Owing to formidable advances in the electronics industry, efficient heat removal in electronic devices has been an urgent issue. For thermal management, electrically insulating materials that have higher thermal conductivities are desired. Recently, nanocelluloses (NCs) and related materials have been intensely studied because they possess outstanding properties and can be produced from renewable resources. This article gives an overview of NCs and related materials potentially applicable in thermal management. Thermal conduction in dielectric materials arises from phonons propagation. We discuss the behavior of phonons in NCs as well. In the t plates . If the −1·K−1) is the commonly used unit for thermal conductivity in the related disciplines. As the heat spreading materials, polymer-based composite materials containing inorganic fillers have been studied quite actively [−1·K−1 [Heat transfer can occur through radiation, convection or conduction. Heat removal (passive cooling) in electronics is typically governed by conduction. The effectiveness of a heat spreading material is related to its thermal conductivity . Thus, tactively ,9,10,11. [−1·K−1 . The orgHeat conduction in metals is dominated by free electrons. In dielectric materials, heat conduction arises from lattice vibrations. By analogy with the photons of the electromagnetic field, the quanta of the lattice vibrational field are referred to as “phonons” ,14,15. ICellulose is the most abundant polysaccharide in nature. Cellulosic materials that have at least one dimension less than 100 nm are referred to generally as nanocelluloses (NCs). NCs are bio-based nanomaterials that are continuously photosynthesized and accumulated in plants. Due to their abundance, biodegradability and renewability, the novel forms of cellulose have been generating much activity in the materials science field ,20,21,22κ) can be roughly estimated by the following equation:Cp is the specific heat capacity, ν is the phonon group velocity, and l is the mean free path of phonons in the material. The mean free path of phonons expresses how far phonons can travel before they are scattered by lattice imperfections, electrons, and other phonons. In bulky materials, internal scattering dominates heat conduction. As the size (characteristic length) shrinks, however, phonon scattering at the structure boundaries or interfaces is enhanced. In nonmetallic systems, the mean free paths at room temperature are from 1 to 100 nm. In nanostructures, their characteristic lengths are smaller or comparable to the intrinsic phonon mean free paths. Such an effect reduces the effective mean free path of phonons and the phenomenon is referred to as “classical size effect” [Recent theoretical and experimental research on nanomaterials has revealed interesting heat transfer phenomena at the nanoscale. As stated above, heat is mainly carried by phonons in dielectric materials. Thermal conductivity due to phonons  and Iβ (monoclinic) [β. Crystallographic studies by Nishiyama and coworkers revealed that cellulose Iβ possesses more compact crystalline structure and is thermodynamically more stable than cellulose Iα [β would be more significant for the present subject.Preceding studies on papers and woods have reported that thermal conductivity of usual cellulosic fibers is in the range of 0.1–0.4 W·m−1·K−1 ,40. That−1·K−1 . Subsequ−1·K−1 ,43. Celloclinic) . The celulose Iα ,46. Polyulose Iα . Since tβ structure observed along two directions. Cellulose sheets composed of the flat ribbon-like chains are stacked in parallel. Because of technical difficulties, direct thermal conductivity measurement of cellulose Iβ single crystals has not been reported to date. However, a prediction by molecular dynamics (MD) simulations was reported recently [−1·K−1 at 300 K. When compared with the ultra-drawn polyethylene nanofibers, the value is not very high. But it is significantly higher than those of usual organic polymers. The thermal conductivity values in other directions are predicted to be less than 1 W·m−1·K−1. In the transverse direction, the cellulose sheets are stabilized by a network of hydrogen bonds [β relies on phonons traveling along the chain direction in its crystal structure.recently . Accordien bonds ,50. In ten bonds . The weaConsidering the aforementioned discussion, the superior thermal conductivity of NCs should stem chiefly from two factors: diminished amorphous regions and fewer frequency of inter-chain interactions. The two major factors are summarized in During the last decade, papers proposing novel heat-dissipation materials containing NCs have drastically increased in number. The related works published in scientific archival journals are summarized and assorted in The thermal conductivity values given are the highest reported in the reference papers. Thermal conductivity values vary depending on many factors such as solids loading, filler sizes, filler alignment, and so on. Comparing the given values naively is not very meaningful.−1·K−1. In 2017, the identical group reported an attempt to align cellulose chains in BNC hydrogels by drawing [In 2015, Uetani and coworkers reported thermal conductivity measurements of non-woven sheets composed of various CNFs . Among t drawing . The BNC−1·K−1, which is about 7 times higher than that of the pure epoxy resin. Following this, several groups have reported their attempts to utilize NCs as thermally conductive fillers [As described in  fillers ,58,59. T fillers . Optical fillers ,62. The  fillers . When we fillers ,58. Chow−1·K−1). Added extra values such as optical transparency and a lightweight property might become the main indices for practical use.In composite materials, the phonon transports are hampered by scattering at filler–matrix interfaces. Thus, the thermal conductivity of polymers filled with NCs cannot surpass the intrinsic value of NCs , it has been intensely studied as thermally conductive fillers [In 2014, Zhu and coworkers reported about the effect of hexagonal boron nitride (h-BN) nanosheets incorporated in CNF sheets . The achnsulator ,68. Sinc fillers ,70,71,72 fillers ,75,76,77 fillers ,79,80,81 fillers . A soy-d fillers . A work  fillers .−1·K−1 [The structural similarity between graphitic carbon and h-BN invoked studies on BN nanotubes (BNNTs) ,86. The −1·K−1 ,88 and t−1·K−1 ,90. In 2−1·K−1 . Nonethe−1·K−1 . Long an−1·K−1) [2·V−1·s−1 at room temperature [2O3 particles between graphene fillers in ternary composites comprising of graphene, Al2O3 and CNF to avoid formation of electrical conduction paths. The resultant composites could be regarded as insulators.Graphene, a form of carbon that consists of a single layer of atoms arranged in a honeycomb lattice, exhibits quite high thermal conductivity (5000 W·m−1·K−1) . It is p−1·K−1) . Song an−1·K−1) ,96,97. Bperature . When elperature . They ar−1·K−1). Nanodiamond (ND) is known as a member of the nanocarbons and has become a subject of active research [2O3 [Diamond is electrically insulating and boasts high thermal conductivity  is known as a high thermal conductivity non-metallic solid . Its int−1·K−1 ,110. Ver−1·K−1 ,112. Alt−1·K−1 . The sur−1·K−1 ,115. ProIn this review, we have presented an overview of NCs as heat-spreading materials. Neat NCs can propagate thermal energy relatively well. Within the thermally conductive nanocomposites, NCs can fulfill roles both of the fillers and the continuous phases. Versatility would be a noteworthy feature of NCs. NC-based materials are also desirable from a sustainable development perspective. In the near term, process refinements would lower the production cost of NCs . As a neAs far as the higher thermal conductivity remains paramount, combining NCs with inorganic fillers should be the most promising scheme. Indeed, as can be seen in As for NCs and related materials, theoretical works, that are essential for understanding nanoscale heat transfer, are scarce. Knowledge accumulated in the phonon engineering discipline might be of help . Additio


In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca2+ oscillations within mammalian eggs. Moreover, studies describing PLCζ “knockout” mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca2 + oscillations and egg activation in mammals, while also discussing the nature of a potential “alternative” sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca Upon spte, PIP2 . The impte, PIP2 .2+ release when microinjected into mouse eggs by ICSI. However, in vitro fertilisation (IVF) with such sperm, produced atypical and delayed patterns of Ca2+ oscillations (lower in number and frequency) with a high degree of polyspermy and activation failure, compared to the robust, physiological pattern triggered by physiological PLCζ-induced egg activation  to disrupt cytoskeletal arrangement were unable to exhibit a rapid Ca2+ wave response upon interaction with sperm, instead exhibiting a much more delayed pattern of release, and failed to prevent polyspermy. Furthermore, the amplitude of subsequent Ca2+ peaks were reduced, exhibiting an effect similar to observations made with sperm from PLCζ-null mice. In starfish, it was suggested that heparin- or age-induced hyperpolymerization of the cortical actin disrupted actin cytoskeleton dynamics at fertilization influenced Ca2+ release . However, these mechanisms are poorly understood in mammals, and require further investigation to fully ascertain.Intriguingly, starfish eggs pre-injected with heparin  with the most elementary domain organization , 2017a.  isozyme . PLCζ do isozyme . All theozyme  have also been reported by 2+ sensitivity, as well as its enzymatic activity.Interestingly, 2+ oscillations , involving artificially mediated Ca release . Ca2+ ioI cycles . Recentl in ICSI . Howeverlization . In factreatment . Thus, P product , culminaman eggs . More imman eggs . FurtherFinally, PLCζ not only represents a promising clinical therapeutic agent, but also a potentially powerful diagnostic biomarker, which may help in determining the criteria and requirements of the fertility treatment of male patients, significantly decreasing the number of cycles needed for a successful pregnancy to occur. Taking into consideration that PLCζ analysis might be beneficial to identify not only cases of ICSI-failure but also cases of male subfertility, a simple immunocytological approach to routinely examine PLCζ protein in sperm is currently widely regarded as a cost-effective approach, which could be easily applied by the majority of IVF clinics worldwide. However, it is essential that studies focus on the reliable investigation of PLCζ parameters in relation to sperm health, using robust protocols and ultra-specific antibodies, before such clinical promise can be achieved. Indeed, significant issues remain regarding such analyses, particularly in humans, where PLCζ antibodies used recognize multiple protein bands in addition to the full-length PLCζ protein. The same antibodies have also been used to identify different immunoblotting profiles  at the Office of Research Affairs (ORA) at the King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. The protocols utilized for the relevant study (RAC-216004) were approved by the ACUC.AS, JK, AT, and MN prepared the first draft of the manuscript, which was revised and approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Castanea to diverse environmental conditions, but also to different management strategies encompassing orchards. The characterisation and description of chestnut populations and cultivars are crucial to develop effective conservation strategies of one of the most important Italian and European fruit and wood species. Chestnut cultivars grown in the same pedoclimatic conditions and on the same clonal rootstock were characterised with sensory, spectrophotometric, and chromatographic analysis to determine the phytochemical composition and nutraceutical properties. A multivariate approach, including principal component analysis and conditional inference tree models, was also performed. The ease of peeling, seed colour, and intensity of sweetness were the sensory descriptors that allowed us to differentiate C. sativa cultivars. Antioxidant capacity ranged from 9.30 ± 0.39 mmol Fe+2 kg−1 DW (‘Bouche de Bètizac’) to 19.96 ± 1.89 mmol Fe+2 kg−1 DW (‘Garrone Rosso’). Monoterpenes represented the main component, reaching 88% for hybrids, followed by polyphenols . A multivariate approach showed that phenolic acids and tannins were the bioactive classes with the highest discriminating power among different genotypes, and that genotype is a significant variable (p < 0.05). In addition, most of the analysed chestnut cultivars showed a content of bioactive compounds similar to or higher than the main hazelnut, walnut, and almond varieties. Chestnut agrobiodiversity could be intended as strictly associated to the genotype effect and underlines the large variability within the genus Castanea, and therefore, the importance of in farm and ex situ conservation of local germplasm is part of a global strategy aimed at increasing the levels of agrobiodiversity.A large species diversity characterises the wide distribution of chestnuts in Asia, North America, and Europe, hence reflecting not only the adaptation of the genus  Castanea spp.) [Landscape modification, habitat loss, and fragmentation stand among the main agrobiodiversity conservation issues that affect the ecosystem structure and functioning with negative effects on plants populations and communities . The conea spp.) .Castanea to diverse environmental conditions, but also to different management strategies encompassing orchards for fruit production, coppices for timber production, and naturalised populations providing several ecosystem services [Castanea spp. and human populations [A large species diversity characterises the wide distribution of chestnut in Asia, North America, and Europe, hence reflecting not only the adaptation of the genus services . Indeed,ulations .Natural and planted forests of sweet chestnut cover the species’ ecological limits , spreadiPhytophthora cambivora (Petri) Buisman and P. cinnamomi Rands, chestnut blight associated with the ascomycete Cryphonectria parasitica (Murrill) M.E. Barr, the emerging nut rot due to Gnomoniopsis castaneae G. Tamietti, and the infestation and control of the Asian gall wasp Dryocosmus kuriphilus Yasumatsu [C. sativa showed the wide plasticity of the species to cope with different environments (water and nutrient uptake efficiency) [However, since the beginning of the 20th century, the growing areas of sweet chestnut have dramatically decreased because of several social, cultural, and environmental changes. Such challenges include the progressive depopulation of mountain areas, diet changes ,11, climasumatsu ,14,15,16asumatsu . The aboasumatsu . Moreoveiciency) .Castanea spp., and on farm and ex situ conservation combined with other initiatives represent the first step to prevent the loss of biodiversity [For these reasons, the characterisation and description of chestnut populations and cultivars  are cruciversity ,4.Moreover, an increased economic interest for sweet chestnut in the food industry increased the demand for selected varieties, which is also driven by several research studies on the potential positive health benefits that can be derived from the consumption of fresh and processed chestnut products ,22. In aIn this study, a combination of different approaches was applied to describe and characterise 18 chestnut cultivars in order to assess their diversity in bioactive compounds and sensory attributes. It is worth noting that the attempt of silencing the exogenous sources of variability on the phenotypical expression maximises the likelihood of detecting the genotype influence on chestnut chemical and sensorial properties, hence providing an element of novelty and added value to the current state-of-the-art method in the subject of chestnut cultivars characterisation. The diversity that was detected in bioactive compound profiles and sensory traits among the chestnut cultivars is likely related to the effect of the genotype. Thus, chemical and sensorial analyses such as the ones that were applied in this study could be, in the future, an important mean of characterisation, while also allowing the evaluation and the selection of new cultivars .Castanea spp. fruits were collected in October 2018 from 18 accessions grown in the collection field (about 5 ha) of the Chestnut R&D Center—Piemonte in Chiusa Pesio, Cuneo Province  . These cultivars were grown under the same pedoclimatic conditions, agronomic management practices and, for the first time, on the same clonal rootstock. The origin and identification codes of the sampled material are listed in Samples of Castanetum” and “hot Fagetum”, following Mayr-Pavari’s classification [The experimental design allowed the assessment of the genotype effect on the chestnut composition while controlling for the potential confounding effect potentially exerted by the above environmental and agronomic variables. This approach resulted in the implementation of an effective tool to distinguish the different cultivars and, in particular, the “Marrone-type” (MT) cultivars from the other categories as sweet chestnut (SC) and Euro-Japanese hybrids (EH). The germplasm repository includes the main local and Italian chestnut cultivars, several European varieties, and accessions from China, Japan, and USA . The arefication . The annfication .Chestnut fruits (1 kg for each plant of each cultivar—three plants for each cultivar) from 18 accessions were randomly collected, hand selected to remove damaged fruits, and divided into two lots for sensory and chemical analysis. The nuts for the sensory analysis were boiled (100 °C for 45 min) and given to the trained panellists for the tasting sessions .Raw nuts used for the chemical analysis were hand-pealed, fragmented in small pieces, dried in an oven  at 40 °C for 2 days, and then ground to a fine powder and subdivided into small portions (500 g for each one) sealed in plastic bags.Sensory evaluation was used to identify and quantify the product organoleptic traits . CurrentSensory analysis was performed in a specific sensory laboratory, by 12 selected panellists  from ONAFrut . Three training sessions were carried out with 12 panellists to ensure a common lexicon of terms for flavour and aroma. During the three training sessions, the panellists worked in a group, but they individually evaluated the samples. After each panel training session, a discussion was held to decide the appropriate set of descriptors to use . Quantitv/v/v). Methanolic extracts were filtered through a membrane microfilter , and then were stored for a few days at normal atmosphere (NA), at 4 °C and 95% RH.All the chemicals/reagents are reported in Monoterpenes, sugars, and organic acids were extracted with 95% ethanol solution. Samples were then stored until analysis in NA, at 4 °C and 95% RH.−1 citric acid, 2 mmol·L−1 ethylenediaminetetraacetic acid (EDTA) disodium salt, and 4 mmol·L−1 sodium fluoride in methanol-water, 5:95, v/v). o-Phenylenediamine (OPDA) solution (18.8 mmol·L−1) was added to 750 μL of extracted samples for dehydroascorbic acid (DHAA) derivatisation to a fluorophore, 3-furo quinoxaline-1-one (DFQ).Ascorbic acid and dehydroascorbic acid were extracted by an extraction solution  of dried weight (DW).Antioxidant capacity in the chestnut fruits was assessed by a ferric reducing antioxidant power (FRAP) assay , and resThe total polyphenol content (TPC) was evaluated following the Folin–Ciocalteu colourimetric method , and theAbsorbance at 595 nm (for antioxidant capacity) and 765 nm (for TPC) with a UV/Vis spectrophotometer  was recorded.An Agilent 1200 High Performance Liquid Chromatograph, equipped with a G1311A quaternary pump, a manual injection valve, and a 20 μL sample loop, coupled to an Agilent GI315D UV-Vis diode array detector , was used for the analysis. 2 column . Different chromatographic conditions were used to analyse the samples according to the methods described by other studies [−1 of DW.Six different chromatographic methods were used to analyse the samples. Chromatographic separations were carried out on a Kinetex—C18 column , and a SphereClone—NH studies ,38, withn = 3) [LT), performed on a subset of 106 permutations [RT), carried out by setting the same permutation number. Sensory and nutraceutical data of 18 chestnut cultivars were subjected to one-way analysis of variance (ANOVA), and the averages were compared with the Tukey’s HSD post-hoc comparison test (n = 3) . Correlan = 3) . A princn = 3) ,40 was cn = 3) ,40. The n = 3) . Based on = 3) ,40. The n = 3) ,40. Poinn = 3)  and Lionn = 3) . The span = 3) . The spautations . Similarp-value correction for multiple comparisons and the 95% criterion to implement the model splits [The effect of the genotype on the chemical fingerprint was tested by fitting a conditional inference tree model ,46 on CAl splits ,45,46.https://apsjournals.apsnet.org/doi/suppl/10.1094/PHYTO-05-15-0112-R) [ANOVA and PCA were performed with statistical software package IBM SPSS Statistics 22.0 , the Mean Distance Randomisation Tests were run with the MDT software (-0112-R) , and the-0112-R)  with the-0112-R) . For allEight sensory attributes were used to characterise, qualitatively and quantitatively, the 18 chestnut cultivars analysed in this study, including three textural and visual  and five flavour descriptors . An overall subjective estimation (subjective judgement) was also expressed in order to describe the panellist personal rating. A 0 to 10 linear scale was used to evaluate the intensity of each attribute.C. sativa showed significant differences (p < 0.05) for all the descriptors, except for intensity of saltiness. The EH group was more homogeneous and samples differed (p < 0.05) only for ease of peeling, seed colour, intensity of flavour, and intensity of bitterness.Although different cultivars showed the same sensory attributes, they differed in terms of intensity . Within SC cultivars achieved high values in terms of seed colour and intensity of sweetness, according to Kunsch et al. , but thep < 0.05) were observed between chestnut samples for ease of peeling and seed colour, two important attributes. ‘Marrubia’ (SC) presented the highest value of ease of peeling (7.00 ± 0.65), while ‘Marrone di Castel del Rio’ and ‘Marrone di Marradi IGP’ showed a good ease of peeling in the MT group  as shown in other studies [Significant differences . Sensory analysis on the three cultivars from this group partially confirmed the results published in previous studies ,50. Theyp < 0.05) among the SC and MT cultivars: ‘Canepina’ presented the lowest value (4.36 ± 1.18) and ‘Marrone di Castel del Rio’ the highest one (7.07 ± 1.59), while there were no significant differences (p > 0.05) among the EH group. Data showed a high correlation level, evaluated by Pearson’s coefficient (r), between intensity of sweetness and sugar content in the analysed cultivars . ‘Marrubia’ showed no intensity of bitterness, recording the lowest value for this attribute, while ‘Marsol’ was the most bitter Euro-Japanese hybrid (0.90 ± 0.74). A high correlation coefficient was also found between intensity of bitterness and tannin content .In particular, the descriptor “intensity of sweetness” varied significantly (p < 0.05) were also observed in intensity of flavour and chestnut aroma (flavour descriptors). The first one is usually measured during the seed breakup and refers to the smell sense, while the other is the expression of chestnut aroma measurable by multiple senses as described in other studies [p < 0.05) than all the other sweet chestnuts. In the case of chestnut aroma, both ‘Marrubia’ and ‘Marrone di Castel del Rio’ significantly differed (p < 0.05) from the other cultivars that showed higher ratings. Euro-Japanese hybrids did not differ significantly (p > 0.05) in terms of chestnut aroma, while significant differences (p < 0.05) were observed in intensity of flavour, which was higher in ‘Marsol’ (6.00 ± 0.23).Significant differences  values than all the other cultivars, while EH chestnuts showed lower but not significant values (p > 0.05) in the group. Data pointed out a good correlation between judgment and sugar content . These values seemed to be lower than the Pearson’s coefficients between intensity of sweetness and sugar content due to the influence of other sensory traits on panellist judgment. Indeed, a significant correlation was also observed between judgment and chestnut aroma, with values similar to the correlation between judgment and sugar content . These results showed that judgment was equally influenced by chestnut aroma and sugar content. Moreover, a significant correlation between intensity of sweetness and chestnut aroma was observed . In any case, further statistical assessments are necessary to confirm this hypothesis. Moreover, a good correlation was found between intensity of sweetness and judgement , suggesting the influence of this sensory parameter on panellist personal preference. Pearson’s correlation data were reported in Panellists were also asked to give a personal preference to each cultivar or hybrid, although not planned in the Quantitative Descriptive Analysis (QDA), in order to assess a subjective general rating. ‘Marrubia’ (7.71 ± 0.76) and ‘Contessa’ (7.10 ± 1.30) for the SC chestnuts, and ‘Marrone di Castel del Rio’ (7.43 ± 1.24) for the MT group, displayed significantly higher , complemented by the measurement of the antioxidant capacity, of the 18 cultivars and hybrids of chestnuts were defined by chemical analysis .GAE kg−1 DW for the French cultivar ‘Bouche Rouge’ to 1.40 ± 0.05 gGAE kg−1 DW for the Italian cultivar ‘Marrubia,’ in agreement with other studies [p < 0.05), expressed as a FRAP assay, were observed among the cultivars with a trend similar to the one observed for TPC levels. Antioxidant capacity ranged from 9.30 ± 0.39 mmol Fe+2 kg−1 DW (‘Bouche de Bètizac’) to 19.96 ± 1.89 mmol Fe+2 kg−1 DW (‘Garrone Rosso’), as shown in Mean TPC and antioxidant capacity values are reported in  studies ,56. Ches studies . The higThe phytochemical composition of the analysed cultivars identified 21 biomarkers by HPLC-DAD. To evaluate the contribution of each class to the total phytocomplex composition, the bioactive compounds were grouped in the following classes: polyphenols , monoterpenes, and vitamin C  .−1 DW and 0.76 g·kg−1 DW, respectively), a cultivar from central Italy, while the highest values of vitamin C were observed in the French cultivar ‘Bouche Rouge’ (0.19 g·kg−1 DW) as shown in Monoterpenes, recognised for their anti-tumour and anti-inflammatory properties , represeWithin the polyphenolic group, differences were observed among chestnut genotypes, but most of the cultivars showed phenolic levels similar to hazelnut ones ,64, and −1 DW). This value was higher than the average of the other samples, as confirmed by Tukey’s test (p < 0.05), which included this sample in a separate group. The same holds true for the catechin class, which was represented mainly by epicatechin (0.15 ± 0.04 g·kg−1 DW). Regarding catechins, the MT varieties were included within the same group, together with ‘Gentile’ and ‘Madonna’ cultivars, that showed relevant quantities of these compounds as shown by De Biaggi et al. (2018) [−1 DW, while French cultivar ‘Bouche Rouge’, with about 0.06 g·kg−1 DW, was classified by the post-hoc test in a separate group (p < 0.05). The presence of tannins in adequate amounts increases the nutraceutical properties of chestnuts since these compounds are free radical quenchers [Data on each bioactive and nutritional compound content are reported in . (2018) . The ide. (2018) . For tanuenchers .−1 DW). Ferulic acid was not detected in all the analysed chestnut cultivars, except in ‘Canepina’ and ‘Madonna’ (about 0.01 g·kg−1 DW). In the flavonol class, no traces of quercetin were detected in any of the analysed samples, and quercitrin and rutin occurred only at low concentrations , except in ‘Marrubia’, which showed a quercitrin value of 0.120 ± 0.001 g·kg−1 DW. The most representative flavonol was isoquercetin, which is detected in large quantities, especially in ‘Tarvisò’ (0.26 ± 0.03 g·kg−1 DW), ‘Canepina’ (0.23 ± 0.03 g·kg−1 DW), and ‘Madonna’ (0.22 ± 0.04 g·kg−1 DW). ‘Tarvisò’ cultivar displayed the highest flavonol content (0.26 ± 0.03 g·kg−1 DW), followed by ‘Madonna’ (0.25 ± 0.04 g·kg−1 DW), and ‘Canepina’ (0.24 ± 0.03 g·kg−1 DW), while ‘Mansa’, ‘Bouche Rouge’, and ‘Marrone di Marradi IGP’ showed lower values. Flavonols quench active oxygen species and inhibit in vitro oxidation of low-density lipoproteins [Caffeic and coumaric acids were quantified in all the samples, although in low quantities (<0.01 g·kgproteins .−1 DW), while gallic acid showed high variability among the samples, except for ‘Marrone della Val Pellice,’ which did not contain gallic acid and, in general, was characterised by the lowest content of benzoic acids. These molecules are very important in human nutrition and are related to many biological properties, including anticancer, anti-atherosclerotic, anti-inflammatory, antihepatotoxic, and anti-HIV replication activities [Ellagic acid was the most abundant benzoic acid in ‘Gabiana’ and ‘Canepina’ (about 0.05 g·kgtivities .−1 DW). Limonene was predominant and reached quantities of 6.35 ± 1.77 g·kg−1 DW in the ‘Canepina’ cultivar. High limonene amounts were also found in the EH group. γ-terpinene was detected in high quantities, in particular in ‘Precoce Migoule’ (0.38 ± 0.70 g·kg−1 DW), ‘Marrone della Val Pellice’ (0.21 ± 0.13 g·kg−1 DW), and ‘Gabiana’ (0.16 ± 0.05 g·kg−1 DW), similar to other studies [As well as polyphenols, the analysed samples also showed different monoterpenes and discrete contents of vitamin C, as reported in  studies ,72. Seve studies . Terpino−1 DW), followed by ‘Marrone della Val Pellice’ (0.18 ± 0.09 g·kg−1 DW), while the minimum amount was detected in ‘Marrone di Marradi IGP’. Among EH chestnuts, ‘Bouche de Bétizac’ provided the largest amount of vitamin C (0.12 ± 0.02 g·kg−1 DW), which was comparable to the values reported in De Biaggi et al. (2018) [Vitamin C was evaluated as the sum of ascorbic and dehydroascorbic acids due to their biological activity in human organisms as reported in other studies ,74. The . (2018) . The maj. (2018) ,75, and . (2018) .p < 0.05) in organic acid and sugar content values were detected among cultivars. Mean values for SC, MT, and EH chestnuts are reported in Large and significant differences (−1 DW), while the chestnut cultivar ‘Garrone Rosso’ showed the lowest values (1.20 ± 0.21 g·kg−1 DW). Citric acid was the most abundant organic acid in the analysed chestnuts, with high levels in ‘Mansa’ and ‘Precoce Migoule’ cultivars , followed by quinic acid , as reported by similar studies [p < 0.05) in the organic acid composition among different cultivars of the SC, MT, and EH groups, leading to the identification of different groups composed by one or a few compounds. This result could be due to the differences associated with the different genotypes but, since organic acids are volatile molecules, other factors could have slightly influenced the results, such as the extraction technique, sample storage, and applied drying technique [High levels of organic acids were observed in hybrids, in particular for ‘Precoce Migoule’ (7.43 ± 0.09 g·kg studies . Quinic  studies . Tukey’sechnique .−1 DW), while the average values ranged from 10.64 ± 2.33 g·kg−1 DW (‘Bouche Rouge’) to 114.80 ± 10.63 g·kg−1 DW (‘Canepina’); chestnut cultivars showed higher values if compared to the sugar levels of other tree nuts such as walnut and almond [−1 DW) similar to other chestnut cultivars, which was in agreement with other studies [p < 0.05) from the other samples, followed by ‘Canepina’, as mentioned above, and ‘Brunette’ and ‘Gentile’, which reported quantities close to 50 g·kg−1 DW. The highest quantity of sugars was observed for the cultivar ‘Mansa’  showed a significant collinearity among variables. The KMO index attained a value of 0.74. The PCA resulted in two principal components accounting for 50.71% of the total variance, with 32.47% explained by PC1, and 18.24% by PC2. The location in the PCs plane of the 18 samples  in relation to phytochemical composition, nutritional properties, and nutraceutical traits is shown in the score plot  in their bioactive content among the different cultivars. Moreover, monoterpenes showed also a good discriminating power among chestnut samples. For this reason, all these molecules could represent the most important markers in order to build a discriminant model between chestnut genotypes, but further studies are necessary to confirm this hypothesis.Phenolic acids and tannins, associated with PC1, were identified as bioactive classes with the most discriminating power among different genotypes; these phytochemical classes included compounds displaying significant differences . The MDRTLT test showed a significant clustering of points associated with MT group (p < 0.05) in the PC plan, while the MDRTRT test showed that the points associated with SC + EH groups are characterised by a significantly dispersed spatial distribution (p < 0.05) in the same PCs plane. These results suggest that the MT cultivars represented a homogenous group with less variable traits than the SC + EH groups  role and may account for most of the differences detected among the chemical fingerprints of the chestnut fruits. In fact, the tree model (p < 0.001) splits resulting in two intermediate and four terminal nodes. The outcomes of the conditional inference tree model pointed out that the genotype plays a significant (ee model  displayep > 0.05), while genotypes included in separate nodes were characterised by significantly different chemical fingerprints (p < 0.05). It is worth noting that while the PCA clearly pointed out the role played by different chemical compounds in profiling, the samples analysed, the conditional inference tree model accounted for the role of the genotype, a categorical variable that cannot be included directly within a PCA, since this can only handle continuous covariates [Each terminal node clustered the genotypes whose overall chemical fingerprint was homogeneous ; followed by polyphenols, which are characterised by antioxidant and anti-bacterial properties (10–25% for EH and SC); and vitamin C in trace (about 2%). Tannins were the main polyphenolic compounds detected in the analysed chestnuts, followed by phenolic acids and flavonols. In particular, ‘Canepina’ presented higher phenolic amounts than almost all the analysed cultivars. Moreover, the majority of the analysed chestnut cultivars showed a content of bioactive compounds, as phenolics and vitamin C, whose levels were similar to, or higher than those reported for the main hazelnut, walnut, and almond varieties.Chromatographic and spectrophotometric data confirmed the high variability within the genus Castanea germplasm. As genetic and phytochemical diversity represent fundamental aspects to ensure the productivity and adaptability of chestnut orchards, different approaches need to be developed to ensure the correct characterisation strategy. Hence, different techniques were combined in this study to define a suitable strategy for the characterisation of the chestnut cultivars as a key prerequisite to allow the conservation of Castanea, and therefore, the importance of in farm and ex situ conservation of local germplasm as part of a global strategy, and also in relation to an active utilisation of agrobiodiversity.The analysed cultivars were selected as part of a core collection that maximises the chestnut agro-biodiversity. The diversity observed among the analysed cultivars could be strictly associated to the genotype effect and underlines the large variability of the genus


The MSHTS system is seldom influenced by contaminants in samples and is able to directly evaluate Aβ42 aggregation inhibitory activity of samples containing various compounds. In this study, to elucidate whether the MSHTS system could be applied to the evaluation of processed foods, we examined Aβ42 aggregation inhibitory activity of salad dressings, including soy sauces. We estimated the 50% effective concentration (EC50) from serial diluted dressings. Interestingly, all 19 commercial dressings tested showed Aβ42 aggregation inhibitory activity. It was suggested that EC50 differed by as much as 100 times between the dressings with the most (0.065 ± 0.020 v/v%) and least (6.737 ± 5.054 v/v%) inhibitory activity. The highest activity sample is traditional Japanese dressing, soy sauce. It is known that soy sauce is roughly classified into a heat-treated variety and a non-heat-treated variety. We demonstrated that non-heat-treated raw soy sauce exhibited higher Aβ42 aggregation inhibitory activity than heat-treated soy sauce. Herein, we propose that MSHTS system can be applied to processed foods.The aggregation and accumulation of amyloid β (Aβ) in the brain is a trigger of pathogenesis for Alzheimer’s disease. Previously, we developed a microliter-scale high-throughput screening (MSHTS) system for Aβ One of the problems facing an aging society is the increase of patients with dementia. While various diseases are known to cause dementia, Alzheimer’s disease (AD) in particular, accounts for the majority of cases ,2,3. FouLamiaceae such as rosemary, perilla, and lemon balm. RA is a known inhibitor of Aβ aggregation [Rosmarinic acid (RA) is a polyphenol found in abundance in plants of the regation ,13. Its regation ,15. Amonregation . Furtherregation . Hsu et regation . Thus, tregation . ThT emi42 aggregation process with a fluorescence microscope using a quantum dot (QD) nanoprobe and developed a microliter-scale high-throughput screening (MSHTS) system for Aβ42 aggregation inhibitors by applying this imaging method [42 aggregates is quantified from standard deviation (SD) value estimated from the variation in fluorescence intensity of each pixel of obtained images and the emission wavelength of QD605 does not overlap with the absorption of almost natural products [42 aggregation as EC50 values. Before, we evaluated the Aβ42 aggregation inhibitory activity of 52 spices using this method and demonstrated that the herb-based spices of the Lamiaceae family exhibited high Aβ42 aggregation inhibitory activity [42 aggregates morphology was affected with seaweed-derived polysaccharide including in boiling water extracts [42 aggregation inhibitory activity. Thus, MSHTS system is useful for quantitative evaluation of Aβ42 aggregation inhibition ability of various natural products. However, it is unclear whether MSHTS system can evaluate Aβ42 aggregation inhibitory activity in foods including various natural substances with many impurities. In this study, to elucidate whether the MSHTS system is applied to processed foods such as salad dressings, including soy sauces, we evaluated Aβ42 aggregation inhibitory activity of dressings using the MSHTS system. We found that all tested commercial dressings showed Aβ42 aggregation inhibitory activity despite there were differences in their activities. Especially raw soy sauce showed the highest inhibitory activity among the tested samples. These results suggest that the MSHTS system is a powerful and useful tool that is expected to be applied and developed in various processed foods.Previously, we succeeded in real-time imaging of the Aβg method ,20. The products . Thus, textracts . Furtherextracts . Screeni42  and Cys-conjugated Aβ40  kits were purchased commercially. Twenty different commercially available salad dressings and soy source brands were purchased from Japanese companies  using catalog shopping in June 2016.Human Aβ2 (QdotTM 605 ITKTM Amino (PEG) Quantum dot; Q21501MP, Thermo Fisher Scientific, Waltham, MA USA) according to our previous reports [2 with 1 mM sulfo-EMCS  in PBS  for 1 h at room temperature. QDAβ concentration was determined by comparing absorbance at 350 nm to that of unlabeled QD-PEG-NH2.The QDAβ nanoprobe was prepared using QD-PEG-NH reports ,20,21,2250 values of various dressings were determined by a modified MSHTS system, as was described in our previous reports [42 in PBS containing 5% EtOH and 3% DMSO were incubated in a 1536-well plate  at 37 °C for 24 h. The QDAβ-Aβ42 aggregates that formed in each well were observed by an inverted fluorescence microscope . Standard deviation (SD) values of fluorescence intensities of 40,000 pixels (200 × 200 pixels) around the central region of each well were measured by ImageJ software Ver 1.53b (NIH). The SD values, which were approximately proportional to the amount of aggregates [The EC reports ,21,22. MAggregates in the 1536-well plate were observed by an inverted fluorescence microscope  using a 4× objective lens  equipped with a color CCD camera .Samples were deposited in 10 µL aliquots onto 200-mesh copper grids and negatively stained with 1% phosphotungstic acid at room temperature. Specimens were examined under an H-7600 TEM  at 60 kV.The ThT assay was conducted according to the method of Levine modified in our laboratory ,21. StatSDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using standard techniques. Soy sauce and raw soy sauce were heated by block incubator at 80 °C for 60 min before electrophorese. For dialysis protocol, soy sauce and raw soy sauce were dialyzed against distilled water. Distilled were changed three times for overnight. Then, the gel was silver-stained by staining kit 42 aggregation inhibitory activity of the liquid dressings using the ThT method was difficult because the absorption wavelength of almost samples overlapped with the excitation and emission wavelengths of ThT.First, in order to assess whether 19 liquid salad dressings could be evaluated for activity by the ThT method, their absorbance spectra were measured using a Nanodrop 2000c (Thermo Fisher Scientific) . The excv/v%) was measured in three conditions; +Aβ42 and +ThT, −Aβ42 and +ThT, −Aβ42 and −ThT . The fluorescence intensity of samples C, D, and E did not show a higher value than the negative control. The color and some components of the evaluated samples may affect the ThT method by absorbing the excitation or emission of ThT. If the sample solution without Aβ42 exhibits a higher fluorescence intensity than the negative control, it is difficult to determine whether the sample solution affects ThT. Therefore, the ThT method might not accurately evaluate the Aβ42 aggregation inhibitory activity of a commercial dressing.To confirm whether the evaluation using ThT method was performed correctly, the fluorescence intensity of five samples at a high concentration  caused an inhomogeneous distribution of fluorescence intensity in images, resulting in an increased standard deviation (SD) value estimated from the variation in fluorescence intensity of each pixel in the images. Therefore, we could estimate the effects of certain aggregation inhibitors by detecting changes in the SD value. Here, we evaluated the Aβ42 aggregation inhibitory activity of 19 commercial dressings using the MSHTS system  contained plant oil while the other 16 samples were non-oil type dressings. We compared the mean value of EC50 value of non-oil and oil type  and performed a statistical analysis. There was no significant difference between non-oil type and oil type . The activity did not depend on the presence of oil, suggesting that oil in the dressing did not affect the Aβ42 aggregation inhibitory activity. Next, to confirm whether Aβ42 aggregation was inhibited by the effect of the dressing, we observed the Aβ42 aggregates at a sample concentration of 0.04 v/v% using TEM . Because the EC50 of sample A is 0.065 ± 0.020 v/v%, it was indicated that 1.8 mM NaCl is included. The NaCl concentration of the 0.4 v/v% and 0.04 v/v% solution samples is 11 mM and 1.1 mM, respectively. Therefore, the NaCl in 0.065 v/v%  soy sauce solution might not affect the formation of Aβ42 aggregates. In fact, as shown in v/v%. Especially, 0.04 v/v% of sample A inhibited the aggregation formation. These results suggest that the Aβ42 aggregation inhibitory activity by soy sauce was due to not NaCl but other components.In general, it is well known that the soy sauce contains a large amount of NaCl and that high salt concentration affects protein aggregation. In order to determine whether the Aβgregates A. We preo 200 mM B. Howeve42 aggregation inhibitory activity of raw soy sauce purchased from NIHON SYOYU KOGYO using the MSHTS system. The Aβ42 aggregation inhibitory activity of raw soy sauce was 0.0045 ± 0.0015 v/v% (Data not shown). This activity is about 15 times higher than that of soy sauce which showed the highest activity among the 19 dressings. Therefore, it is likely that the difference in the aggregation inhibitory activity of Aβ42 between soy sauce and raw soy sauce is caused by the raw material-derived protein and the microorganism-derived enzyme, which are lost by heating.As shown in 42 aggregation inhibitory activity, each sample was subjected to SDS-PAGE after heat treatment  and/or dialysis treatment and their band patterns were compared using silver staining (To examine whether proteins and/or low molecular weight compounds in soy sauce and raw soy sauce affect Aβstaining A. As rev42 aggregation inhibitory activity of soy sauce and raw soy sauce (42 aggregation inhibitory activity of soy sauce was not changed by heating. This result was consistent with the SDS-PAGE banding pattern (42 aggregation inhibitory activity of raw soy sauce was reduced by about 30 times or by about half after heat treatment and dialysis, respectively. Dialysis treatment did not affect the 100–200 kDa band that was reduced by heat treatment, i.e., the 100–200 kDa protein did not contribute to the Aβ42 aggregation inhibitory activity of raw soy sauce. The heat treatment after dialysis decreased Aβ42 aggregation inhibitory activity by about 440 times, which was consistent with the SDS-PAGE result in which many bands were reduced (42 aggregation inhibitor found in soy sauce and raw soy sauce is a low molecular weight compound that is removed by dialysis and a protein that is thermally denatured by heat treatment at 80 °C for 60 min. Since the Aβ42 aggregation inhibitory activity of raw soy sauce was greatly reduced after heat treatment, the proteins found in raw soy sauce are considered to be particularly important for the inhibition of Aβ42 aggregation.Next, using the MSHTS system, we assessed whether heating and dialysis treatment would affect the Aβoy sauce B. Furthe reduced A, lane 842 aggregation inhibitory activity. The main raw materials of soy sauce, soy, and wheat, are decomposed into amino acids, peptides and saccharides in the manufacturing process. It is known that soybeans have many physiologically active ingredients such as soy protein and isoflavone [In general, proteins and peptides are denatured by heating, then lose their physiological activity. It is possible that the presence or absence of heat treatment in the fermentation process may be involved in the physiological activity of dressings such as soy sauce, which contains Aβoflavone ,26,27, soflavone . Genisteoflavone . It was oflavone ,29 Furthoflavone ,31,32. W42 aggregation inhibitory activity of 19 commercial liquid dressings using the MSHTS system. All tested dressings exhibited Aβ42 aggregation inhibitory activity, suggesting that the MSHTS system can be applied to processed food containing various impurities. Japanese traditional liquid dressings, soy sauce, exhibited the highest inhibitory activity. However, these findings are limited to in vitro conditions. The physiological activity of the dressings should be clarified through animal experiments, taking into account dynamics such as intestinal absorption and metabolism, particularly the permeability of the blood-brain barrier. Although there is a strong demand for functional food products that help maintain and improve brain function, it is not realistic to subject all food products to animal testing. We are confident that we can progress quickly to a second screening stage such as animal testing by using the MSHTS system as a first screening tool to discover food materials with high Aβ42 aggregation inhibitory activity. Recently, we confirmed that aggregation of various amyloid proteins Aβ42, tau, and α-synuclein could be visualized using nonlabelled QD and succeeded in the evaluation of aggregation inhibitory activity of RA [In this work, we evaluated Aβty of RA . MSHTS sty of RA  and non-


Introduction. Environmental exposure of the developing offspring to cigarette smoke or nicotine is an important predisposing factor for many chronic respiratory conditions, such as asthma, emphysema, pulmonary fibrosis, and so forth, in the exposed offspring. Studies showed that electroacupuncture (EA) applied to maternal “Zusanli” (ST36) acupoints during pregnancy and lactation protects against perinatal nicotine exposure- (PNE-) induced lung damage. However, the most effective time period, that is, prenatal vs. postnatal, to attain this effect has not been determined.  To determine the most effective developmental timing of EA's protective effect against PNE-induced lung phenotype in the exposed offspring. μl) and EA was administered to bilateral ST36 acupoints once daily during the specified time-periods. At the end of the experimental periods, key hypothalamic pituitary adrenal (HPA) axis markers in pups and dams, and lung function, morphometry, and the central molecular markers of lung development in the offspring were determined.  Pregnant rats were given (1) saline (“S” group); (2) nicotine (“N” group); (3) nicotine + EA, exclusively prenatally (“Pre-EA” group); (4) nicotine + EA, exclusively postnatally ; and (5) nicotine + EA, administered both prenatally and postnatally (“Pre- and Post-EA” group). Nicotine was injected once daily  and serum corticosterone (Cort) levels increased, in line with the known PNE-induced lung phenotype. In the nicotine exposed group, maternal hypothalamic corticotropin releasing hormone (CRH) level decreased, but pituitary adrenocorticotropic hormone (ACTH) and serum Cort levels increased. In the “Pre- and Post-EA” groups, PNE-induced alterations in lung morphometry, lung development markers, and HPA axis were blocked. In the “Pre-EA” group, PNE-induced changes in lung morphometry, GR, and maternal HPA axis improved; lung PPARγ level decreased, but glucocorticoid receptor (GR) and serum corticosterone (Cort) levels increased, in line with the known PNE-induced lung phenotype. In the nicotine exposed group, maternal hypothalamic corticotropin releasing hormone (CRH) level decreased, but pituitary adrenocorticotropic hormone (ACTH) and serum Cort levels increased. In the “Pre- and Post-EA” groups, PNE-induced alterations in lung morphometry, lung development markers, and HPA axis were blocked. In the “Pre-EA” group, PNE-induced changes in lung morphometry, GR, and maternal HPA axis improved; lung PPAR After nicotine exposure, alveolar mean linear intercept (MLI) increased, but mean alveolar number (MAN) decreased and lung PPAR Maternal EA applied to ST36 acupoints during both pre- and postnatal periods preserves offspring lung structure and function despite perinatal exposure to nicotine. EA applied during the “prenatal period” affords only limited benefits, whereas EA applied during the “postnatal period” is ineffective, suggesting that the EA's effects in modulating PNE-induced lung phenotype are limited to specific time-periods during lung development. Despite well-established dangers of tobacco to human health, exposure of pregnant women to mainstream or sidestream smoke remains extremely high . Althougγ (PPARγ). PPARγ is centrally involved in alveolar and airway development [γ knockout mice show enlarged alveolar sacs, increased apoptotic cells, and an enlarged lung volume, highlighting PPARγ's indispensable role in lung development [γ, drives alveolar lipofibroblasts to transdifferentiate to myofibroblasts, which are the hallmarks of all chronic lung conditions including the perinatal nicotine exposure- (PNE-) induced lung damage [γ agonists blocks and/or reverses the PNE-induced lung damage in the exposed offspring [Nicotine's effects on the developing lung have been largely attributed to a disruption in epithelial-mesenchymal paracrine signaling, the central component of which is the nuclear transcription factor peroxisome proliferator-activated receptor-elopment –20 and ielopment , 21. Lunelopment , 20. Nicg damage , 23. Supffspring , 25.Hypothalamic pituitary adrenal (HPA) axis, by regulating the production of glucocorticoids, also performs an essential role in lung development and maturation . GlucocoCurrently, there is no clinically safe and effective pharmacologic intervention to prevent or treat PNE-induced lung damage , 33–39. Approval was obtained from the Beijing University of Chinese Medicine experimental animal Ethics Committee in 2017 and all animal procedures were performed in accordance with the “Guide to the Care and Use of Experimental Animals” of the China Animal Welfare Commission. Thirty female and ten male speciﬁc pathogen-free Sprague-Dawley rats (11 weeks old) without prior mating history were obtained  2006-0002). Animals were housed at a constant temperature and humidity environment with 12 hours of alternate light and dark cycle, with the provision of ad lib food and water. The feeding cages and water bottles were regularly disinfected.μl volume once daily and nicotine: 1 mg/kg in 100 μl volume once daily) were started on E6, and continued throughout pregnancy and lactation, that is, up to PND21 (except on the day of delivery). The saline group (“S” group) was injected saline once daily. The nicotine group (“N” group) was injected nicotine once daily. For the prenatal EA group (“Pre-EA group”), nicotine injection was the same as in the “N” group, but these dams were also administered EA to bilateral ST36 acupoints from E6 to the day of delivery. For the postnatal EA group (“Post-EA” group), nicotine injection was the same as in the “N” group, but these animals were administered EA to bilateral ST36 acupoints from PND1 to PND21. The prenatal and postnatal EA group (“Pre- and Post-EA” group) was administered nicotine similar to the “N” group, but these animals also received EA at bilateral ST36 acupoints from E6 to PND21 (except on the day of delivery). On PND21, pulmonary function testing was performed before sacrificing pups for lung tissue and serum collection and dams for the hypothalamus, pituitary, and serum collection.In line with a well-established model , 32, salThe ST36 acupoints were identified at the posterolateral side of knee-joint about 5 mm below the head of the fibula, as detailed in “Experimental Acupuncture Science” . DisposaPulmonary function testing was performed by the Respiratory Function Instrument with Buxco FinePointe software . The pups were intraperitoneally injected with 2% pentobarbital (5.5 mg/100g) for anesthesia, tracheotomized, cannulated, and connected to a ventilator for plethysmography. After a period of steady breaths, the lung resistance (RL), dynamic compliance (Cdyn), minute ventilation volume (MV), and peak expiratory flow (PEF) were recorded.μm slices, which for lung morphometry were stained with hematoxylin and eosin (H&E). Subsequently, lung tissue morphology was assessed by determining mean linear intercepts (MLI) and mean alveolar numbers (MAN) using previously described methods [At sacrifice, pup lungs were fully inflated with 4% paraformaldehyde (PFA) in PBS with constant pressure; after ligation, the lungs were submerged in 4% PFA for about 5 h, followed by immersion in 30% sucrose in PBS. The left lung was used for paraffin embedding, cut into 5  methods .γ , GR , CRH , and ACTH  using ELISA as per manufacturer's instructions.Offspring lung tissue and maternal hypothalamic and pituitary tissues were homogenized and the supernatants were collected for detecting PPARSerum Cort levels in the mother and offspring rats were performed using radioimmunoassay as manufacturer's instructions .γ and GAPDH have been described previously [The method for RNA extraction, Real-time PCR, and the primers information of PPAReviously .P < 0.05 was considered statistically significant.The data are expressed as mean ± SD. Statistical analysis was performed using SPSS statistical software . One-Way ANOVA-Bonferroni test was used for the comparison of differences between groups, and P < 0.01, <0.01, and <0.05, respectively), while RL increased (P < 0.01) significantly. Compared with the “N” group, in the “Pre-EA” group, Cdyn increased (P < 0.01) and RL decreased (P < 0.01) significantly; however, the MV and PEF were not different (P > 0.05). Compared with the “N” group, the RL decreased significantly (P < 0.01) in the “Post-EA” group; however, the Cdyn, MV, and PEF were not significantly different (P > 0.05). Furthermore, the Cdyn, MV, and PEF increased , while RL decreased (P < 0.01) significantly, in “Pre- and Post-EA” group vs. the “N” group , accompanying a lower MAN (P < 0.01), and in parts ruptured and fused alveolar walls. Compared with the “N” group, the “Pre-EA” and the “Pre- and Post-EA” group had smaller alveolar volumes  and more alveoli (P < 0.01 vs. the “N” group), the rupture and fusion of alveolar walls improved; however, the “Post-EA” group was not different from the “N” group (P > 0.05 vs. the “N” group)  .γ mRNA . Both of these changes were blocked in the “Pre- and Post-EA” group ; although the “Pre-EA” group showed a significant increase, it did not reach statistical significance (P > 0.05 vs. the “N” group); however, the “Post-EA” group was not different from the “N” group in both PPARγ mRNA and protein levels (P > 0.05 for both).Using Real-time PCR and ELISA, compared to the “S” group, PPARγ mRNA  and protγ mRNA  levels dP < 0.05, and <0.01, respectively), which normalized in the “Pre- and Post-EA” group . Furthermore, in the “Pre-EA” group, compared with the “N” group, though the lung GR decreased significantly (P < 0.05), serum Cort was not significantly different (P > 0.05). Furthermore, the “Post-EA” group was not different from the “N” group in both (lung GR and serum Cort levels) of these parameters (P > 0.05).The results showed that serum Cort  and lungP < 0.05, P < 0.01, P < 0.01, P < 0.01, <0.05, and <0.05, respectively), “Post-EA” , and “Pre- and Post-EA”  group.The results showed that compared to the “S” group, the levels of maternal hypothalamic CRH decreased , pseudoglandular (E13–18.5), canalicular (E18.5–20), saccular (E20-PND4), and alveolar (PND4–21) stages. Specific growth factors and signaling mechanisms regulate each stage and drive its progression to the next stage . By compγ is a ligand-activated transcription factor that plays a key role in regulating lipid storage and metabolism in various organs including the lung [γ expression in the developing lung along with the associated nicotine-induced pulmonary structural and functional phenotype [γ protein levels, in conjunction with blockage of the perinatal nicotine-induced pulmonary structural and functional changes. Interestingly, EA applied exclusively during the “prenatal period” only slightly blocked the PNE-induced decrease in pulmonary PPARγ protein levels, which, not surprisingly, was accompanied by incomplete protection against PNE-induced pulmonary effects; that is, although the lung morphology improved, it only partially blocked nicotine's effects on pulmonary function. In contrast, administration of EA exclusively during the “postnatal period,” neither improved pulmonary PPARγ protein levels nor nicotine's effects on lung structure and function.PPARthe lung –57. Expehenotype , 58. EA β-HSD-2 activity, and compromises the placental barrier to maternal glucocorticoids, which leads to fetal overexposure to maternal glucocorticoids, which in turn affects fetal HPA axis and growth [To understand the mechanism of EA's effects on nicotine-induced pulmonary morbidity in the developing lung, it is important to understand nicotine's effects on maternal and fetal HPA axes and how these are affected by EA. Glucocorticoids are key players in mediating stress response on the HPA axis, both before and after birth , 60. In d growth , 30. In d growth , 32. Pred growth . It is lγ and serum Cort levels at PND21 following prenatal EA. We also found that although EA applied to ST36 acupoints during lactation modulated maternal HPA axis, it had no apparent effect on offspring rats. It is likely to be due to relatively limited transfer to maternal glucocorticoids via breast milk to offspring. A previous study showed that PPARγ agonists administered during lactation (PND1- PND21) could reverse nicotine-induced lung damage in rat offspring [γ agonist was directly administered to rat pups, whereas in the present study, the protective effect was dependent upon transmission of protective factors via breast milk. Overall, our data support that for the optimal benefit of EA at ST36 acupoints against perinatal nicotine-induced lung damage, it needs to be administered both pre- and postnatally.Our data suggest that maternal EA during pregnancy can have lasting effects on the maternal HPA axis, that is, at least until the end of lactation. Long lasting effects after acupuncture have been demonstrated in other conditions as well , 65. Forffspring . The conIn conclusion, in an experimental rat model, maternal EA applied to ST36 acupoints, during both “pre- and postnatal periods,” preserves offspring lung structure and function despite perinatal exposure to nicotine. This effect is accompanied by blockage of PNE-induced changes in HPA axes in both the mother and the offspring, thus preventing offspring exposure to excessive maternal glucocorticoids, which occurs with perinatal nicotine exposure. Maternal EA at ST36, administered exclusively during the “prenatal period,” affords only limited benefit, while its administration exclusively during the “postnatal period” does not afford obvious protection.


However, for adults already consuming 0.8–1.3 g·kg−1·d−1, the preponderance of evidence supports that consuming at least one meal that contains sufficient protein quantity to maximally stimulate muscle protein synthesis, independent of daily distribution, is helpful to promote skeletal muscle health.There is a shift in thinking about dietary protein requirements from daily requirements to individual meal requirements. Per meal, stimulation of muscle protein synthesis has a saturable dose relationship with the quantity of dietary protein consumed. Protein intake above the saturable dose does not further contribute to the synthetic response; the “excess” amino acids are predominantly oxidized. Given that daily dietary protein intake is finite, finding protein distribution patterns that both reduce amino acid oxidation and maximize their contribution towards protein synthesis  could be “optimal” and is of practical scientific interest to promote beneficial changes in skeletal muscle-related outcomes. This article reviews both observational and randomized controlled trial research on the protein distribution concept. The current evidence on the efficacy of consuming an “optimal” protein distribution to favorably influence skeletal muscle-related changes is limited and inconsistent. The effect of protein distribution cannot be sufficiently disentangled from the effect of protein quantity. Consuming a more balanced protein distribution may be a practical way for adults with marginal or inadequate protein intakes (<0.80 g·kg After the retrospective analysis estimating the saturable dose limit in adults was published [Prior to the acute randomized controlled trials that popularized the protein distribution concept known today ,35,36, tublished , Gingricublished  followedublished ; howeverublished  is more ublished  reportedublished , is moreublished  speculatublished , a more Physical activity improves muscle strength in adults and “sensitizes” the muscle to protein feeding, enhancing MPS for up to 48 h after exercise . In lighTo our knowledge, two retrospective cohort studies have been published documenting relationships between protein distribution (CV) and changes in lean mass and physical function. Using baseline and 2 y follow-up data from the Quebec Longitudinal Study on Nutrition as a Determinant of Successful Aging study ,26, FarsCollectively, four ,24,25,26n = 341); 1 meal containing ≥30 g protein (n = 560); 2+ meals containing ≥30 g protein (n = 172). Loenneke et al. [−1. Compared to consuming 0 meals, consuming 2 or 3 meals per day, but not 1 meal, with adequate protein content was associated with lower risk of physical disability among 4 (30 g criterion) and 2 (0.4 g·kg−1 criterion) of the 15 outcomes assessed. Notably, the reference groups for each study [−1·d−1. Although total daily protein quantity was controlled for in the analyses, these results may reflect negative consequences of consuming less than the protein recommended dietary allowance on muscle mass and strength and not the benefits of frequently consuming moderately high-protein-containing meals.The previous section compared and contrasted studies that defined protein distribution by the degree of “balancing”. That is, protein distribution was quantified on a continuous scale between 0 and 1 . Another method of exploring the protein distribution concept is to assess whether the number of meals people consume that reach a target MPS threshold result in different muscle-related responses. Hypothetically, persons that consume more meals that elicit maximal MPS responses would have more favorable skeletal muscle-related outcomes . In essence, these studies are assessing muscle-related outcomes by moving from a less to more optimal distribution. Two cross-sectional studies by Loenneke et al.  and Lopre et al.  and Lopre et al.  both repe et al.  further e et al.  used thech study ,28,29 we−1 and ~78 g·d−1, respectively. It is possible that Mishra et al. [−1); similar to the studies by Loenneke et al. [−1). Conversely, it is possible that Loenneke et al. [−1 or ≥2.5 g leucine was not associated with leg strength, leg power, and hand grip strength.A more recent analysis by Mishra et al.  using 20a et al.  would hae et al.  and Lopre et al.  , consuming an unbalanced protein distribution with a higher-protein diet >1.0 g·kg−1·d−1, where at least one meal contains sufficient protein quantity to maximally stimulate MPS, is helpful to promote skeletal muscle health; consuming meals with “adequate” protein more frequently  was not associated with further skeletal muscle-related benefits.Collectively, three ,28,29 of−1 at breakfast, lunch, or dinner, respectively. That is, they were assessing whether specific meals with “inadequate” protein were associated with functionality: does the meal in which protein “inadequacy” occurs matter? Using the 30 g criterion, inadequate protein intake at lunch was associated with both low and middle scores for activities of daily living. Using the 0.4 g·kg−1 criterion, only inadequate protein intake at dinner was associated with middle scores for instrumental activities of daily living. The authors [−1 at three meals compared to those that did not consume ≥0.24 g·kg−1 in at least one meal [In contrast to comparing the skeletal muscle-related outcomes among groups consuming incrementally more meals that reach a target threshold, the protein distribution concept can be approached by assessing the contrapositive: studying the deviation away from an optimal protein distribution. Gayatán Gonález et al.  assessed authors  argued tone meal . A diffeone meal ,33 appar−1·d−1, consuming a relatively more balanced protein distribution may be superior to an unbalanced protein distribution to promote skeletal muscle-related outcomes. When total protein intakes are less than 0.8 g·kg−1·d−1 and more balanced, consuming a higher unbalanced protein distribution may be more favorable because at least one meal may contain sufficient protein to support muscle anabolism. However, encouraging people to consume a more balanced distribution may be advised to increase their total protein intakes at or above. 0.8 g/kg.Collectively, the limited inconsistent results among the observational studies suggest that when total protein intakes are above 0.8 g·kgCompared to consuming an unbalanced protein distribution, more proteins may be synthesized by consuming an optimal protein distribution; this seems intuitive: consuming more meals that stimulate a higher MPS rates should result in more synthesized muscle protein. To our knowledge, there are five acute studies that test the within-day protein distribution hypothesis  34,35,3,43,44. TCrudely, the protein distribution concept is supported when testing is performed during energy balance in younger , but notApart from age, the inconsistent results could be explained by differences in both protein source and quantity, which consequently impact protein synthesis. In the retrospective analysis by Moore et al. , the satEven when consuming protein supplements, sufficient protein doses still need to be achieved. In energy balance, Murphy et al.  provided−1·d−1 over four whole-food meals. As mentioned above, a blunted rise in postprandial plasma amino acid concentration from consuming whole-food protein sources, although protein intake was higher per meal, could have led to the discrepant results. Again, a higher protein dose may be needed with whole foods than with protein supplements.In energy restriction, Murphy et al. ,44 preseThe findings from Kim et al. ,43 and MWhile it is known that the quantity and quality of the protein consumed influences MPS, we still do not know the contribution of each individual postprandial MPS response to the daily synthesized protein pool. There are many stimuli that can affect MPS; under the most favorable feeding conditions, it is likely the anabolic effect of feeding only partially contributes to total daily protein synthesis. In laboratory settings, we assume the aggregate MPS response over the measurement period results from the sum of the individual postprandial MPS responses. The research by Mamerow et al.  suggest When performing these types of acute studies without titration, we do not know definitively when an individual or group of participants have maximized their ability to synthesize proteins; we only assume based on previous research under similar conditions. Consequently, we make evidenced-based assumptions when meals likely did not provide enough protein to meet or exceed the saturable dose estimates; often, this occurs as an explanation for why consuming a balanced protein distribution did not result in higher MPS rates. In practical terms, real-world meals typically contain a variety of whole-food animal- and plant-protein sources. While we have reasonable saturable dose estimates for isolated protein sources, future research should address how much protein is needed when consumed in a mixed meal with whole-food plant and animal protein sources. We could then design more ecologically valid studies to test whether an “optimal” protein distribution exists. By extension, we need studies in both younger and older cohorts; they should be designed to detect the potential for sexual dimorphisms; they should be tested in a range of energy balance states. Of course, while testing each of these factors in a single study would be ideal, it is likely not practical. With this in mind, we should strive for designing multiple studies with similar criteria to address these questions.When we consider the protein quantities within meals relative to the age of the participants within each study, it is clear that a cursory tallying of the results is not sufficient. Hindering a uniform conclusion, we have several studies reporting a null effect ,36,43 piThe notion that the time of day, frequency, and distribution of daily dietary protein intake could influence protein metabolism dates back to the late 1930s. Researchers at the time manipulated dietary protein consumption frequency and distribution to effect (1) protein/amino acid balance and 2) nitrogen balance. These two balances are thought to be connected through the free amino acid pool. Constituting only a fraction of the total nitrogen pool, the free amino acid pool is maintained within narrow limits; therefore, when the body is in nitrogen balance (input = output), then we presume protein synthesis and breakdown are in balance  nitrogen,58,59,60For most the 20th century, highly controlled nitrogen balance studies were considered the gold standard for studying protein metabolism; in fact, results from nitrogen balance studies still inform adult protein requirements . Althougp = 0.16). The authors highlight the difference in nitrogen balance between groups was 23 mg N·kg fat-free mass−1·d−1 ; this was similar in magnitude to the difference reported in the older females  [Arnal et al.  were thealanced) ,39.These results from Arnal et al. ,39 are cAcute human clinical studies often provide the foundational science used to design longitudinal studies. Longitudinal randomized controlled trials have the benefit of inherently capturing daily fluctuations in hormonal concentrations, energy intakes, and exposure to stress; these factors can have profound impacts on both phenotypical and functional outcomes. Chronic feeding studies are necessary to assess the applicability and efficacy of acute studies in real-world settings. Only five longitudinal studies to date (to our knowledge) have investigated the effect of protein distribution on body composition ,72,73,74p = 0.056); there was no effect on appendicular lean mass changes. Notably, the balanced group (high-protein breakfast) had a lower total protein intake (1.3 g·kg−1·d−1) than the unbalanced group . However, due to small samples sizes in both groups, albeit a large effect size (d = 0.7), this study is likely more proof of concept. Only one other study [−1·meal−1); this should have provided a “safety margin” of ~0.1 g·kg−1 [−1) and quality (whole foods and supplement) may have been adequate to achieve a saturable dose and promote an optimal distribution. Although Hudson et al. [Among all randomized controlled trials, only one study supports consuming a balanced protein distribution to support lean mass gains . In thiser study  seemingler study , Hudson er study  did not er study , Hudson er study  prescrib1 g·kg−1 . As ment1 g·kg−1 . Mixed-n1 g·kg−1 , contain1 g·kg−1 . Undersc1 g·kg−1  found th1 g·kg−1  prescribn et al.  prescribn et al. ,62: theyp = 0.067) towards losing less lean mass when they consumed an unbalanced protein distribution (−0.5 kg) versus consuming a balanced protein distribution (−1.5 kg). These results [The research by Bouillanne et al.  in hospi results ,74 seemi results  and Lopr results  and the  results : consumiWhile protein quantity within meals may partially explain the null results, the interplay between the anticipated effect size and the studies durations could provide further context. Per annuum, lean mass decreases by approximately 1%–2% on average in adults who typically consume an unbalanced protein distribution ,76. We aAn exercise stressor has been proposed to provide the “optimal environment” for protein distribution to induce differential changes in lean mass, including skeletal muscle, over a few months. Compared to protein ingestion while being sedentary, resistance training “sensitizes” the muscle to promote greater MPS . Theoretvastus lateralis. While MPS does not vary by either anatomic location or fiber type [As discussed, postprandial MPS partly determines changes in the skeletal muscle protein pool; however, we must acknowledge that both acute and chronic studies have several limitations. First, skeletal muscle changes in response to repeated exposures to both catabolic and anabolic stressors. These stressors include, but are not limited to, hormonal concentrations, energy status, and exercise training status. Their influence on lean mass changes cannot be accurately predicted from acute studies because they fluctuate and persist beyond the durations measured in laboratory settings. Second, the saturable dose estimates were established by sampling a single muscle—the ber type , lean maber type ,81. ThisWe argued that the lack of evidence in support of an optimal protein distribution may be explained by both inadequate protein quantity and quality within meals; this resulted in balanced but not optimal protein distributions. The saturable dose estimates we used to critique meal protein quantity are based on isolated intact proteins. However, importantly, the vast majority of protein consumed comes from protein-rich foods, not supplements. Establishing the relative saturable doses using whole-food animal- and plant-protein sources will facilitate conducting optimal distribution studies while participants consume varied protein-rich foods.−1·meal−1 to promote muscle retention [−1·d−1, which is 50% to 125% higher than the recommended dietary allowance and ~15% to 110% higher than the average daily protein intake amongst older adult men and women [−1·d−1 in a balanced protein distribution to promote muscle size, strength or quality. There is evidence these quantities of protein intake do promote beneficial changes in lean mass; however, this is compared to consuming lower-protein diets [Recommendations in the scientific literature are made for older adults to consume 0.4–0.6 g·kgetention . This eqnd women . Currentin diets ,84,85,86Arguably, research designed to measure muscular function outcomes may be more meaningful than those measuring lean mass. Skeletal muscle strength and function, rather than mass, is often associated with health and longevity among aging adults . ShiftinWhile balanced versus unbalanced protein distributions have been experimentally tested, there is likely only one study assessing an “optimal” protein distribution . The dis−1·d−1), to better balance their daily protein intake, by consuming more protein at breakfast and lunch meals, may be a practical way to achieve a moderately higher total protein diet and promote skeletal muscle health. However, recommending individuals who consume a low-protein diet to balance protein distribution without increasing their total protein intake to become adequate is ill-advised. Among individuals who consume adequate total protein (0.8–1.3 g·kg−1·d−1), the preponderance of evidence suggests that consuming at least one high-protein meal per day may be sufficient to support skeletal muscle-related outcomes even if the distribution is unbalanced. There seems to be a valid theoretical rationale to optimize protein distribution to influence muscle-related outcomes. However, the current available evidence is too limited and inconsistent to make a definitive conclusion about whether changing dietary patterns from consuming an unbalanced distribution to consuming an “optimal” protein distribution pattern will positively influence muscle-related outcomes. The underlying rationale for promoting an optimal protein distribution throughout the day remains intriguing but, from the available literature, it appears more important to ensure adequate total daily protein intake. Because typical protein distribution patterns are skewed towards the dinner meal, encouraging adults, especially older adults with marginal or inadequate protein intakes (<0.80 g·kg


Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines “09CR500” and “09CR501”, respectively. The resistance of “09CR500” to Plasmodiophora brassicae pathotype “Banglim” was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. BanglimPbBrA08 was identified as having a logarithm of odds value of 7.9–74.8, and a phenotypic variance of 26.0–97.1% with flanking marker “09CR.11390652” in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. BanglimPbBrA08 was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the “09CR.11390652” marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in  Plasmodiophora brassicae, is a soil-borne contagious disease that leads to decreases in crop productivity, including that of Chinese cabbage and other relatives in Cruciferae.The Cruciferae family includes various crops that provide sources of daily vegetables and oils for humans and forage crops for animals worldwide. Clubroot, caused by P. brassicae infections, various crop cultivation methods have been introduced, such as management of soil pH and soil humidity; treatments with trace elements such as a calcium, antagonists, and fungicides; and crop rotations with nonhost crops  ×100, where n2 through n6 represent the numbers of plants with symptoms and NT represents the total number of plants tested [To analyze QTLs, the scale values were converted to a disease index. It was calculated according to the following formula: disease index = [ markers that were used in map construction between “Chiifu” and “Kenshin” ,50, and The nomenclature for the new markers followed the Bakus–Naur form described in Marcotty and Ledgard : <3-lett® EP1™ system using 96.96 Dynamic Array™ IFC , performed in accordance with Choi et al. [Crr1a, Crr2, Crr4, CRc, CRk, CRs, CRa, Crr3, PbBa3.1, PbBa3.3, and CRb) [B. rapa A-genome chromosomes, were used to screen for polymorphisms in the parental lines  and an HT DNA 1K LabChip® kit , or analyzed on 6% polyacrylamide gel electrophoresis. The genotyping of SNPs was performed using two methods: the LightScanner System , based on a high-resolution melting (HRM) analysis, performed in accordance with Pang et al.  and Li ei et al. , in whicand CRb) ,60,61,62al lines . The PCRLinkage map construction was performed using JoinMap version 4.0 ,64. LinkFor the QTL analysis, WinQTL cartographer 2.5 software using the composite interval mapping (CIM) was employed . The walTo validate false-positive QTLs, we performed additional analysis with IciMapping 4.1 and confirmed that detection of QTLs is reproduced . The misBrassica database (BRAD), which is based on genome version 3.0 [To predict candidate genes in the QTL region, the putative functions of genes were obtained from the sion 3.0 . The domThe SNP data information generated using BRAD v1.2 were further converted to the advanced reference genome v3.0  to compaCrr1, Rcr9, and CRs [To confirm that the newly identified locus was novel and different from the adjacent resistance loci  and CRs ,27,28, s and CRs , and we  and CRs . The synB. rapa. To achieve this goal, we compared the candidate genes sequences of newly identified QTL regions with those of a previous report using whole-genome sequencing of resistant line “09CR500”. The QTL analysis mapped a novel locus BanglimPbBrA08 for Banglim pathotype resistance on chromosome A08. This locus exhibited structural differences and expression variations compared with earlier reported QTLs, and the plant’s responses to the pathotype were also found to be different, suggesting that BanglimPbBrA08 is a novel resistance loci to Banglim. The results of this study add to the available genetic information that can be used to elucidate the genetic mechanism of clubroot resistance. The newly developed marker “09CR.11390652” for BanglimPbBrA08 will be beneficial in Brassica breeding programs for Banglim pathotype-specific resistance and gene pyramiding by allowing accurate phenotype selection.The prime aim of this study was to identify Banglim pathogen-specific resistant loci in