L*) decreased. Moreover, a* parameter increased with the increment of WL in the formulation, leading to a redder and less yellow-hued fat-replaced muffin. In conclusion, WL could effectively replace fat in the 25–50% range in muffins, achieving a final product with reduced calories, a higher dietary fiber content, higher volume, and promising sensory aspects.The current study evaluates the prospect of wine lees (WL), a costless by-product from Amarone winemaking, as a fat replacer in muffin formulation. WL have elsewhere replaced sunflower oil, allowing the creation of 0, 25, 50, 75, and 100% fat-substituted muffins named ML0, ML25, ML50, ML75, and ML100, respectively. Batter rheology, in addition to the textural and colorimetric characteristics, the pore dimension, and the sensory aspect of the different formulations were evaluated. The batter consistency (K) of fat-replaced muffins was lower than that of the control, while the hardness and chewiness of the end products were higher. ML25 and ML50 samples reached the highest volume, while the baking loss decreased due to WL’s fiber components. ML25, ML50, ML75, and ML100 accounted for caloric reductions of 9, 18, 22, and 26%, respectively, compared to full-fat muffins. Muffins with WL showed a darker crust and crumb as lightness ( Non-communicable diseases (NCDs), such as obesity and type 2 diabetes, are related to an unbalanced diet ,2,3. TheIndeed, fat replacers have recently undergone significant development, driven by consumers’ demand for low-fat foods . Fat repUse of the discarded grape by-product, such as grape pomace and lees from the fermentation process, could promote a circular economy and responsible use of natural resources, and also prevent environmental degradation, following the principles of the ONU Agenda 2030 . MoreoveWheat flour was supplied from Macinazione Lendinara , and sunflower oil, salt, sugar, fresh eggs, and baking powder were commercially available. The wheat flour’s composition was: total carbohydrates 71 g/100 g, protein 11 g/100 g, fat 1.2 g/100 g, and total dietary fiber 2.3 g/100 g (as reported on the label). The WL were obtained after the production of Amarone wine in Valpolicella and kindly provided by Cantina Sperimentale of Verona University . The WL were stored in plastic bottles in a −18 °C refrigerator and heated up to 25 °C upon use for WL characterization or muffin preparation. The muffins were prepared according to the formulation reported in The proximate composition determination for the muffin and WL included DM method 930.15), ash (method 942.05), total sugars , crude protein (method 976.05), crude lipid (method 954.02), and total starch (method 996.11), using thermostable amylase (Megazyme cat. no. E-BSTAA), amyloglucosidase (Megazyme cat. no. E-AMGDF), and tartaric acids . The total, soluble and insoluble dietary fiber contents  were determined according to method 991.43. . Analyse30.15, as−1 range. The results were fitted to the Power Law model Equation (2) by the software program Rheotex .A rheometer  equipped with an MSDIN-11 system was used for rheological analyses. Fifteen ml of batter were poured into the vessel and let rest for 1 min to reach a constant temperature of 25 °C. Shear stress was a function of shear rate over the 10–300 sThe moisture content of the muffins was evaluated using AACC method 44-15A . To deteOnce the muffins had cooled down, their texture profile analysis (TPA) was conducted using a texture analyzer TX700  with a flat probe measuring 50 mm in diameter. For the TPA analyses, two cm of each muffin was cut from the bottom and the upper part was discarded. Seven measurements were performed for each batch. Hardness, cohesiveness, chewiness, and resilience values were collected. Hardness was calculated as the maximum force of the 1st cycle of compression, cohesiveness was the ratio of the work of the first compression and the second, and chewiness was a product of hardness, cohesiveness, and springiness, while resilience was the ratio between the time to reach the maximum peak force of the second cycle and the time of the first cycle.2 in area. The scanner captured images of the muffin sections at a resolution of 600 DPI. Images were first converted to greyscale and binarized with ImageJ (version 1.8.0) to obtain the pore area fraction; the density of pores; the average pore diameter; D1,0 and D3,2; the pore perimeter; and the pore circularity [Muffins were cut to obtain a slice of 1–2 cm in height and about 20 cmcularity . AnalyseL* a* b*. Measurements were taken at five different points within the crumb and crust areas.The color was measured by a reflectance colorimeter (Illuminant D65)  based on the color system CIE—A quantitative descriptive sensory analysis (QDA) was conducted in the sensory test room of the University of Verona; the QDA was designed according to UNI ISO 8589 standards. The panelists were carefully selected based on their sensory sensitivity and ability to describe and communicate sensory perceptions. The panelists, consisting of 18 people  between the ages of 22 and 28, recruited from the staff and students of the Department of Biotechnology at the University of Verona, were trained for 12 sessions of one hour each to evaluate the specific sensory attributes of muffins.The panelists generated 10 sensory terms and were qualified to identify them. The following sensory attributes were evaluated: color intensity, egg taste, wine taste, wine odor, sweetness, acidity, chewiness, elasticity, hardness, and astringency. All sensory attribute definitions are reported in p < 0.05 was employed for comparing the means. Pearson’s correlation tests and statistical analyses were performed using XLSTAT Premium Version  software.All data are presented as mean values ± standard deviation, based on at least three measurements (n = 3). Analysis of variance (ANOVA) followed by a post-hoc Tukey test at a significance level of The chemical composition of WL is reported in 2 higher than 0.997. All batter formulations showed a shear-thinning (pseudoplastic) behavior (n < 1) over the studied shear rate (10–300 s−1), suggesting that the apparent viscosity decreased with the increase in shear rate. Bianchi et al. (2022) reported a similar performance in muffin batters made with grape pomace powder [Batter rheological data are reported in ear rate –300 s−1,ML25, ML50, and ML75 showed similar consistencies, but were all lower than the full-fat sample, while ML100 had a higher consistency, resembling the behavior of 100% pectin as a fat replacer in cakes, as reported by Psimouli & Oreopoulou et al. (2012) . The conThe higher consistency indicated a higher ability to entrap air during the beating process, resulting in a lower density of batters and lower volume development. If the K value is too low, cake or muffin volumes shrink because batters lose effectiveness in retaining air bubbles ,33. ThisThe chemical composition of the samples is reported in The oil replacement in muffins influenced most of the technological parameters, as depicted in In addition, the greatest volume was recorded for ML25 and ML50, increasing by 8.32 and 4.3% compared to the control, respectively, suggesting that the optimal substitution of fat with WL to achieve a better muffin volume should not exceed 50%. Higher fat replacement provoked a volume decrease of about 16% compared to the control. This could reflect the role of WL in capturing air during the beating step of batter preparation, stabilizing the air cell, and preventing their coalescence during the cooking, but only below the 50% threshold of replacement . Even thML0 accounted for the highest baking loss in the study, whereas ML100 and ML75 showed the lowest ones. Bender et al. (2017) argued that the hydration capacity of the fiber fractions in fortified muffins led to a decrease in the baking loss when Riesling skin or Tannat skin powder was added . The prePersea puree [The image analyses showed that the muffin had no significative differences in pore area fraction and pore circularity . Indeed,ea puree , while Rea puree .The ML25, ML50, and ML75 samples had higher pore densities than did the control or the ML100 sample. These data, for WL replacement < 100%, are in line with previous studies reporting that muffins with defatted sunflower seed flour, pecan nut expeller, and grape pomace powder had higher pore densities than did their controls ,45. MarcThe pore perimeter values D1,0 and D3,2 showed a similar trend when replacing fat with Amarone WL. Indeed, ML100 led to a pore density similar to ML0, and thus to a more open crumb with a lower volume.p < 0.05) the color of the muffins (L and b* parameters decreased (p < 0.05), both in crust and crumb, following the substitution level of the fat with WL, while the a* parameter significantly increased (p < 0.05). Bianchi et al. (2022), Rainero et al. (2022), and Acun et al. (2014) obtained similar results, respectively, in muffins with distilled grape pomace powder, breadsticks with grape pomace powder, and cookies fortified with different grape products [Consumers typically attribute darker crusts and crumbs to whole-grain baked goods. The addition of WL significantly changed , who stated that this increase might be due to a denser crumb cell structure in fat-replaced cakes . Indeed,Chewiness values for fat-replaced samples were statistically higher than that of the control sample. Martínez-Cervera et al. (2011) obtained the opposite results in a muffin with cocoa fibers used as a fat substitute . HoweverIn the present study, all the samples had resilience values lower than 0.6, reflecting the presence of softening ingredients, such as sugars and fat, in the formulations. Indeed, resilience increased in muffins with lower fat content, underlying their role as tenderizers in bakery products.Overall, hardness, cohesiveness, chewiness, and resilience increased with the incremental increase of WL used as a fat replacer in muffins.The replacement of oil with WL significantly influenced most of the selected sensory attributes, as depicted in p < 0.05). The WL substitution reduced the egg taste perception and raised the wine taste. Noticeably, the egg perception was strongly affected by the WL addition, decreasing from a score of 7.9 for ML0 to less than 4.8 for the fat-reduced muffin. Wine odor and acidity were influenced by the oil replacement with WL as well. Bianchi et al. (2022) reported a similar effect in muffins fortified with grape pomace powder. The chewiness and hardness increased in muffins with the increased use of fat replacer. Indeed, the chewiness and hardness attributes of ML100 and ML75 were higher and statistically significant  reported an increase in the perceived color of muffins fortified with distilled grape pomace powder, due to grape pigments. The WL’s inclusion affected almost all aroma- and taste-based sensory attributes  as a sustainable and effective ingredient used to partially replace fat ingredients in muffin formulations. Overall, nutritional, technological, and sensorial analyses indicated that the suggested amount of Amarone WL as a fat replacer in wheat-based muffins was between 25% and 50%, causing a calorie reduction and a higher dietary fiber content of the products without drastically compromising technological and sensorial aspects.However, further optimization of the muffin formulas with 75% and 100% of WL by the addition of ingredients or alternative mixing and whipping strategies may improve their texture and sensory properties. Overall, this study highlights the potential of using WL as a sustainable fat-substitute in baked goods. Further research is also needed to explore the potential of WL as fat-replacer in different cereal-based food formulations.


Gastric cancer (GC) ranks among the most common malignancies worldwide. This study aimed to find critical genes/pathways in GC pathogenesis.Gene interactions were analyzed, and the protein–protein interaction network was drawn. Then enrichment analysis of the hub genes was performed and network cluster analysis and promoter analysis of the hub genes were done. Age/sex analysis was done on the identified genes.Eleven hub genes in GC were identified in the current study , mostly involved in mitochondrial functions. There was no report on the ATP5D, ND6, NDUFS3, RPL8, and RPS16 in GC. Our results showed that the most affected processes in GC are the metabolic processes, and the oxidative phosphorylation pathway was considerably enriched which showed the significance of mitochondria in GC pathogenesis. Most of the affected pathways in GC were also involved in neurodegenerative diseases. Promoter analysis showed that negative regulation of signal transduction might play an important role in GC pathogenesis. In the analysis of the basal expression pattern of the selected genes whose basal expression presented a change during the age, we found that a change in age may be an indicator of changes in disease insurgence and/or progression at different ages.These results might open up new insights into GC pathogenesis. The identified genes might be novel diagnostic/prognostic biomarkers or potential therapeutic targets for GC. This work, being based on bioinformatics analysis act as a hypothesis generator that requires further clinical validation. Gastric cancer (GC) is the fifth most common cancer and the fourth cause of cancer-related death worldwide, with more than 1 million new cases in 2020 and an estimated 769,000 deaths, equal to one out of each 13 deaths worldwide [Carcinogenesis is a complicated process involving several genetic and epigenetic alterations . AdenocaThere are many investigations on alterations of the genes in GC. However, they are not still enough to provide an elucidated picture of the molecular pathogenesis of GC . TherefoIn our previous studies –14, we uhttp://string-db.org) [CytoHubba software (version 0.1) was used to identify hub proteins between all nodes [CytoHubba plugin.STRING is a database of predicted and known interactions of proteins. To evaluate the interactions among the genes listed in Table -db.org)  with a mll nodes . Four toThe enrichment analysis of subnetwork genes of the hub genes was done based on the KEGG and GO, including molecular function (MF), cellular components (CC), and biological process (BP) at the web-based application of STRING .CytoCluster (version 2.1.0) was used for the clustering of network nodes. The IPCA (Identifying Protein Complex Algorithm) algorithm was used for cluster analysis of the subnetwork. This algorithm is density-based, which identifies dense subgraphs in protein interaction networks. IPCA determines the weight of an edge by calculating the common neighbors of its connected nodes and calculates each node’s weight through a sum up of the weights of its incident edges. . The thrhttps://asia.ensembl.org/info/data/biomart/index.html). Conserved motifs on the sequences were identified using MEME Suite (version 5.4.1) (meme.nbcr.net/meme/intro.html) [http://meme-suite.org/tools/tomtom) [http://meme-suite.org/tools/gomo) was also used to find potential roles for motifs [The 1 kbp upstream flanking regions (UFRs) of hub genes were extracted from Ensembl BioMart web services (ro.html)  with its/tomtom)  was usedr motifs .For each gene selected in the previous steps, we downloaded expression data from the GTEx portal  accessed.In the present study, bioinformatics analysis of differentially expressed genes (DEGs) was performed in GC based on the identified genes from our previous studies –14. The 2As shown in Table GO analysis is a well-known method for gene and gene products and identification of typical biological aspects of high-throughput genome or transcriptome data, including molecular function, cellular components, and biological process , 25. TheThe predominant (≥ 50% observed genes) GO terms found for BP are significantly enriched in cellular process, metabolic process, cellular metabolic process, organic substance metabolic process, primary metabolic process, nitrogen compound metabolic process, transport, organonitrogen compound metabolic process, cellular biosynthetic process, organic substance biosynthetic process, cellular nitrogen compound metabolic process, cellular component organization or biogenesis, organonitrogen compound biosynthetic process, cellular nitrogen compound biosynthetic process, and cellular component organization Fig. a.The predominant (≥ 50% observed genes) GO terms found for CC are intracellular, cytoplasm, organelle, intracellular organelle, membrane-bounded organelle, protein-containing complex, intracellular membrane-bounded organelle, membrane, organelle membrane, and organelle envelope Fig. b.The predominant (≥ 20% observed genes) GO terms found for BF are nucleic acid binding, RNA binding, oxidoreductase activity, transporter activity, proton transmembrane transporter activity, structural molecule activity, and electron transfer activity Fig. c.In addition, the GO results in terms of BF showed the significance of oxidoreductase activity, electron transfer activity, and proton transmembrane transporter activity which may all contribute to the mitochondrial function of ATP synthase that has been discussed in previous sections.http://www.genome.jp/) is a database for allocating certain pathways to groups of DEGs, therefore, linking omics data with higher-order functional data [KEGG  pathway was considerably enriched, and the cluster analysis confirmed it.The UFRs (1000 bp) of DEGs were analyzed to identify the conserved motifs and consensus cis-regulatory elements (CREs). The UFRs were extracted using Ensembl BioMarts, a hub for data retrieval across taxonomic space . The MEMGene Ontology indicated that these motifs participated in potassium ion transport, negative regulation of neuron apoptosis, neuron fate commitment, and inner ear morphogenesis. However, involvement in the negative regulation of signal transduction was the most common biological process among all motifs. Therefore, this biological process may play an important role in GC pathogenesis. In addition, the predominant GO terms found for CC were significantly enriched in transcription factor complex and dendrite. Moreover, these motifs involved molecular functions, including chromatin binding, protein homodimerization activity, protein heterodimerization activity, potassium ion binding, and transcription activator activity. Therefore, these biological processes play an important role in the pathogenesis of GC.The age analysis revealed the presence of a few genes whose expression presents some changes considering time intervals and in particular around 60 years: MTRNR2L12, EIF4G2, and EIF4B.In recent years, different investigations in the area of multi-center genomics research from gene to systems level and next-generation sequencing, clarified the various mechanisms specifically involved in the tumor progression . This stEleven hub genes were identified in this study, most of which were involved in mitochondrial functions. Recently, many cancer investigations have focused on the mitochondria for further elucidation of the molecular mechanisms of carcinogenesis . MitochoAlthough the observation of mitochondrial dysfunction in various cancers, the concept of the involvement of both mitochondrial glycolysis and metabolism in cancer cells is controversial. On the other hand, the critical role of the mitochondria in the maintenance of cancer is inevitable. Consequently, there have been several discussions and investigations about the alterations in the function of the mitochondria and their protein expression in various human cancers . Based oMitochondrial quality control and biogenesis are enhanced in tumors. In addition, mutations in the enzymes of the nuclear-encoded mitochondrial tricarboxylic acid (TCA) cycle have been reported in some tumors leading to the production of oncogenic metabolites. However, negative selection is exploited for pathogenic mutations of the mitochondrial genome . It has . introduced ATP5A1 as one of the up-regulated genes in GC [. found that high ATP5B expression in tumor tissues of GC is positively correlated with age, tumor size, the TNM stage, lymph node metastasis, and patients’ poor prognosis. They reported that ATP5B overexpression in GC cells caused ATP-promoting migration, invasion, and proliferation. In their study, an increase in MMP2 expression results in the phosphorylation of FAK, and phosphorylated AKT was observed by ATP5B overexpression in GC cells. Also, an increased level of extracellular ATP happens after ATP5B overexpression through the intracellular ATP secretion, and the FAK/AKT/MMP2 pathway is activated. Activation of the ATP5B-induced downstream pathway is activated through the P2X7 receptor. Inhibition of P2X7, FAK, AKT, and MMP2 results in the suppression of proliferation, migration, and invasion of GC cells. In conclusion, studies by Wang et al. showed that ATP5B involves in GC tumor progression through FAK/AKT/MMP2 pathway [The respiratory chain, located in the mitochondrial membrane, is composed of 5 complexes, including Complex I or CxI (NADH dehydrogenase), Complex II (SDH), Complex III (cytochrome bc1), Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) . In our es in GC . ATP5B i pathway . TherefoMT-ATP8, as one of the components involved in the OXPHOS pathway, is a mitochondrial gene encoding membrane subunit 8 of ATP synthase. Several genetic syndromes that contributed to mitochondrial dysfunction depicted by a reduced OXPHOS ability have been explained . In addi. found that overexpression of PSMB10, VPS13D, NDUFS8, ATP5D, POLR2E, and HADH were correlated with adverse overall survival in acute myeloid leukemia (AML) [ATP5D encodes the subunit delta of the catalytic core of ATP synthase, F1. Wei et alia (AML) .NDUFS3, MT-ND4, and MT-ND6, as three other hub genes identified in this study, encode subunits of CxI. MT-ND6 encodes NADH dehydrogenase 6, part of CxI in the mitochondria. CxI is involved in the first step in the electron transport process, in which the electrons are transferred from NADH to ubiquinone. Electrons are subsequently thrown from ubiquinone through various other enzyme complexes leading to the preparation of energy for ATP generation .. showed that the MT-ND6 mutation resulted in the suppression of CxI activity and induction of ROS production, leading to the stimulation of metastasis in breast and lung cancer cells [Ishikawa et aler cells . There ier cells .MT-ND4 encodes NADH dehydrogenase 4, a part of CxI, one of the enzyme complexes involved in OXPHOS. In the whole mitochondrial genome sequencing study in GC, it has been reported that the tumor has remarkably more variants in the MT-ND4 region .NDUFS3 encodes one of the iron-sulfur protein components of mitochondrial NADH: ubiquinone oxidoreductase (CxI). Recent studies in GC showed the prognostic capabilities of Nicotinamide N-methyltransferase (NNMT). They found that NNMT expression was positively connected to clinical pathologic stage, tumor size, and lymph node status in GC. Silencing the NNMT gene resulted in the inhibition of proliferation, invasion, and migration of the GC cells. These results represent the potential of NNMT as a beneficial prognostic marker of GC. It has been observed in neuroblastoma that the presence of NNMT could meaningfully reduce the death of SH-SY5Y cells, and the effects of NNMT were positively correlated with the elevated intracellular ATP content, ATP/ADP ratio, and CxI activity, as well as a decrease in the degradation of the NDUFS3 subunit of CxI . There a. showed that increased expression of COX7A2 is a characteristic feature of intestinal metaplasia in the esophagus [Among the identified genes in this study, COX7A2 and COX6C, encode cytochrome c oxidase subunits. COX7A2 encodes subunit 7A2 of the cytochrome c oxidase, which is the terminal module of the respiratory chain in the mitochondrial and catalyzes the transfer of electrons from the reduced cytochrome c to oxygen. Data extracted from MALDI-MSI experiments have represented that COX7A2 expression is correlated with the survival curve in GC . Elsner sophagus .COX6C encodes subunit 6c of the COX complex, which catalyzes the final step of the electron transfer chain. Recently, many investigations have reported the unusual level of COX6C in different cancerous and non-cancerous disease conditions such as diabetes, uterine leiomyoma, prostate cancer, melanoma tissues, breast cancer, and follicular thyroid cancer. It has been reported that the overexpression of NDUFA4 leads to substantial upregulation of the COX6C, which subsequently promotes GC cell proliferation and reduces apoptosis in these cells .RPL8 and RPS16 were also identified in the present study. RPL8 encodes ribosomal L8 protein, which is one of the components of the 60S subunit. Based on a bioinformatics analysis-based study, among the genes highly expressed in one of the clusters, few were associated with ribosomal protein-encoding genes such as RPL8 . RPS16 eThe oxidoreductase activity which was considerably enriched in our GO analysis has been investigated in various cancers, including the breast, liver, gastrointestinal tract, and kidney. Also, it has been shown that Xanthine oxidoreductase (XOR) expression is negatively correlated with poor prognosis in these malignancies .The OXPHOS pathway which was considerably enriched in our KEGG and cluster analyses produces energy in most cells, and as the final stage of cellular respiration, is composed of two parts: the electron transport chain (ETC) and chemiosmosis. In the ETC, the electrons are transferred from one molecule to the other, and the released energy from this transfer is applied in the formation of an electrochemical gradient. In chemiosmosis, ATP is made from the stored energy in the gradient. ATP synthase exploits the produced gradient of the proton to generate ATP after the phosphorylation of ADP. At the end of the ETC, oxygen accepts the electrons and makes water with protons . Four geRecent studies presented that expression levels of several genes, such as NDUFB7, UQCRC2, and UQCRQ in the OXPHOS pathway, were meaningly down-regulated in GC. In addition, studies showed that the enhanced expression of the genes in this pathway, such as UQCRQ, NDUFB7, and UQCRC2, positively correlated with a better prognosis . It has It has been reported that besides the role of the oncogenes and tumor suppressors in the expression control of the metabolic enzymes, mitochondrial energy metabolism is also affected by environmental conditions, and it has been shown that mitochondrial dysfunction is one of the characteristics of tumor cells . Under vAssociation of mitochondrial dysfunctions with OXPHOS complexes content was also reported in a few studies . A decreFeichtinger et al. investigated the expression of OXPHOS complexes in GC using immunohistochemistry. They found that CxI expression was significantly reduced in the intestinal type of GC (but not the diffuse type of GC). Higher expression of complex I and II was seen in larger tumors, and higher expression of complex II and III was observed in higher grades [. found that all of the identified DEGs involved in OXPHOS were down-regulated. They also reported that GC has substantially changed metabolic processes, including NADH dehydrogenase complex assembly and tricarboxylic acid cycle, consistent with our KEGG analysis results [The switching of cellular energy production from OXPHOS by mitochondria to aerobic glycolysis, named the Warburg effect, happens in several cancer types . One of r grades . On the  results . TherefoMitochondrial changes have been also reported to be correlated with tumor cell migration, invasion, and resistance to chemotherapy . The priMany of the enriched pathways in our KEGG analysis were also related to neurodegenerative diseases and based on the previous studies , SirutinThe results of pathway enrichment analysis in our study also showed that the metabolic processes are the most affected in GC. Recent investigations have focused on the elucidation of the correlation between metabolic reprogramming and the pathogenesis of cancer. Specifically, regulation of the metabolism and cancer investigation is more brought into intense attention with the development of metabolomics . MetabolOur results also showed the potential significance of RNAs in GC pathogenesis, confirming recent studies focused on elucidating the role of RNAs in the pathogenesis of this cancer . Using amt2a, fn1, hgf, col1a1, col1a2, and mmp2. In their study, overexpression of signaling pathways, including extracellular matrix organization, cell division, and cell cycle, as well as downregulation of ion transport and digestion pathways were also indicated. They also reported that their identified hub genes in gastric adenocarcinoma participated in platelet activation, focal adhesion, gastric acid secretion, cell cycle and HPV infection pathways [By integrated bioinformatics analysis in 2022, Zhang et al. studied the stomach adenocarcinoma and introduced seven hub genes  as related genes to occurrence of stomach adenocarcinoma . Throughpathways . In 2018pathways .We also found three genes that present significant changes in the elder population: MTRNR2L12, EIF4G2, and EIF4B. MTRNR2L12 has no annotations available, so we suggest further analyzing this possible target. EIF4G2, as reported in the STRING database appears to play a role in the switch from cap-dependent to IRES-mediated translation during mitosis, apoptosis and viral infection . EIF4B aGEPIA  web server is an important resource for the analysis of gene expression based on cancerous and normal samples from the GTEx and the TCGA databases and GEPIA2 is an improved version . The KapEleven hub genes  in GC were identified in the current study, mostly involved in mitochondrial functions. There was no previous report on the ATP5D, ND6, NDUFS3, RPL8 and RPS16 in GC. The GO and pathway analysis showed that the metabolic processes are most affected in GC. These analyses also confirmed the potential significance of the mitochondrial role in GC pathogenesis. Although many investigations have been focused on the mitochondria further to elucidate the potential contribution of it to carcinogenesis, however, the characteristics of respiratory and metabolic changes in GC have not been fully elucidated. In addition, KEGG analysis and cluster analysis results showed that most of the affected pathways in GC were the pathways also involved in neurodegenerative diseases. KEGG pathway analyses also showed that the OXPHOS pathway was considerably enriched, and the cluster analysis confirmed it. Also, the promoter analysis results showed that negative signal transduction regulation might play an important role in GC pathogenesis. The results of this study might open new insights into GC pathogenesis. In addition, the identified genes in this study might be promising diagnostic and prognostic biomarkers or therapeutic targets for GC. This study confirms previous findings that mitochondria metabolism and its related protein alteration occur in various cancers. This work is based on bioinformatics analysis and tried to introduce important genes and pathways in GC pathogenesis. However, validation in a large number of clinical GC samples is suggested for future studies to evaluate the prognostic or therapeutic value of the identified markers in this study. Furthermore, additional studies are required to explore the inherent mechanisms and associated pathways of these genes in GC pathogenesis. Considering the complexity of cancer and the involvement of various molecular mechanisms in the pathogenesis of cancer, a single pathway cannot be used as a description of cancer pathogenesis .


Ziziphus jujuba Mill.) is an economically important tree in China and provides a rich source of sugars, vitamins, and bioactive components, all of which are indispensable and essential for the composition and participation in life processes of the human body. However, the location of these metabolites in jujube fruits has not been determined. This study applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate the spatial distribution of sugars, organic acids, and other key components in jujube fruits at different developmental periods. Soluble sugars such as hexoses and sucrose/maltose significantly increase with fruit ripening, while organic acids show an overall trend of initially increasing and then decreasing. Procyanidins and rutin exhibit specific distributions in the fruit periphery and peel. These findings suggest that MALDI-MSI can be used to study the spatial distribution of nutritional components in jujube fruits, providing insights into the changes and spatial distribution of substances during jujube fruit development. This technique offers a scientific basis for jujube breeding, utilization, and production.Chinese jujube (also called Chinese date,  Ziziphus jujuba Mill.) is a native economic tree species in China with a long history of cultivation , white-ripened , half-red , and fully red  a. The coFor sample preparation and matrix coating, the frozen tissue samples were first fixed in three drops of distilled water during the cutting stage. The tissue samples were then sectioned at a thickness of 90 μm using a Leica CM1950 cryostat  at −20 °C. Due to the presence of nuclei and cavities and the larger fruit size of “Jingzao 39”, the lower part of each fruit was selected based on actual observations, and fan-shaped tissue sections were intercepted for subsequent analysis b. The tyThe desiccated tissue sections mounted on ITO glass slides were then spray coated using an HTX TM sprayer  with a solution of 15 mg/mL DHB  dissolved in a mixture of 90% acetonitrile and 10% water. The sprayer temperature was set to 60 °C, with a flow rate of 0.12 mL/min and a pressure of 6 psi. Thirty passes of the matrix were applied to the slides, with a drying time of 5 s between each pass.m/z 50 to 1300 Da. The imaging spatial resolution for the tissues was set to 50 μm, and each spectrum consisted of 400 laser shots. The tissue samples were detected under the same laser energy, and the laser beam was irradiated to the tissue area on the target plate through the grating. The samples were scanned continuously, with the tissue samples being ionized and resolved within the matrix under the excitation of the laser beam, and the released molecules were identified using the mass spectrometer to obtain the mass-to-charge ratio (m/z) information of each pixel of the samples, the peak raw data of each pixel of the samples, and the peak intensity.MALDI timsTOF MSI experiments were conducted using a prototype Bruker timsTOF flex MS system  equipped with a 10 kHz smartbeam 3D laser. The laser power was initially set to 70% and remained fixed throughout the entire experiment. Mass spectra were acquired in the positive mode, covering a mass range from m/z at each spatial point, and the data were transformed into pixels on the imaging thermogram. The acquired MALDI mass spectra were normalized using the root mean square (RMS) method, and the signal intensity in each image was presented as the normalized intensity [The raw data were imported into SCiLS Lab software v. 2022a  for smoothing and root mean square (RMS) normalization to obtain the relative intensity information of different ntensity . That isThe target peaks of the target substance (MS1 information) were subjected to on-tissue in situ fragmentation to obtain on-tissue MS/MS spectra (MS/MS fragment ion information). Substance identification was carried out by comparing the MS/MS spectra obtained with those of the reference standards in a self-built database MWDB . For target peaks (MS1 information) with low intensity that could not be subjected to MS/MS fragmentation, substance identification at the MS1 level was performed based on the detected molecular weight. If the mass error range between the detected molecular weight and the theoretical monoisotopic molecular weight under the adduct mode of the target substance was within 10 ppm, the substance was identified at the MS1 level. A total of 18 substances were identified in this project, with all 18 substances identified at the MS/MS level. The distribution of soluble sugar metabolites on the tissue samples was obtained by imaging all the identified target peaks.The selection of the matrix is crucial for the success of MALDI-MSI. Commonly used matrices for screening include DHB , CHCA 2, 9-AA 2, DMCA 22, and NEm/z 365.1053, isomer) increased significantly with an increase in fruit ripeness , ascorbic acid, procyanidin B, and procyanidin C were detected as Na+ adducts , sucrose, and maltose  are shown in m/z 136.0622), citric acid and isocitric acid , quinic acid (m/z 210.0890), ascorbic acid (m/z 199.0212); strong antioxidant substances including procyanidins A1, A2 , B1, B2, B3 , C1 (m/z 889.1980), and rutin (m/z 633.1424); plant hormones such as Indole-3-acetic acid  and 6-Benzylaminopurine ; and the secondary maps of these substances are shown in the In this study, MALDI-MSI was performed to analyze the spatial distribution of soluble sugars, organic acids, plant hormones, vitamins, procyanidins, and flavonoid in jujube fruits at four different maturity stages . The average mass MS spectra of the jujube tissues at different periods are shown in ripeness . In the  adducts . In this adducts . The idem/z 203.0529) and sucrose/maltose (m/z 365.1053) in jujubes at four different developmental stages was determined. As shown in Sweetness is an important indicator that determines fruit quality and market value. Sugars such as glucose, fructose, and sucrose are the major sugars found in most fruits, including peaches, apples, watermelons, and cherries . Each tym/z 136.0622 was distributed most abundantly, followed by citric acid/isocitric acid at m/z 215.0169, while quinic acid at m/z 210.0890 was distributed the least , one of the major polyphenolic compounds in jujubes, possess various physicochemical properties and biological activities, including antioxidant, anticancer, and cardiovascular activities . They com/z 198.0534 and 6-BA at m/z 226.1054 during jujube fruit development was analyzed. The results showed that the relative intensity of IAA exhibited a decreasing trend followed by an increasing trend (Plant hormones are an indispensable part of regulating fruit development and ripening. Indole-3-acetic acid (IAA) and 6-benzylaminopurine (6-BA) belong to auxin and cytokinin, respectively. They are natural endogenous plant hormones that are associated with fruit development and ripening ,46. Indong trend . It was ng trend . TherefoMALDI-MSI technology is a mature imaging technique that has been used in research on food ,16,17,34In this study, MALDI-MSI technology was used to identify and locate major metabolites, including soluble sugars, organic acids, procyanidins, rutin, and plant hormones, in jujube fruit sections at different developmental stages. The relative intensities and spatial distributions of these metabolites in jujube fruits at four different stages were compared. The results showed that soluble sugars such as hexoses and sucrose/maltose significantly increased with fruit ripening, while organic acids showed an overall trend of initially increasing and then decreasing. Procyanidins and rutin exhibited specific distributions in the fruit periphery and peel, which is related to their specific functions in the fruit. The findings of this study contribute to a better understanding of the distribution of various nutrients in jujube fruits at different developmental stages and provide a reliable basis for jujube breeding and fruit utilization.


COVID-19 isolation recommendations have evolved over the course of the pandemic. Initially, the US Centers for Disease Control and Prevention required 10 days of isolation after a positive test result. In December 2021, this was reduced to a minimum of 5 days with symptom improvement, followed by 5 days of mask wearing. As a result, several institutions of higher education, including the George Washington University, required persons testing positive for COVID-19 to either submit a negative rapid antigen test (RAT) with symptom resolution to leave isolation after 5 days or to maintain a 10-day isolation period in the absence of a negative RAT and the presence of continued symptoms. RATs are tools that can be used both to shorten isolation periods and to ensure that persons testing positive for COVID-19 remain in isolation if infectious.The purpose of this analysis is to report on the experience of implementing RAT policies, examine the number of days that isolation was reduced via RAT testing, determine the factors that predicted uploading a RAT, and determine RAT positivity percentages to illustrate the utility of using RATs to end isolation.In this study, 880 individuals in COVID-19 isolation at a university in Washington, DC, uploaded 887 RATs between February 21 and April 14, 2022. Daily positivity percentages were calculated, and multiple logistic regression analyses examined the odds of uploading a RAT by campus residential living status , student or employee designation, age, and days in isolation.A total of 76% (669/880) of individuals in isolation uploaded a RAT during the study period. Overall, 38.6% (342/887) of uploaded RATs were positive. Uploaded RATs were positive 45.6% (118/259) of the time on day 5; 45.4% (55/121) on day 6; 47.1% (99/210) on day 7; and 11.1% (7/63) on day 10 or beyond. Adjusted logistic regression modeling indicated cases living on campus had increased odds of uploading a RAT , whereas primary student affiliation  and days in isolation  had decreased odds of uploading a RAT. Of the 545 cases with a negative RAT, 477 were cleared prior to day 10 of their isolation due to lack of symptoms and timely submission, resulting in a total of 1547 days of lost productivity saved compared to all being in isolation for 10 days.RATs are beneficial, as they can support a decision to release individuals from isolation when they have recovered and maintain isolation for people who may still be infectious. Future isolation policies should be guided by similar protocols and research to reduce the spread of COVID-19 and minimize lost productivity and disruption to individuals’ lives. More than 2 years into the COVID-19 pandemic, research and policy recommendations continue to evolve. Initially, the US Centers for Disease Control and Prevention (CDC) required 10 days of isolation after a positive test result, contingent upon being fever-free for 72 hours with symptoms improving .Rapid antigen tests (RATs) are used to detect a COVID-19 infection, having the advantage of providing quick results and the ability to be used at home . HoweverAs a result, several institutions of higher education ,11, inclDue to rising infection rates during the first Omicron wave, GW started the first 2 weeks of the spring (January 2022) semester virtually and required that university members obtain a PCR test or RAT when arriving on campus. GW mandated that all those on campus be tested twice monthly for those who were fully vaccinated and weekly for those with a vaccine or booster exemption. The GW Public Health Laboratory provided COVID-19 PCR testing on campus; however, university community members could report external PCR or RAT positive results to the internal GW medical portal.On January 28, 2022, GW modified the required isolation time from 10 to 7 days per guidance from DC Health . EffectiThe purpose of this analysis is to report on the experience of implementing these policies and examine the number of days that isolation was reduced via RAT testing; this analysis also aims to examine the factors that predicted uploading a RAT and determine RAT positivity percentages to illustrate the utility of using RATs to end isolation.The GW COVID-19 case management system embedded in the Student Health medical record system was used to evaluate the data we collected relevant to RAT testing for people who were in isolation. There were 955 cases identified between February 21 and April 14, 2022. Cases with the following attributes were removed from the analysis: a positive test uploaded more than 7 days into the isolation period (n=34), false positives (n=2), no initial positive listed in the patient chart (n=2), individuals lost to follow-up (n=8), individuals who uploaded a PCR for purposes of isolation clearance (n=3), those with a positive test within 90 days of a previous positive test (n=4), and those who were medically released before 10 days based on verbal confirmation of negative antigen tests (n=22). The final analytic sample was 887 RAT results among 880 cases.Descriptive statistics were computed for all variables. Categorical variables, including campus residential living status and student or employee designation, were described using frequencies and percentages. Continuous variables, including age and number of days in isolation, were described using medians and IQRs. Next, we calculated positive percentages for the day a RAT was submitted to identify if individuals were eligible to be released before the end of the 10-day isolation period. Then, simple logistic regressions were run to compute the odds of uploading a RAT by individual independent variables, including campus residential living , student or employee designation, age, and days in isolation. A final multivariable logistic regression model computed the odds of uploading a RAT with the independent variables simultaneously, including campus residential living status (on or off campus), student or employee designation, age, and days in isolation. SAS (version 9.4) was used for all analyses.All community members provided informed consent to participate in the GW COVID-19 surveillance program, and the GW Institutional Review Board concluded that these were nonresearch-related activities.The sample included 43.6% (384/880) of persons living on campus and 56.4% (496/880) of persons living off campus. Students accounted for 87.8% (773/880) of the sample, and 12.2% (107/880) were employees. The median age was 21.8 (IQR 20.3-25.7) years, and the mean number of days in isolation was 8.5 days. A total of 76% (669/880) of individuals uploaded a RAT during the study period with the majority  uploading 1 test, 19.2% (169/880) uploading 2 tests, and 2.7% (24/880) uploading 3 or more tests. Overall, 39% (342/887) of all RATs were positive. Assuming all cases were cleared from isolation by a medical provider the same day they uploaded a negative antigen test, those who uploaded a negative result on days 5-9, a total of 477 cases were cleared and were not required to isolate for the full 10 days. This resulted in 1547 days or approximately 4.2 years of productivity, classes, or days “saved” and not in isolation. For example, a case who uploaded a negative RAT on day 6 and was subsequently cleared would result in 4 days “saved” that, without the test-out protocol, would have remained in isolation . By day This analysis sought to evaluate the utility of using RATs to end isolation periods more safely prior to the full 10 days of isolation. Rapid antigen tests are beneficial, as they provide valuable insight to guide isolation and treatment, allowing health care providers to release individuals from isolation when they are no longer contagious and maintain isolation for people who may still be infectious. In this analysis, the percentage of positive RATs uploaded on day 5 of isolation was 45.6% (118/259). If isolation were set to only 5 days, most likely a proportion of the 45.6% who uploaded a positive RAT would still be infectious and able to spread COVID-19 on campus and in the community despite advice to employ one-way masking . This reAfter adjusting for confounders, living status, days in isolation, and primary affiliation were all significantly associated with uploading a RAT. All those living on campus were students, and they may have had higher odds of uploading a RAT because they were provided RATs in person. Off-campus cases had the option to either pick RATs up from an on-campus location or have a testing kit mailed to them, possibly resulting in delays, which may have made it more challenging for certain off-campus cases to complete a RAT when recommended. Employees were more likely to upload RATs, possibly due to reasons such as wanting to return to work or running out of pandemic leave or sick time. Additionally, occupational health followed up with cases through phone calls multiple times during their isolation, whereas the student health center was unable to do so. Certainly, a testing out of isolation strategy should ensure that all members of the population have equitable access to RATs as well as follow-up medical supervision.Although this is not the first study to analyze a COVID-19 RAT protocol, this study has two unique strengths. First, the sample size was significantly larger than several similar studies, which ranged from 40 to 323 ,10,16,17There are several limitations to this study. First, this study relied on cases self-reporting the results of their RATs, and we do not know why 24% (211/880) of our cases did not do so . Almost certainly, more cases took a RAT than GW was aware of, which could skew the results, depending on why the RATs were not uploaded. For those with asymptomatic infections, either at the outset or over the entire course of the illness, it is difficult to ascertain when they acquired their infection . Thus, aThe most important issue is how to best isolate people with COVID-19 to balance public health protection with sparing individuals’ needless days in isolation. This study, and many others, support the notion that automatically ending isolation at 5 days is risky in terms of sending many who are infectious back into the community, in fact, that 7 days may be too short as well. Yet many can come out of isolation earlier with RAT testing. Many questions remain, including the efficacy of one-way masking for those coming out of isolation who still are RAT positive (as occurs with the current CDC protocol) and the need to document transmission of COVID-19 from people who have been isolated fewer than 10 days. Additionally, for both asymptomatic and symptomatic cases, RATs may not be the best measure of infectiousness ,15,20,21


Breastmik is considered the optimal source of nutrition in early infancy. However, recommendations and practices for when and how complementary food should be introduced in the first year of life vary worldwide. Early introduction of allergenic foods may prevent food allergies, but if early food introduction influences infant feeding practices is less known.We sought to assess infant feeding practices in the first year of life and to determine if early interventional food introduction influences breastfeeding and dietary diversity.1) control, 2) skin intervention, 3) introduction to 4 allergenic foods between 3 and 4 mo of age: peanut, cow milk, wheat, and egg, as small tastings until 6 mo, and 4) combined skin and food interventions. Dietary data were available from at least one of the 3-, 6-, 9-, and 12-mo questionnaires in 2059 infants. In the present analysis, groups 1 and 2 constitute the No Food Intervention group, whereas groups 3 and 4 constitute the Food Intervention group.Dietary intake was assessed in infants from the population-based clinical trial Preventing Atopic Dermatitis and ALLergies (PreventADALL) in children study. A total of 2397 infants were cluster-randomized at birth into 4 different groups: We used the log-rank test and Cox regression to assess the impact of food intervention on age of breastfeeding cessation. Mixed effects logistic regression was used to compare dietary diversity, defined as the number of food categories consumed, between intervention groups.P < 0.001) and 0.7 units  higher at 12 mo (P < 0.001) compared to the No Food Intervention group.At 3, 6, 9, and 12 mo, 95%, 88%, 67%, and 51% were breastfed, respectively, and breastfeeding duration was not affected by the food intervention. In the No Food Intervention group, mean age of complementary food introduction was 18.3 wk . In the Food Intervention group, the dietary diversity score was 1.39 units  higher at 9 mo (Early food intervention did not affect breastfeeding rates and increased dietary diversity at 9 and 12 mo. The first year of life is a critical period to establish feeding practices, taste preferences, and long-term dietary habits. Breastmilk is considered the optimal source of nutrition for infants ,2. ExcluCompared to other countries, Norway has high rates of breastfeeding initiation at birth 98%), with 80% of infants still breastfeeding at 6 mo, and many continuing to breastfeed throughout the first year of life 8%, with , 12]. BrThroughout the years, recommendations regarding the introduction of complementary foods have changed and continue to be a topic of debate ,16. ThesA diverse diet and timely introduction of complementary foods may also ensure an adequate supply of nutrients and influence both short- and long-term health outcomes . As longGiven the conflicting advice and cultural differences in infant feeding practices, more research into the timing of complementary food introduction is warranted. Therefore, the primary aim of this substudy, from the Scandinavian RCT and birth cohort PreventADALL, was to assess infant feeding practices, including the timing of complementary food introduction. The secondary aim was to determine if early food intervention  impacted breastfeeding rates and dietary diversity in the first year of life.In the present study, we analyzed dietary data from infants in the PreventADALL RCT, a multicenter, prospective, general, population-based mother–child birth cohort, aiming at primary prevention of allergic disease. Study design, recruitment, inclusion criteria, and baseline characteristics are described in detail elsewhere . BrieflyNewborns were randomly assigned to 4 intervention groups: no intervention (control group), skin intervention, food intervention, or the combined interventions . The conclinicaltrials.gov (NCT02449850). Informed consent was given upon enrollment and inclusion of the infant at birth.The PreventADALL study was approved by the Regional Committee for Medical and Health Research Ethics in South-Eastern Norway (2014/518) and in Stockholm, Sweden (2014/2242-31/4). The study was registered at Dietary data were obtained through electronic weekly diaries (2–26 wk of age) and questionnaires . Breastfeeding was assessed at 3, 6, 9, and 12 mo of age by parents reporting for the last 3 mo if the infant had received breastmilk and the time of breastfeeding cessation. If the infant was not exclusively breastfed, the amount of breastmilk consumed was categorized as most of the diet, the same amount as other food, or as a small part of the diet. At 6 mo, parents reported the amount of breastmilk consumed as no breastmilk, a small part of the diet, approximately half of the diet, or most of the diet. Consumption of porridge, how often it was given, at what age it was first introduced, and what it was made of  was reported at 3 and 6 mo. Questions about dairy intake at 6, 9, and 12 mo included the time of first introduction and what kind of dairy products were given to the infant. At 6, 9, and 12 mo, parents were asked to indicate how often the infant consumed several solid foods. The categories included: bread/cookies/waffles/cakes and other baked goods, fruit or berries, root vegetables , other vegetables, peanut (as a spread or incorporated in other foods), pure egg , egg in other foods , fatty fish , other fish, shellfish, poultry meat, and other meat. We also asked about how much of the infant food was home cooked compared with commercially prepared , as well as the content of organic food in the infant’s diet.All questions about diet were mandatory, assuring a complete set of values in all questionnaires. Please see the Supplementary data section and In the present analysis, we included all infants who were randomly assigned to the food intervention  into thComplementary food introduction was defined as the age at which any food or drink other than breastmilk, formula, and water was given for the first time to the infant.Primary outcome: Breastfeeding duration was defined as the number of months an infant was breastfed. Breastfeeding rates included exclusively breastfed infants and partially breastfed infants (receiving breastmilk and other sources of nutrition).Secondary outcome: The dietary diversity score is defined as the number of different food groups given to the infant during the last 3 months at 6, 9, and 12 mo of age. The calculated score was based on the following food groups: bread and other baked goods, milk, fermented dairy products, cheese, other dairy products, fruits and berries, root vegetables, other vegetables, peanuts, other nuts, egg (pure and egg in other foods), fatty fish, other fish, shellfish, poultry, and other meat. Summing the number of foods introduced at 9 and 12 mo, the maximum score was 16. At 6 months, the maximum score was 10 because intervention foods, including all dairy products, peanuts, and egg, were excluded from the dietary diversity score. Infant porridge was not included in the score because we did not include questions on infant porridge in the 9- and 12-mo questionnaires. The dietary diversity score did not consider frequency or portion sizes.Categorical variables are presented as numbers and percentages. Continuous variables, including frequency and intake of various foods, are presented as means, SD, and minimum (min) – maximum (max). To analyze breastfeeding rates in the first year of life, we used Cox regression. Kaplan–Meier curves were estimated for the FI and NFI groups, and differences in cessation rates between intervention groups were compared using the log-rank test. The proportional hazards assumption was tested and met. These analyses were performed using IBM SPSS statistics version 25.We used R version 3.6.0 for dietary diversity analysis, including complete case  and modified intention to treat analysis . For dietary diversity assessment at each time point between the intervention groups, we analyzed dichotomous end points using mixed effects logistic regression with the interventions and their interaction as fixed effects and randomization period and residential postal code as random effects.For the intention to treat analysis, missing primary outcome data were imputed using multiple imputations by chained equations. The number of multiple imputations was 15, and the scalar giving the number of iterations was 20. Complete case analysis was conducted as a sensitivity analysis.The background characteristics of the 1022 infants in the FI group and 1037 infants in the NFI group (N1037) were similar, as shown in In the Swedish population, breastfeeding rates at 3 mo and 6 mo were 92% and 80% compared with 95% and 88% in the Norwegian part of the study population, respectively. At 12 mo, 22% breastfed their children in Sweden and 51% in Norway.In the NFI group, the mean age of complementary food introduction was 18.3 wk , whereas infants randomly assigned to food intervention were introduced complementary foods from 12 wk of age. Porridge was the most common first food, other than the interventional foods . In the total study population, porridge was introduced in 9.6 % of infants by 3 mo of age, 45.7% at 4 mo, and 25.6% at 5 mo. By 6 mo of age, 81.1% infants had been given porridge. The most frequent porridges to be introduced before 6 mo were oat (62.2%), wheat (40.5%), and corn (34.1%). The following porridge types were given less frequently: millet (18.3%), whole meal (13.3%), spelt (4.1%), Sinlac (4.2%), and other types (5.1%).The cumulative proportion of infants introduced to other complementary foods is listed in At 6 mo, 45% of infants were reported to consume mostly or only commercially prepared infant foods, whereas 9% consumed only home-cooked foods. By 12 mo, the proportion of home-cooked foods in the infant’s diet increased. The proportion of infants mainly receiving organic food declined from 6 to 12 mo .P = 0.96), as shown in Breastfeeding rates from birth until 12 mo did not differ significantly between the FI  and the NFI group (P < 0.001). At 9 and 12 mo, the percentage of infants consuming these foods remained lower (P < 0.001) in the NFI group compared with the FI group (P = 0.032) and more infants consumed bread and other baked goods at 9 mo (P = 0.043). Intake of commercially prepared infant foods compared with home-cooked foods was similar in both groups, as was the intake of organic foods, except more infants in the FI group consumed little or no organic food at 12 mo.Significantly fewer infants in the NFI group consumed dairy products, peanuts, other nuts, egg foods, and pure egg at 6 mo compared with infants in the FI group (FI group . IntakesP < 0.001), as shown in Excluding interventional foods, the mean dietary diversity score (number of food categories consumed) was similar in the FI compared with the NFI group at 6 mo . However, the dietary diversity score was significantly higher in the FI group compared with the NFI group at 9 and 12 mo of age  guidelines . The ratBreastfeeding rates in our study were not affected by the food intervention, similar to 2 other large early allergenic food introduction RCTs, the Enquiring About Tolerance study in a general population  and the The finding that early introduction of complementary foods was associated with significantly increased dietary diversity at 9 and 12 mo of age is novel, to the best of our knowledge. Dietary diversity in infancy has gained significant attention because of its role in modulating allergic disease outcomes. Given the complexities and challenges of dietary diversity research, the European Academy of Allergy and Clinical Immunology Task Force recently provided an expert consensus regarding multiple aspects of diet diversity research and highlighted the need for more clinical trials with agreed definitions .Major strengths of the present study include the prospective design and robust sample size from a general population and the systematic collection of detailed questionnaires, which limits the likelihood of recall bias and ensures high accuracy of timing of solid food introduction and milk-feeding practices . ResponsThe Norwegian Directorate of Health recommends exclusive breastfeeding for 6 months, in line with the WHO ,8. TheseAn infant’s readiness to start complementary feeding will depend on the individual’s development ,4. EvideAs breastfeeding is the best for the infant, early introduction of solid foods should not come at the expense of breastfeeding. The results from our study suggest that early complementary introduction is beneficial for increased dietary diversity with no negative impact on breastfeeding rates. Further studies are needed to address the potential impact on disease prevention, as well as potential underlying mechanisms.In summary, in this general population-based mother–child birth cohort, most infants were breastfed throughout the first 6 mo of life, and half were still breastfed at 12 mo. The mean age of complementary food introduction was 4.5 mo, and most infants received complementary foods by 6 mo. Our data provide novel information that introducing small amounts of complementary foods from 3 mo of age increased dietary diversity but not at the expense of breastfeeding rates or breastfeeding duration.The authors’ responsibilities were as follows – CMS, EMR, KLC, HOS, MHC: designed research; CMS, EMR, KLC, CMJ, MLB, BJ, HOS, CS, RV, MHC: conducted research; CMS, MLB, RV: analyzed data or performed statistical analysis; CMS: wrote paper, CMS, EMR, MHG: had primary responsibility for final content; and all authors: read and approved the final manuscript.CMS reports personal fees from Libero outside the submitted work. ML reports personal fees from MSD outside the submitted work. EMH reports personal fees from Sanofi-Genzyme, Novartis, Leo-Pharma, Perrigo, and The Norwegian Asthma and Allergy Association outside the submitted work. All other authors report no conflicts of interest.Østfold Hospital Trust, by unrestricted grants from the Norwegian Association of Asthma and Allergy, The Kloster foundation, Thermo-Fisher, Uppsala, Sweden  and Fürst Medical Laboratory, Oslo, Norway , Norwegian Society of Dermatology and Venerology, Arne Ingel’s legat, Region Stockholm , Forte, Swedish Order of Freemasons Foundation Barnhuset, The Sven Jerring Foundation, The Hesselman foundation, The Magnus Bergwall foundation, The Konsul Th C Bergh’s Foundation, The Swedish Society of Medicine, The King Gustaf V 80th Birthday Foundation, KI grants, The Cancer- and Allergy Foundation, The Pediatric Research Foundation at Astrid Lindgren Children’s Hospital, The Samaritan Foundation for Pediatric research, Barnestiftelsen at Oslo University Hospital, Roche, The Frithjof Nansen Institute. The funding sources had no influence on study design, conduct, or analyses.The PreventADALL study was supported by the following sources: The Regional Health Board South East, The Norwegian Research Council, Oslo University Hospital, The University of Oslo, Health and Rehabilitation Norway, The Foundation for Healthcare and Allergy Research in Sweden – Vårdalstiftelsen, The Swedish Asthma- and Allergy Association’s Research Foundation, The Swedish Research Council – the Initiative for Clinical Therapy Research, The Swedish Heart-Lung Foundation, SFO-V Karolinska Institutet, Data described in the manuscript, code book, and analytic code will be made available upon request pending application and approval.


Modified surfaces like siliconized glass are commonlyused to supportprotein crystallization and facilitate obtaining crystals. Over theyears, various surfaces have been proposed to decrease the energeticpenalty required for consistent protein clustering, but scarce attentionhas been paid to the underlying mechanisms of interactions. Here,we propose self-assembled monolayers that are surfaces exposing fine-tunedmoieties with a very regular topography and subnanometer roughness,as a tool to unveil the interaction between proteins and functionalizedsurfaces. We studied the crystallization of three model proteins havingprogressively narrower metastable zones, i.e., lysozyme, catalase,and proteinase K, on monolayers exposing thiol, methacrylate, andglycidyloxy groups. Thanks to comparable surface wettability, theinduction or the inhibition of nucleation was readily attributed tothe surface chemistry. For example, thiol groups strongly inducedthe nucleation of lysozyme thanks to electrostatic pairing, whereasmethacrylate and glycidyloxy groups had an effect comparable to unfunctionalizedglass. Overall, the action of surfaces led to differences in nucleationkinetics, crystal habit, and even crystal form. This approach cansupport the fundamental understanding of the interaction between proteinmacromolecules and specific chemical groups, which is crucial formany technological applications in the pharmaceutical and food industry. Self-assembled monolayers (SAMs) allowfor precise controlof surface chemistry and roughness. In this study, the crystallizationof three model proteins, i.e., lysozyme, catalase, and proteinaseK, was performed on SAMs to investigate heterogeneous nucleation.We show that the nucleation time, the number of crystals, and thecrystal form can be modified depending on the terminal groups of SAMs. This superficial treatmentleads to a well-rounded drop shape thanks to moderate surface hydrophobicity,thus allowing slow water removal. Few studies have investigated theeffect of surfaces other than the siliconized glass on the crystallizationof proteins.8 A chronological summary of proteins crystallized in the presenceof heterogeneous substrates together with the proposed crystallizationmechanism was reported by Fermani and co-workers9 in 2013, showing the diversity of possible interpretationsof the results. Furthermore, surface patterning has been proven tobe sufficient to induce nucleation.13Vapor diffusion (VD) crystallizationguarantees slow supersaturationrates thanks to the progressive removal of water from a drop containingprotein and precipitants. The nucleation and growth of protein crystalscan thus occur under mild conditions, favoring good-quality crystals’harvesting. Over the years, complementary strategies for modifyingthe water evaporation rate have been proposed, including the use ofcapillaries,14 SAMs turned out to have a dramatic impact onthe nucleation step, both in terms of nucleation kinetics and preferentialorientation of crystal faces nucleated on the surface. The use ofuniform and flat surfaces as SAMs allows for the avoidance of surfaceheterogeneities in local charge or chemical composition, which canboth affect nucleation.15 As far as biological macromoleculesare concerned, the action of surfaces on nucleation is even more complexthan for small molecules, since the effective alteration of the nucleationpathway of biomolecules by surfaces can only be achieved under specificconditions. In the metastable area of the phase diagram, only heterogeneousnucleation and crystal growth are allowed, thus excluding the overlapwith homogeneous nucleation.In our recent publication,we investigated the action of self-assembledmonolayers (SAMs) immobilized on glass on the crystallization of amodel active pharmaceutical ingredient (API), i.e., aspirin.16 Conversely, the energetic demand increases with the contact anglebetween the surface and the drop of protein plus precipitant solution.17 Studies on hydrophobic SAMs made of hydrocarbonchains of variable length and different terminal groups have shownthat SAMs promoted faster crystallization rates and expanded the rangeof crystallization conditions compared to conventional coverslips.This ability was attributed to the inhibition of amorphous precipitation.19 The use of SAMs has also been reported to support the assembly ofmicroscale crystals of redox proteins for constructing bioelectronicdevices.20 Also, alternative approachesto foreign surfaces have been reported to promote crystallization.For example, the rational engineering of a protein surface to reducethe conformational entropy has been proposed.21The interplay between differenttypes of interactions, includingelectrostatics, hydrogen bonding, van der Waals, and hydrophobic interactions,determines the protein behavior in solution and the extent of interactionwith external surfaces. In this scenario, the surface properties ofthe heteronucleant become of the utmost importance. Surface functionalizationcan often come along with altered surface roughness, which impedesa rigorous study of the effect of specific functional groups on proteindynamics. Surface topography plays a key role since roughness decreasesthe energetic penalty required by heterogeneous nucleation.In the present paper, highly smooth SAMs have been used assupportsfor protein crystallization and to study the action of surface chemistryon nucleation phenomena avoiding surface roughness effects. Lysozyme,catalase, and proteinase K were crystallized on SAMs exposing variousterminal groups to unveil the main mechanism governing surface–proteininteraction.22.1–1·cm–1 as extinctioncoefficients for HEWL, catalase, and proteinase K, respectively.All of the reagents employedfor functionalizing coverslips made of borosilicate glass  were purchased from Sigma-Aldrich . 3-Mercaptopropyltrimethoxysilane ,3-glycidyloxypropyltrimethoxysilane , 3-(trimethoxysilyl)propylmethacrylate , hydrogen peroxide , sulfuric acid , anhydroustoluene , toluene ,and ethanol  were used forthe synthesis of self-assembled monolayers. For the crystallizationtrials, lysozyme obtained from chicken egg white , catalase from bovine liver, and proteinaseK  were selected.Before each crystallization test, protein concentration was measuredvia UV/vis spectrophotometry at 280 nm using 2.65, 1.48, and 1.42mL·mg2.222 Briefly, all of the steps of thesynthesis were carried out at room temperature. First, coverslipswere sonicated twice in ethanol for 10 min and dried with nitrogen.Then, they were incubated in a 5:1 (H2SO4/H2O2) piranha solution for 1 h and thoroughly rinsedwith deionized water. After drying, coverslips were immediately immersedin 0.054 M silane solutions in toluene for 15 h. To complete the synthesis,three washing steps with toluene, toluene/ethanol (1:1), and ethanoland blow-drying with nitrogen were performed to remove unreacted silanesand solvent residues. SAMs topography was measured by Atomic ForceMicroscopy in tapping mode. The scanned area was 1 μm2, at 256lines per scan. SiN4 tips were used, and the cantileverfrequency was 0.8 Hz. Gwyddion software  was used to process the images after the analyses.The detailed synthesisoptimization and characterization of SAMs is reported in our previousstudy.2.315 Equal volumesof protein and precipitant solutions were mixed to form 6 μLdrops. The reservoir was filled with 5 mL of precipitant solution.As reported in 3 in 25 mM Na citrate or 1.7 M (NH4)2SO4. The crystallizer was sealed withvacuum grease, incubated at 20 °C, and periodically checked  with an optical microscope for the presence of crystals.All of the protein solutions were filtered with0.22 μm pore-size low protein-binding membrane filters. Initially,the crystallization conditions of three proteins, i.e., HEWL, catalase,and proteinase K, were investigated in the absence of SAMs insidemushroom crystallizers.Once the optimal conditions had been identified, proteincrystallizationwas performed on functionalized surfaces. One untreated coverslipand three coverslips functionalized with different SAMs were carefullyplaced inside mushroom crystallizers according to the setup illustratedin Catalase crystals were diffracted at the ID23-1and ID30A-3 beamlinesof the European Synchrotron Radiation Facility (ESRF), Grenoble, France.Crystals were fished out from the drops with the help of LithoLoop, immediately flash cooledin liquid nitrogen, and stored for data collection.2.4Further tests inside mushroom crystallizerswere carried out with HEWL and catalase varying the pH of the precipitantsolution. Experiments were performed both without and with SAMs. Inregard to stock solutions, the HEWL concentration was 60 mg/mL in50 mM Na acetate at pH 4.5, the precipitation cocktail was made of4 wt % NaCl in 400 mM Na acetate at a pH between 4.0 and 6.0. Dropswere equilibrated against 4 wt % NaCl aqueous solution. The catalaseconcentration was set at 3.7 mg/mL. The precipitation cocktail wascomposed of 15 wt % PEG4000 in Milli-Q water for the reservoir and15 wt % PEG4000 in 400 mM K phosphate at pH 6.0–8.0 for theprecipitant solution of the drops. Experiments were performed in duplicate.3Figure S1). Alternative surfacechemistries, such as amino-terminated SAMs, were excluded becauseof the instability of surface chemistry over time.22 Depending on the nature of the protein and its phase diagram, studyingthe action of surfaces on crystallization may be more or less challenging.Heterogeneous nucleation can only be studied when crystallizationconditions fall in the metastable zone of the protein phase diagram.Therefore, the width of the metastable zone is related to the probabilityof observing heterogeneous nucleation events. In the present study,we selected three handleable model proteins, HEWL, proteinase K, andcatalase, having different metastable zones, as qualitatively describedin previous nucleation studies.23 HEWLhas a wide metastable zone. Conversely, proteinase K has a very narrowmetastable zone, thus being in principle insensitive to the presenceof foreign surfaces. Catalase was selected as a protein showing anintermediate behavior.In the presentstudy, self-assembled monolayers (SAMs) of silanesexposing thiol, methacrylate, and glycidyloxy terminations were graftedon glass. The chemical groups exposed by SAMs were selected so asto design an ideal platform to study nucleation. The three selectedchemistries guaranteed highly reproducible and stable surface functionalization,comparable wettability, and subnanometer roughness, as determinedby AFM .As expected, the increase in the ionic strength shortened the nucleationtime: few crystals were grown with 4 wt % NaCl after 3 days, whereasmassive crystallization was obtained with 15 wt % NaCl after a fewhours. It was believed that the extent of interaction between proteinand salt under the latter conditions would prevail over the interactionbetween protein and foreign surfaces. Therefore, the optimal saltconcentration for crystallization on SAMs was set at 4 wt %.The crystallization of HEWLon modified surfaces was first studied since such a protein representsa well-established model for nucleation studies and has a wide metastablezone. As a first step, screening over the nucleation time of HEWLon an m-glass surface was carried out inside mushroom crystallizersto identify the optimal nucleation kinetics and transpose such conditionsto experiments involving modified surfaces. HEWL concentration wasincreased from 5 to 80 mg/mL in 50 mM Na acetate at pH 4.5 and crystallizedwith 4 wt % NaCl. The nucleation time was defined as the time elapsedto observe the first crystals in the drops by optical microscopy observation.As outlined in Having selected the proper protein concentrationrange, two crystallizationexperiments were performed on THIOL, GLY, and ACR SAMs setting theHEWL concentration at 45 and 57 mg/mL. Thanks to the relatively highprotein concentration and low salt concentration, the crystallizationconditions allowed for the presence of a significant amount of macromoleculesavailable to interact with the surfaces. Two reference surfaces wereselected to compare the results: EtOH-cleaned glass coverslips (u-glass)and the mushroom glass (m-glass). Such a need was due to the differentcontact angle displayed by the protein solution on the two types ofglass: approximately 40° and 60° contact angles were obtainedon u-glass and m-glass, respectively. Surface hydrophobicity affectsthe drop shape and, thus, the evaporation rate. From this perspective,all of the SAMs had a comparable contact angle to u-glass to rigorouslycompare the nucleation kinetics regardless of the differences in evaporationrates due to surface hydrophobicity.The nucleation time obtainedon different SAMs and on the two referenceglasses is reported in 25 We hypothesized that the charge–charge interactions betweenTHIOL SAMs  and HEWL  could attract more protein molecules toward thesurface and increase the local supersaturation. ACR and GLY SAMs,on the other hand, carried fewer negative charges on the surface  and had lessimpact on nucleation.The inducing ability of THIOL SAMs was attributedto the earlyaggregation of HEWL macromolecules thanks to the decrease in energeticpenalty required for nucleation promoted by the surface. It has beenpreviously reported that when aggregates are formed on a surface andthe bond angles between protein molecules match those of a proteincrystal, they can evolve into nuclei and grow.Figure S4). The HEWL nucleation timeon m-glass and on THIOL SAMs as a function of pH is reported in In order to get further insight in thehypothesized mechanism ofnucleation induction of THIOL SAMs on HEWL crystallization, experimentswere repeated varying the pH. In this sense, the pH of the proteinbuffer was varied from 4.0 to 6.0, while keeping constant the restof the conditions. To test the suitability of such a system for HEWLcrystallization, we preliminarily carried out an HDVD screening, whichconfirmed the appearance of well-faceted crystals at all pH valuessee S4. The H3.2Catalase was the secondprotein tackled in this study. Catalase crystallization is more challengingsince the protein can easily undergo oxidation and lose its activity,resulting in a more difficult process to control. As for HEWL, a preliminaryscreening on nucleation time was performed. However, a precise tuningover nucleation time was not achieved as, after 1 day, crystals wereobserved in all of the drops prepared starting from 1.8 to 3.7 mg/mLcatalase and 15 wt % PEG4000. Further reduction in protein concentrationhindered the crystal formation, rather than tuning nucleation time,and it would be detrimental to ensure protein/surface interaction.Low supersaturation conditions were tested on the surfaces to maximizethe impact of SAM chemistries .After 12 h, crystals were obtained on all of the surfaces, excepton THIOL SAMs (observation time = 10 days). The average nucleationtime was by far shorter than for HEWL and, when nucleation occurred,it did in a comparable time window on all the surfaces. However, themorphology of the crystals was different, pointing to a clear effectof the SAMs on catalase crystallization. Elongated bar-like crystals(300–400 μm long) were generally obtained on m-glass,GLY, and ACR SAMs. A second population of thin plate-like crystalswas observed only on GLY and ACR SAMs . Underthese conditions, bulk nucleation prevailed, and only bar-like crystalswere obtained, even on THIOL SAMs. Catalase was also crystallizedvarying the pH between 6.0 and 8.0. Results, reported in Table S1, show that fast crystallization (within12 h) occurred both on m-glass and THIOL SAMs for pH above 7.0, whereaslonger nucleation times were required to obtain crystals at pH 6.0.At this pH, crystals appeared first on THIOL SAMs (3 days) and thenon m-glass (5 days). Thus, the inhibitory effect observed with THIOLSAMs at low supersaturation was overturned in an inducing effect athigh supersaturation. This apparently contradictory result was explainedconsidering that, at pH 6.0, catalase net charge is close to 0 coulomb. In the absence of charge–chargerepulsion, which is dominant at higher pH, nucleation could be inducedby the nonspecific interactions.When higher supersaturation conditions were employed, massive crystallization was obtained onall of the surfaces  pushed the system within the metastablezone with much higher waiting time .Because of the narrow metastability zone of proteinase K, bulk nucleationalways prevails, masking the effect of surfaces, as already observedin the case of agarose.23The crystallizationof proteinase K was last investigated. Preliminary HDVD screeningtests were performed with sodium nitrate or ammonium sulfate to lookfor the most appropriate crystallization conditions to analyze SAMseffects. However, as shown in 4In the present study, theaction of surface chemistry on the nucleationof three model proteins, i.e., lysozyme, catalase, and proteinaseK, was investigated. SAMs displaying thiol, methacrylate, and glycidyloxygroups were used because of their extremely high surface smoothnesscoupled with controlled chemistry. These surface features mitigatedthe potential influence of surface roughness decoupling the chemicaland physical influence of the surface on nucleation. The interactionbetween the exposed chemical groups and the protein macromoleculeswas exploited to tune the nucleation step. We demonstrated that surfacescannot be generally applied to any kind of protein since the interactionis specific for each protein and depends on the protein itself, thecharge (pH), and the level of supersaturation. The action of surfaceswas clearly discerned for lysozyme. With a wide metastable zone, thisprotein was an ideal model for studying specific interactions withSAMs. THIOL SAMs strongly promoted nucleation thanks to electrostaticmatching, as confirmed by crystallization experiments over a widepH range. When the direct tackling of nucleation was not feasible,as in the case of catalase, we analyzed the crystal population’smorphology to correlate the surface’s action to nucleation.Such a technique may support the discovery of new polymorphs thanksto the early stabilization of prenucleation clusters by a selectiveinteraction with the surface and help the fundamental understandingof the interaction between proteins and specific chemical groups.The change of the dynamic of the drop allowed us to get a third polymorphin the case of catalase extending the benefits of using SAMs. Lastly,when the metastability zone was even narrower, as for proteinase K,the action of SAMs was completely leveled off.