The genetic mechanisms that regulate biological processes, such as skeletal muscle development and growth, or intramuscular fat deposition, have attracted great interest, given their impact on production traits and meat quality. In this sense, a comparison of the transcriptome of skeletal muscle between phenotypically different pig breeds, or along growth, could be useful to improve the understanding of the molecular processes underlying the differences in muscle metabolism and phenotypic traits, potentially driving the identification of causal genes, regulators and metabolic pathways involved in their variability.biceps femoris muscle in growing pure Iberian and Duroc pigs. The study comprised 59 Iberian (IB) and 19 Duroc (DU) animals, who started the treatment at an average live weight (LW) of 19.9 kg. The animals were kept under identical management conditions and fed two diets with different energy sources (6% high oleic sunflower oil or carbohydrates). Twenty-nine IB animals were slaughtered after seven days of treatment at an average LW of 24.1 kg, and 30 IB animals plus all the DU animals were slaughtered after 47 days at an average LW of 50.7 kg. The main factors affecting the muscle transcriptome were age, with 1832 differentially expressed genes (DEGs), and breed (1055 DEGs), while the effect of diet on the transcriptome was very small. The results indicated transcriptome changes along time in Iberian animals, being especially related to growth and tissue development, extracellular matrix (ECM) composition, and cytoskeleton organization, with DEGs affecting relevant functions and biological pathways, such as myogenesis. The breed also affected functions related to muscle development and cytoskeleton organization, as well as functions related to solute transport and lipid and carbohydrate metabolism. Taking into account the results of the two main comparisons (age and breed effects), we can postulate that the Iberian breed is more precocious than the Duroc breed, regarding myogenesis and muscle development, in the studied growing stage.This experiment was conducted to investigate the effects of developmental stage, breed, and diet energy source on the genome-wide expression, meat quality traits, and tissue composition of  Moreover, this breed is characterized by high residual feed intake and a peculiar fatty acid (FA) profile [Pig meat is widely consumed worldwide and comprises one of the main sources of protein for human nutrition. Modern pig production is mostly established on highly selected genotypes, which are produced in intensive systems, focused on the efficient acquisition of meat. Nevertheless, in the south of Europe, this production system exists alongside autochthonous, less efficient breeds, as is the case of the Iberian pig breed. The traditional-based production systems are attaining important technical and commercial interest because of their use in the production of high-quality meat and derived products , and the profile , with a  profile ,5 and thNowadays, the Iberian pig may also be reared under intensive conditions, based on local agriculture, and with a high standard of quality characteristics and welfare conditions. In this case, both purebred Iberian and Duroc pigs may be used as terminal sires, with the aim of improving reproductive and growth performances, and primal cut yields ,6. It haIMF and FA compositions are highly dependent on breed and diet ,11. At tbiceps femoris (BF) muscle is the main lean mass within the ham and is characterized by high oxidative ability, due to the relatively higher content of type I muscle fibers than other muscles, such as the Longissimus dorsi (LD) muscle [Both developmental timing and metabolic and physicochemical features differ between muscles . The bic) muscle ,21, alsoversus fatty pig breeds in different developmental stages. Some studies compared different Chinese fatty pig breeds to lean western ones, such as the comparison between Tongcheng and Yorkshire pigs across 11 developmental stages [Several transcriptomic studies have compared lean l stages , betweenl stages , or betwl stages . Additiol stages  or RNA-Sl stages ,24, havel stages , we studThe present work employed the same experimental animals  and evalComunidad de Madrid animal welfare and protection committee, with reference number PROEX-007/15.All experiments were carried out in accordance with the Spanish Governance for Protection of Animals used in Research, RD 53/2013, which covers the European Union Directive 2010/63/EU about the protection of animals used in experimentation. The project was approved on 20 March 2015, by the 2 pig−1), with a concrete floor and straw bedding. Iberian animals were distributed in 16 pens (8 for CH diet and 8 for HO diet) and Duroc animals were distributed in 4 pens (2 for CH diet and 2 for HO diet ). Temperature was recorded with a mean of 23.8 °C throughout the experiment. Twenty-nine Iberian pigs (HO diet n = 14 and CH diet n = 15) were slaughtered after seven days of treatment (transition piglets), at an LW of 24.1 kg (SD = 3.3 kg). Thirty Iberian pigs (HO diet n = 17 and CH diet n = 13) and all nineteen Duroc pigs (HO diet n = 10 and CH diet n = 9) were slaughtered after forty-seven days of treatment (grower pigs), at an LW of 50.2 kg (SD = 7.5 kg) and 51.2 kg (SD = 5.09 kg) for Iberian and Duroc pigs, respectively. Feed composition is shown in The present study was carried out at the facilities of the Pig Test Center ITACYL  and comprised a total of 78 animals, including 59 Iberian  and 19 Duroc males born in 32 contemporary litters (26 Iberian litters and 6 Duroc litters), who started the experiment at 19.9 kg (standard deviation (SD) = 3.8 kg) (LW). Iberian litters had 2 to 4 male piglets and Duroc litters had 3 to 5 male piglets. At 10 weeks of age (SD = 1.6 days), the animals were allocated to two experimental groups, by distributing the animals coming from the same litter into the two groups, and fed two different isocaloric and isoproteic diets . Both diets differed in the energy source; the experimental diet was enriched with 6% high oleic sunflower oil (HO) and the standard diet contained carbohydrates as the energy source (CH). Feed and fresh water were provided ad libitum. Pigs were kept under identical management conditions, housed in 20 pens within the same experimental shed  were taken after slaughter. A slice of the muscle was taken from its central part (60 ± 2 mm) and divided into two slices (30 ± 5 mm); one slice was for the composition analysis, another for textural analysis and a small portion of 100 mg for RNA extraction. The slices were cut perpendicular to the longitudinal axis of the muscle. The IMF content of BF muscle was quantified using the method proposed by Segura et al., 2014 [l., 2014 , based ol., 2014  using a Regarding meat quality analysis, a portion of each fresh muscle from 49 grower pigs (30 Iberian and 19 Duroc) was divided into several parts for the subsequent determinations. The samples were vacuum packed in nylon/polyethylene bags and stored at −20 °C for thawing and cooking loss, instrumental color  and cooked meat shear force determinations. The meat quality analyses were carried out following the procedures described in .2 sections. The sections were fixed in 10% neutral buffered formalin. Then the specimens were embedded in paraffin, cut at 4 μm and stained with hematoxylin and eosin for routine examination [Regarding the histological analysis, a portion of each muscle tissue from 49 growers (30 Iberian and 19 Duroc) was divided into 2 to 4 cmmination . The mea® Pro 13  and the rest of the analyses were carried out using the MIXED procedure of SAS 9.4 . The results were considered to be significant at p-value < 0.05 and data were log transformed when necessary to meet normality and homoscedasticity criteria.The influence of breed, age and diet on FA composition was separately analyzed for each FA or FA index, within each lipid fraction. The analysis of the different effects on the FA composition, meat quality determinations, IMF content and size of adipocytes of BF muscle was performed with a linear model fitting as fixed-effects age, diet and the age–diet interaction , and breed, diet and the diet–breed interaction , and litter, pen and residual as random effects (in both models). Pen was nested to (Diet × Age) and (Diet × Breed) in the respective models. The animal was the experimental unit for all analyses. FA composition analyses were carried out using JMPModel 1 is as follows:Model 2:Muscle samples were maintained at −80 °C until gene expression analysis. For the transcriptome study, 36 muscle samples were used, corresponding to 12 Iberian animals belonging to the first slaughter  and 24 animals belonging to the second slaughter . The animals were randomly selected to perform transcriptomic analysis, representing all available litters. Total RNA was isolated from 50–100 mg samples of BF using the RiboPureTM RNA isolation kit , following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment , and the RNA quality was assessed with an Agilent 2100 bioanalyzer device  and submitted to the CNAG_CRG . Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit  according to manufacturer’s protocol. Each library was paired-end sequenced (2 × 75 bp) using TruSeq SBS Kit v3-HS, on a HiSeq2000 platform .www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 13 March 2021) was employed to assess the quality of raw sequencing data. TrimGalore (version 0.5.0)  was used to qualitatively trim data with default settings, and to remove the Illumina sequencing adapters and poly A and poly T tails  keeping only paired-end reads where both pairs were longer than 40 bp.FastQC software (version 0.11.8)  using TopHat (v.2.1.0)  with BowIn order to confirm that the mapping had been carried out correctly, quality control was performed in all samples using two tools, Samstats  and QualResulting BAM files were analyzed with HTSeq-count (version 0.11.1)  to countp-value ≤ 0.05 for the breed effect, BH-adjusted p-value ≤ 0.1 for the diet effect and BH-adjusted p-value ≤ 0.01 for the age effect and FC ≥ 1.5 in all of them. This software supports complex experimental designs in addition to simple two-group setups. With DESeq2 software, RNA-seq read counts were modeled by generalized linear models including the breed, age and diet effects. The age and breed effects were tested including the diet effect in the model. The diet effect was evaluated within each breed and at each age.The differential expression analyses were carried out in the R environment (R Core team) with the “DESeq2”package  that offp-values < 0.05 were considered statistically significant.RNA obtained from the 36 samples used in the RNA-Seq assay was used to perform the technical validation of the differential expression of 12 genes that were affected either by the breed or by the age. This technical validation was performed by studying the Pearson correlation between the expression values obtained from RNA-seq data (counts) and the normalized gene expression data obtained by RT qPCR. To validate the global RNA-Seq results, the concordance correlation coefficient (CCC)  was calcFASN, ME1, NR4A3, PON3, LEP, MSTN, IGF2, PVALB, DAPK3, GPX2, MTUS2 and MGLL was quantified. Primer pairs were designed using Primer Select software  from the available GENBANK and/or ENSEMBL sequences. Primer pairs covered different exons to assure the amplification of the cDNA. Information on primer sequences and efficiency are indicated in GAPDH, ACTB, TBP, PPIA, and B2M were selected for data normalization. The stability of the endogenous genes was tested with the Genorm and Normfinder softwares [ACTB and PPIA genes were selected as the most stable endogenous genes. Normalization of gene expression data was performed with Genorm software.The expression of the genes oftwares ,38. The Ingenuity Pathway Analysis software  was used to identify and characterize biological functions, canonical pathways and regulatory and causal networks affected by the DEGs . IPA sofThe effects of breed and diet on carcass traits and adipose tissue composition have been previously described ,12. Brieversus 70.10 microns, respectively; p-value < 0.0001). Although there are histological studies comparing the adipocytes of fat and lean breeds [In the present work, muscle cellularity, composition and meat quality traits have been studied. The size of the IMF adipocytes in the BF muscle was studied in the growing Iberian and Duroc animals, with larger adipocytes being observed in the Iberian breed than in the Duroc breed . A higher IMF content in Iberian pigs has already been observed when comparing pure IB versus IB × Du crossed newborn piglets  in BF muscle [versus 2.87, respectively) in LD muscle [versus IB × Du crossbred animals, also in BF muscle [In agreement, a higher IMF content was observed in the BF muscle from Iberian grower pigs than Duroc grower pigs  , tenderness (shear force), and color parameters , were determined in Iberian and Duroc growing pigs. Regarding the breed effects on tenderness determinations, the cooking loss percentage and shear force were higher in Duroc pigs and the yellowness (b*) was higher in Iberian pigs. Diet only slightly affected the cooked meat shear force, being higher in animals fed the CH diet, although this difference did not reach the statistical significance threshold ( = 0.07) .Meat tenderness is the sensory quality trait preferred by consumers. It is influenced by the interaction between genotype, age, diet, and many other factors ,43,44. Fn = 78; 29 Iberian transitional piglets, 30 Iberian growers, and 19 Duroc growers), allowing the evaluation of the effects of age, breed, and diet. The effect of diet on the FA profile of the BF muscle was separately explored in the two age groups. Moreover, age–diet (in Iberian) and breed–diet (in growers) interactions were studied. In all the lipid fractions , the FA composition showed significant effects on the three main tested factors , observed in the Duroc pigs. The main finding regarding FA composition is the higher SFA observed in the Iberian pigs compared to the Duroc pigs, and the unexpectedly higher MUFA content observed in the Duroc pigs . This result is in agreement with the very intense de novo fat synthesis in the Iberian pig tissues, which predominates over FA desaturation in this developmental period, leading to a higher SFA content and a dilution effect for other FAs, such as MUFA and PUFA. On the other hand, the higher levels of PUFA found in the NL and FL fractions in the BF muscle of Duroc pigs, which were also previously observed in the dorsal and ham subcutaneous fat, could be explained by higher ability of the Duroc genotype to store dietary unsaturated lipids in its tissues .It is interesting to remark that the main diet effects on FA composition were detected very early during the treatment, in the first slaughter (7 days of treatment), and moderate changes in the magnitude of differences between the diets were observed along the trial. These results are in agreement with our previous results in a similar diet trial by Ovilo et al., 2014 .semimembranosus muscle, but not on biceps femoris. In the present study, the comparison between two diets with different sources of energy (high oleic acid or carbohydrates) showed significant differences in the FA profiles of BF muscle, which, in general, reflected the composition of the diets received, with a higher MUFA content and lower SFA content in the HO group. These differences were mainly due to the higher oleic (C18:1n-9) content in the HO diet. The content of SFA was higher in the CH group, although the CH diet included a lower proportion of palmitic and stearic acids, indicating de novo synthesis of FA from the available carbohydrates in this group. All these results are consistent with the deposition of dietary FA and the induction of lipogenesis by dietary CH. These results are, in general, very similar to those found in our previous work when analyzing the effect of diet on the FA profiles of subcutaneous backfat and subcutaneous ham fat [Some studies suggested that the diet FA composition led, in general, to a homogeneous response in the different animal tissues ,53. Howec C18:1n- content Previous works of oleic acid supplementation have been performed in the Iberian breed, focusing on backfat, LD muscle, and liver , and on In the present work, RNA-Seq was used in 36 BF muscle samples, in order to study the effect of developmental stage, breed, diet, and interactions on the transcriptome profile in Iberian and Duroc growing pigs. An average of 45 million sequence reads were obtained for each individual sample, and were assembled and mapped to the annotated Sscrofa11.1 genome. All the samples passed the quality control test and 93–95% of the reads were mapped to the porcine reference sequence. An average of 15,314 genes out of 22,452 annotated genes were expressed in the studied samples. Regarding mapping quality values (MAPQ), an average of 96% of the reads showed MAPQ ≥ 30 in the different samples .p-value < 0.01) , also known as fetuin-A , and ENSSSCG00000023048 , which is a misc_RNA or non-coding RNA. AHSG is a multifunctional molecule that participates in different processes, such as energy expenditure, appetite control, insulin resistance, and regulation of adipogenesis [The effect of developmental stage on the muscle transcriptome was studied in Iberian pigs, allowing the identification of 1832 DEGs between transitional piglets and growing pigs (FC ≥ 1.5 and BH-adjusted  < 0.01) . We deteogenesis .MYOD1, MYMK, AKT1, and E2F1, were upregulated in transition piglets  plays a major role in regulating muscle differentiation [MYMK  regulates the myogenic fusion process [AKT1  is crucial for myoblast proliferation, but is not necessary for differentiation [E2F1  is a member of the E2F family of transcription factors and has a key role in activating primary fiber muscle formation [MEF2A, MDFIC, MSTN, JAK2, FOS, FOSB and AKIRIN1 genes, mostly involved in the formation and proliferation of myofibers and their hypertrophy, were upregulated in growers’ muscles; for example, MSTN  is a relevant gene that is an inhibitor of skeletal muscle growth [MSTN upregulation in grower animals may inhibit the over-proliferation of myoblasts when the primary and secondary fibers are forming.Different genes involved in muscle differentiation were upregulated in transition piglets and in grower pigs. The DEGs mainly involved in the early steps of the myogenesis process, such as  piglets . The MYOntiation . MYMK . The dynamic balance between cell death, proliferation, and differentiation is an important condition for maintaining the development and homeostasis of tissues. Therefore, cell proliferation must be carefully balanced with programmed cell death (apoptosis) to maintain a constant number of cells in adult tissues [p-value = 3.56 × 10−6, with 238 DEGs implicated), dynamics of the microtubules , organization of cytoskeleton , and organization of cytoplasm   .p-value ≤ 0.01; p-value ≤ 0.01), such as oxidative phosphorylation and cell cycle control of chromosomal replication signaling pathways, which suggests greater mitochondrial activity in the muscle of young animals with high energy requirements and a very active cell cycle, indicating the occurrence of mainly proliferation and differentiation in muscle cells. On the contrary, sixty-two pathways were significantly activated in the growers . Out of them, twenty-two canonical pathways were related to cell proliferation and differentiation  is important for postnatal myoblasts to progress through differentiation to form adult skeletal muscle [The  piglets . Oxidatil muscle . Furtherl muscle ,71. Thusp-value < 0.05; The upstream analysis  and regulator effects tools of the IPA package were employed to identify potential transcriptional regulators that may explain the differential patterns of expression observed between ages, and allowed the identification of 333 candidate regulators , mainly related to cell growth, proliferation, and differentiation. On the other hand, 28 upstream regulators were activated in the growing animals . Moreover, the MEF2D isoform was predicted to be an activated upstream regulator in the growing animals  and is also responsible for a causal network  is an important transcription factor involved in the development of skeletal muscle, since it plays a key role in the differentiation of muscle cells . In diffSeveral regulator effects networks were also identified. Interestingly, a network was identified and predicted to be activated in transition piglets, which was involved in nine activated functions in the growers, related to growth . The genp-adj = 0.0001), which is implicated in cell differentiation and transformation [In addition, another interesting regulatory network was identified and predicted to be activated in growers, which is involved in the size of the body, vasculogenesis, migration of cells, and organization of the cytoskeleton, and includes the transcriptional regulators CAMK4 and GNA12 . CAMK4 cormation .Skeletal muscle development and growth are complex processes, highly organized and regulated by the interaction of muscle cells with their extracellular microenvironment. These processes require specific cytoskeletal, membrane and adhesion proteins. Our results of functional enrichment and activation  showed rKIF2A, KIF3A, KIF5B, KIF12, etc.), myosins (MYO6), dyneins (DYNLT3), centrosomal proteins , coiled-coil domain-containing proteins , and others [JAK2) and integrins (ITGVA and ITGA4), and is also upregulated in growers. However, intriguingly, several α and β tubulins were upregulated in the transition piglets , most likely suggesting an increase in microtubule networks, leading to greater cell division and proliferation in the piglets, providing muscle cells for adhesion and later differentiation [The cytoskeleton is composed of a polymeric arrangement of proteins and structures organized in the following three specific systems: microfilaments, intermediate filaments, and microtubules ,79. All d others ; all of d others . Additiontiation .ITGA1 and ITGA5, which were positively regulated in the transition piglets. Moreover, ITGAV and ITGA4 were positively regulated in the growers, which suggests sequential activation of the different integrins involved in the process of skeletal muscle development. Collagens and laminins are found abundantly in the ECM environment, and are vitally important for the mechanical support of tissues, in addition to cell adhesion and differentiation [COL4A3, COL6A6, COL8A1, and COL17A1, which were upregulated in the growers, and COL20A1 was upregulated in the transition piglets. Additionally, the laminin LAMA2 was upregulated in the growers. This laminin is a major component of the basement membrane, and is thought to mediate the attachment, migration, and organization of cells into tissues during embryonic development by interacting with other ECM components [MSTN) with its receptor, activin receptor type IIA (ACVR2A) [p-adj = 0.0009 and FC = 1.57, p-adj = 0.005, respectively).The ECM of skeletal muscle is a dynamic mixture that has both mechanical support and active signaling roles . The ECMntiation . Severalmponents . The ECM(ACVR2A) , both ofAltogether, our results of differential expression and functional interpretation between growing stages are compatible with the transition piglets showing preponderance of early differentiation events (from satellite cells to myoblast), and also proliferation and hyperplasic processes, while the grower animals show preponderance of differentiation of muscle cells, leading to hypertrophy and growth of skeletal muscle.p-value < 0.05), of which 505 genes were overexpressed in IB pigs (FC ranging from 1.5 to 56) and 550 DEGs were overexpressed in DU (FC ranging from 1.5 to 15)  and PCK2 . The CLCA1 gene is thought to act as a signaling molecule for the innate immune response, through the activation of macrophages, and, consequently, increases pro-inflammatory cytokine release . Additionally, it has relevance in the early innate immune response in murine models [PCK2 gene encodes a mitochondrial enzyme that catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the presence of guanosine triphosphate (GTP) [PCK2 may be associated with muscle anabolism and cell proliferation [PCK1 gene and has also been associated with meat quality traits in pigs [DESeq2 identified 1055 annotated DEGs between breeds (FC ≥ 1.5 and BH-adjusted 5 to 15) . The gene models . Intereste (GTP) . In addiferation . A cytos in pigs .ME1, FASN, G0S2, and PDK4, have been identified in the present study, which is in agreement with the breed’s adipogenic trend. The malic enzyme  gene is involved in fatty acid synthesis, encoding a cytosolic enzyme that produces NADPH (dihydronicotinamide adenine dinucleotide phosphate) [ME1 gene was also observed in our previous results obtained by qPCR and RNAseq from subcutaneous fat samples [FASN)  is a central enzyme for fatty acid synthesis, which incorporates acetyl-CoA and malonyl-CoA, derived from glucose or other carbon precursors, to form palmitate [G0S2)  gene is a negative regulator of lipolysis, whose activation is known to downregulate ATGL expression. There is evidence that illustrates the critical roles of this gene in modulating energy metabolism, cell growth, and apoptosis in a multitude of cell types [PDK4 is overexpressed in Iberian pigs , controlling the activity of PDC in skeletal muscle, and contributes to glucose and FA metabolism (FA oxidation and de novo FA biosynthesis) regulation [In Iberian pigs, overexpressed genes with known biological functions related to lipid and protein metabolism and energy homeostasis, such as osphate) ,97. High samples ,9,98. Thll types . The mitll types . In partgulation . In factgulation . This gegulation . These rgulation .IGF2 gene , which encodes for a member of the insulin family of polypeptide growth factors, which participate in growth and cell differentiation. IGF2 stimulates the proliferation and differentiation of myoblast and satellite cells. Moreover, it is a paternally imprinted gene, linked to heart size, fatness, and muscle growth. In swine, a causal mutation in IGF2 intron 3 has been identified (g.3072G > A) [IGF2 expression is associated with the mutation [IGF2 in Duroc pigs is in concordance with previous observations and with the presence of alternative alleles in both the studied breeds.Among the genes overexpressed in Duroc pigs, and coinciding with our results in ham subcutaneous adipose tissue , we foun72G > A) ,107. Thi72G > A) ,109. Themutation , and, thDLK1 gene was found among the DEGs overexpressed in the Duroc pigs . DLK1 is expressed in preadipocytes, but is absent in mature adipocytes [It is interesting to note that the ipocytes , suggestipocytes . This alp-value < 0.01)  in the dataset of 1055 DEGs (Following gene ontology (GO) enrichment, 299 biological functions were predicted to be affected by breed ( < 0.01)  and twen055 DEGs , with si055 DEGs .p-value = 6.32 × 10−6), development of vasculature , quantity of cells , organization of the cytoskeleton , and microtubule dynamics  functions, and growth hormone and GM-CSF signaling pathways . This is in agreement with the findings reported by Ayuso et al., 2016 [The functions activated (z-score < −2) or enriched (z-score with a negative sign) in the Iberian breed, and the activated pathways, were mainly related to development and growth, cytoskeleton organization, and adipogenesis, such as development of the body trunk . Triacylglycerols are the main form of fat storage in the tissues, and are also responsible for thermogenesis [p-value = 5.63 × 10−5), indicating greater catalytic oxidation of fatty acids.Regarding FA metabolism-related functions, in the Iberian fatty breed, we observed that the concentration of triacylglycerol function and the triacylglycerol biosynthesis pathway were enriched    and glucose tolerance , which are also activated functions in the Duroc breed [On the contrary, the synthesis and metabolism of acylglycerol function   was acti = 0.02) . These poc breed ,114.retinol biosynthesis and LXR/RXR activation signaling pathways) (Two pathways activated in the Duroc breed (the athways)  are intip-value < 0.01; An in silico prediction of potential transcriptional regulators allowed the identification of 73 regulators or molecules that may explain the differential expression patterns observed between the breeds , and ITGA4 and ITGA8 . It has been proposed that integrins also regulate angiogenesis [p-value = 1.5 × 10−5, and z-score = −1.063, p-value = 5.6 × 10−7, respectively), with important genes, such as ANGPT1 , overexpressed in this breed. There is evidence of the close relationship between the development of adipocytes and vasculature; hence, the influence of integrins on angiogenesis may have an impact on adipogenesis as well. In fact, adipogenesis and angiogenesis are reciprocally regulated [p-value = 3.1 × 10−7), with CEBPD, a key transcriptional regulator of adipogenesis, also overexpressed in the IB breed . Moreover, in the muscle, ITGAs bind to collagens and both types of molecules interact in the maintenance of the ECM [ITGA2, which is overexpressed in DU, has collagens as ligands and five different collagen genes were also overexpressed in the DU pigs , and one collagen (COL17A1) was overexpressed in the IB pigs. In addition, other ECM-related proteins, such as FBN2  and FMOD , were overexpressed in the DU pigs. The results of ITGA2 and collagen expression are in agreement with our findings in a previous study in crossbred DU × IB pigs [Most of the differences found between the DU and IB pigs, in functions and pathways , were inogenesis . In factegulated ,118, and the ECM . Specifi IB pigs . Moreove IB pigs . The gre IB pigs ,123, and IB pigs .The transport of materials across membranes is a vital process for all aspects of cellular function, including growth, metabolism, and communication between cells . ProteinNOS1, CTSL, TRIM23, and TRIM24 . NOS1 and CTSL differential expression was consistent with previous findings, but different types of TRIM genes were differentially expressed in comparison to previous work [Muscle mass markedly increases along postnatal development, through the process of hypertrophy that takes place when the overall rates of protein synthesis exceed the rates of protein degradation, leading to protein accretion. The whole-body protein turnover and protein synthesis/protein deposition ratio differ between Iberian and other lean breeds, such as Landrace ,128. It n = 190) were all coincident to the DEGs overexpressed in the DU breed, and the common DEGs overexpressed in the growers (n = 207) were all coincident to the DEGs overexpressed in the IB pigs. Many of these common genes were closely related to muscle development and growth, such as the MYOD1, MYBL2, FOS, JAK2, E2F1, FOXM1, MSTN and DLK1 genes, and were also related to the organization of the cytoskeleton and microtubule dynamics, such as ATXN1, CDC20, JAK2, KAT2B, and several kinesin-like genes; for instance, our data show higher expression of myostatin (MSTN) in the grower animals in comparison to the transition piglets , and higher expression in the IB pigs in comparison to the DU pigs . On the other hand, MYOD1 is an example of the opposite situation, being overexpressed in the transition piglets  and in the DU pigs . Other relevant genes involved in the early proliferative processes of muscle, such as the MYBL2, E2F1, FOXM1, DLK1 and NFATC4 genes, followed this same pattern of expression, being overexpressed in the transition piglets and in the DU animals. On the other hand, key genes in the processes of differentiation and hypertrophy, such as JAK2, FOS, GHR, FOXN3, and CTF1, followed the contrary pattern of expression, being overexpressed in the grower pigs and in the IB animals, indicating that these animals could be in a more advanced stage of muscle development. The differential expression pattern of all the common genes, affected by age and breed, would support the hypothesis stating that both breeds progress with different paucity regarding muscle development, with the Iberian breed being in a more advanced stage of development than the Duroc breed, or, in other words, that the Iberian breed is more precocious in muscle development than the Duroc breed, at this specific growing stage. Similar hypotheses have been postulated, proposing that myogenesis starts earlier, but progresses more slowly, in local fat pigs than in lean pigs [It is interesting to note that out of the 1832 DEGs affected by age in Iberian growing pigs and 1055 DEGs affected by breed at the growing stage, as many as 397 DEGs were common , which wean pigs ,17.MYL4 gene), although the differential expression was opposite in the two breeds (upregulated in the HO diet in the Iberian pigs and in the CH diet in the Duroc pigs).The diets employed in the present study were formulated to represent a standard pig diet with carbohydrates as the energy source (CH diet), and an alternative diet, including high oleic sunflower oil (HO diet), which is employed in Iberian pig intensive production, to mimic the traditional extensive production system in which animals are fed acorns, rich in oleic acid, and grass. The diets were isocaloric and isoproteic, and, thus, they differed in several ingredients besides oleic acid, such as fiber and SFA. Therefore, it is not possible to study the isolated effect of one particular nutrient, but the aim of this study was to be as close to the practical scenario as possible and deepen the understanding of the molecular events differing in the animals fed a standard carbohydrate diet vs. a high oleic acid isocaloric diet. The effect of diet on the muscle transcriptome was studied in the two development stages in the Iberian animals. In addition, the effect of diet was also studied in the Duroc growing animals. We detected eight DEGs that were conditional on diet in the transition Iberian piglets and eight DEGs that were conditional on diet in the grower Iberian animals. No common genes were found between both breeds. In addition, three DEGS that were conditional on diet were detected in the growing Duroc pigs . One comThe effect of diet on the muscle transcriptome was smaller than the effect observed in these same experimental animals on the subcutaneous ham adipose tissue, with only a few genes affected by diet at both development stages in the Iberian animals and in the Duroc growing pigs. This lower responsiveness of muscle tissue to diet interventions, at the gene expression level, was also observed in previous studies focused on candidate genes in animals fed different diets ,130,131.p-adj < 0.1): NEFH, LY6G6C, and SCN10A. The first gene codes a neurofilament heavy polypeptide that is a structural constituent of the cytoskeleton, and is related to cytoskeleton organization, the transmission of nerve impulses, and axon development [In order to deepen the potential interaction effects, we used the DESeq2 tool to perform a joint analysis using a full model, fitting the age–diet and breed–diet interactions . This anelopment . The secelopment . The laselopment . All of p-adj < 0.1): MYL4, MYL5, and CD36. Myosins are involved in several functions, such as muscle contraction, cytokinesis, cargo transport, cell adhesion and migration, and the formation and stabilization of actin-rich structures. MYL4 is involved in muscle filament sliding and in the positive regulation of ATPase activity. The MYL5 gene encodes one of the myosin light chains, a component of the hexameric ATPase cellular motor protein myosin [CD36 appears to be a food-sensitive lipid sensor in the gustatory circumvallate papillae, although it is not only expressed in the taste buds, but also in other tissues. Lipid-regulated alteration in lingual CD36 expression might modify the interest for fat during a meal, which would initially be high and would then progressively decrease after feed intake. This lipid-mediated regulation is reminiscent of sensory-specific satiety. Our findings apparently support the role played by CD36 in the oro-sensory perception of dietary lipids [This analysis also yielded the following three genes with significant breed–diet interaction effects . Selection was performed from the lists of DEGs for the breed and age effects. The selected DEGs were technically validated, showing significant results in the correlation between the RNA-Seq and qPCR results (−11). In order to assess the technical validation in high-throughput transcriptomic studies, we also calculated the CCC coefficient, and it is interesting to highlight that a value of 0.80 was obtained, which supports a considerable general relationship between the RNA-Seq and qPCR expression values [With the purpose of validating the reported observations from the RNA-Seq analysis, we selected 12 representative genes, and their relative expression was analyzed by qPCR in all the studied samples  and mechanisms  involved in the regulation of the progression of muscle development and intramuscular fat accretion, which also explain the phenotypic differences between the breeds. Taking into account the results of the two comparisons (age and breed effects), we can postulate that both breeds progress with different paucity regarding muscle development, proposing that myogenesis starts earlier, but progresses later and more slowly, in Iberian pigs. In other words, the Iberian breed is more precocious in muscle development than the Duroc breed at this specific growing stage. Our results highlight the deep influence of age and breed on muscle metabolism during growth, suggesting specific needs across the different phases of the growing period, which should be key in the design of breed- and age-specific productive feeding programs.We found a strong effect of age and considerable effect of breed on the muscle transcriptome. Nevertheless, the effect at the transcriptome level of the two employed diets at this particular developmental stage was small. Most differences in the transcriptome profiles between the transition Iberian piglets and grower Iberian animals, and between the Iberian and Duroc growing pigs, were related to growth, muscle development, and cytoskeleton and ECM organization. In addition, the differences between the breeds were also related to solute transport, and lipid, carbohydrate and protein metabolism. The results provide key genes (such as


Perimenopausal depressive disorder (PDD) is an affective disorder involving endocrine, neurological, immune, which seriously endangers the physical and mental health of human. Selective serotonin reuptake inhibitors (SSRIs) are the current first-line clinical treatment, have limited efficacy and serious side effects. Acupuncture combined with SSRIs therapy has been widely used clinically because it increases efficacy and reduces side effects. There is a lack of high-quality evidence to assess its efficacy and safety. This study evaluated the effectiveness and safety of acupuncture combined with SSRIs in the treatment of PDD by meta-analysis.All randomized controlled trials articles about acupuncture combined with SSRIs treatment of PDD will be searched in databases, such as PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Wan Fang, Wei Pu from the construction of the library to December 16, 2021. According to Cochrane 5.1 Handbook criteria, two researchers independently screened the literature, extracted data, and evaluated the quality of included studies. Meta-analysis was performed by using RevMan 5.4 and STATA 16.0 software.This study will summarize the current evidence to evaluate the effectiveness and safety of acupuncture combined with SSRIs for the treatment of PDD.The results of this study will provide clinicians with new treatment ideas and bring benefits to most patients.INPLASY2021120080 (DOI number: 10.37766/inplasy2021.12.0080). Perimenopausal depressive disorder (PDD) is a mood disorder in which women experience persistent depressed mood, anxiety and depressive disorder, and vegetative nervous disorders during perimenopause.,5 Women are about twice as likely as men to be depressed, and perimenopausal women are about twice as likely as premenopausal women to be depressed. Studies showed a 36% prevalence of depressive disorder in perimenopausal women, and the trend is increasing year by year. If patients with PDD are not treated promptly or given inappropriate treatment, it increases the risk of suffering and suicide. Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for depressive disorder, it is also used clinically for the treatment of PDD. However, patient adherence to SSRIs is low for two main reasons:Perimenopause is the physiological transition period from the onset of menstrual disorders to 1 year after menopause in women,1)SSRIs act solely on the serotonin system and do not address the multisystem disorders of PDD;2)–12SSRIs have side effects such as gastrointestinal distress, weight changes, sleep disturbances, and sexual dysfunction. Acupuncture treatment upholds a holistic concept, so it can regulate and treat PDD at multiple levels, links, and targets, such as regulation of sex hormones, neurotransmitters, hypothalamic-pituitary-adrenal axis, hypothalamic-pituitary-ovarian axis, oxidative stress, signaling pathways, and other cellular events. Acupuncture can compensate for the inability of SSRIs to address multisystem disorders in PDD. Unluckily, acupuncture is difficult to achieve short-term results for severe depressive disorder. Acupuncture as a physical stimulus to accelerate the onset of action of SSRIs and reduce the dose and side effects of antidepressant applications. The combination of acupuncture and SSRIs in the treatment of PDD can take advantage of the wide range of acupuncture effects, and take advantage of the precise pharmacology and specific effects of SSRIs to achieve the effect of increasing efficacy and reducing side effects. Studies showed acupuncture combined with antidepressants had excellent efficacy in treating PDD. There is no high-quality evidence to assess the efficacy and safety of acupuncture in combination with SSRIs. This study investigated the effectiveness and safety of acupuncture combined with SSRIs by meta-analysis to provide clinicians with a better basis for clinical decisions.The combination of acupuncture and SSRIs in the treatment of depressive disorder has been proven to have increased efficacy and reduced side effects, which may provide a new direction for clinical treatment.22.1The systematic review protocol was registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols. The registration number: INPLASY2020110072. The consent of this protocol report is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMAP) statement guidelines.2.2From the construction of the library to December 16, 2021, all randomized controlled trials (RCTs) articles about acupuncture combined with SSRIs treatment of PDD will be searched in databases, such as PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Wan Fang, and Wei Pub by advanced search. The key search terms used are (Perimenopause or Climacteric or Menopause) And (Depressive Disorder or Mood Disorders) And  And . The search strategy for PubMed is shown in Table 2.3The participant, intervention, comparison, and study design (PICOS) principle has been applied in the study design.2.3.1We will include clinical RCTs, which will be categorized by language in Chinese and English. Any other types of studies, such as animal studies, case reports, case series and reviews, will be excluded.2.3.2Women were diagnosed with PDD. The diagnostic criteria for depressive disorder were in accordance with the Chinese Classification and Diagnostic Criteria of Mental Disorders, 3rd edition (CCMD-3), American Psychiatric Association DSM-5, American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders; diagnostic criteria for perimenopause in accordance with the Diagnostic Criteria for Gynecological Diseases, the diagnostic criteria for TCM evidence should be in accordance with the “Internal Medicine of Chinese Medicine” or “Diagnostic and Efficacy Criteria for TCM Medical Evidence”.2.3.32.3.3.1All patients used received acupuncture combined with SSRIs in the experimental group. The SSRI drugs were same as the control group.2.3.3.2Patients used were treated with SSRIs in the control group.2.3.42.3.4.1The primary outcome are the Hamilton depressive disorder scale and clinical efficiency.2.3.4.2The primary secondary outcome are the self-rating depressive disorder scale, quality of life scale and Estradiol levels.2.4We selected a PRISMA flow chart showing the processing of selected literature throughout the study Fig. . Prior t2.4.1 A third author will help reconcile any differences between the 2 authors.Two authors will independently extract the relevant information from included trials, such as first author, time of publication, group, sample size, patient age, intervention, duration of treatment, outcome indicators, method of randomization, blinding, concealment, allocation, and duration of follow-up. Gathering out specific information about acupuncture, such as the acupoints selected, the type of needles, the method of needle insertion, and the duration of needle retention according to the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICA).2.4.2 After 2 authors have completed their assessments, the results are cross-checked if there is disagreement, a third author discusses arbitration.The Cochrane Collaboration tool was used to assess the risk of bias in randomized trials, including the generation of randomized sequences, allocation concealment, implementation of blinding (blinding of subjects and implementers and blinding of study outcomes), data integrity, selective publication, and other biases, all of which could be classified as “low risk”, “unclear” and ‘high risk”. “unclear” and “high risk”.2.4.3If data from included studies are incomplete, we will try to contact the author of the article by sending an email or making a phone call. We will exclude studies with incomplete data if the author cannot be contacted.2.4.4We will use Review Manager Software (RevMan) V. 5.4  for data analysis and quantitative data synthesis. If no statistical heterogeneity is found, a fixed-effects model is used for data synthesis. If there is significant statistical heterogeneity, we will use a random effects model.2.4.5I2 statistic methods. When P < .1 or I2 > 50%, there was heterogeneity between studies; when P > .1 or I2 < 50%, there was no heterogeneity between studies.Heterogeneity in the forest diagram was determined by Q statistic or 2.4.6When heterogeneity occurs in the analysis, different drugs in SSRIs can be analyzed in subgroups.2.4.7In this analysis, once more than 10 trials were included, funnel plots could be used to test for reporting bias. When more than 20 studies were included, Egger regression was used to test reporting bias.2.4.8Sensitivity analysis was performed to exclude tests with quality defects and to ensure the stability of analytical results based on sample size, study design, heterogeneity, methodological quality, and statistical models.2.4.9This study will use a quality-of-evidence rating method to assess the results obtained from this analysis. GRADE is generally applied to a large body of evidence. It has 4 evaluation levels, such as high, moderate, low, and very low. GRADE is used to assess the bias, inconsistency, discontinuity, and inaccuracy of test results. In the context of systematic reviews, quality reflects our confidence in the validity of the assessment.2.5The approval of an ethical committee is not required to conduct this study. Patients and the public were not involved in the design and conception of this study. The results of this meta-analysis will be disseminated through publication in a peer-reviewed academic journal or relevant conference.3The incidence of PDD is increasing with the increase of social pressure, but there are no clinically recommended drugs for the treatment of PDD. Medications and hormone replacement therapy are generally used to treat depressive disorder, but the results do not meet expectations and there are serious side effects, there is an urgent need to seek new treatment methods. The combination of acupuncture with SSRIs is not only synergistic, but also reduces the side effects associated with SSRIs.The combination of acupuncture with SSRIs has been widely tried clinically and achieved good efficacy. The current lack of high-quality evidence validation has led to limited global replication. Although some potentially low-quality raw RCTs may affect the accuracy of the study, there is still a need to investigate them.Conceptualization: Zhan Gao.Data curation: Tingting Song, Xiangju Gao.Formal analysis: Zhan Gao, Mingzhou Gao.Funding acquisition: Jieqiong Wang.Methodology: Zhan Gao.Project administration: Mingzhou Gao, Jie Gao, Mingqi Qiao.Supervision: Jie Gao, Mingqi Qiao.Validation: Zhan Gao, Jieqiong Wang, Mingzhou Gao, Mingqi Qiao.Writing – original draft: Zhan Gao, Mingzhou Gao, Tingting Song.


Without timely diagnosis and treatment, tachycardia, also called tachyarrhythmia, can cause serious complications such as heart failure, cardiac arrest, and even death. The predictive performance of conventional clinical diagnostic procedures needs improvement in order to assist physicians in detecting risk early on.We aimed to develop a deep tachycardia onset prediction (TOP-Net) model based on deep learning  for early tachycardia diagnosis with easily accessible data.2) acquired continuously by wearable embedded systems, and electronic health records, containing age, gender, admission type, first care unit, and cardiovascular disease history. The model was trained with a large data set from an intensive care unit and then transferred to a real-world scenario in the general ward. In this study, 3 experiments incorporated merging patients’ personal information, temporal memory, and different feature combinations. Six metrics  were used to evaluate predictive performance.TOP-Net leverages 2 easily accessible data sources: vital signs, including heart rate, respiratory rate, and blood oxygen saturation (SpO2) statistical information.TOP-Net outperformed the baseline models on the large critical care data set  when predicting tachycardia onset 6 hours in advance. When predicting tachycardia onset 2 hours in advance with data acquired from our hospital using the transferred TOP-Net, the 6 metrics were 0.965, 0.955, 0.881, 0.937, 0.793, and 0.680, respectively. The best performance was achieved using comprehensive vital signs (heart rate, respiratory rate, and SpOTOP-Net is an early tachycardia prediction model that uses 8 types of data from wearable sensors and electronic health records. When validated in clinical scenarios, the model achieved a prediction performance that outperformed baseline models 0 to 6 hours before tachycardia onset in the intensive care unit and 2 hours before tachycardia onset in the general ward. Because of the model’s implementation and use of easily accessible data from wearable sensors, the model can assist physicians with early discovery of patients at risk in general wards and houses. Tachycardia, a heart rhythm disorder, is defined as an adult resting heart rate that exceeds 100 bpm . Spontan2) [Recently, several hospitals have attempted to utilize wearable devices for continuous monitoring of vital signs such as heart rate, respiration rate, and oxygen saturation (SpO2) ,10. The 2) , machine2) -14. Fork2)  leverage2) . Lee et 2)  develope2)  utilizedWith nonlinear computation and flexible feature extraction, deep learning models show strong performances in representation learning and exploration of unknown information . Researc2 has also been shown as a diagnostic marker of acute heart failure [Cardiovascular diseases are complex and heterogeneous; multiple factors such as genetics, environment, age, and gender can affect the occurrence and severity of cardiovascular disease ,31. Age  failure . HoweverThe aim of this study was to develop a bidirectional long short-term memory (BiLSTM) model—TOP-Net—that is applicable to both intensive care units and general wards , leveragWe leveraged a large data set from the Medical Information Mart for Intensive Care III (MIMIC-III)  and its 2; (2) patients’ personal information from electronic health records, which represents their individual health status when admitted to the hospital, including age, gender, admission type, first care unit, and history of cardiovascular disease.We combined 2 types of data to develop TOP-Net: (1) information from biological sensors (wearable), including heart rate, respiratory rate and SpOBiLSTM , a sequeWe used a BiLSTM algorithm to represent the relationship between the multiple timeseries collected by biological sensors. Data from an observing window before tachycardia onset were used to train the model. Inspired by convolutional-LSTM model , we desi1), aggregation function of autocorrelation (ƒ2), and measurement of discrimination power (ƒ3)—that are commonly used to describe the timeseries characteristics. Herein, we focus on explaining the calculation process of ƒ1, ƒ2, and ƒ3.We explored 8 types of statistical features—mean, standard deviance, slope, quantiles, sum, absolute energy  [where al to l) .We extracted the previously mentioned static patient information which was merged with the statistical features. The concatenated vectors were normalized and input to the BiLSTM model.In this step, TOP-Net determines a real-time risk score that evaluates an individual risk probability of tachycardia onset. When the risk score continuously exceeds the threshold set by the doctor for a period of time, the caregiver is alerted.admissions, patients’ hospital admission information; icustays, ICU transfer (in and out) information; patients, individual birth and death dates; and diagnoses_icd, diagnosis codes during hospitalization. All of the tables can be associated with subject_id, a unique identity of patients. The waveform database includes the header files  and segments of recordings . MIMIC III is a large, publicly available critical care database (version 1.4 ), with 32 monitoring (1 Hz) based on near-infrared spectroscopy, and a posture recognition sensor using a 3-axis accelerometer. These signals are collected and stored in a data logger. The logger has an ultra–low power Wi-Fi module and supports long-term data transmission by relying upon hospital networks. The central monitoring system receives information, processes data, and delivers and displays information. The algorithms deployed on the system included signal quality evaluation, signal processing, real-time abnormal event monitoring and early prediction, and patients’ health assessment, which were packaged as a toolkit (Midas). The accuracy, stability, and effectiveness of our system have been validated in previous studies [The use of general ward data was approved by the ethics committee of the General Hospital of PLA (S2018-095-01). In the general ward, we utilized a SensEcho medical-grade monitoring system, which can monitor patients anytime and anywhere. SensEcho contains 3 parts : a weara studies -46.patient_id, a unique identification of patients similar to subject_id in MIMIC III.Patients admitted to the hospital were assessed by a doctor using the system. Continuous monitoring physiological signals were transmitted to the hospital server and the data in numeric format were acquired based on the waveform processing function in Midas. The clinical information was stored separately in the hospital information system. Data from the different sources were linked  using paDiagnostic tachycardia onset criteria were determined by 3 clinical experts from the Emergency Department, the general ward, and surgical ICU. A tachycardia event was defined as any of the following: (1) heart rate above 100 bpm sustained over 30 minutes; (2) heart rate above 130 bpm sustained over 20 minutes; (3) heart rate above 150 bpm sustained over 5 minutes. The initial timepoint meeting of any of these conditions was recognized as tachycardia onset.2 recordings. The size of the observing window was chosen as 2 hours, which was used to extract the statistical features. The negative sample set was built by extracting information in the observing window with a 1-hour sliding step throughout monitoring for patients without tachycardia. The positive sample set was acquired by selecting the same features in the observing window before the occurrence of tachycardia with a forecast range. To balance the ratio of positive and negative samples, we kept extracting positive samples with a 5-minute delay based on the former (for target replication), which is a method used in a previous study [In the ICU environment, we selected 5699 patients with the following criteria: age over 18 years old, admitted to the hospital and ICU for the first time, monitoring data longer than 14 hours with heart rate, respiratory rate, and SpOus study . The datIn the general ward, we deployed the wearable grade monitoring system a in a ca-4 to 1-2 with an interval of 1-4 and training epochs from 5 to 100 with an interval of 10. The best hyperparameters were determined by minimizing validation loss. We retrained the model using the optimal hyperparameters on the training set, and the performance of the model was assessed on the test set.In the early prediction model, developed from the MIMIC-III data set, predictions (forecast ranges) with TOP-Net were explored from 0 hour to 6 hours with a 2-hour interval. A total of 21 statistical features were included . The siz2.To further investigate the performance of TOP-Net, we designed subexperiments 1, 2, and 3 to obtain a comprehensive assessment. In subexperiment 1, the model was acquired without considering personal information and bidirection memory functions. That is, LSTM and convolutional neural network models were obtained in a total cohort without considering the personal information of patients. The structure of the LSTM was consistent with that of a BiLSTM, and the convolutional neural network model had 2 convolutional layers. In subexperiment 2, conventional machine learning methods, including extreme gradient boosting , multilaPrediction performance was measured with 6 metrics: sensitivity, specificity, accuracy, F1 score, precision, and area under the receiver operating characteristic curve (AUROC).The performance of TOP-Net was validated using the data collected in the general ward  by the SensEcho system. A transferrable model suitable for non-ICU patients was acquired by finetuning the ICU scenario model. The model performance was also assessed with the 6 metrics using 5-fold cross-validation due to the small sample size.We utilized PostgreSQL  to extract the clinical data. All data processing and analyses, model development, and result visualization was performed with Python (version 3.7.1) and CUDA (version 10.0).We leveraged 5-fold cross-validation to select optimal hyperparameters with the training set and assessed the performance of the model on the test set. The hyperparameter values that we selected were learning rate =0.0002, epoch=20, and batch size=64. Although the 95% CI in subexperiment 1 overlaps, TOP-Net has better performance than LSTM and convolutional neural network in each prediction range above 0.5%-1%. Therefore, fusing patient personal information and bidirection memory makes the prediction model more accurate and robust. In subexperiment 2, TOP-Net was consistently superior to the other machine learning models, especially 6 hours before tachycardia onset; TOP-Net performs well .2 (n=15), and statistical features of all vital signs (n=21) are shown. For 2- to 6-hour forecast ranges the model with all of the features input has the best performance with highest AUROC values. The performance is slightly reduced when inputting heart rate and respiratory rate, or heart rate and SpO2. The performance was the worst when including only heart rate statistical features. The statistical characteristics of heart rate play a dominant role in real-time diagnosis. Furthermore, we employed the extreme gradient boosting algorithm to rank the importance of 21 designed features for a forecast range of 6 hours. The top 8 features —were ranked third and fourth, respectively. The respiratory feature resp_c3 was ranked sixth.In features  were hr_We assessed the performance of the model 2 hours before tachycardia onset because the interval between the tachycardia onset and the admission time to the department was short in our scenario of the general ward. Given the limited training data, we used the transfer learning method to finetune the model. The parameters were learning rate=0.0002, epoch=18, and batch size=32. The 5-fold cross-validation was also used to assess the performance and prevent possible overfitting. The retraining results can be seen in In this study, we developed a model using a publicly accessible data set and transferred it to a real clinical scenario. The performance of TOP-Net for predicting tachycardia onset 0 to 6 hours in advance was better than that of the baseline models ; TOP-Net outperformed benchmarks of 2 deep learning models, 2 ensemble, and 1 neural network models for predictions 6 hour in advance.Many continuous monitoring physiological status studies have indicated the deterioration of vital signs occurred more than 6 to 12 hours before serious adverse events . ContinuIn recent years, some novel models for early risk prediction of adverse events have been developed based on electronic health records or physiological signals. Pan et al  utilizedIt is necessary for clinicians to combine a patient’s current symptoms, basic information, and past medical history to diagnose disease severity . For exaRisk prediction is a core task in the artificial intelligence–assisted medical domain. Cardiovascular disease prediction models based on electronic health record analysis have been studied -60. DoctThis study had some limitations. Because SensEcho was deployed in the clinic for only 1 year after our research project began, the limited data collected prevented us from directly developing a general ward model. Moreover, interventions such as beta-blocker medication may affect the occurrence of tachycardia onset and cause it to not be captured by the input features. Electronic health records contain rich information such as laboratory tests, clinical orders, and nursing notes that can characterize a patient’s health status and depict the trajectory of diseases. Further studies involving the integration of multivariate timeseries from electronic health records are expected to improve the prediction performance of tachycardia onset, and more data from the general ward for TOP-Net performance evaluation are required.TOP-Net for real-time evaluation and early prediction of the risk of tachycardia onset, which made it possible to achieve an early forecast of tachycardia onset 6 hours in advance with clinically acceptable performance. TOP-Net was assessed using 6 metrics, 3 subexperiments, different prediction times from 0 to 6 hours. The comparison between the TOP-Net and the other 5 approaches  showed that TOP-Net was superior to the other models. The model with personal information from electronic health records had better performance than those without. The easily accessible input data of the model  and the good performance of the transferred model in the general ward indicated the early prediction of tachycardia onset using wearable sensors is possible in hospitals or houses.


Aedes aegypti virus, has been occurring during the monsoons every year. The prevalence and incidence of dengue fever and death due to its complications have been increased drastically in these recent years in Bangladesh, Philippines, Thailand, Brazil, and India. Recently, dengue had spread in an epidemic form in Bangladesh, Thailand, and Philippines. Although the infection affected a large number of people around the world, there is no established specific and effective treatment by synthetic medicines. In this subcontinent, Malaysia could effectively control its incidences and death of patients using alternative medication treatment mainly prepared from Carica papaya L. leaves along with proper care and hospitalization. Papaya leaves, their juice or extract, as well as their different forms of preparation have long been used traditionally for treating dengue fever and its complications to save patients’ lives. Although it is recommended by traditional healers, and the general public use Papaya leaves juice or their other preparations in dengue fever, this treatment option is strictly denied by the physicians offering treatment in hospitals in Bangladesh as they do not believe in the effectiveness of papaya leaves, thus suggesting to patients that they should not use them. In Bangladesh, 1,01,354 dengue patients have been hospitalized, with 179 deaths in the year 2019 according to information from the Institute of Epidemiology, Disease Control, and Research as well as the Directorate General of Health Services of Bangladesh. Most of the patients died because of the falling down of platelets to dangerous levels and hemorrhage or serious bleeding. Therefore, this paper aims to critically review the scientific basis and effectiveness of Carica papaya L. leaves in treating dengue fever based on preclinical and clinical reports. Thrombocytopenia is one of the major conditions that is typical in cases of dengue infection. Besides, the infection and impairment of immunity are concerned with dengue patients. This review summarizes all the scientific reports on Carica papaya L. for its ability on three aspects of dengue: antiviral activities, prevention of thrombocytopenia and improvement of immunity during dengue fever.Dengue, a very widespread mosquito-borne infectious disease caused by  Aedes egypti, a flaviviridae virus  and clinical study reports. Besides, other plants with anti-dengue effects with the same constituents and toxicity study reports of Papaya leaves extracts or other preparations were also extensively reviewed in this paper.In this study, we tried to depict the dengue situation in the current world, the mechanism of thrombocytopenia in dengue, the role of papaya leaves extracts and their other preparations in alleviating thrombocytopenia, cytotoxic  activities, and immunomodulating properties with comprehensive study reports from preclinical  is listed below  where, before 1970, only nine countries had a severe experience with dengue. Countries with severe cases of dengue are located in the regions of Africa, the Americas, the Eastern Mediterranean, South-East Asia, and the Western Pacific, whereas the most seriously affected countries are in the American, South-East Asian and Western Pacific regions. In 2018, about one to two million cases of dengue incidents were reported in the Americas, South-East Asia and Western Pacific, whereas in 2016, the numbers recorded were 3.34 million , Aug 09.In 2015, the number of cases of dengue incidents in the Americas was 2.35 million, with 1,181 deaths due to dengue . In 2016In the year 2018, a total of 264,262 probable cases of dengue in Brazil were reported with the incidence rate of 126.7 per 100,000 population where in 2017, this incidence rate was at 115.3 cases per 100,000 population , Feb 22.In 2018, 44,825 cases of dengue were reported in Columbia with 176 probable deaths due to dengue and an incidence rate of 179.9 cases per 100,000 population at-risk , Feb 22.In Honduras, 42,346 cases of dengue were observed as of EW 30 in 2019 with a fatality rate of 0.19% and an incidence rate of 462.39 per 100,000 population. Within the same period of the year, a total of 12,081 suspected cases with 47 deaths due to dengue were reported in Guatemala , Aug 09.In the western pacific region, dengue is widespread in Cambodia, Malaysia, Lao PDR, the Philippines, and Vietnam. According to WHO, a total of 3,130 dengue incidents have been suspected in Cambodia during the year 2017 . A totalIn the year of 2017, a total of 80,805 dengue cases with 169 deaths were reported in Malaysia, which was less than that in 2016 where 97,041 cases with 229 deaths due to dengue were reported . In 2018In this region, Bangladesh, India, Indonesia, Sri Lanka, and Thailand have the highest numbers of dengue incidents. The first epidemic of dengue fever in Bangladesh was recorded in 2000 when 5,551 dengue infections were reported from Dhaka, Chittagong, and Khulna cities with 93 deaths . IncreasIn Indonesia, a total of 59,047 cases of dengue fever were reported with 444 deaths due to dengue in 2017. The incidence rate this year was 0.75% . In 2016In the year of 2018, Sri Lanka observed 48,000 cases of dengue fever with 50 deaths . HoweverDue to the increasing prevalence of death in Bangladesh, Philippines, and other Asian countries by dengue, the unavailability of effective treatment in modern medicine and the prospective role of Papaya leaves from its traditional use in managing dengue fever, the current review was aimed at exploring and analyzing its therapeutic potential and safety/risk based on Pharmacological, Toxicological, and Clinical study reports. Besides, this study presents the etiology and pathogenesis of dengue prior to the mechanism of action, pharmacological, and clinical effectiveness of Papaya leaves to better understand it.Aedes aegypti, is known as a Flaviviridae virus and has four serotypes numbered as Dengue Virus-1, Dengue Virus-2, Dengue Virus-3, and Dengue Virus-4 that stimulate the production of different antigens  is a cytokine that is responsible for regulating the production of megakaryocytes. TPO cytokines are produced in the response of low platelets, which bind with the TPO receptor, activate JAK and STAT pathways as well as stimulate the production of megakaryocytes . TPO levMolecular mimicry between viral proteins such as NS1, prM, and E with host platelet, endothelial cells and blood clotting molecules play a key role in the direct destruction of platelets  through In addition, dengue infection causes endothelial cells to activate, which in turn causes platelet adhesion and activation as well as express P-selectin on its surfaces . The expPlatelet dysfunction has been reported in many investigations. DV has been reported to activate platelets and suppress platelet aggregation . PlateleSeveral activities of platelets are influenced by a dengue infection. Anti-inflammatory properties have been observed to be exerted by the activation of platelets due to dengue infection. Activation of platelets causes the interaction of CD40 and CD40L, leading to an increase production of IL-10 and suppression of TNF-α by monocytes . IL-10 hDisseminated intravascular coagulation (DIC) is activated during dengue infection as DENV activates fibrinolysis and coagulation as well as changes in coagulation and fibrinolytic parameters . It has +CD16+ monocyte even if DENV does not cause platelet infection directly  responsible for antithrombocytopenic activity of C. papaya L. leaves are alkaloids, particularly the “carpaine” alkaloid, rather than phenolic compounds (p < 0.001) and NS1 proteins (p < 0.001) was observed in DENV-infected THP-1 cells after aqueous extract of the plant leaves was administered. In the study, IFN-α expression was reported to be 1.6-fold higher in DENV-infected THP-1-cells treated with the leave extract. Significant reduction in erythrocyte damage and hydrogen-peroxide-induced lipid peroxidation (p < 0.001) has been also observed after treatment with papaya leave extract  in platelet aggregation has been observed in platelet-poor plasma collected from dengue patients after treatment with papaya leaves  and Dd2 strains (IC50 2.19 ± 0.60 μg/ml) (Treatment of dengue infected AG129 mice with papaya leaf juice for three consecutive days after 24h of dengue virus inoculation resulted in increased plasma CCL2/MCP-1 level during the peak of viremia . No effea leaves . Carpain in vivo . Carpain0 μg/ml)  due to aAedes aegypti, it was found that the 2nd and 4th stage mosquito larvae had 83 and 86% mortality, respectively, against aqueous extract of papaya seeds. Meanwhile, the mortality percentage of 2nd and 4th stage larvae was 96 and 100%, respectively, against ethanolic extract of the seeds   . The moraya peel .in vitro study model, aqueous extract of papaya leaves have been found to reduce the production of IL-2 and IL-4 and enhance the production of IL-12p40, IL-12p70, IFN-γ, and TNF-α  induced allergic asthma (p < 0.05) isopentenyl pyrophosphate induced TNF-α production in LPS-induced dendritic cells  in platelet count has been observed within five days of therapy sessions, for three times a day, in the intervention group treated with the papaya leave extract tablets  for three times a day (p < 0.01) platelet count was observed (p < 0.01) in platelet count has been also reported in a study where 30 patients with dengue fever associated thrombocytopenia were given Carica papaya L. leaf extract (CPLE) (p < 0.001) in platelet count was observed in the group treated with the juice  . Venugopt (CPLE) . In an ihe juice .A 23 year-old male dengue associated thrombocytopenic patient has been reported to have increased platelet count with every administration of the extract. Platelet count increased from 28000/micro liter to 138000/micro liter by the end of the treatment duration of five days . In anotp = 0.005]). These extracts were also found to decrease the duration of hospital stay   .p = 0.15) in children aged one to 16 years old with dengue and platelet counts from 1.5 × 105 per μL to 50 × 103 per μL. In the study, 30 children were divided into control and test groups where the test group received 1,100 mg tablet (>12 years), syrup of (275 mg/5ml) 10 ml (between 6 and 12 years) and 5ml for (<6years), which were given thrice daily for five days along with routine symptomatic treatments (p < 0.01) in platelet count has been reported in a study where each dengue patient of 100 cases was either in the study or the control group. The study group was treated with papaya leaf extract at the dose of 500 mg thrice a day while the control group received placebo capsules in the same frequency for five consecutive days (p < 0.05) in WBC and platelet counts  (p < 0.05) (eatments . A signiive days . In an it counts . Similart counts . Papaya t counts . In a stt counts . In a stctively) . In a st < 0.05) .Several mechanisms  have beeCarica papaya L. leaves has been found to inhibit NS2B-NS3 serine protease, which is essential for dengue viral assembly  leaf on fingerlings of African catfish CLarias gariepinus was observed, behavioral changes such as air gulping, erratic swimming, discoloration, loss of reflex, and skin peeling as well as a decrease in packed cells volume (PCV), hemoglobin (Hb), red blood cell (RBC), mean corpuscular haemoglobin concentration (MCHC), and an increase in the mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were recorded in a time-and dose-dependent manner. The extract was given for four days at doses of 4.40, 8.80, 13.20, 17.60, and 22.00 ml/L and compared with the control group. In terms of behavioral change, gasping occurred in all five doses and other behavioral changes occurred in doses ranging from 13.20 to 22.0 ml/L. Considerable hematological changes occurred especially at the doses of 13.20, 17.60, and 22.00 ml/L when compared with control. LC50 value was found to be at a concentration of 10.9 ml/L  in hemoglobin (HGB), hematocrit (HCT), red blood cell (RBC), and total protein suggesting dehydration was observed  in male Wister rats. In the study, normal sperm count and biochemical profile were observed at the dose of 100 mg/kg body weight 50 of the virus  behind the role of papaya leaves to improve the platelet count. If the exact mechanisms are known, papaya leaves extract could be optimized for better effectiveness as well as therapeutic preparations that could be formulated targeting the same pathway as papaya leaves. Also, the anti-thrombocytopenic potential of papaya leaves is not very widely known. Since there is no randomized controlled clinical trial with significant patients available, its use is still neglected and not approved by the authorities including US-FDA. Thus, more clinical studies with a large number of patients should be conducted for the clinical and therapeutic role of papaya leaves as a curative agent in dengue to confirm and establish its use for dengue patients. Also, approved marketed preparation of papaya leaves in different dosage forms such as extract, syrup, tablet, etc. should be made available for dengue patients as a supplemental remedy, especially in countries with frequent dengue cases, to control the severity of this viral fever with multiple clinical complications and no remedy.Considering the four varieties of viruses responsible for dengue fever, one person can suffer from dengue multiple times although being infected once with a particular serotype provides long term immunity against that serotype. Dengue infection arrays a wide range of symptoms including fever, rash, extreme muscle pain, and headache as well as clinical manifestations such as multiple organ failures and thrombocytopenia, and an abnormal lowering of platelet count. Thrombocytopenia is associated with severe cases of dengue and most cases of fatality due to this infectious fever. Dengue virus reduces platelet count of the host directly or indirectly by changing the environment of bone marrow, influencing different factors involving platelet production, destroying and replicating into platelets and reducing the circulating platelets as suggested by different investigations. Though dengue has been observed to turn into epidemics every year in many countries with numerous cases of fatalities, preventive or curative treatment has yet to be found. Nonetheless, papaya leaves have been seen effective to improve the platelet count in investigations on dengue patients and Currently available products based on Papaya leaves are lacking the standardization, dose optimization, therapeutic regimen, extensive/long term toxicity study reports, and proper approval of products from the regulatory authority. However, further research and monitoring on Papaya leaf-based products are required to overcome the above limitations.Carica papaya L. leaves for the effective and safe treatment against dengue fever and to save thousands of lives around the world.We performed this comprehensive review to enlighten the scientific community about the therapeutic potential of Papaya leaves so that further analysis and investigations can be made to establish Papaya leaves preparations as a potential possible therapeutic agent for the effective and safe treatment of dengue fever; thus to prevent the death of many lives due to dengue fever, especially in developing countries. Therefore, extensive clinical trials and further investigations for the establishment of proper mechanism of action of Papaya leaves preparations along with the confirmation of its reported and identified bioactive compound(s) are required for the establishment of different preparations of


Norway has not implemented universal varicella vaccination, despite the considerable clinical and economic burden of varicella disease.® or Varilrix®) and a quadrivalent vaccine against measles-mumps-rubella-varicella (ProQuad® or PriorixTetra®), with the first dose given with a monovalent vaccine at age 15 months, and the second dose with either a monovalent or quadrivalent vaccine at either 18 months, 7 or 11 years. Costs were considered from the perspectives of both the health care system and society. Quality-adjusted life-years saved and incremental cost-effectiveness ratios relative to no vaccination were calculated. A one-way sensitivity analysis was conducted to assess the impact of vaccine efficacy, price, the costs of a lost workday and of inpatient and outpatient care, vaccination coverage, and discount rate.An existing dynamic transmission model of varicella infection was calibrated to age-specific seroprevalence rates in Norway. Six two-dose vaccination strategies were considered, consisting of combinations of two formulations each of a monovalent varicella vaccine (Varivax® at 15 months and 7 years (payer perspective) and two doses of Varivax® at 15 months and 18 months .In the absence of varicella vaccination, the annual incidence of natural varicella is estimated to be 1,359 per 100,000 population, and the cumulative numbers of varicella outpatient cases, hospitalizations, and deaths over 50 years are projected to be 1.81 million, 10,161, and 61, respectively. Universal varicella vaccination is projected to reduce the natural varicella incidence rate to 48–59 per 100,000 population, depending on the vaccination strategy, and to reduce varicella outpatient cases, hospitalizations, and deaths by 75–85%, 67–79%, and 75–79%, respectively. All strategies were cost-saving, with the most cost-saving as two doses of VarivaxAll modeled two-dose varicella vaccination strategies are projected to lead to substantial reductions in varicella disease and to be cost saving compared to no vaccination in Norway. Varicella, or chickenpox, is caused by the varicella zoster virus and is one of the most common infectious diseases in children. Varicella infections are characterized by a generalized pruritic vesicular rash, which, while usually mild, can result in serious complications and, in rare cases, death . Almost Varicella vaccines are well-tolerated and effective in preventing varicella. Where introduced, universal childhood vaccination has led to significant declines in varicella disease –7. GlobaVaricella vaccination is recommended only for selected risk groups in Norway  and is nTo support national vaccination policy decisions, the study objective is to quantify the long term clinical and economic impact of universal varicella vaccination in Norway under different vaccination scenarios using a dynamic transmission model.The deterministic, compartmental model of varicella infection used for this analysis has been described in detail elsewhere . It is aThe model has several components: demographic, epidemiologic, vaccination, and economic. The demographic component of the model defines the age structure of the population simulated and describes how persons enter, age, and exit the model . DemograAlso, as in the previous work, we incorporated the effects of exogenous boosting on herpes zoster incidence , and theThe model is calibrated to the population of Norway using population-specific initialization parameters, including the age distribution of the population, age-specific fertility, varicella seroprevalence (in the absence of vaccination), and herpes zoster incidence by age. Age-specific incidence rates of varicella were obtained by finding a best fit to the seroprevalence rates . The ageThe varicella vaccination component of the model implements a structure similar to that described elsewhere . VaccineThe economic component of the model calculates healthcare usage and costs associated with varicella infection, with costs expressed in NOK . The time horizon was 50 years . Direct Six two-dose vaccination strategies were modeled, labelled A through F, with the first dose at 15 months and the second dose at 18 months, 7 years, or 11 years . The ageA one-way sensitivity analysis was conducted from the societal perspective, in which variations in economic parameters and in parameters related to vaccine properties were analyzed. In the one-way sensitivity analysis, vaccine price and the costs of a lost workday, and of outpatient and inpatient care, were varied by ± 20%. Vaccination coverage for doses 1 and 2 were varied by ± 5% . VaccinIn the absence of varicella vaccination, the projected annual incidences of natural varicella and wild-type herpes zoster in Norway are 1,359.4 per 100,000 population and 303.8 per 100,000 population, respectively.With universal vaccination, depending on the vaccination strategy, the natural varicella incidence rate at 50 years is projected to vary from 48.4 to 58.9 cases per 100,000 population  and the In the absence of vaccination, the cumulative numbers of varicella outpatient cases, hospitalizations, and deaths over 50 years are projected to be 1.81 million, 10,161, and 61, respectively. At 50 years, universal vaccination is projected to have reduced outpatient cases by 75%-85%, hospitalizations by 67%-79%, and deaths by 75%-79%. Strategies D and E result, respectively, in the lowest and highest percent reductions in outpatient cases, hospitalizations, and deaths .The cost-effectiveness of the six varicella vaccination strategies compared with no vaccination is graphed in In the one-way sensitivity analysis of strategy A , the cosIn this study, all modeled two-dose vaccination strategies are projected to reduce natural varicella incidence by 95% to 96% relative to no vaccination in Norway. At the 50-year time horizon, all strategies are projected to reduce outpatient cases and hospitalizations by 67% to 85%, and deaths by 75% to 79%. A varicella vaccination program in Norway under all modelled strategies would be cost saving compared to no vaccination from the perspectives of both the healthcare system and society. From the societal perspective, most of the projected cost savings are due to reductions in lost workdays. Among the vaccination strategies, those with two doses of V-MSD, with the second dose at 18 months or 7 years , were the most cost saving from the perspectives of both the healthcare system and society. The least impactful strategy in terms of cost-effectiveness and resource utilization was strategy D (V-GSK at 15 months and MMRV-GSK at 11 years).These results are consistent with applications of the same dynamic transmission model to other countries , 27, 39.The effect of universal childhood varicella vaccination in Norway was modeled in a previous analysis . This moMost (73%) of the annual healthcare costs of varicella virus infection in Norway are associated with herpes zoster, primarily due to hospitalization costs . Our modAs discussed above, the present model incorporated an exogenous boosting effect. Models of universal varicella vaccination that include exogenous boosting generally predict a transient increase in herpes zoster in adults , 41–43. In observational studies, the relationship between circulating varicella virus and herpes zoster epidemiology is unclear. A review of studies of different types was equivocal, in that the magnitude of any exogenous boosting effect was indeterminate . More reFrom the perspectives of both society and the healthcare system, strategies with two doses of V-MSD (strategies A and E) were the most effective in reducing costs and making gains in quality of life. The cost of a caregiver’s lost work time had the greatest effect on costs from the societal perspective, though varying this cost by ± 20% resulted in only a ± 5.0% change in the ICER with strategy A. Varying vaccine price by ± 20% resulted in a ± 2.4% change in the ICER for strategy A from the societal perspective.This analysis is subject to several limitations. The modeled vaccine administrations at 15 months, 7, and 11 years match Norway’s existing childhood vaccination program . We modeled a strategy that included a vaccination at 18 months (strategies E and F). However, there is no existing immunization visit at 18 months, which was the modelled timepoint for the second vaccine dose in strategies E and F. Any costs associated with this currently unscheduled visit were not included in the model, thus, the actual costs may be higher than estimated in the present study. Due to a lack of country-specific data for caregiver costs, the model employs published estimates from other countries. This is a conservative estimate of ICERs as the analysis utilizes list price for vaccines and not tender price. There are several other potential limitations to the modeling approach taken . The modIn conclusion, universal childhood varicella vaccination with a two-dose schedule is projected to lead to substantial reductions in varicella incidence and mortality in Norway, with associated reductions in healthcare use and in direct and indirect costs. All modeled two-dose varicella vaccination strategies are cost saving, but the most cost-effective strategies are with V-MSD at both the first and second doses, with the second dose administered at either 18 months or 7 years . PolicymS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(ZIP)Click here for additional data file.