their coexpressed mRNAs. GO term analysis showed that the mRNAs coexpressed with the upregulated lncRNA DN12058 were significantly enriched in the term autophagy, and KEGG pathway analysis showed that the mRNAs coexpressed with the upregulated lncRNA DN12058 were significantly enriched in the proteasome signaling pathway. These data suggested that two pathways, ATP1, ATP6, ND4, ND5, EEF1A, ND5, htpG (HSP90α), COX3, UBC, petF, acpP and PABP.Our results showed that the mRNAs coexpressed with the upregulated lncRNA DN12058 included ATP1 and ATP6 are ATP synthase subunits and key enzymes in mitochondrial energy metabolism in organisms, and they are involved in oxidative phosphorylation19. COX3 is a cytochrome C oxidase (COX) subunit and plays an important role in oxidative phosphorylation through the mitochondrial respiratory chain20. ND4 and ND5 are NADH dehydrogenase subunits and two of the seven subunits of respiratory complex I (NADH dehydrogenase) that span the inner membrane of mitochondria. They participate in the first step of mitochondrial oxidative phosphorylation through the electron transport chain21. Ferredoxin (petF) is an electron carrier active in the electron transport chain in mitochondria22. It was speculated that the lncRNA DN12058 is likely to facilitate the oxidative phosphorylation process by orchestrating the upregulation of these coexpressed mRNAs and then providing the ATP necessary for the rapid morphological changes during cyst formation. HtpG, called heat shock protein-90α (Hsp90α), is a member of the HSP90 family23. Hsp90α is secreted by a variety of cell types in response to extracellular stress signals and plays an important role in cell repair24. Hsp90α also participates in a variety of key cellular reactions, including signal transduction, cell cycle modulation and transcriptional regulation25. These observations suggest that lncRNA DN12058 probably participates in the abovementioned functions to facilitate the development of cysts by upregulating the coexpressed HSP90α.EEF1A is involved in cellular processes such as proteasomal protein degradation26 and cytoskeletal rearrangement27, which suggests that upregulated expression of EEF1A in tandem with lncRNA DN12058 increases protein degradation and cytoskeletal rearrangement during encystment to facilitate rapid changes in cell morphology and the formation of compact rounded cysts.PAB) binds directly to the poly(A) tail of mRNA and plays a critical role in the initiation of eukaryotic translation and stabilization/degradation of mRNA29. Acyl carrier protein (acpP) is a fundamental component of fatty acid biosynthesis and is a cofactor in many other biochemical pathways, such as those mediating the synthesis of polyketides, phospholipids, and cell wall polysaccharides30. The polyubiquitin gene ubiquitin C (UBC) is considered to be a stress-protective gene and is upregulated under various stress conditions, which is probably a consequence of an increased demand for ubiquitin to remove toxic misfolded proteins31. Therefore, we speculated that the lncRNA DN12058 can increase the levels of encystment-related substances  as well as the ability of cysts to defend against stress.Several studies have shown that polyadenylate-binding protein  is a membrane-bound protein and an important member of the zinc transporter family that has essential roles in zinc homeostasis by regulating the transport of zinc across membranes into the cytoplasm. ZIP9 participates in the regulation of intracellular zinc homeostasis and is involved in the processes of morphogenesis and differentiation in eukaryotes40. The coexpression of ZIP9 with the upregulated lncRNA DN20924 may facilitate the transformation of vegetative cells to into cysts and maintain intracellular zinc homeostasis during encystment.A large amount of evidence indicates that zinc is an essential metal that has many vital functions in eukaryotes and often exerts cell-specific effects on morphogenesis, differentiation and development. Zinc acts as a catalytic cofactor for more than 300 enzymes and also functions as an intracellular signaling molecule. Zinc deficiency can lead to various adverse effects on physiological processes. Zinc transporter member 9 (TOP3), are ubiquitous enzymes that catalyze topological structural changes in DNA necessary for many steps in DNA processing, such as replication, transcription, repair and recombination. TOP3 plays roles in maintaining genomic stability and participates in various cellular processes. TOP3 can promote cyst formation by inducing gene expression in nonciliate protozoa42. Several studies have documented that nuclear changes, macronuclear chromatin reorganization and DNA modifications such as demethylation occur during ciliate encystment44. These results indicated that coexpression of TOP3 with the upregulated lncRNA DN20924 stimulated cyst formation by promoting DNA recombination and transcription and inducing cyst wall protein gene expression in P. cristata. Upregulated expression of TOP3 may also promote DNA repair and maintain genome stability to enhance the resistance of cysts.Recent studies have shown that DNA topoisomerases, including DNA topoisomerase III  can produce polyglutamine for an original posttranslational modification of tubulin. TTLL is widely found in protozoa45. Studies have shown that polyglutamine may play a key role in regulating microtubule structure or the interaction between microtubule-associated dynein and microtubules47. Considering that microtubules in the ciliature are absorbed during the encystment of P. cristata37, it was proposed that coexpression of TTLL11 with the downregulated lncRNA DN30855 could result in poor microtubule transport functions and facilitate the entry of P. cristata into dormancy.The lncRNA DN30855 and all of its coexpressed mRNAs, primarily MELK) is a cell cycle-dependent protein kinase, and its expression is specific to proliferating cells48. Intestinal cell (MAK-like) kinase (ICK) plays a positive role in the regulation of cell proliferation and differentiation. Suppressed or downregulated expression of ICK can effectively inhibit cell proliferation and regulate the transition of cells from proliferation to differentiation49. Downregulation of the lncRNA DN3085 with coexpression of MELK and ICK may inhibit cell proliferation and in turn promote dormancy.Maternal embryonic leucine zipper kinase  was provided by Professor Fukang Gu from East China Normal University. P. cristata specimens were collected from paddy field water in southern Anhui, China, in May 2005. The ciliate cells were cultured in 10-cm Petri dishes with treated pond water and incubated at 25 °C. The pond water was filtered with filter paper produced from cotton linters and was then treated by autoclaving  for 30 min at 121 °C. One or 2 boiled wheat grains were added to the culture to enrich bacteria, as the diet of ciliate cells. When the cultured vegetative cells reached a high density, the wheat grains were removed, and most of the vegetative cells formed cysts within 3–4 days. Both assays in our RNA-Seq study were performed with two biological replicates and technical replicates.4 cells were collected and centrifuged at room temperature at 3000 rpm for 2 min for extraction of RNA. Total RNA was extracted using a mirVana miRNA Isolation Kit (Ambion-1561). RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer . RNA integrity was assessed using an Agilent 2100 Bioanalyzer . Quality-qualified RNA was processed with a TruSeq Stranded Total RNA with Ribo-Zero Gold Kit  to eliminate ribosomal RNA. Then, libraries were prepared using an Illumina TruSeq Stranded Total RNA LT-(with Ribo-Zero Gold)-Set A/B  according to the manufacturer’s instructions. After quantification using the Agilent 2100 bioanalyzer, the libraries were sequenced with the Illumina HiSeq X Ten platform. The raw data have been submitted to the Sequence Read Archive (SRA) (https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?) database under accession numbers SRR11413602 and SRR11413601.Approximately 2 × 1050 software by the following steps: (1) removal of adaptor sequences; (2) removal of low-quality reads; and (3) elimination of 3′ and 5′ low-quality bases via different methods. Then, the original amount of sequencing reads, effective quantity of sequencing reads, Q30 values and guanine and cytosine contents were determined and used to comprehensively evaluate the quality. Next, contamination of reads from the vegetative cells and the dormant cysts was assessed using BLAST and the NT library (fp://fp.ncbi.nih.gov/blast/db) with the criteria of an E value < 1e-10 and coverage > 80%, which confirmed that the samples were not contaminated.To avoid data error, quality control of the raw reads was completed with Trimmomatichttp://CPC.cbi.pku.edu.cn)51, Coding-Non-Coding Index 52, the protein families database 53, and predictor of lncRNAs and messenger RNAs based on an improved k-mer scheme 54 were used to filter transcripts with coding potential. Finally, the intersection of the CPC, CNCI, Pfam, and PLEK results was selected. The expression levels of the samples were calculated with the FPKM method55. FPKM, considers the impact of sequencing depth and unigene length on the fragment count and is currently the most commonly used expression level estimation method.De novo transcriptome splicing was used to connect overlapping reads into longer sequences without relying on the reference genome, followed by splicing into transcripts through continuous extension. The assembled transcripts were selected for further analysis. Then, the retained transcripts were filtered according to the following process. (1) Transdecoder software was used to identify the coding region in the transcript. (2) The candidate sequence annotation information was combined to filter out the annotation sequences in the coding library. (3) The Coding Potential Calculator  was used as the reference gene, and all of the primers used are listed in Supplementary Table −ΔΔCt method59. Student’s t-test was performed, and the results were considered to be significant at p < 0.05.To verify the reliability of the HiSeq results, we randomly selected and validated the expression of 16 lncRNAs in vegetative cells and dormant cysts by qRT-PCR analysis. Total RNA was extracted using a MiniBEST Universal RNA Extraction Kit  according to the manufacturer’s instructions. Total RNA was transcribed into cDNA by using a PrimeScript RT Reagent Kit  following the manufacturer’s guidelines. The primers for qRT-PCR verification were designed by Sangon Biotech . qRT-PCR analysis was performed using a TB Green Premix Ex Taq II Kit . Briefly, the 20-μl qRT-PCR mixtures consisted of 10 μl of TB Green Premix Ex Taq II (Tli RNase H Plus), 0.8 μl of PCR forward primer (10 μM), 0.8 μl of PCR reverse primer (10 µM), 0.4 μl of ROX Reference Dye II, 2 μl of cDNA and 6 μl of nuclease-free water. The amplification reactions were incubated at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (Supplementary Information


Roasted yellow split pea (YSP) flours were used to substitute wheat flour, at 10–20% (flour basis) in wheat bread formulations. Rheometry showed that roasted YSP flour addition increased elasticity and resistance to deformation and flow of the composite doughs, particularly at 20% substitution; instead, at 10% addition (either raw or roasted YSP flour), there were no effects on dough rheology and bread textural properties. Breads fortified with roasted YSP flour at levels >10% exhibited lower loaf-specific volume and harder crumb compared to control (bread without YSP flour). Moreover, only breads with 20% roasted YSP flour displayed a significantly higher staling extent and rate, compared to control, as assessed by large deformation mechanical testing and calorimetry (starch retrogradation) of crumb preparations. This formulation also showed a large increase in β-sheets and β-turns at the expense of α-helix and random coil conformations in protein secondary structure as assessed by FTIR spectroscopy. Roasting of YSP effectively masked the “beany” and “grass-like” off-flavors of raw YSP flour at 10% substitution. Overall, roasted YSP flour at the 10% level was successfully incorporated into wheat bread formulations without adversely affecting dough rheology, bread texture, and shelf-life, resulting in final products with a pleasant flavor profile. Wheat is one of the most important crops in terms of global production and use in various food items. More specifically, the increased consumption of wheat-based products has been associated with the adoption of a “western lifestyle” . The whoDry pulses mainly consist of carbohydrates (60–70% on dry weight basis), and they are superior sources of both dietary fibers and lysine-rich proteins ; apparenConsidering the potential role of pulses consumption in preventing chronic diseases, and in order to improve the nutritional quality of grain foods, there have been many attempts to incorporate legumes into bread and other widely consumed cereal-based products, such as biscuits, pasta, snacks, etc., with limited success, mostly leading to products of inferior technological and sensory characteristics . IncorpoAs expected, the modifications in dough rheological behavior greatly influence bread volume and crumb textural characteristics, which are important indicators of bread quality attributes, from the consumers’ point of view. Legume flour addition at levels higher than 10%, in most cases, adversely affects specific volume and crumb hardness, as it has been observed with the use of various legume flours in breadmaking, such as chickpea ,24,25, lPretreatment of legumes prior to milling might have an important impact on flavor improvement of the end products; e.g., bread made with roasted peas was found to have a less intense aroma and pulse flavor compared to that made from untreated peas . VolatilFinally, bread staling is an important determinant of bakery products shelf-life, since unfavorable changes in texture during storage often precede microbial spoilage. The increase in protein and fiber content in the fortified products might reduce water availability to gluten, but it might also limit the water migration from the crumb to the crust and/or decrease amylopectin retrogradation. For example, an anti-staling effect of lupin protein isolates as evidenced by delaying bread firming during storage for up to 48 h has been reported ; this ouAdditional research is needed to understand the physicochemical properties of legume flours, in order to promote their incorporation into different types of baked items, especially in bread with improved nutritional attributes, but also with acceptable sensorial characteristics and adequate shelf-life. In the present study, roasting of yellow split pea seed before milling was explored as a quick and cost-effective process for eliminating the undesirable legume off-flavors in the final product and thereby creating products with acceptable sensorial attributes to the consumers. Roasted yellow split pea, usually containing 25–30% of protein, was chosen for partial substitution of wheat flour in bread formulation due to its high nutritional value, aiming at providing a comprehensive investigation of its effect on dough thermal and rheological properties as well as physicochemical, sensory, and nutritional characteristics of the end-product, including the staling events. To our knowledge, for the first time, a multi-instrumental analytical approach including calorimetry, FTIR spectroscopy, and texture analysis was employed for the investigation of quality parameters of both fresh and staled products in an attempt to shed some light on the mechanisms of staling kinetics of pulse flour-supplemented wheat doughs and bread; this approach was also combined with an assessment of the potential nutritional benefits and sensorial attributes, which are important complementary aspects of product acceptance by consumers, besides the shelf-life.Wheat flour (WF) with extraction rate of 55% (Type 55), and 11 and 0.70% protein and ash content, respectively, was gifted by Flourmills Thrakis I. Ouzounopoulos, SA , whereas yellow split pea  and dry instant yeast  were purchased from the local market. 50 (median diameter) of 220, 230, and 95 μm, respectively (Yellow split peas (YSP) were roasted in a dry nut roaster  at 150 °C for 25 min in 3 batches of 5 kg each, under continuous mixing to ensure uniform thermal treatment, and then, the roasted YPS seeds from all batches were combined; the color and moisture content of the roasted legumes were determined as described below, and the obtained values from each batch were used as indicators to ensure the repeatability of the roasting treatment. Raw and roasted yellow split pea were milled into flour using a household stone mill , stored at −18 °C until used and designated as yellow split pea flour (YSPF) and roasted yellow split pea flour (RYSPF), respectively. Particle size distribution of the obtained flours and of WF was determined by sieve analysis, according to Sereti et al. , using 2ectively .The RYSPF preparation was incorporated into breads at 10, 15, and 20% substitution levels of the wheat flour. The following flour samples were used for breadmaking : CON  were used to determine the optimum water level of the different dough formulations following the ICC method ; other tDough rheological properties  were also determined with a rotational Physica MCR 300 rheometer , using a Paar Physica circulating bath  and a controlled peltier system  for temperature control. The doughs were prepared as described above, using the same formulations and procedure with breadmaking, but omitting the yeast addition. After mixing, the doughs were wrapped with a plastic membrane to avoid moisture loss and left to rest for 20 min at room temperature, before any rheological measurement. The oscillatory measurements and creep-recovery tests were performed using a parallel plate geometry , with a solvent trap to avoid moisture loss during measurements. After dough loading on the rheometer, the specimen was left to rest for 15 min prior to any measurement. The temperature was regulated at 25 (±0.1) °C. The doughs for these measurements were tested in triplicate.Frequency sweep (oscillatory) measurements  were performed from 0.1 to 20 Hz under a constant strain (0.1%), which fulfilled the linear viscoelasticity requirements as shown by preliminary strain sweep tests. Storage modulus, G′, loss modulus, G″, complex viscosity, η*, and damping factor, tan δ, (G″/ G′) were recorded. Creep-recovery tests were carried out by applying a constant stress (50 Pa) for 60 s on the dough and allowing strain recovery for 180 s after removal of load. The compliance curve data from the creep-recovery tests were analyzed using the supporting software  of the rheometer and fitted to the Burgers model as described elsewhere .The moisture of raw materials and final products (bread crumb and crust) and protein content were determined according to the American Association of Cereal Chemists International official methods 44-15.02  and 46-3Total dietary fiber assays were carried out based on the AACC method 32-05  and the Digestible and resistant starch of the breads was determined using the respective assay procedure of Megazyme (K-DSTRS), which is based on a modified method of Englyst et al. . Accordiab) and Chroma (C*ab) parameters were calculated as described elsewhere [Color parameters  of both bread crumb and crust were measured using a Chromameter  calibrated with a white tile . Additionally, the hue angle . Three bread samples were measured from each breadmaking batch.A texture Analyzer TA.XT plus from Stable Micro Systems  was used for crumb and crust texture analysis. For crumb analysis, a circular shape cutter was employed to obtain cylindrical bread crumb specimens (40 mm diameter × 30 mm height). The samples were submitted to texture profile analysis (TPA) using a 75 mm diameter plate probe. The test was performed at 60% applied deformation, 0.8 mm/s test speed, and 5 s delay time between first and second compression cycles. The TPA parameters, namely, hardness, cohesiveness, springiness, resilience, and chewiness, were calculated according to Armero and Collar . A puncto), the peak temperature (Tp), and the melting enthalpy (ΔHgel) of starch gelatinization (endothermic transition) were determined from the respective thermograms. For estimation of starch retrogradation, samples of about 12 mg of lyophilized crumb from breads stored for 0, 1, 2, and 4 days at 25 °C were mixed with distilled water (flour: water 30:70) and conditioned as described above. Then, the crucibles were heated from 5 to 120 °C (heating rate 5 °C/min). The onset temperature (Toret), the peak temperature (Tpret), and the melting enthalpy (ΔHret) of the retrograded amylopectin were determined from the respective thermograms (endothermic transition). Moreover, the retrogradation index (RI), which represents the percentage of retrograded amylopectin in relation to that of granular starch undergoing gelatinization, was calculated according to Correa and Ferrero [Differential scanning calorimetry (DSC) was performed using the DSC 3 calorimeter . For starch gelatinization, about 12 mg of flour samples were mixed with distilled water (flour: water 30:70), and the slurries were hermetically sealed into DSC stainless steel crucibles and conditioned (proper hydration of flour particles) for 2 h at room temperature; then, the crucibles were heated from 25 to 120 °C (heating rate 5 °C/min). The onset temperature  with a 3-Reflection Diamond/ZnSe Performance Crystal Plate, and to attain good contact between flour particles and cell surface, a constant pressure was applied by the pressure tool. Spectra were obtained in triplicate. To avoid significant interference to the spectra signal due to the water high FTIR signal, freeze-dried bread samples were instead used for the FTIR analyses [2, H2O, and ATR corrections were performed in this order with the aid of Spectramanager v.2.15.15, JASCO. The corrected spectra were then subjected to Savitsky–Golay smoothing  and baseline correction  by the software SpectraGryph v.1.2.13 . The Amide I region between 1580 and 1700 cm−1 [−1 have been assigned to the β-sheet structure, peaks centered in the region at 1660–1685 to β-turn structure, peaks centered in the region at 1652–1660 cm−1 to α-helix conformation, and peaks centered in the region of 1644–1652 cm−1 to random coil structures [Fresh bread samples (2 h after baking) and those obtained from breads following storage for 1, 2, and 4 days were freeze-dried and used for FTIR analysis to evaluate the effect of storage on protein secondary structure and starch chain reordering. Wheat and raw and roasted YSP flours, as well as freeze-dried crumb of all bread samples, were used to obtain their absorption spectra (32 scans) by a FTIR spectrometer  at a resolution of 4.0 cmanalyses . The CO2700 cm−1  was furtructures ,46. The In vitro starch enzymatic digestibility of the bread crumb was assessed according to Lazaridou et al. [3 and placed in 35 mL of sodium phosphate buffer . The pH of the suspension was adjusted to 1.5 (with HCl) and first digested with pepsin  (575 units/g starch) for 30 min at 37 °C. The pH of the mixture was subsequently re-adjusted to pH 6.9 (with NaOH), the volume of the liquid was made up to 50 mL with a solution of sodium phosphate buffer , and porcine pancreatic α-amylase (Megazyme) (110 units/g starch) was added. The suspension was transferred to a dialysis tube , which was placed in a screw cap glass bottle containing 450 mL of sodium phosphate buffer  for 5 h at 37 °C under mild stirring in a water bath shaker . Aliquots (1 mL) of dialysates were taken at 20, 60, 120, 180, 240, and 300 min in duplicate. The aliquots were incubated with amyloglucosidase , and the released glucose was measured using the GOPOD reagent. For evaluation of in vitro starch digestibility, the area under the curve (AUC) of released glucose over 300 min of digestion was calculated. The analysis was performed in triplicate.u et al. . BrieflyA preliminary sensory analysis was carried out at the beginning of the present study to figure out whether the sensory characteristics of the fortified breads with flour from raw or roasted YSP were acceptable by the panel. Thus, 20 individuals were selected to perform a paired preference test in order to choose the most preferable sample between the fortified with 10% raw YSP flour and that with 10% roasted YSP flour. The assessors were selected as potential consumers of the product, based on their responses in a previously distributed questionnaire, indicating that they daily consume wheat bread. Approximately 75% (data not given) of the assessors preferred the formulation with the addition of 10% roasted YSP flour, and therefore, this type of flour was chosen for studying its impact on sensory characteristics of the fortified products at higher levels of supplementation (up to 20%). Quantitative descriptive analysis of the final products was carried out by a trained panel of 18 assessors, focusing on flavor characteristics, such as “wheat bread”, beany, green (grass-like), earthy, roasted, and over-roasted (burnt). The process included several meetings in order first to define the perceived flavors and followed by training to familiarize the panelists with the sought flavor notes. The mean values of all analyzed parameters were compared using Tukey’s test at α = 0.05 significance level.p) was at 63.76 °C (p < 0.05) onset (To) and peak (Tp) gelatinization temperatures, compared to wheat flour alone. Higher gelatinization temperatures have been previously found for starch of different legume species than that of cereal species, attributed to the higher amylose content of starch, i.e., ~30–40% for the former than ~20–25% for the latter [gel) of YSPF or RYSPF was not significantly different compared to that of WF  was higher than the loss modulus (G″), while both moduli slightly increased and complex viscosity (η*) sharply decreased with frequency a. As shop < 0.05) lower maximum creep compliance (Jmax) and viscoelastic compliance (Jm) in the creep phase. The YSP10 and RYSP20 were also characterized by significantly lower maximum creep strain compared to control. The RYSP20 dough showed the highest zero shear viscosity (ηo), which along with the lowest Jmax, describes a dough with a greater resistance to flow and deformation, in agreement with the highest η* and G’ values of this sample, respectively. In previous studies, shifting of the maximum creep strain to lower values and ηo to higher values have been related to stronger doughs [m) compared to control. The rheological properties of dough inevitably affect the textural characteristics of the final product, influencing bread quality, since the dough must be sufficiently elastic to allow the formation of the three-dimensional deformable network and strong enough to maintain the bubble structures formed during proofing and baking. Lupin fiber inclusion in a wheat dough has been found to exhibit a similar pattern, since at increased concentration (15% on flour basis), there was a significant decrease in maximum deformation, compared to lower levels of substitution [Regarding the creep-recovery test, the obtained rheological responses were typical of viscoelastic behavior, as expected for wheat-based doughs, showing the creep compliance data to slightly decrease with increasing roasted legume flour level b. All rhr doughs . It is wtitution . Overalltitution ,17, whilThe appearance of the fortified wheat breads with yellow split pea flours was evaluated by loaf-specific volume measurements and crust and crumb color parameters. Loaf-specific volume was negatively affected by the higher level of legume flour addition, as the RYSP15 and RYSP20 exhibited significantly lower specific volume values than the CON sample . Additiow values in crumb, ~0.97 [The crust and crumb color parameters of the examined breads are summarized in b, ~0.97 . Similarb, ~0.97 . Consumeb, ~0.97 . p < 0.05) hardening rate compared to YSP10 and RYSP10, indicating a quicker staling process. The increased crumb firmness of breads with the highest yellow split pea level is consistent with its reduced loaf volumes and rather compact crumb structure  and peak (Tpret) retrogradation temperatures did not show any differences among the tested samples, probably because the substitution levels were relatively low to have an impact. The ΔHret, corresponding to the melting enthalpy of retrograded amylopectin, reflects the extent of retrogradation upon storage  [−1 is characteristic of the presence of protein molecules and is attributed to the Amide I  and Amide II  bond vibrations [g modes) . As was brations ,62. The −1, assigned to the β-sheet structure, peaks centered at 1668, 1677, and 1683 cm−1, assigned to the β-turn structure, peaks centered at 1653 and 1660 cm−1, assigned to α-helix structure, and a peak centered at 1646 cm−1, assigned to random coil structures. The RYSP20 sample revealed peaks centered at 1622, 1629, and 1693 cm−1 for β-sheet structure, peaks centered at 1671, 1678, and 1686 cm−1 for β-turn structure, peaks centered at 1656 and 1664 cm−1 for α-helix structure, and a peak centered at 1647 cm−1 for random coil structure [Deconvolution of the Amide I peak of the flour spectra yielded several peaks attributed to different secondary structures of the protein components b–e. CON,tructure ,45,46. Ttructure ,64. Raw tructure ,65. Roasp < 0.05) increased estimates of β-turns. As reported by other investigators, for protein structures in a complex system, such as bread, conformational stabilities are very sensitive to interactions with the water molecules and presence of polysaccharides, mainly due to gluten dehydration upon kneading and baking [The evolution of the CON, YSP10, RYSP10, and RYSP20 protein secondary structures was also monitored with FTIR spectra through storage time . The prod baking ,45,72. Sd baking , suggest−1 are attributed to the carbohydrate region and are associated with starch. The peaks around 1010–1020, 1080, and 1150 cm−1 have been attributed to the coupled C-O and C-C stretching vibrations of the polysaccharide molecules [−1 have been previously assigned to short-range molecularly ordered or crystalline structures and amorphous forms of starch, respectively [1047/1022) of the intensities at 1047 and 1022 cm−1 has been adopted as an indicator of the relative level of starch chain ordering [1047/1022 values for the flours used in the present study. Crystallinity calculated by this index was not significantly (p > 0.05) different between wheat and raw YSP flours being 0.455 for both samples; this value is lower than those reported for wheat and waxy maize starches  [p < 0.05) lower R1047/1022 ratio (0.432) compared to YSPF probably due to the thermal processing, which may have caused some disordering of organized chains in starch granules. The spectral features of flour and bread samples a around olecules . The higectively ,74, and ordering . Table 7ctively)  due to t1047/1022 has been proposed as a relative index for monitoring starch retrogradation during bread storage and, thus, product staling [−1 band) and the loss of amorphous chain domains (corresponding to 1022 cm−1). As expected, the increase in starch molecular ordering and the decrease in the amorphous starch structure, due to retrogradation events, were evidenced by the increased R1047/1022 values calculated for the crumb during storage, although such an increase was pronounced only for the RYSP20 bread  reduced “wheat bread” flavor, as that typically recorded for the CON bread preparation. Sample YSP10, which contained the untreated (raw) YSP flour, had higher scores for “green, grass-like” and “beany” flavors, while flour from roasted YSP, when added at 10%, seemed to effectively reduce the aforementioned attributes, and at the same time, a characteristic, pleasant, “roasted” flavor was developed. Inclusion of 20% of flour from YSP increased the “roasted” perception and the “beany flavor” as well, suggesting that the roasting pretreatment of YSP seeds could mask completely, only the “green, grass-like” in the final product when this legume was added to the bread formulations at relatively high levels. Low volume, compact crumb structure, and “beany” and “grass-like” off-flavors are the major defects of legume flour inclusion into bread formulations. The flavor profile analysis, as presented in It has been suggested that the use of gluten or carboxymethylcellulose can significantly improve the volume and crumb characteristics of breads fortified with high levels of legumes, providing breads with accepted textural characteristics . On the Addition of YSP flours improved the nutritional quality of breads, to a certain degree, depending on the fortification level. Protein content adequately increased, only in the case of RYSP20, while all legume-fortified breads had higher total dietary fiber (TDF) content compared to control ; all thein vitro enzymatic starch digestion of breads. Inclusion of roasted YSP at 20% level into the bread formulation significantly decreased the areas under the curves (AUC), calculated over 300 min of digestion, compared to control bread  flour into bread formulations at 10, 15, and 20% substitution level of wheat flour has been investigated in this study. Multi-instrumental analysis of doughs and breads as well as sensory evaluation of the final products were employed for the assessment of dough rheological properties and textural, nutritional, and sensorial attributes of breads as well as for evaluation of bread staling events. Rheometry showed that the addition of flour from roasted YSP at a 20% level exhibited a significant effect on dough rheological parameters by increasing storage modulus and zero shear viscosity as well as decreasing maximum creep compliance, indicating a more elastic dough with a greater resistance to flow and deformation. Parameters derived from farinographs showed that both higher levels (15 and 20%) of legume flour inclusion significantly increased development time and decreased stability of the dough. Moreover, extensographs showed that stretch energy and ratio of resistance to extension/extensibility of dough fortified with 20% roasted legume flour was reduced compared to control formulation made solely by wheat flour, implying a poor baking performance for the composite flours. Inclusion of 10% raw or roasted YSP flour into bread formulations did not have any significant effect on loaf specific volume or on crumb textural properties as evaluated by texture profile analysis. Instead, breads with 15 and 20% roasted YSP exhibited significantly lower specific volume and higher crumb hardness than control, while a product with 20% YSP addition level also had higher chewiness and lower resilience of bread crumb, higher crust moisture content, and lower moisture loss rate of crumb upon bread storage, presumably due to presence of higher dietary fiber and protein concentrations. Therefore, it appeared that the high resistance to deformation and flow of dough fortified with 20% YSP flour resulted in low loaf-specific volume (18% lower than control), which subsequently led to a harder bread crumb and a more compact macrostructure. Moreover, for the latter bread sample, a disruption of the gluten network was revealed by the FTIR analysis of the protein secondary structure, as shown by the large increase in β-sheets in the crumb of this formulation, associated with dehydration of the gluten network, probably due to the presence of high amounts of dietary fiber and legume globulins into the composite dough system; migration of water from gluten to other bread components including starch could also contribute to observed increases in crumb hardening and starch retrogradation rates upon bread storage. Breads formulated with 20% roasted YSP flour exhibited the highest changes in these properties. in vitro testing. Overall, substitution of wheat flour with roasted YSP flour at 10% level was the most successful treatment, since it did not adversely affect dough rheology, bread texture, and staling kinetics, as well as the product sensory attributes. It is obvious that flours from roasted YSP can be effectively incorporated into wheat bread formulations in order to improve quality and nutritional characteristics of the final product when introduced at relatively low levels of fortification. Sensory evaluation using a trained panel revealed that roasting of YSP seeds before milling can effectively reduce the “beany” and “grass-like” off-flavors often detected for the fortified breads with raw YSP flour; however, both attributes were eliminated only in the case of 10% roasted YSP addition. Therefore, breads with 10% roasted YSP could be appealing to a wider diet-conscious consumer group. Finally, inclusion of YSP flours into wheat bread formulation enhances the nutritional quality of the product by increasing the protein and the total dietary fiber content, as well as reducing starch digestibility rates as monitored by


P < 0.001). Among APOE ɛ4 carriers with elevated amyloid, remaining lifetime risk of dementia at age 65 years was greater in women [74%  high and 58%  moderate amyloid], than men [62%  high and 44%  moderate amyloid]. Overall, the hazard and absolute risk of dementia varied considerably by predictor group. The absolute risk of dementia associated with predictors characteristic of Alzheimer’s disease was greater in women than men while at the same time the combination of APOE ɛ4 non-carrier with normal amyloid was more protective in women than men. This set of findings may be attributed in part to different biological effects and in part to lower mortality rates in women.Dementia and mortality rates rise inexorably with age and consequently interact. However, because of the major logistical difficulties in accounting for both outcomes in a defined population, very little work has examined how risk factors and biomarkers for incident dementia are influenced by competing mortality. The objective of this study was to examine long-term associations between amyloid PET, APOE ɛ4, sex, education and cardiovascular/metabolic conditions, and hazard and absolute risk of dementia and mortality in individuals without dementia at enrolment. Participants were enrolled in the Mayo Clinic Study of Aging, a population-based study of cognitive ageing in Olmsted County, MN, USA. All were without dementia and were age 55–92 years at enrolment and were followed longitudinally. Predictor variables were amyloid PET, APOE ɛ4 status, sex, education, cardiovascular/metabolic conditions and age. The main outcomes were incident dementia and mortality. Multivariable, multi-state models were used to estimate mortality and incident dementia rates and absolute risk of dementia and mortality by predictor variable group. Of the 4984 participants in the study, 4336 (87%) were cognitively unimpaired and 648 (13%) had mild cognitive impairment at enrolment. The median age at enrolment was 75 years; 2463 (49%) were women. The median follow-up time was 9.4 years (7.5 years after PET). High versus normal amyloid , APOE ɛ4 , older age and two additional cardiovascular/metabolic conditions  were associated with the increased hazard of dementia (all  The objective of this study was to examine long-term associations between amyloid PET, APOE ɛ4, sex, education and cardiovascular/metabolic conditions, and hazard and absolute risk of dementia and mortality in individuals without dementia at enrolment. Overall, the hazard and absolute risk of dementia varied considerably by predictor group.  Prior work has demonstrated that the predictor variables we evaluated—amyloid PET, APOE, age, sex, education and CMC—are related to the risk of dementia.Because both incident dementia and mortality increase with advancing age, failure to account for the competing risk of death impacts interpretation of the effects of risk factors and biomarkers on dementia incidence. The Mayo Clinic Study of Aging (MCSA) is a longitudinal observational study that is uniquely positioned to address this.16First, we were able to capture the two primary outcomes in both active participants and those who discontinued in person follow-up visits. In the latter group, clinical status could be determined by review of their medical records, owing to the unique design of the Rochester Epidemiology Project. Longer observation periods lead to greater cumulative withdrawal which in turn introduces greater selection bias in the remaining cohort. Therefore, this unique feature mitigated selection bias.Second, because we ascertained both incident dementia and death in the same defined population, we were able to calculate absolute risks for dementia, which are more interpretable, and more relevant to patients, than relative rates [i.e. hazard ratios (HRs)], the norm in Alzheimer’s biomarkers studies. Further, we applied analytic methods—multivariable, multi-state models—that are unique in their ability to portray competing outcomes.17–27 However, neuropathological changes leading to dementia evolve slowly8 and long-term follow-up is needed to fully capture associations between outcomes and upstream predictor variables.A third distinguishing feature was a long-term follow-up after enrolment . Much of the existing literature relating amyloid PET to clinical outcomes in individuals without dementia has focused on cognitive change over short-to-medium observation periods (∼1–5 years).This study was approved by the Mayo Clinic and the Olmsted Medical Center Institutional Review Boards. All participants provided written informed consent.28 A clinical diagnosis was determined for each participant at enrolment and for all subsequent visits using clinical criteria alone.28 All participants in the present study were 55 years of age or older and without dementia at enrolment, including both mild cognitive impairment (MCI)29 and cognitively unimpaired (defined as not MCI and without dementia)30 participants.All study participants were enrolled in the MCSA, a population-based epidemiological cognitive ageing study among a stratified random sample of a geographically defined population, Olmsted County, MN, USA see .28 A cli32 All participants had an initial study visit including at minimum a clinical evaluation and blood draw between November 2004 and September 2020. MCSA participants without a medical contraindication were invited to participate in imaging studies.Predictor variables were age, sex, education, APOE genotype, the first available amyloid PET and the first available composite CMC score. CMC score is the sum of the presence or absence of seven vascular-health-related conditions. A higher score indicates worse cardiometabolic health see .31,32 Al33 using previously described methods  and highly elevated (referred to as high) ranges on the Centiloid scale.36 The cut point separating normal and moderate amyloid was Centiloid 22 [standardized uptake value ratio (SUVR) 1.48] which is the value beyond which rates of amyloid PET reliably increase.34 The value separating moderate from high amyloid was Centiloid 68 (SUVR 2.0) which corresponds to the global maxima of the amyloid PET SUVR by delta amyloid curve.38Amyloid PET imaging was performed with Pittsburgh Compound Bhods see .34 The c30 and mortality. Participants were followed from enrolment through all MCSA visits until an event or censoring occurred . Outcomes in study participants who had previously discontinued in person study follow-up visits were ascertained through semiannual reviews of the electronic medical record39 using the Rochester Epidemiology Project medical records-linkage system  and 42 illustrated in The primary analysis was based on a multi-state intensity model,Each of the three transition rates (represented by the arrows in predicted state curves) and the probability of ever passing through the dementia state before death (i.e. remaining lifetime risk). Tests comparing two predicted state curves were based on the area under the curve with jackknife standard errors. Tests for the comparison of two remaining lifetime risk estimates were based on a jackknife standard error.For an individual without dementia at a given age with certain predictor variables, the fitted rate model also leads to estimates of the probability of being in each of the three states at a future age  were diagnosed as cognitively unimpaired and 648 (13%) as MCI at enrolment . The parMortality rates, both with and without dementia, increased exponentially with age and were higher in men than women, 1.31-fold (95% CI 1.12–1.54) for those without dementia and 1.43-fold (95% CI 1.28–1.60) for those with dementia . Rates oOf the 712 incident dementia events and the 1852 deaths, 418 (59%) and 1066 (58%), respectively, occurred in participants who had previously discontinued in person study follow-up visits and thus were identified through medical record abstraction. Additionally, 434 (61%) incident dementia events and 1359 (73%) deaths occurred 5 or more years after enrolment.Using the overall study population average as the reference, the HRs associated with different predictor variables are shown in Among women, the hazard of incident dementia increased monotonically with increasing amyloid level; 2.91 (95% CI 1.37–6.17) for moderate versus normal amyloid and 2.83 (95% CI 1.61–4.99) for high versus moderate amyloid. Among men, the hazards of incident dementia were similar for moderate versus normal amyloid , but were greater for high versus moderate amyloid . Averaging across men, women and APOE, the HR associated with high versus normal amyloid was 2.11 (95% CI 1.43–2.79).Within each amyloid group, the hazard of incident dementia was higher for APOE ɛ4 carriers than non-carriers for women  and men . The APOE ɛ4 effect was 1.63 (95% CI 1.20–2.23) times greater for women than men, averaged over all three amyloid groups.Two additional CMC increased the hazard of incident dementia by a factor of 1.37 (95% CI 1.22–1.53). While greater education was mildly protective, the association was not significant.The hazard of death among those without dementia was not associated with amyloid level or APOE ɛ4 status but was higher in men than women . More education was slightly protective . Two additional CMC increased the hazard by 1.75 (95% CI 1.61–1.91).The hazard of death among those with dementia was not associated with amyloid level, APOE ɛ4 status or education but was higher in men than women . Two additional CMC increased the hazard by 1.22 (95% CI 1.08–1.39).In We performed sensitivity analyses of HRs and lifetime risk ; however, here we also estimated the absolute risk of dementia  and 4. H43–45 Prior studies examining age and sex alone as predictors most often observed that the remaining lifetime risk of incident dementia was constant or declined modestly with increasing age in both sexes.43–45 We examined remaining lifetime risk associated with various predictor variables beyond age and sex and found that remaining lifetime risk remained relatively constant across different starting ages for the highest risk groups, particularly those with high amyloid, but declined slightly with older starting age for lower risk groups  from our study with prior epidemiological literature. Therefore, we first estimated overall incident dementia and death rates by age and sex alone. Reported dementia incidence rates vary overall  which is56–61 and APOE ɛ462–69 are associated with cognitive decline and dementia. More interesting were the complex relationships between sex and both amyloid and APOE ɛ4.HRs of incident dementia varied considerably by predictor variable group . Higher 70 some studies have identified a stronger association between APOE ɛ4 and dementia or cognitive decline in women than men.71 However, β-amyloid and APOE ɛ4 are closely related to each other. APOE ɛ4 increases the likelihood of and lowers the age of onset of both amyloidosis and dementia.73 To gain more comprehensive understanding, it is necessary to examine the separate sex × amyloid and sex × APOE ɛ4 interactions. Doing so, we found that APOE ɛ4 had a stronger association with incident dementia in women than men across all amyloid levels. At the same time, the combination of APOE ɛ4 non-carrier with low amyloid was selectively more protective in women than men   . One pos83 Monoclonal antibodies that target fibrillar forms of β-amyloid can effectively decrease amyloid load.85 While our data are observational and therefore cannot prove that removing amyloid would reduce incidence rates or absolute risk of dementia, the results show that those with higher amyloid progress to dementia at faster rates and have significantly higher lifetime risk for dementia than those with normal levels. At present, anti-amyloid clinical trials require an abnormal amyloid biomarker study for inclusion. The fact that hazard and remaining lifetime risk of incident dementia varied dramatically by the subgroups examined suggests that it might be useful to take a more granular approach to inclusion and stratification based on combinations of sex, APOE ɛ4 and amyloid level.The fact that higher amyloid PET levels increased the hazard, and more importantly, the remaining lifetime risk of incident dementia  is relevThe US Food and Drug Administration recently approved aducanumab for individuals in the MCI or mild dementia phases of Alzheimer’s disease. Most individuals in this study were cognitively unimpaired at baseline and therefore results of this study are only somewhat relevant to current clinical care considerations. Information in this study nonetheless has practical clinical value in life planning for elderly individuals without dementia, value for assessing the utility of combined biomarker and genetic screening of individuals without dementia, and value for assessing the potential public health impact of interventions.This study has some limitations. Including the entire MCSA cohort over age 55 years rather than only those with amyloid PET studies allowed us to strengthen estimates of the associations between other predictor variables and outcomes as shown in the sensitivity analyses . Howeverde facto demographics of Olmsted County, MN, USA of which the majority is non-Hispanic White. Results may differ in populations with different patterns of social determinants of health86–88; however, the predictor variables examined in this study do exist in all populations.This cohort is from a population-based sample and so by design reflects the 89–91 Future work should assess if similar associations are found between plasma biomarkers and the absolute risk of dementia reported here with amyloid PET.Ascertainment of amyloidosis was based on amyloid PET which may not be available in some settings. However, recent reports indicate high correlation between amyloid PET and plasma biomarkers.fcac017_Supplementary_DataClick here for additional data file.


The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote—when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes’ parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived  chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived  ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain “imprinting” of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent’s age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual’s gametes with those in chromosomes inherited from different individuals’ gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual’s reaction norm. Telomeres are repetitive DNA sequences capping the ends of linear chromosomes and featuring a specific structure. Telomeres consist of a varying number of tandemly repeated hexanucleotides, shelterin proteins and telomere repeat-containing RNAs ,2,3,4,5.Telomeres are among the most dynamic genome structures. Telomere length (TL) remains in continuous dynamic equilibrium during ontogenesis and the transfer of genetic information from one generation to another. On the one hand, telomeres inevitably shorten during cell divisions ,14,15,16TL has been extensively investigated in human somatic cells ,23,24,25We examined TL in 28 triploid zygotes obtained from 22 couples. For ethical reasons, diploid zygotes could not be included in our study. However, there is some evidence suggesting that critical developmental events in triploid human embryos are similar or identical to those in normal ones. Triploid human embryos are capable of implantation and even a full-term development . KatteraThe participants’ demographic information and semen parameters, and the parental origin of pronuclei in the zygotes are presented in After fertilisation, the male and the female pronuclei are not formed in the zygote simultaneously. Pronucleus formation normally occurs within several hours after the second polar body extrusion but may vary significantly in different oocytes. After we registered three pronuclei in a zygote, and therefore once its further culturing was no longer practical, we added colchicine to the culture medium to achieve the synchronisation necessary for an objective comparative assessment of TL in pronuclei of different parental origin. This approach enabled us to evaluate the TLs of oocyte- and sperm-derived chromosomes in zygotes at the same cell cycle stage—the metaphase of mitosis. We analysed metaphase chromosomes from 84 parental pronuclei across 28 zygotes. In some of the zygotes, parental chromosomes were visible as separate haploid sets ; in otheThe parental origin of chromosomes was determined immunocytochemically with the use of specific anti-5-methylcytosine (5mC) and anti-5-hydroxymethylcytosine (5hmC) antibodies ,49,50. Tp = 0.28).TL in the metaphase chromosomes was assessed through hybridisation signal fluorescence intensity after quantitative fluorescence in situ hybridisation (Q-FISH), which is the optimal technique for single-cell analysis. In view of the fact that chromosomes inherited from the sperm undergo protamine replacement with histones in the zygote, their condensation level is different from that of chromosomes inherited from the oocyte. To account for the gap, we calculated relative TLs instead of absolute values. Relative TLs were calculated by dividing telomere fluorescence by the fluorescence of the reference hybridisation signal. As a reference, we used the subtelomeric region of the short arm of chromosome 16 (16p), which is characterised by exceptionally low, verging on negligible, interindividual variability. To evaluate the possible influence of the chromosome’s parental origin on the 16p subtelomere’s fluorescence intensity, we compared this parameter for paternal and maternal chromosomes using the Wilcoxon test, which did not show a significant difference , two measurements were conducted on each chromosome set: one on each sister chromatid. The arithmetic mean for 16p subtelomere fluorescence was then calculated. The mean relative telomere fluorescence value for each chromosome set was calculated by dividing its mean telomere fluorescence by the mean 16p subtelomere fluorescence. p < 0.0001), attesting to the applicability of our approach . Therefore, in human triploid zygotes, TL in paternal chromosomes is significantly higher than in maternal ones.We carried out a comparative analysis of mean relative TLs in the chromosomes of paternal and maternal origin across 28 zygotes. The comparison involved 24 zygotes with one female and two male chromosome sets, and four zygotes with one male and two female sets, totalling 56 comparison pairs . In mostp = 0.5167) or between the mean relative TL in maternal chromosomes and the maternal age  (p = 0.60), normal sperm morphology  or progressive sperm motility   . Moreove = 0.87) . TherefoTo assess the input of heredity, we compared intraindividual (intercellular) and interindividual TL variability.p = 0.32), we combined the values into one group and determined the indicators of intraindividual (intercellular) variability: a variance of 0.09, a standard deviation of 0.3, a standard error of the mean of 0.04 and a coefficient of variation of 22.66%. Intraindividual (intercellular) variability was assessed based on TL comparison across pronuclei inherited from different gametes of the same individual. Based on parental origin analysis, we calculated the number of pronuclei formed from each individual’s gametes in all of the obtained zygotes and treated this number as the number of gametes obtained from each respective individual . This nup = 0.97), we grouped the values calculated for opposite-sex individuals and determined indicators reflecting interindividual variability: a variance of 0.42, a standard deviation of 0.65, a standard error of the mean of 0.1 and a variation coefficient of 36.8%. To assess interindividual variability, we estimated the fold change between the relative TL across all of the pronuclei in the same individual and the mean value for all individuals within the female and the male groups . Based on the absence of differences between the female and the male groups as per the Mann–Whitney U test (p = 0.0003) (p < 0.0001). We compared fold change values characterising intraindividual (intercellular) and interindividual variability with the non-parametric Mann–Whitney U test, which showed a higher interindividual variability ( 0.0003) . The varTherefore, interindividual variability significantly exceeds intraindividual (intercellular) variability, attesting to the role of heredity in determining TL. We assessed TLs in the zygotes after the completion of the S-phase of the cell cycle and before the first cleavage division, in metaphase chromosomes comprising two chromatids each. To investigate interchromatid differences reflecting changes in the telomere regions of the daughter chromatid compared to the mother chromatid, we calculated TL ratios between sister chromatids. The calculation was carried out for chromosomes 1, 9 and 16, which could be easily identified due to their large heterochromatic regions. To calculate the ratio between sister chromatids, we divided the higher value of the telomere fluorescence intensity by the lower value. Two values were calculated for each of the chromosomes 1, 9 and 16: the ratio between TLs in the sister chromatids in the short chromosome arms and those in the long chromosome arms, except for cases of overlapping chromosomes, in which telomere fluorescence could not be reliably measured. We then grouped the values obtained by parental origin: 274 values in chromosomes from 52 paternal pronuclei and 162 values in chromosomes from 32 maternal ones. As a baseline group, we assessed TLs in the sister chromatids of chromosomes 1, 9 and 16 of PHA-stimulated lymphocytes of 11 adult individuals in 22 metaphases . p = 0.91). However, they were significantly higher in both maternal and paternal pronuclei compared to adult lymphocytes   . TherefoWe identified two noteworthy phenomena in our analysis. On the one hand, the low TL variability across the gametes of one individual and the high TL variability across different individuals’ gametes attest to a dominant role of heredity in determining TL in paternal and maternal pronuclei at the zygote stage. Multiple studies on the lymphocytes of monozygotic and dizygotic twins ,52,53, aOn the other hand, we established that telomeres of paternal chromosomes in the zygote are longer than maternal ones. This premise suggests a certain “imprinting” of TLs. In other words, TL in a zygote is determined not only by the traits of the individual from whom they were inherited but also by the individual’s sex. Therefore, there is a possibility of sex-specific TL changes during spermatogenesis or oogenesis. Most likely, however, these changes occur only within the reaction norm of this trait.As our study suggests, the impact of a parent’s sex on TL in the zygote is considerably more pronounced than that of a parent’s age. Although the effect of age has been observed in male gametes ,43,44,45In addition, our study did not find evidence of any correlation between TL in the male pronucleus and such parameters of sperm quality as concentration, progressive motility or normal morphology. While some studies registered correlations between TL in spermatozoa and total sperm count ,55, sperLonger telomeres in the paternal chromosomes of a zygote could be explained by the fact that, during cleavage divisions, in the absence of active telomerase, the zygote needs a quick mechanism preventing telomeres from shortening at the least and elongating at the most. The most likely mechanism is ALT, as telomerase, which is most active at the blastocyst stage ,33, workOur findings suggest that at least two factors determine TL in human zygotes: heredity and parental origin. The input of other factors is possible within the reaction norm determined by individual traits. Therefore, the length of telomeres in a newly formed organism is, on the one hand, under strict genetic control, which is most likely closely linked to the epigenetic reprogramming of the embryo genome, and, on the other hand, retains a certain degree of variability, which is influenced by internal and external factors—directly or via cell biosensors, such as epigenetic mechanisms . The sex2 for 3 h. Routinely prepared sperm was then added for IVF. The presence and the number of pronuclei were examined after 20 h. A total of 28 triploid zygotes from 22 couples were included in the study.This study included human triploid zygotes that were routinely subjected to negative selection in IVF protocols. Zygotes were produced through conventional IVF at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology . Shortly after, cumulus–oocyte complexes were retrieved after controlled ovarian hyperstimulation as described earlier . OocytesThe peripheral blood lymphocytes were donated for the study by 11 healthy karyotypically normal volunteers aged 18–54 years. The lymphocytes were cultured for 72 h at +37 °C in the presence of phytohemagglutinin (PHA)  according to the standard protocol with minor modifications used in our laboratory .For chromosome preparations from the tripronucleate zygotes, 5 µL of 0.1% colchicine (Merck) was added to the culture medium at the point of visualisation of three pronuclei. After the pronuclei had dissolved , the zygotes were fixed on glass slides as described previously , with miPreparations of metaphase chromosomes from the PHA-stimulated lymphocytes were made according to standard techniques with minor modifications repeatedly used in our laboratory ,76,77. TFor detection of telomeric regions, fluorescence in situ hybridisation (FISH) with telomeric probes  was carried out on the chromosome preparation slides according to the manufacturer’s recommendations, with minor modifications. The slides were incubated in two changes of TBS, pH 7.5  for 5 min each at room temperature. Then, the slides were treated with pepsin solution supplemented with 2 M HCl  for 5 min at +37 °C and fixed in 3.7% formaldehyde in a TBS buffer for 5 min at room temperature. The preparations were washed twice with TBS for 5 min each, rinsed in distilled water, dehydrated in an ethanol series  and air dried. Denaturation (10 min at +88 °C) and hybridisation with telomeric probes  (18–20 h at +37 °C) were carried out using the ThermoBrite System . After hybridisation, the preparations were washed in Rinse Solution  for 1 min at room temperature, washed in Wash Solution  for 5 min at +62.5 °C in a shaking bath, rinsed in distilled water and air dried. Then, the preparations were dehydrated in an ethanol series , air dried and mounted in DAPI-containing VECTASHIELD Antifade . The preparations were stored in the dark at +4 °C until the time of microscopic analysis and photoimaging. After photoimaging, the detection of the 16p subtelomeric region (the reference region for TL measurements in our study) was carried out using FISH with a TelVysion SpectrumGreen 16p DNA probe . The coverslips were removed; the preparations were washed with cold water, dehydrated in an ethanol series  and air dried. Denaturation (10 min at +78 °C) and hybridisation with a 16p subtelomeric probe  (18–20 h at +37 °C) were carried out using the ThermoBrite System . After hybridisation, the preparations were washed in 4xSSC supplemented with Tween 20 and in two changes of 4xSSC at +37 °C in a shaking bath. Then, the preparations were rinsed in distilled water, dehydrated in an ethanol series , air dried and mounted in DAPI-containing VECTASHIELD Antifade . The preparations were stored in the dark at +4 °C until the time of microscopic analysis and photoimaging. The immunodetection of 5hmC and 5mC was carried out on the chromosome preparations from zygotes using primary antibodies against 5hmC  and 5mC  and secondary goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555  antibodies according to the protocol repeatedly used in earlier studies ,78,79. TFluorescence images of chromosomes after FISH and after immunodetection of 5mC and 5hmC were acquired using the Leica DM 2500 microscope, the Leica DFC345 FX camera and the Leica Application SuiteV.3.8.0 software. Fluorescence images of chromosomes after hybridisation with telomeric DNA probes were acquired using the following acquisition options: exposure time—1.8 s, gain—×3, gamma—3.44. Fluorescence images of chromosomes after hybridisation with the 16p DNA probe were acquired using the following acquisition options: exposure time—2.0 s, gain—×2, gamma—2.0.The telomeric and subtelomeric FISH signal intensity was evaluated on the digital photoimages using the Image J 1.48v software. Each fluorescent signal area was selected manually with the Freehand Selection tool, and the intensity of the fluorescence in relation to the signal area was measured. Statistical analysis was carried out with GraphPad Prism, Version 6.01. The pairwise comparison of TLs between paternal and maternal chromosome sets in zygotes was carried out using the Wilcoxon test. The correlation coefficients were calculated using the non-parametric Spearman test. Non-parametric variables were compared using the Mann–Whitney U test. The variances were compared in IBM SPSS Statistics 23 using Levene’s test. The α-level was set at 0.05.


To our knowledge, this is the first study to utilize LC–MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC–MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement  and a strong correlation (Pearson’s  O-methyltransferase (COMT) and monoamine oxidase (MAO) . Of . Of 4]. Until now, few studies have evaluated the sensitivity and specificity of HVA and VMA in neuroblastoma patients at disease onset, but there are no studies regarding utility of HVA and VMA quantitation in spot urine for monitoring of neuroblastoma patients ,15,16.Among 1209 measurements, 20 were performed for patients at onset of neuroblastoma, and diagnosis was confirmed by histopathology and/or imaging techniques . The othConcentration of urine creatinine showed severe variation in the range of 5.1 mg/dL to 121.3 mg/dL; thus, normalization of HVA and VMA results with concentration of urine creatinine was inevitable. Volume of 24 h urine also showed variation with range of 48 mL to 1600 mL, which implies that the collection procedure might not be accurate, affecting the validity of HVA and VMA results in 24 h urine.Among 20 neuroblastoma patients, 14 tested positive both for HVA and VMA, two tested negative both for HVA and VMA, and four showed discrepant results between HVA and VMA . Among 1Among the four patients with discrepant results, three patients tested positive for HVA and negative for VMA. These patients were all stage 4 patients with MYCN amplification, and the VMA in 24 h urine was negative patients 17–19). It is interesting that all of them had mild (MIBG) involvement, which implies that VMA production by neuroblastoma is low in these patients –19. It i,16.Three  of 20 newly diagnosed patients were ganglioneuroblastoma patients. Although ganglioneuroblastoma is a less malignant tumor, it can present as advanced cancer when patients have poor prognostic factors, as in patient 8 in our case. Patient 8 was stage 4 ganglioneuroblastoma and tested positive for HVA and VMA; the patient had a 17q gain on fluorescence in situ hybridization (FISH) analysis, which is a poor prognostic factor.5 and VMA-d3 were purchased from CDN Isotopes . Lyphocheck Urine Quality control (QC) samples were obtained from Bio Rad .HVA and VMA standards were purchased from Sigma Aldrich . LC–MS/MS-grade acetonitrile, methanol, and distilled water were purchased from Burdick & Jackson . Formic acid and hydrochloric acid were purchased from Sigma-Aldrich . Mass Spect Gold Human Urine (MSG5000) was purchased from Golden West Diagnostics . HVA-dv/v) methanol; these were frozen and stored at −80 °C until use. The stock solutions were diluted with blank urine (Mass Spect Gold Human Urine) to prepare the calibrators at six concentrations . Stock solutions of internal standards (IS) (2 mg/mL) were prepared by dissolving HVA-d5 and VMA-d3 in methanol. Quality control (QC) samples were prepared at two concentrations  with 0.05% hydrochloric acid as per the manufacturer’s instructions.Stock solutions (1 mg/mL) were prepared by dissolving HVA and VMA in 70%  including IS (5 μg/mL) were added to Eppendorf tubes. The tubes were sealed and vortexed for 30 s and then centrifuged at 20,600× Chromatographic separation was carried out using an Agilent 1260 HPLC system  coupled to an Agilent 6460 tandem mass spectrometer equipped with a polar embedded C18 with trimethylsilane endcapping LC column . Mobile phases consisted of water containing 0.1% formic acid (mobile A) and acetonitrile containing 0.1% formic acid (mobile B). The flow rate was 300 μL/min. Gradient elution is summarized in 5 and VMA-d3, 5 μg/mL). Because the urine samples contained endogenous VMA and HVA, the peak areas of the analytes needed to be adjusted by subtracting the endogenous peak area from the peak area of the analytes. Then, adjusted peak area was divided by the area of pure analyte in distilled water to calculate the matrix effect. The matrix effect was observed due to ionization suppression. After normalizing the peak area to the area of IS, matrix effects were considered acceptable as matrix effect bias (%) with IS normalization was within ±15%. Extraction recovery was calculated as the ratio of the peak areas before and after spiking. Carryover was evaluated with a blank sample after running the highest concentration calibrator containing 100 mg/L of HVA and VMA. The responses of analytes in blank samples were <20% in comparison with those in the LLOQ sample. Accuracy was evaluated with proficiency testing materials (13 College of American Pathologists (CAP) and six Korean Association of External Quality Assessment Service (KEQAS) materials). For method comparison, VMA concentrations in spot urine were measured using the Agilent 1200 HPLC system  coupled to a Coulochem® III ECD . The data were analyzed using MedCalc Statistical Software version 19.0.3 .We validated an LC–MS/MS assay for simultaneous measurement of HVA and VMA according to CLSI guidelines ,23,24. C® 800 .From October 2019 to September 2020, 1209 analyses for 357 neuroblastoma patients were performed to quantitate HVA and VMA in spot urine at Samsung Medical Center. HVA and VMA were reported as mg/g creatinine after measuring urine creatinine with the automatic kinetic Jaffe method on the Cobas INTEGRADemographic and clinical information of sex, age, and laboratory test results was obtained by reviewing the patients’ electronic medical records. We analyzed HVA and VMA results in spot urine for neuroblastoma patients at the onset of disease.In the present study, our simple and rapid LC–MS/MS method allowed simultaneous quantitation of HVA and VMA in spot urine in large cohort of patients with neuroblastoma. Analysis of clinical data revealed that HVA and VMA measurement in spot urine is a useful biomarker in clinical practice for diagnosis and monitoring of neuroblastoma patients. To our knowledge, this is the first approach to analyze the large-scale clinical application of HVA and VMA in spot urine of neuroblastoma patients using LC–MS/MS.