Metabriggsia ovalifolia W. T. Wang is one of the first-class national protected plants endemic to Karst areas in China. In this study, the complete chloroplast genome was sequenced using Illumina pair-end sequencing data. The chloroplast genome was 153,333 bp in length, consisting of a pair of inverted repeats , separated by a large single copy region  and a small single copy region . The chloroplast genome encodes 131 genes, including 87 protein-coding genes, 36 transfer RNA, and eight ribosomal RNA genes. The overall GC content of the chloroplast genome was 37.55%. M. ovalifolia phylogenetic analysis indicated close relationship with Oreocharis mileensis of Gesneriaceae family. These genomic resources will be helpful for conservation of this endemic plant species. Metabriggsia W. T. Wang is an endemic genus of China belongs to subfamily Didymocarpoideae and Trichosporeae tribe of Gesneriaceae, consists of only two species. Metabriggsia ovalifolia W. T. Wang is a perennial herb, endemic to Guangxi and Yunnan provinces of China , an emergency rescue plan for threatened species in China  as a reference genome following Geseq and further manually verified and visualized in Geneious Prime v.2019.1.3 , Chinese Academy of Sciences, Guilin, Guangxi, China and voucher specimen (MYP2020010) was deposited in the Herbarium of Guangxi Institute of Botany (IBK). These plants were introduced from Libo County, South of Guizhou province, China. Genomic DNA was isolated using Tiangen plant genomic DNA kits  and sequenced on the Illumina Hi-Seq 2500 platform. A total of 6 Gb of 150 bp pair end raw reads were obtained and used for assembling the chloroplast genome using GetOrganelle pipeline  region of 84,381 bp and a small single copy (SSC) region of 18,056 bp, with a pair of inverted repeat (IR) regions of 25,448 bp each. The overall GC content of the chloroplast genome was 37.55%. In total, the chloroplast genome contained 131 genes, consists of 87 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes.M. ovalifolia and previously published 11 chloroplast genomes from the Gesneriaceae family were used to investigate the phylogenetic relationships using Streptocarpus ionanthus as outgroup. A maximum-likelihood tree with 1000 bootstrap replicates was inferred by RAxML-HPC2 Workflow on XSEDE (v.8.2.12) using the CIPRES online portal (Miller et al. M. ovalifolia grouped with Oreocharis mileensis. Our results provide fundamental genetic resources for conservation and future evolutionary studies of this endemic species of China.The complete chloroplast genome sequence of


The acute phase reaction is a systemic response to acute or chronic inflammation. The serum level of C-reactive protein (CRP) is the only acute phase biomarker widely used in routine clinical practice, including its uses for prognostics and therapy monitoring in cancer patients. Although Interleukin 6 (IL6) is a main trigger of the acute phase reactions, a series of acute phase reactants can contribute . However, the experience from patients receiving intensive chemotherapy for hematological malignancies has shown that, besides CRP, other biomarkers  also have altered systemic levels as a part of the acute phase reaction in these immunocompromised patients. Furthermore, CRP and white blood cell counts can serve as a dual prognostic predictor in solid tumors and hematological malignancies. Recent studies also suggest that biomarker profiles as well as alternative inflammatory mediators should be further developed to optimize the predictive utility in cancer patients. Finally, the experience from allogeneic stem cell transplantation suggests that selected acute phase reactants together with specific markers of organ damages are useful for predicting or diagnosing graft versus host disease. Acute phase proteins may also be useful to identify patients (at risk of) developing severe immune-mediated toxicity after anticancer immunotherapy. To conclude, future studies of acute phase predictors in human malignancies should not only investigate the conventional inflammatory mediators  but also combinations of novel inflammatory parameters with specific markers of organ damages. Serum levels of C-reactive protein (CRP) have been used for several decades as a marker of the acute phase reaction that can be induced both by acute and chronic states as a systemic reaction to inflammation, injury, or infections ,2. HowevThe acute phase reaction is characterized by increased levels  of several proteins in response to inflammation, infection, or tissue injury, but the name is misleading because the reaction can also be seen in chronic diseases with fluctuating conditions , or be a chronic or long-lasting response that is maintained during the chronic disease ,6. The rThe acute phase reaction was initially described as a reaction involving various serum proteins, including increased levels of several complement factors, coagulation factors, antiproteases, transport proteins, CRP, serum amyloid A, ferritin, as well as proinflammatory cytokines ,3. HowevAccording to the definition of acute phase proteins many cytokines, soluble cytokine receptors, and soluble adhesion molecules can be classified as acute phase proteins. The final acute phase reaction  reflects both the initial step/mechanisms for induction of the reaction (see above) and altered serum levels of associated proteins responding to the inducers. However, the induction and the reaction proteins are overlapping, as can be illustrated by the CRP example. Serum CRP production and release can be induced by IL6, IL8, and TNFα , but at The complexity of the acute phase reaction is illustrated by the observations from patients with febrile chemotherapy-induced neutropenia . Even thA similar complexity of the acute phase reaction is also observed for immunocompetent individuals. This is illustrated by a recent study investigating patients with sepsis . The autThe molecular structure and the biological functions of CRP have been described in several recent reviews ,28,29,30vβ3 integrins, respectively. Monomeric CRP effects can also be mediated via lipid rafts, and integrins than αvβ3 can also bind monomeric CRP. Furthermore, recent studies suggest that CRP can also exist as fibril-like structures . Th. Th43]. Inflammatory biomarkers have a prognostic impact on several malignancies; mainly solid tumors see . HoweverAllogeneic stem cell transplantation is used in the treatment of aggressive hematological malignancies, and antileukemic effects can then be mediated both as a leukemia-specific graft versus leukemia reactivity and as a consequence of the general GVHD reaction directed towards antigens that are expressed both by normal and malignant recipient cells ,133. ThuWill CRP have a role in future cancer treatment? In our opinion, the answer is yes. CRP is already a useful biomarker especially for prognostication and diagnosis of complications to anticancer treatment. However, we believe that CRP may become even a more useful diagnostic or prognostic biomarker if it can be used in combination with other acute phase markers as a part of an acute phase profile. The biological functions of CRP are now being explored, and some studies suggest that CRP may even become a therapeutic target for anti-inflammatory treatment .Recent reviews of epidemiological studies suggest that inflammation can predispose to cancer, and targeting of inflammation and the molecular mechanisms involved in the inflammatory processes may therefore represent a possible strategy for cancer prevention ,135. ThiWhat can we then learn from the hematological experience with regard to the acute phase reaction in clinical oncology? First, broad acute phase (cytokine) responses can be detected even in severely immunocompromised patients receiving the most intensive anticancer therapy. Second, hematological biomarkers of inflammation  can be used together with molecular acute phase biomarkers for prognostication and therapy monitoring in cancer patients. Alternative markers should be further investigated as markers of the acute phase reaction, including systemic levels of alternative single molecules, biomarker profiles, and monocyte subsets. The optimal biomarker or biomarker combination to use for prognostication or therapy monitoring will probably differ between various malignancies and possibly also between patient subset with the same malignant disease. Third, the GVHD experience shows that based on an initial screening of several inflammatory biomarker it is possible to identify a few optimal biomarkers and an optimal time point for prognostic evaluation of patients. A similar strategy should be tried to identify relevant biomarkers to use in patients receiving anticancer immunotherapy. A major goal would then be to try to identify proinflammatory biomarkers that can distinguish between the likelihood of an anticancer effect versus the risk of severe immune-mediated complications. The possibility of combining inflammatory biomarkers and organ-specific markers should also be considered in anticancer immunotherapy.


Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt. Habowski et al. develop a sorting protocol for purification and comparative analysis of mouse colon stem cells and their progeny. By proteomic and transcriptomic analysis, they determine that lineage commitment is accompanied by a greater change in mRNA splicing and polyadenylation than in gene expression and they characterise signalling pathways involved in differentiation. Constant replacement maintains homeostasis and proper absorption of water and nutrients, but the fast timescale of birth-to-death places great demand on both stem and daughter cells. Stem cells are by necessity non-quiescent and rapidly dividing, and progenitor cells exhibit rapid loss of stemness and commitment to differentiation. Multiple studies have shown how absorptive and secretory cell types can respond to wounding by de-differentiation and repopulation of the stem cell compartment5. Although this de-differentiation process occurs promptly, it is unknown if these reverse changes in cell state and gain of stemness occur on a similar rapid timescale as loss of stemness.The intestinal crypt is a good model for studying how stem cells support a rapidly renewing tissue. Crypts are invaginating structures of single-layer epithelium in which stem cells reside in a supportive niche at the base where they produce daughter cells (progenitors). Progenitors move up the crypt to differentiate and replace mature cells that are dying at the mucosal surface—a process with an average lifespan of only 4–5 days6. The progenitor’s first round of cell division and commitment to either an absorptive (AbsPro) or secretory (SecPro) lineage happens nearly simultaneously with entrance into this zone. These changes occur within minutes-to-hours of each other, suggesting that loss of stemness and choice of cell lineage are connected and directed by processes that occur on this timescale.Quantitative imaging and lineage-tracing tools have shown that newly produced progenitor cells lose stemness as they move from the stem cell niche into a compartment called the transit amplifying zone (TAZ)7. In addition, Notch signaling balances commitment to either the absorptive or secretory lineage through lateral inhibition signaling4. There has been a longstanding expectation that stem cells are defined by a unique transcriptome and that loss of stemness and lineage commitment are similarly defined by unique signatures. However, although signaling systems are capable of altering transcription, it is not known how much of the rapid changes in cell state are due to the turning ON/OFF of signal-targeted gene programs versus more immediate processes of co- and post-transcriptional processing, such as alternative mRNA splicing and alternative polyadenylation (APA)10. Each of these processes can quickly modify the nascent transcriptome and its attendant proteome by altering the coding sequences of mRNAs, the localization or interactions of mRNA and proteins, or by changing protein abundance through alterations in mRNA stability and/or protein translation rates14.Several signal transduction systems are important for the early changes in cell state. A decrease in Wnt signaling and activation of the unfolded protein response (UPR) correlates with loss of stemness16. For example, the transgenic stem cell lineage marker Lgr5-EGFP enables purification of GFP-bright stem cells, but a mosaic expression pattern of the transgene in the intestine has made it difficult to confidently separate daughter cells from GFP-negative stem cells and differentiated cells18. Single-cell RNA-sequencing captures the diversity when analyzing mixed cell populations and has been useful for defining intestinal lineage trajectories and diversity of mature cells 23. However, its low sequencing depth misses moderate-to-lowly expressed transcripts and mRNA splicing and polyadenylation analyses are not yet reliable. Therefore, the transcriptome and proteome basis for loss of stemness and early commitment is unknown.To study how transcription and post-transcription processes contribute to stemness and differentiation, it is necessary to separate stem cells, daughter cells, and their differentiated progeny. Multiple cell sorting protocols have been optimized to isolate stem cells, but each lack resolution of these three cell typesHere, we developed a new cell sorting protocol for purification and comparative analysis of colon stem cells, their immediate daughters , and their differentiated cell types, including tuft cells, EEC, and enterocytes (Ent). The protocol can be used with non-transgenic mice of any strain and importantly, when coupled to bulk RNA sequencing and mass spectrometry-based global proteome profiling, can provide a deeper analysis of cellular transcriptomes and proteomes. Using this protocol, we found that while the transcriptome and proteome of each cell type are generally correlated, deeper analyses of the bulk RNA-seq data reveal that loss of stemness and lineage commitment are accompanied by a greater change in mRNA splicing and polyadenylation than in gene expression, a pattern that largely resolves as progenitor cells mature. Sequencing analysis also enabled higher resolution of signal transduction systems , environmental sensing pathways, and patterns of lineage distinction, including prostaglandins and Fgf signaling pathways. These patterns were seen at both the RNA and protein level and are likely key to understanding the processes of homeostasis, namely: (i) loss of stemness, (ii) lineage commitment, and (iii) signaling connections between mature cell types. We relate how these findings are relevant to the earliest events that happen during loss of stemness and we highlight ways in which mature cells might de-differentiate to re-acquire the state of stemness.16  and from mature enterocytes . Secretory progenitors were identified as SecPDG as this population contains mostly secretory progenitors and deep crypt secretory cells, with a possible minor contribution of goblet cells, a cell type that is largely missing from our isolated cells . For each of the isolated cell types we identified strongly associated biomarkers, including novel highly expressed proteins confirmed via proteomic analysis and immunohistochemistry  protocol with new methods for global proteome analysis of small numbers of cells <200 cells) enabled us to compare the transcriptome and proteome for all six cell populations00 cells Lgr5, Smoc2, Cd44, Cdca7, Notch1, and Rnf43, which show elevated expression in stem cells, but are well expressed in the other cell populations  to the final steps of differentiation  and Wnt signaling targets , but newly identified markers are connected to epigenetics processes , regulation of the cell cycle  are highest in stem cells, but interestingly, their protein products are readily detectable in differentiated cells, thus highlighting inconsistencies between mRNA and protein biomarkers of proliferation , are shown in Fig. Although these data suggest that colon crypt stem cells have few specific markers, our analysis identified a set of 16 highly enriched mRNAs that distinguish stem cells from all other cell populations  events  has known functions in adhesion as well as Wnt and Notch signaling , Wdhd1 (DNA replication), and Cby1 ) show significant increases in distal polyA choice and lengthening of the 3′-UTR  has four RRM RNA-binding domains and functions in splicing and transcription regulation, including suppression of Notch and activation of Wnt signalingRNA Fig. . Delta-cing Fig. . A C-terUTR Fig. . InteresUTR Fig. .47. Consistent with this, our analysis revealed that exon 25 has the highest inclusion in stem cells, and the lowest in SecPDG and Ent  are present in the crypt with Itga6 isoform A (inclusion of exon 25) being more abundant in the base of the crypt, and isoform B (skipping of exon 25) being more abundant near the top of the crypt  that activate expression of lineage-specific genes. Significantly, in addition to common splicing and APA changes in both lineages, our analysis detected 469 and 207 lineage-specific changes in alternative mRNA splicing , whereas nearly half of the Eif4a2 mRNA in the stem, AbsPro, and Ent populations encodes a truncated protein isoform  is largely missing in AbsPro mRNA (34% inclusion) but mostly present in SecPDG mRNA (71% inclusion) , whereas polyadenylation of Ihh mRNA is shifted to a more distal site in SecPDG (Wnt enhanced). Alternate processing of these genes could potentially contribute to the skewing of Wnt and Notch signaling activities in cells55.Although the functional consequences of many distal polyA choices are not known, two striking examples of changes in polyA choice in absorptive versus secretory lineage are shown in Fig. Dll1 and Dll4, as well as a third ligand Nov  and lineage commitment steps , but also identified expression patterns suggesting additional autocrine/paracrine signaling that could impact lineage choice. For example, Notch signaling is known to direct lineage choice via lateral inhibition signaling in small intestinal crypts. Our RNA-seq data indicate that secretory lineage  cells express high mRNA levels for Notch ligands Nov Fig. . Stem an7. Although our analysis indicates activation of UPR in colon crypt progenitors, we observe that UPR signaling is lineage-skewed and most active in secretory populations  were detected in the secretory lineage, the downstream target genes for two of them—Atf6, Ern1 (Hspa5 and Hsp90b1) displayed the highest expression in this lineage. These targets promote ER expansion and survival from stress. Taken together, the increased expression of sensors and downstream targets in the secretory lineage suggests a sensitization to UPR stress that might play a role in lineage choice and/or stabilization. Interestingly, ER stress can slow migration, consistent with recent observations that secretory progenitors migrate up the crypt at a slower rate than absorptive cells58.UPR directs cellular responses to ER stress such as growth arrest, apoptosis and/or survival, and can trigger loss of stemness as intestinal stem cells exit their nicheons Fig. . Active lls Fig. 57. Furth60. Our transcriptional profiling indicated that only a few Fgf ligands are expressed by the epithelia , and predominantly by EECs. Fgf receptors, in contrast, are broadly expressed across the different cell types with Fgfr3 detected at the highest level in the secretory lineages of SecPDG and tuft , which converts arachidonic acid into prostaglandin H2  and the prostaglandin transporter Abcc4 are expressed in all cell types  for prostaglandins and an enzyme that degrades these molecules (Hpgd), suggesting that enterocytes might act as sinks for prostaglandin-mediated signals.Gene expression analysis also uncovered potential for lineage-specific autocrine/paracrine activities in prostaglandin signaling Fig. . ConsistPDG Fig. . EnterocTcf7 and Tcf7l2 are the predominant family members in stem cells, but Tcf7l1 and Tcf7l2 are even more highly expressed in progenitor and mature populations along with negative regulators, such as the Tle repressors  are broadly expressed, whereas the direct negative regulator Insm1 is elevated in EECs  have a well-established role in maintaining the intestinal stem cell niche and allowing for differentiation of progenitor cells upon exit from that niche. Wnt transcription factors ors Fig. a–d, 27a.ECs Fig. 62. InterEnt Fig. . BindingEnt Fig. . Express66, yet their expression patterns suggest that there is potential for additional cross-talk signaling with the rare tuft and EEC cell types. Previous work has suggested that Kit , the receptor for kit ligand  that directs cell survival pathways in stem cell niches, is specific for Paneth cells in the small intestine and DCS/goblet cells in the colon65. Although we observed highly expressed Kit mRNA in SecPDG and tuft populations  and secretory (Kit receptor) cell populations suggest that Kit could be an intra-cryptal signal from the absorptive lineage to tuft cells. This is mainly a soluble signal since the dominant spliced isoform of Kitl (inclusion of exon 6) is the  secretable isoform , and its negative regulators . Other Egf receptor family members, Erbb2, and Erbb3, are highly expressed in all cell types including stem cells  are robustly expressed in all colon crypt populations. These expression patterns show that subsets of known stemness regulators are a broadly shared feature of all intestinal crypt cell types.Finally, the intestinal crypt is known for its impressive plasticity to rapidly regenerate stem cells at the base of wounded crypts. Multiple studies have shown that the epithelial cell populations, including Ent, tuft, EEC, and progenitor cells have the capacity to de-differentiate into stem cells and restore the nicheors Fig. , includiors Fig. . ImportaThis study presents a high-resolution cell sorting protocol for mouse colon crypt epithelia, an advance that permitted deep RNA-seq and proteomics analyses of multiple cell types including progenitor cells for absorptive and secretory lineages Fig. . A key fGlobal analysis of gene expression in all six sorted cell populations enabled a more precise identification of stem cell markers Fig. , reveali6. The shared changes in RNA processing in the AbsPro and SecPDG populations might therefore represent events that occur during these earliest steps of transition. Indeed, changes in splicing and polyadenylation were detected in regulators of Wnt, Notch, and other known regulators of intestinal stem cells. For example, alternative RNA splicing of delta-catenin mRNA removes an exon that encodes a Numb binding domain in progenitor populations, and it removes an exon for RNA binding domain in Split ends (Spen) mRNA in stem cells  cell leaves the stem cell niche and enters the TAZlls Fig. . Numb isSUMOylation is the most significant ontology category associated with commonly processed mRNA targets Fig. . SUMO prPtger4  are most highly expressed in the absorptive lineage, implying that prostaglandins have different roles in the two lineages (Supplement 25d)72. The overall expression pattern of prostaglandin genes suggests that tuft cells could provide prostaglandin precursors to all cryptal cell types for conversion and whole-cryptal production of PGE2 , a potential form of “crowd-sourcing” of a signal known to be important for responding to wounding Cle/J, Stock Number 008875)24, agouti mice , and NSG mice , which were purchased from The Jackson Laboratory. FVB/NCrl mice (Strain Code 207) and BALB/cAnNCrl mice (Strain Code 028) were purchased from Charles River. A detailed step-by-step procedure is available through Nature Protocol Exchange77. In brief, mouse colons (cecum to rectum) were removed, flushed, and linearized. Tissue was dissociated at a slow rotation at 4 °C for 1 hr in a solution of 2 mM EDTA and 10 µM Rock inhibitor. Aggressive shaking of the tissue solution, filtering (using 100 µm followed by 40 µm filters), and centrifugation (500–1000 × g for 5–10 min at 4 °C depending on the step) were performed to isolate single cells. Data in the Supplementary Fig. All mouse work was performed in accordance with NIH guidelines and was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, approval numbers AUP- 17-053. Male C57BL/6 N(NJ), obtained from the KOMP repository, mice aged 5–7 weeks were used unless otherwise noted . Following a wash step, cells were incubated for 30 min in FACS buffer ) with the following pre-conjugated validated flow antibodies: CD45-BV510 , CD31-BV510 , CD326-eFluor450 , CD44-PerCP-Cy5.5 , CD24-PECy7 , and CD117-APC-Cy7 . Following wash steps, cells were resuspended in FACS buffer and Live/Dead Aqua (Thermo Fisher # L34957). An alternative CD45-APC  antibody was used in the Supplementary Fig. Cells were bulk sorted on a BD FACS Aria Fusion using a 100 µm nozzle (20 PSI) at a flow rate of 2.0 with a maximum threshold of 5,000 events/sec. The sample chamber and collection tubes were kept at 4 °C. Following exclusion of debris and singlet/doublet discrimination, cells were gated as demonstrated in Supplementary Fig. RNA was extracted from TRIzol samples using a Direct-zol RNA Micro-Prep kit (Zymo #11-330 M) and associated guidelines. Sorted samples of each cell type were pooled as needed at the start of RNA preparations to ensure a minimum of 2,500 cells per sample. RNA sample quality and concentration was evaluated using an Agilent Bioanalyzer on an RNA high sensitivity pico chip. RNA samples were then pooled as needed to allow 1 ng library preps with Clontech Low Input Pico Kit (Takara #634940). Following confirmation of library quality by Agilent Bioanalyzer DNA high sensitivity chip, a total of 22 samples were sequenced . Samples were multiplexed and sequencing was performed with 100 bp paired-end run on Illumina HiSeq 4000.78. Gene expression significance was determined by DESeq2 Wald P value test with a padj < 0.01 with a minimum mean of 50 normalized counts. Heatmaps and PCA plot were generated in RStudio (version 1.0.153) with R (version 3.6.1) using pheatmap  and plotPCA, respectively, of r-log-transformed (regularized log) DESeq2 data. r-log-transformation is a robust way to transform the count data, used in differential gene expression analysis, to a log2 scale in a way which minimizes differences between samples and normalizes with respect to library size, it is also a standard function for downstream analysis such as clustering or linear discriminant analysis. Bar graphs of gene expression data were generated in GraphPad Prism (version 6.01) with normalized read counts (output of DESeq2) and error bars defining standard deviation. Supplementary Data #Paired-end sequencing reads were trimmed of adapter sequences and analyzed for quality using Fastqc (version 0.11.7). Data were aligned to the mouse genome (UCSC mm10 from Illumina iGenome) using STAR (version 2.5.2a), converted to bam files and merged (samtools 1.3) and read counts were generated using HTSeq . Differential gene expression analysis was done in RStudio (version 1.0.153) with R (version 3.6.1) using default setting of the DESeq2 pipeline for statistical analysis Merged bam files were sorted and indexed (samtools 1.3) for downstream analysis. Alternative splicing was investigated using rMATS Turbo (rMATS.4.0.1) with STAR 2.5.2a, Samtool 1.3, and enthought_python 7.3.2 comparing two cell types at a time using UCSC mm10 gtf. MAJIQ (v1.1) was also run for alternative splicing with anaconda 3–2.0.1 and recommended mm10 ensembl gff3 reference with type = strand-specific followed by VIOLA for visualization. DaPars (v0.9.1) was used for alternative polyadenylation analysis with recommended mm10 UCSC reference files and python 2.7.15, bedtools 2.25.0, R 3.4.1, and the following settings . rMATS significance was defined in three different levels of significance: FDR < 0.05, FDR < 0.01, FDR < 0.01 with ±25% dpsi. Similarly, DaPars significance was defined in three different levels of significance: FDR < 0.05, FDR < 0.01, FDR < 0.01 with ±25% PDUI. Alternative processing gene lists are provided in Supplementary Data #79 (http://pantherdb.org/) and Enrichr81 (https://amp.pharm.mssm.edu/Enrichr/).Gene ontology and gene enrichment analysis of mRNA-seq data were performed on specified gene lists using Pantherg for 10 min at 4 °C to keep the cells at the bottom of the tube to avoid potential cell loss. In all, 2 µL of 0.1% n-Dodecyl β-D-maltoside in 25 mM ammonium bicarbonate was added to each PCR tube with gentle shaking. Intact cells were lysed using sonication five times at 1-min intervals over ice and then centrifuged for 3 min at 3000 × g. Samples were then incubated on a thermocycler for denaturation at 75 °C for 1 h. In all, 1 µL and 2 µL of 10 ng/µL trypsin (Promega) in 25 mM ammonium bicarbonate was added to the PCR tubes at a total amount of 10 ng for <1000 cells and 20 ng for >1000 cells. Samples were digested for overnight (~16 h) at 37 °C with gentle sharking at ~500 × g. After digestion, 2 µL of 5% formic acid was added to the tube to stop enzyme reaction. The final sample volume was reduced down to ~20 μL using SpeedVac and the sample PCR tube was inserted into the Liquid chromatography vial for direct liquid chromatography–mass spectrometry (LC-MS) analysis. The processed samples were either analyzed directly or stored at −20 °C for later LC-MS analysis.Prior to sample processing PCR tubes were centrifugated at 1000 × 6, a maximum ion trap time of 100 ms. Top-10 precursors were isolated with an isolation window of 2, an AGC target of 2 × 105, a maximum ion injection time of 250 ms , and then fragmented by high energy collision with an energy level of 32%. A dynamic exclusion of 30 s was used to minimize repeated sequencing. MS/MS spectra were scanned at a resolution of 17,500.The cell subpopulation digests were analyzed using a commonly available Q Exactive Plus Orbitrap MS . The standard LC system consisted of a PAL autosampler , two Cheminert six-port injection valves , a binary nanoUPLC pump , and an HPLC sample loading pump . Both SPE precolumn  and LC column  were slurry-packed with 3 µm C18 packing material (300-Å pore size) . Sample was fully injected into a 20 µL loop and loaded onto the SPE column using buffer A (0.1% formic acid in water) at a flow rate of 5 µL/min for 20 min. The concentrated sample was then separated at a flow rate of 150 nL/min and a 75 min gradient of 8–35% buffer B (0.1% formic acid in acetonitrile). The LC column was washed using 80% buffer B for 10 min and equilibrated using 2% buffer B for 20 min. Q Exactive Plus Orbitrap MS (Thermo Scientific) was used to analyze the separated peptides. A 2.2 kV high voltage was applied at the ionization source to generate electrospray and ionize peptides. The ion transfer capillary was heated to 250 °C to desolvate droplets. The data-dependent acquisition mode was employed to automatically trigger the precursor scan and the MS/MS scans. Precursors were scanned at a resolution of 35,000, an AGC target of 3 × 1083 and MS/MS spectra were searched by Andromeda search engine against the against mouse UniProt database (fasta file dated 12 April 2017) . Search results were processed with MaxQuant and filtered with a false discovery rate ≤1% at both protein and peptide levels. For label-free quantification, the match between runs (MBR) function was activated with a matching window of 0.4 min and the alignment window of 20 min. The quantitation results were extracted from MaxQuant outputs based on at least two valid values in one sample type by using Peruses (Version 1.5.8.3)84. Supplementary Data #The freely available open-source MaxQuant software was used for protein identification and quantification. The MS raw files were processed with MaxQuant (Version 1.5.1.11)http://www.proteinatlas.org. Tissue in these images are from the intestine and labeled with the specific location including duodenum, small intestine, colon, or rectum86.All protein staining images are from the Human Protein Atlas and readily available at More than 200 mice were used to optimize and validate the flow sorting procedure and perform mRNA sequencing and proteomics. For proteomics three biological replicate were collected for each cell type, each biological replicate is treated as one sample during data analysis. These biological replicates are from independent mice and independent flow sorts. For mRNA-sequencing additional mice had to be used and pooled in order to isolate enough cells for sequencing, particularly for rarer cell types. In total we sequenced the following number of biological replicates (aka samples) per cell type stem = 3, AbsPro = 3, SecPDG = 4, tuft = 5, Ent = 5, EEC = 2. These biological replicates are from independent mice (sometimes sets of pooled mice) and independent flow sorts. Pooling of independent sorts was done as needed to ensure >2,500 cells for RNA preparation as described in the “RNA Preparation and RNA-seq” method section. The number of independent mice for each of the biological replicates per cell type is as follows: stem = 1,1,1; AbsPro = 1,1,1; SecPDG = 2,2,4,4; tuft = 4,4,4,4,4; Ent = 5,5,6,5,5; EEC = 8,10.t test); standard deviation quantitation for mRNA expression graphs  were used for statistical analysis as specified in the appropriate methods section. GraphPad Prism version 6.01) was used for additional analysis including: Fig. .01 was uFurther information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Description of Additional Supplementary FilesReporting SummaryPeer Review File


Childhood obesity has become a global public health issue. Today, there are opportunities to promote health through technological devices such as serious games. Despite the major advancement of this field of research, the use of serious games as a validated intervention in clinical practice requires further clarifications on some methodological aspects. In this perspective article, we report the pros and cons of existing serious games. Besides, we attempt to propose a new methodology of design of a serious game that could help to cope with childhood obesity. The proposed idea consists of a serious game in virtual reality based on enjoyment, movement, education, and executive functioning (EF) training. Longitudinal studies and solid research protocol would certainly ensure consistency and aid interpretation. The prevalence of obesity among children is freighting, and is constantly raising in both developed , while in a sitting or reclining posture”  posture” . Notablyposture” . With reposture” .While the multidisciplinary approach had a clinically significant impact on body weight, physical fitness, and psychosocial wellbeing, its long-term success was modest and not sustained . This siIn the context of obesity, there are four types of serious games: The first type aimed to decrease energy intake by improving knowledge concerning healthy nutrition and changing children’s attitudes about food . The second type aimed to reinforce the weight control process and apply nutrition knowledge in daily life (serious games based on EF training). The third type aimed to increase the energy expenditure . The last type aimed to provide knowledge, enhance motivation, and encourage behavior change related to healthy eating, PA, and stress coping . Current studies showed that playing serious games might contribute to tackling childhood obesity . DespiteSerious games based on nutrition knowledge and/or dietary change were designed primarily to reduce energy intake by (i) improving knowledge concerning healthy nutrition, and (ii) changing children’s attitudes about food. Despite the relevance of these findings, there is a need to standardize how serious games were assessed. Another critical point is that the “source” of eating habits in the majority of studies is not the children but rather the parents . Hence, Executive functioning regroups higher cognitive processes allowing flexible behavior to environmental circumstances in essentially all facets of daily living . EF is rTaking into account all the above, a burgeoning field of research suggests that training EF may be a promising strategy to aid in obesity treatment . DespiteExergaming is an emerging technology that allows the players to interact physically with onscreen avatars through a variety of gross motor movements such as jumping, kicking, punching, and ducking . Today, Systematic reviews and meta-analyses comparing the effects of playing exergames to engaging in another form of sedentary screen time on obesity-related outcomes reported inconsistencies in the literature . Some laRecently, the Youth Compendium of Physical Activities reported PA intensities from exergames play based on the specific type of exergames, age, and gender . AccordiIt is worth mentioning that only a few studies examined the possible effects of exergames on snacking behavior during activity. The study by To date, four research groups took the initiative to develop multidisciplinary serious games including different game modules that can be used to promote healthy behaviors in children with obesity. Three of them were recently published , while oCritically, the available multidisciplinary serious games focused more on nutrition knowledge and dietary change and much less on PA, which was lightly involved in the game design. The latter also clearly lacked elements of EF training.Serious gaming could be a promising new way to tackle childhood obesity. In this perspective article, we discuss relevant trials on serious games for four main purposes: (1) decrease energy intake, (2) increase energy expenditure, (3) reinforce the weight control process, and (4) encourage behavior change related to healthy eating, PA, and stress coping. Still, none of the published serious games combined all these purposes into the same game design, whereas all of them are essential for successful weight control . From ouApplying our new model would be possible by incorporating both healthy and unhealthy eating messages (nutrition education), into the exergames instead of a film game. Moreover, as EF plays an essential role in both learning processes and children’s attitudes about food , we suggIn respect to the movement dimension, the active gaming mode must contain a properly planned program of PA instead of non-controlled movements to ensure the reliability of the program as well as a child’s safety . It is important to highlight that the intensity of serious game must range from moderate to vigorous activity. This will help to both prevent and treat childhood obesity .To ensure the enjoyment dimension and reduce stress induced by PA, the use of Virtual Reality appears to offer a good solution due to its fun character . EnjoymeTechnically, to ensure that each dimension of our model is properly designed, guidelines in the literature must be followed . In addiTo summarize, creating a serious game in virtual reality based on enjoyment, movement, education, and EF training may have the potential to help treat childhood obesity in clinical practice. Longitudinal studies and standardized protocols would certainly ensure consistency and aid interpretation.The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.MB wrote the manuscript. AA-Z and YS revised it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Choniomyzon copepod obtained from a longlegged spiny lobster Panulirus longipes longipes , collected from Hualien Port, Eastern Taiwan. We illustrated morphological features of the specimen to determine its taxonomic identification. The new species reported here is the first record of Choniomyzon species from spiny lobster in Taiwanese waters.This study reports an undescribed species of the Choniomyzon taiwanensis n. sp. is described based on specimens collected from examining external egg masses of spiny lobster Panulirus longipes longipes , obtained from Hualien, Taiwan. The new species differs from its congeners in possessing the following characteristics: (1) small prosome (about 0.84 mm); (2) armature of antennule being 1, 1, 2, 2, 1, 1, 1, 2, 1, 1+1 (aesthetasc), 4, 6+1 (aesthetasc); (3) five-segmented antenna; (4) second segment of antenna bearing 1 inner seta; (5) two-segmented maxilla. Based on the evidence of distinctive morphological features and host preference, Choniomyzon taiwanensis n. sp. is a new species. Until now, four species of Choniomyzon have been known living on decapods, and the new species reported here is the first record of Choniomyzon species from spiny lobster in Taiwanese waters. Choniomyzon inflatus Wakabayashi, Otake, Tanaka & Nagasawa, 2013, simulates the external eggs of its host in its prosome shape and size, and its caudal rami are similar to its host’s egg attachment filaments [Nicothoid copepods are small, highly specialized parasites that live on other crustaceans ,2. So failaments .Choniomyzon Pillai, 1962, is a small genus of siphonostomatoid copepod in the family Nicothoidae Dana, 1849, and only three species have been recognized [Choniomyzon panuliri Pillai, 1962, parasitic on the abdomen of spiny lobsters, Panulirus homarus Linnaeus, 1758, and Panulirus versicolor Latreille, 1804; Choniomyzon libiniae Santos & Björnberg, 2004, on the external eggs of an epialtid crab, Libinia spinosa Guérin, 1832; and C. inflatus on the external egg masses of the smooth fan lobster, Ibacus novemdentatus Gibbes, 1850  of the lobster were measured. The external egg mass of the lobster was examined for the presence of parasitic copepods, and the copepod specimens were picked up by using insect forceps. A total of 37 female copepods were collected from the lobster. One live non-ovigerous female and one live ovigerous female copepod were photographed on a Motic BA210 compound microscope . Subsequently, all the copepod specimens were preserved in 70% ethanol.An egg-bearing female longlegged spiny lobster Morphological observations of parasitic copepods were performed as described by Humes and Gooding in 1964  for the Four copepod specimens were transferred to 4% buffered glutaraldehyde for further analysis in scanning electron microscope (SEM). The specimens were dehydrated by an ethanol gradient (70% → 85% → 95% → 100%) and transferred on aluminum stubs with a drop of hexamethyldisilazane (HMDS) for critical point drying. The stubs were sputter-coated with gold  and then observed using a Hitachi TM4800 SEM .Order Siphonostomatoida Burmeister, 1835Family Nicothoidae Dana, 1849Choniomyzon Pillai, 1962Genus Choniomyzon taiwanensis n. sp.Figure 4 and Figure 5Materials examinedHolotype: one ovigerous female (NTUM-Inv-10017). Paratype: one ovigerous female (NTUM-Ivn-10018). Types were deposited in the National Taiwan University Museum (NTUM). Other materials: 30 females, deposited in the Department of Fisheries Production and Management, National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan.Type host:P. longipes longipes . Morphometric measurement: total body length: 193.84 mm, abdomen length: 108.13 mm, cephalothorax length: 85.71 mm, cephalothorax width: 50.46 mm, and abdomen width: 40.52 mm  share the greater number of common characteristics compared with the other two species   are phylogenetically close to each other. Several characteristics  found in the former two species are also present in C. inflatus (I. novemdentatus) belongs to the same infraorder (Achelata) as Panulirus. On the other hand, C. libiniae  shows some unique characteristics  , and theinflatus , and itsC. taiwanensis n. sp. is confirmed a new species and the first record of Choniomyzon species from spiny lobster in Taiwanese waters.Based on the evidence of distinctively morphological features and host preference,


The tumor immune microenvironment (TIME) has been recognized to be an imperative factor facilitating the acquisition of many cancer-related hallmarks and is a critical target for targeted biological therapy. This research intended to construct a risk score model premised on TIME-associated genes for prediction of survival and identification of potential drugs for ovarian cancer (OC) patients.The stromal and immune scores were computed utilizing the ESTIMATE algorithm in OC patient samples from The Cancer Genome Atlas (TCGA) database. Weighted gene co-expression network and differentially expressed genes analyses were utilized to detect stromal-and immune-related genes. The Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was utilized for additional gene selection. The genes that were selected were utilized as the input for a stepwise regression to construct a TIME-related risk score (TIMErisk), which was then validated in Gene Expression Omnibus (GEO) database. For the evaluation of the protein expression levels of TIME regulators, the Human Protein Atlas (HPA) dataset was utilized, and for their biological functions, the TIMER and CIBERSORT algorithm, immunoreactivity, and Immune Cell Abundance Identifier (ImmuCellAI) were used. Possible OC medications were forecasted utilizing the Genomics of Drug Sensitivity in Cancer (GDSC) database and connectivity map (CMap). TIMErisk was developed based on ALPK2, CPA3, PTGER3, CTHRC1, PLA2G2D, CXCL11, and ZNF683. High TIMErisk was recognized as a poor factor for survival in the GEO and TCGA databases; subgroup analysis with FIGO stage, grade, lymphatic and venous invasion, debulking, and tumor site also indicated similar results. Functional immune cells corresponded to more incisive immune reactions, including secretion of chemokines and interleukins, natural killer cell cytotoxicity, TNF signaling pathway, and infiltration of activated NK cells, eosinophils, and neutrophils in patients with low TIMErisk. Several small molecular medications which may enhance the prognosis of patients in the TIMErisk subgroup were identified. Lastly, an enhanced predictive performance nomogram was constructed by compounding TIMErisk with the FIGO stage and debulking.These findings may offer a valuable indicator for clinical stratification management and personalized therapeutic options for OC patients and may be a foundation for future mechanistic research of their association. Globally, human ovarian cancer (OC) is associated with the highest lethality among the gynecological malignancies; the mortality accounts for approximately 5% of all female cancer-related deaths . Patient+ and CD8+ T cells into the tumor has been associated with improved overall and progression-free survival in OC patients (The tumor immune microenvironment (TIME) has been known to be an important factor facilitating the acquisition of many cancer-related hallmarks. Successful peritoneal metastasis which entails the co-evolution of stromal and cancer cells has been recognized as the key cause for high risk of recurrence and mortality –7. In OCpatients .The TIMEpatients , a perfehttps://www.gtexportal.org/) and TCGA databases . TCGA database was utilized to examine the possible TIME- and prognosis-related genes and premised on it the TIMErisk was then evaluated. An aggregate of 375 OC patients from TCGA, median age was 59 years (30 to 87), were randomly categorized into two cohorts in the ratio of 7:3 utilizing the “caret” package in R software; these were considered to be the training and testing cohorts, in that order. The training cohort consisting of 263 samples was utilized to detect the TIME regulators and build a prognostic model. The testing set with 112 samples was utilized to evaluate the model’s performance. GSE53963 and GSE140082 cohorts were accessed from the GEO dataset (https://www.ncbi.nlm.nih.gov/geo/) and utilized for independent external validation of the model. Stromal and immune scores were computed utilizing the “estimate” package in R software premised on the ESTIMATE algorithm  value  <0.05 and | Log2 [Fold Change (FC)] | >2 were considered to be DEGs. WGCNA was utilized to detect the co-expressed gene modules strongly related to the stromal and immune scores. The gene modules of maximal |correlation coefficient were regarded as strong stromal and immune correlated modules. The intersection of stromal and immune correlated DEGs and strong stromal and immune-correlated gene modules was utilized as the input in the Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis.LASSO is a descending dimension technique for regularization which could be utilized for examining biomarkers for survival analysis in combination with the Cox model . The strhttp://www.immport.org). The possible enriched biological functions associated with TIMErisk were detected utilizing the gene set enrichment analysis (GSEA) technique and annotated via Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) datasets. In GSEA, the FDR value < 0.05 was judged to be a statistically significant enrichment.The levels of infiltrating stromal and immune cells were computed utilizing the CIBERSORT algorithm . Single-To assess the independent prognostic significance, multivariate and univariate Cox regression analyses for TIMErisk and clinical variables were performed. Based on the independent prognostic significance a nomogram was built for the training set to forecast the survival of EOC patients. In the nomogram score system, each of the variables was allotted a point; total points were calculated by summation of points for each sample to predict survival. Using calibration plot, and decision curve analysis (DCA) the discriminating ability, and predictive performance of the nomogram score system were assessed. The nomogram was also employed in the testing set and the whole set to validate the results.https://www.cancerrxgene.org). Estimation of the drug response was done by computing the half-maximum inhibitory concentration (IC50). Possible small molecule drugs for UCEC were forecasted utilizing the Connectivity map  comprising of genome-wide transcriptional expression data from small molecule drugs; premised on the links between genes, drugs, and diseases via the variation in gene-expression profiles potential candidates were predicted. This was premised on DEGs between low- and high-risk cohorts with | log2fold change (FC) | > 2 and FDR < 0.05.The chemotherapeutic response in OC patients was evaluated utilizing a public dataset, Genomics of Drug Sensitivity in Cancer  and validation cohort (112 participants) in a ratio of 7:3 ratio. The basic features for the TCGA training and validation cohorts, such as FIGO stage, age, grade, lymphatic and venous invasion, days to the new tumor, and debulking, were not significantly different . Through multivariate Cox regression analysis, Immunescore, FIGO stage, and debulking were identified as the prognostic significances  using “rms” and “survival” packages in R  receptor, PTGER3, exerts multiple effects including invasion, epithelial cell growth, and immune regulation . Our fincetylase . CTHRC1 cetylase . Aberrancetylase , 41. In Interestingly, resting or naïve immune cells such as CD4 naive T cells, resting dendritic cells and resting NK cells were elevated in the patients with higher TIMErisk, whereas a large number of functional immune cells, including activated NK cells, eosinophils, and neutrophils, corresponding to more incisive immune reactions, including antigen processing and presentation, chemokines, interleukins, natural killer cell cytotoxicity, and BCR and TNF signaling pathways showed higher infiltration in the low TIMErisk patients. The findings were consistent with the conclusion that immunosuppression was the primary cause for the greater risk of malignant progression and death. Based on the above results, we speculated that the predictive model based on TIMErisk may be a reliable biomarker for cancer therapy.Fortunately, we identified 21 compounds with 20 MoAs, including adrenergic receptor antagonists, a cyclooxygenase inhibitor, and a serotonin receptor antagonist. The PPAR receptor agonist (clofibrate) and chloride channel blocker (benzbromarone), in particular, could offer possible advantages for high-risk cohort patients. Cyclooxygenase inhibitor (Sc-560) and leukotriene receptor antagonist (tomelukast) showed greater sensitivity for patients with low TIMErisk. Clofibrate is widely used in the treatment of tumors including pancreatic cancer , colon cCurrently, FIGO staging is the most extensively utilized technique to examine the malignancy potential as well as the disease progression in OC. However, this technique has its drawbacks since it is mainly contingent on the distant metastasis, lymph node invasion, size, and location of the tumor. It does not consider the heterogeneity of tumor, age, and other clinical characteristics. Therefore, it is costly to construct an integrated prognostic model consisting of clinical properties and high-risk gene signatures. Our results showed that FIGO stage, debulking, and TIMErisk of the analyzed EOC samples in the TCGA training cluster were independent high-risk factors associated with poor survival. In addition, the nomogram based on the prognostic signature showed a higher net benefit and better predictive accuracy than the FIGO stage system and FIGO stage & debulking system. These results may guide the individualized treatment for OC patients.While our model was valuable in examining prognosis and carrying out therapies for OC patients, it ought to be prospectively confirmed by large-sample clinical studies. The context of TIME, such as remodeling stroma, the status of immune cells infiltration, immunoreaction, and the molecular mechanisms mediated by TIMErisk was explored through bioinformatic analysis only. Thus, additional experimental validations are required to corroborate these findings. Additionally, we have identified several potential drug compounds for TIMErisk subgroups. Unfortunately, there is a lack of available research to confirm their efficacy in ovarian cancer. Further investigations are needed to contrast the TIMErisk scores with present biomarkers and examine the relationship between TIMErisk and the potential drugs in OC patients.To summarize, our research detected a risk prognostic signature encompassing 7 genes related to TIME. The TIMErisk can forecast the OS of OC patients and indicated the situation of stroma remodeling and immune response. Candidate drugs for TIMErisk subgroups were identified. Lastly, an enhanced predictive performance nomogram by compounding TIMErisk with the FIGO stage and debulking was constructed. Ultimately, these findings could offer a valuable indicator for clinical stratification management and personalized therapeutic options for OC patients and may be a foundation for forthcoming mechanistic research projects of their association.The original contributions presented in the study are included in the article/JY, SH, and LH were responsible for the design, analysis, and draft of the research. JL, YW, and XZ plotted all figures in this manuscript. ZW and LG helped in data analysis. All authors approved the final version of this manuscript and agreed to be accountable for all aspects of the work.This research is sponsored by Hubei Province’s Outstanding Medical Academic Leader Programme and the Fundamental Research Funds for the Central Universities (No. 2042021kf0125).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vitamin K2 activates vitamin K-dependent proteins that support many biological functions, such as bone mineralization, the inhibition of vascular stiffness, the improvement of endothelial function, the maintenance of strong teeth, brain development, joint health, and optimal body weight. Due to the transformation of food habits in developed countries over the last five decades, vitamin K and, specifically, vitamin K2 intakes among parents and their offspring have decreased significantly, resulting in serious health implications. The therapeutics used in pediatric practice (antibiotics and glucocorticoids) are also to blame for this situation. Low vitamin K status is much more frequent in newborns, due to both endogenous and exogenous insufficiencies. Just after birth vitamin K stores are low, and since human milk is relatively poor in this nutrient, breast-fed infants are at particular risk of a bleeding disorder called vitamin K deficiency bleeding. A pilot study showed that better vitamin K status is associated with lower rate of low-energy fracture incidence. An ongoing clinical trial is intended to address whether vitamin K2 and D3 supplementation might positively impact the biological process of bone healing. Vitamin K2 as menaquinone-7 (MK-7) has a documented history of safe and effective use. The lack of adverse effects of MK-7 makes it the ideal choice for supplementation by pregnant and nursing women and children, both healthy and suffering from various malabsorptions and health disorders, such as dyslipidemia, diabetes, thalassemia major (TM), cystic fibrosis (CF), inflammatory bowel diseases (IBD), and chronic liver diseases. Additionally, worthy of consideration is the use of vitamin K2 in obesity-related health outcomes. Vitamin K1 and K2 are fat-soluble vitamins. Vitamin K1 is found mainly in leafy green vegetables. Vitamin K2 (menaquinones) contains an unsaturated aliphatic side chain with a variable number of prenyl units. The number of prenyl units indicates the respective type of menaquinone. For example, MK-4 contains four prenyl units and MK-7 contains seven prenyl units. Vitamin K2 is mostly produced by bacteria, except for MK-4, which can be developed by tissue-specific conversion from vitamin K1 in animals. A traditional Japanese dish called “natto,” consisting of fermented soybeans, holds the highest content of K2, particularly MK-7.gamma-glutamylcarboxylase (GGCX) and in the carboxylation process of vitamin K-dependent proteins (VKDPs) [Both vitamins K1 and K2 act as essential cofactors for the enzyme  (VKDPs) . VitaminIn addition to participating in the activation of VKDPs, vitamin K has been reported to operate as an anti-inflammatory and antioxidant agent independent of its GGCX cofactor activity ,5. A recVitamin K2 is also a transcriptional regulator of bone-specific genes that functions through the steroid and xenobiotic receptor (SXR) to facilitate the expression of osteoblastic markers . MoreoveThe role of VKDPs in processes beyond coagulation has been discovered and well demonstrated in the past few decades. The evidence from numerous clinical trials clearly supports the benefit of high vitamin K2 consumption for human health. Vitamin K2 meets all criteria for a bioactive substance to be considered for a recommended daily intake (RDI). Many countries have an RDI for vitamin K1 based on earlier research. As vitamin K2 is very important for extrahepatic tissues, the recommendations should combine vitamin K1 and K2 intakes. The procedures by which vitamin K2 levels can be assessed call for standardization, and an RDI for vitamin K2 based on current research needs to be established and accepted worldwide.The objective of this paper was to perform a systematic review of research papers. We also evaluated clinical trials that examined the role of vitamin K2 for children’s health and whether vitamin K2 supplementation influences markers of bone disease such as bone mineral density.Vitamin K2 activates proteins performing crucial biological functions that range from bone mineralization and healthy teeth, through promoting cardiovascular health, to maintaining brain development, joint health, and optimal body weight .Osteocalcin is a VKDP synthesized by osteoblasts and is thought to be related to bone mineralization. Vitamin K is necessary for the activation of osteocalcin. Moreover, vitamin K2 has been shown to inhibit resorption of the bone by suppression of the prostaglandin E2 synthesis in osteoclasts . ChildreAccording to the traditional view, dental caries are considered a tooth de-mineralizing process that takes place solely in the oral cavity. Attributed to a new perception of oral/systemic links is the view that dental caries are an uncontrolled inflammatory response regulated by the brain and mitigated through the hypothalamus/parotid axis of the endocrine system. Tooth vulnerability or resistance is determined by a signaling factor constituted by the role of free radicals in the hypothalamus. This systemic caries paradigm puts nutrition at the forefront of prophylaxis as it concentrates on the root of the disease rather than traditional symptom-focused prevention efforts .Vitamin K2 works in concert with calcium and vitamin D and appears to have a significant antioxidant role. Therefore, vitamin K2 can help to considerably reduce tooth decay while also appearing to play a prospective saliva-buffering role in the exocrine parotid and other salivary glands .While cardiovascular incidents are rare in children, clinical observations show that arterial calcification and atherosclerosis is a progressive process that accumulates over decades ,16. ReseSolid evidence exists that vitamin K2 plays important roles in the nervous system. Vitamin K2 is necessary to activate VKDPs in the brain, such as growth arrest-specific gene 6 protein (Gas6) and protein S, which are connected to cognitive processes. Gas6 and protein S influence different procedures in the brain, such as apoptosis, cell growth, and myelination . MoreoveIn the brain, vitamin K2 occurs mainly as MK-4. The highest concentrations of MK-4 were found in midbrain and pons medulla, and the lowest concentrations were observed in the cerebellum, olfactory bulb, thalamus, hippocampus, and striatum. Although MK-4 is the primary form in the brain, it was found that the supplementation of MK-7 increases the level of MK-4 in brain tissue .Vitamin K is also involved in the synthesis of sphingolipids, crucial for the development of the functional integrity of the nervous system. Animal studies speak in favor of vitamin K2′s role in the biosynthesis of sphingolipids. Initially recognized for their role as building blocks of cell membranes, sphingolipids are now known to be involved in cell signaling, division, differentiation, and apoptosis. There are some studies, which showed the connection between alterations in sphingolipid metabolism and cognitive decline as well as neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases .Moreover, animal and human studies suggest that optimal vitamin K status is important for psychomotor behavior and cognition. Additionally, in one report, vitamin K deficiency, due to administration of warfarin treatment during pregnancy, was associated with warfarin embryopathy. Some infants exhibited optic atrophy, developmental delay, and seizures after treating pregnant mothers with warfarin .Vitamins K1 and K2 both activate VKDPs in the liver . The defWarfarin during pregnancy might impact newborn health. The resulting abnormalities include the cartilage and joints , which iTherefore, the optimal level of vitamin K is important for joint development. Moreover, it has been shown that VKDPs, inclusive of the mineralization inhibitor MGP, are detected in joint tissues, including cartilage and bone. It has been previously indicated that low vitamin K status is correlated with higher risk of osteoarthritis (OA) .In human OA cartilage, MGP was found to be mainly inactive. Conversely, MGP is primarily carboxylated in healthy articular cartilage, suggesting the carboxylation of MGP is associated with OA .Respiratory tract infections (RTIs) are common in children, and vitamin D deficiency was shown to be associated with increased risk of RTI. Thus, vitamin D3 supplementation was accepted by medical community as an important nutrient, which reduces the risk of respiratory problems in children. It is, however, important to remember that vitamin D3 should be supplemented with vitamin K2 .Currently, highest international interest is concentrated on the COVID-19 pandemic and potential ways of reducing its mortality rate. Researchers have recently shown that optimizing blood vitamin D and K levels could offer a solution for the reduction of mortality rate. Researchers proved the synergistic interplay between vitamins D and K on bone and cardiovascular health. Moreover, some scientists suggest that supplementation with vitamin D3 can be considered safe when it is combined with vitamin K2. New randomized controlled trials are needed to prove the discussed evidence .During adolescence, the incidence of forearm fractures in children reaches its highest point as exercise increases. At the same time, cortical bone mass decreases due to increased calcium requirements in skeletal growth.Physical activity and adequate nutrient intake have favorable influence on the bone quality. Hungarian researchers demonstrated that changes in bone mineral status among 10–12-year-old children, assessed by ultrasound method, are correlated with the amount of intense physical activity as well as with optimal vitamin K intake .Vitamin K, as the γ-carboxylase cofactor, takes part in bone metabolism. Childrens’ requirements for this vitamin are among the greatest; its deficiency may have an impact on the peak bone mass formation, as well as on osteoporosis risk in adulthood.The beneficial role of vitamin K, especially vitamin K2, in bone health is well established scientifically, based on epidemiological as well as on interventional studies published over the past decade. Of particular significance is the comprehensive attitude to bone health in children of all ages that includes optimal intake of calcium, vitamins D and K, balanced diet, and exercise. It has been shown that subclinical vitamin K and D deficiency is present in healthy pediatric population with low-energy bone fractures .Further, a pilot study, which enrolled 20 children with radiologically confirmed low-energy fractures and 19 healthy children as a control group, showed that low vitamin K status, represented by the percentage of the active form of osteocalcin (UCR), correlates statistically with the low-energy fracture risk .Based on a previous study, researchers assessed the impact of vitamin K2 on fracture repair. This clinical trial was accepted by the ethical committee and is ongoing. What has been learned so far is that low vitamin K status increases the fracture risk in kids. At this stage the positive effect of vitamin K2 on bone healing is only a hypothesis, but researchers are looking forward to the future results that will give us the answer whether vitamin K2 and D3 supplementation might positively impact this biological process .Several authors have linked vitamin K status to fat and glucose metabolism. What lies behind obesity in childhood and adolescence is the development of insulin resistance that causes metabolic, structural, and functional changes, which may lead to increased risk of cardiovascular diseases and type 2 diabetes. Adolescence is also a crucial period when the so-called ‘adiposity rebound’ may occur, which means that adiposity increases after its lowest point in childhood. Children with a higher childhood BMI tend towards having a higher BMI, waist, and hip circumference in adulthood .The results of a three-month animal study demonstrated that mice supplemented with vitamin K2 on the top of a high-fat diet gained less weight, less body fat, and showed decreased serum glucose and leptin in comparison with the control group on a high-fat diet only . In humaSo far, few studies have investigated the relationship between body weight and vitamin K intake. Nonetheless, it is acknowledged that suitable micronutrient intake is crucial to maintain different metabolic functions of the body, while inadequate intake is one of the top 10 risk components for the overall sickness burden globally . These oThalassemia major (TM) is also called Cooley’s anemia. People suffering from this most dangerous form of beta thalassemia have severe symptoms and life-threatening anemia. Their hemoglobin does not produce enough beta protein; therefore, they need regular blood transfusions and other medical treatment. Thalassemic osteopathy (TOSP) represents a salient factor determining risk for morbidity in patients with Cooley’s anemia and manifests as osteopenia/osteoporosis. A pilot study conducted among children with TM clearly showed that vitamin K2 and calcitriol combination positively affects bone mineral density (BMD). The authors suggested that patients with TOSP might benefit from K2 supplementation, although more clinical trials are needed to study the effects of treatment .People with cystic fibrosis (CF) may develop fat malabsorption due to pancreatic insufficiency, and this may also be linked to deficiencies of fat-soluble vitamins like vitamin K. Studies show that vitamin K deficiency is common in CF infants and toddlers and can even be detected in children receiving recommended supplementation. Moreover, a strong association between vitamin K status and clinical outcome in CF patients was found. Vitamin K intake was found to be an independent predictor of osteoblastic activity markers like: Glu-OC, Gla-OC, PICP , and PINP . Supplementation with vitamin K2 appears to be a good solution to improve the level of vitamin K in children with CF, but agreement needs to be reached on the appropriate dose and frequency of use of this nutrient ,43,44.Vitamin K deficiency was frequent in pediatric IBD patients, and more common in Crohn’s Disease (CD) than ulcerative colitis (UC) in pediatric patients . There iThe reason for low vitamin K status in IBD remains unknown. It has been suggested that vitamin K deficiency is an effect of malabsorption caused by IBD, as well as by current treatment.This is confirmed by the fact of the simultaneous deficiency of vitamin E in IBD and vitamin K metabolism disorders caused by antibiotics ,52,53. IIn addition to the disease mechanism itself, the diet plays a key role in the development of vitamin K deficiency. Patients suffering from IBDs frequently consume inadequate amounts of the minerals and vitamins, resulting in nutritional deficiencies ,56. GoodPediatric IBD patients seem to be prone to vitamin K deficiency due to ongoing inflammation. Consistent with the effects of vitamin K on bone metabolism, numerous clinical studies in adults showed that low vitamin K status contributes to low BMD in CD patients ,59. ClinDue to malabsorption of fats and an inadequate diet, children with chronic liver disease are exposed to ongoing vitamin K deficiency, which increases the risk of hemorrhages and fractures and is related to degree of cholestasis and severity of liver disease. Low vitamin K status was shown to be common in children with mild to moderate chronic cholestatic liver disease, even despite vitamin K supplementation ,63. AdeqThe researchers in Japan found that severely disabled children suffer from deficiencies of various nutritional compounds. There are many factors that contribute to low vitamin K status such as malnutrition, malabsorption, changes in microbiota, and also hepatic dysfunction.The researchers showed that more than 40% of severely disabled children have vitamin K deficiency. In the group with vitamin deficiency, the majority of children demonstrated a bleeding tendency, more than 60% developed vitamin K deficiency in association with infection, and over 40% of them were treated with antibiotics. The good news is that all showed a good response to vitamin K2 supplementation, which seems to be especially important for disabled children in case of long-term antibiotic treatment .A significant number of pediatric patient visits each year end with the prescription of antibacterial drugs. Antibiotic therapy affects vitamin K levels in children. It has been shown that there are changes in gut microbiota due to antibiotics that alter intestinal vitamin K production. The level of K2 in the liver is lowered in people on antibiotics, especially on the cephalosporins, due to impairment the recycling of vitamin K.Vitamin K deficiency is known to cause coagulopathy and bleeding in children on prolonged antibiotic treatment, therefore, in seriously ill patients on extended periods of antimicrobial drugs and inadequate diet, vitamin K2 prophylaxis is suggested to prevent morbidity and mortality ,65,66.For children who suffer from chronic diseases, the immunosuppressive and anti-inflammatory properties of oral corticosteroids constitute an inevitable treatment option. Their prolonged use may lead to various adverse drug reactions (ADRs), such as significant reductions in bone formation through the inhibition of osteoblasts. Moreover, corticosteroid use increases calcium excretion in the urine. Therefore, osteoporosis is considered a particular complication of chronic childhood illnesses cured with glucocorticoids (GCs) ,68.The adverse effects of GCs on bone formation are more pronounced in the growing skeleton, where trabecular and cortical bone growth is negatively affected. Decreased bone quality has been found in different disorders that require GCs, and a clinical study reported increased fracture risk in children who require more than four courses of GCs .Japanese researchers found that vitamin K2 drugs might be effective to prevent bone fracture in glucocorticoid-induced osteoporosis (GIOP), because they increase bone strength independently of BMD .Th authors of a systematic review, who critically evaluated the treatment options used in the management of bone loss associated with GC use among children, established that vitamin K2 (menatetrenone) combined with alfacalcidol has a beneficial effect on BMD in children who were treated with GCs for a longer time .It was also reported by a team of Japanese researchers that treatment with alfacalcidol and vitamin K2 (as MK4) is beneficial for supporting bone health in children with skeletal unloading. The results of this pilot study showed that vitamin K2 as MK4 effectively and safely improves lumbar BMD in long-term prednisolone-treated children .Vitamin K2 treatment has been shown to decrease the osteoporotic bone loss including GIOP . It is aIn recent decades, vitamin K2 (menaquinone) has been specifically highlighted as a crucial cardiovascular and bone health nutrient .Since the 1950s, many children and adults get less of this important nutrient than they should, and substantially lower vitamin K intakes may have serious health implications. Two cohorts of 4-year-old children were compared. A cohort born in the 1950s was compared in terms of dietary intake and sources of vitamin K with children born in the 1990s and showed that dietary vitamin K intake was significantly higher in the 1950s (39 mcg/day) than in the 1990s (24 mcg/day). Between the 1950s and the 1990s, people started to consume less vegetables and more fats and oils, therefore, sources of vitamin K intake have changed remarkably. Due to general changes in food habits , vitamin K intakes of children on Western diets have been on a significant decline since 1950s .During the sharp increase in pubescence children seem to be extremely exposed to forearm fractures. Factors responsible for this regularity are closely related to each other and include skeletal growth, elevated need for calcium, increased physical activity, and decreased cortical bone mass. In recent years researchers aimed to check whether and how this regularity has changed. The outcome of a population-based study that was conducted in Minnesota over four time periods, 1969–1971, 1979–1981, 1989–1991, and 1999–2001, demonstrated that yearly incidence rates of forearm fractures per 100,000 escalated from 263.3 in 1969–1971 to 322.3 in 1979–1981, and to 399.8 in 1989–1991 before stabilizing at 372.9 in 1999–2001. Age-adjusted incidence rates per 100,000 were 32% higher among males in 1999–2001 compared with 1969–1971, and 56% greater among females in the same time periods .Considerable noteworthy and correlatory evidence emerges from the comparison of the two sets of data—the decreased intake of vitamin K over a 40-year period (from the 1950s to the 1990s) and the data from Minnesota inquiry presenting an increase in broken forearms over a similar 30-year period—as vitamin K intake lessened in the youth population, the potential forearm fractures in children heightened .The research also showed that children have the highest tissue-specific vitamin deficiency. Vitamin K status was most frequently evaluated by the percentage of active osteocalcin (cOC), vitamin K-dependent protein, important for bone.When the vitamin K status of bone in healthy children was contrasted with that of adults for the needs of a cross-sectional study, a noticeable elevation of the ratio of uncaroboxylated (inactive) osteocalcin (ucOC) into carboxylated (active) osteocalcin was observed in children, thus indicating a poor vitamin K status. Moreover, in the children’s group the researchers also found a considerable correlation between bone markers for bone metabolism and ucOC and cOC. These deductions imply markedly low levels of vitamin K in the bone during growth .A separate study found that the largest tissue-specific vitamin deficiency is common among children and adults above 40 years, and thus menaquinone-7 (MK-7) supplementation may have a beneficial influence on improving the extra-hepatic vitamin K status .Much research demonstrates that children need more vitamin K than adults due to various health disorders and therapeutics used in pediatric practice. For instance, vitamin K deficiency often goes hand in hand with disorders of fat malabsorption. Shortage of vitamin K manifests itself with easy bruising and bleeding, and children suffering from malabsorption also have tendencies to poor bone health and osteoporosis ,80. SuppLow vitamin K status is much more common in neonates than adults, due to both endogenous and exogenous deficiency. The endogenous case has been associated with poor intestinal colonization by bacteria  and a deThe reasons for the exogenous insufficiency arise from limited vitamin K transport across the placental barrier and its low accumulation in breast milk.The main exogenous source of vitamin K in newborns, which is almost exclusively milk, is not able to suitably equalize the insufficient endogenous production, because mother’s milk contains 1 to 4 mcg/L of vitamin K1 (and a much lower concentration of vitamin K2). Throughout the first 6 months of life the median amount of vitamin K that entirely breast-fed infants receive has been described to be below 1 mcg/day. Surprisingly, this amount in infants, who, as their meal get a typical supplemented formula, is approximately 100-fold higher. This argument should encourage breast-feeding mothers to seriously consider supplementation with vitamin K2 ,84,85,86The content of vitamin K in breast milk depends on the geographical region . ResearcThe second endogenous culprit of the neonates’ deficiency of vitamin K at birth is the poor transport of this nutrient across the placenta from mother to infant. Particularly in preterm pregnancies, little vitamin K actually crosses the placenta from mother to infant . HoweverHealthy newborn infants have a very fragile vitamin K coagulation status . In the Based on epidemiological studies of the prevalence of late VKDB, researchers have demonstrated that China, Japan, countries within Southeast Asia, such as Thailand and Vietnam, and also Australia have greater rates of VKDB than in the remaining parts of the world.Vitamin K insufficiency in Thai mothers (represented by detectable PIVKA-II levels) was due to reduced dietary K intakes during gestation period .The Japanese nationwide surveys found that the 87.7% of VKDB cases reported involved exclusive breastfeeding. Guidelines for expectant mothers in Japan who use drugs that could impair the absorption of vitamin K (excluding warfarin) say that they should be provided regular doses of 15–30 mg vitamin K daily 2 to 4 weeks before delivery or their newborn should receive 0.5–1 mg IM vitamin K2 administration ,103,104.The infant’s intake of vitamin K is a subject to regional variations, and it also depends on the fact whether the mother is supplemented with any form of vitamin K (K1 or K2).In the USA and Canada, the adequate intakes (AIs) for babies are based on the calculated average vitamin K1 intake of healthy breastfed infants and the expectation that newborns receive prophylactic vitamin K1 at delivery, as recommended by American and Canadian pediatric societies . In US, The European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) recommends as follows: healthy new-born infants should either receive 1 mg of vitamin K1 by intramuscular injection at birth; or 3 × 2 mg vitamin K1 orally at birth, at 4 to 6 days and at 4 to 6 weeks; or 2 mg vitamin K1 orally at birth, and a weekly dose of 1 mg orally for 3 months .In Japan an AI of 4 mg/day for infants aged 0–5 months was determined by multiplying the average milk intake and the average vitamin K content of milk, and assuming the presence of the oral administration of vitamin K just after birth in clinical settings . HoweverVitamin K2, as MK-7, has a documented history of safe and effective use in children, as well as in adults. When reading product labels, one can notice that MK-7 is already mentioned as a form of vitamin K. The absent side effects of MK-7 also precluded many authorities, such as The Food and Nutrition Board of the Institutes of Medicine (IOM), the European Commission, the Expert Group on Vitamins and Minerals in UK (UK EVM), and the World Health Organization (WHO)/Food and Agriculture Organization (FAO), from establishing a tolerable upper intake level for any form of vitamin K. This is because exceeding the adequate intake is safe, even when someone additionally ingests MK-7 from foods and supplements. The only possible contradiction is the use of anticoagulant drugs, such as coumarins, which may interfere with vitamin K cycle.Vitamin K2, especially as MK-7, which lasts longer in the body than MK-4, has important roles to play in the health of children, including the performance of various physiological functions such as coagulation, promoting bone mineralization, and a healthy cardiovascular system. Furthermore, vitamin K-dependent matrix-gla protein (MGP) helps inhibit arterial calcification (which may begin in childhood), so early supplementation with vitamin K2 may contribute to good cardiovascular health in infancy, puberty, and beyond. The current adequate intake level of vitamin K for pregnant and nursing women is 90 mcg. Likewise, research suggests that 45–50 mcg/day MK-7 is an appropriate intake range for children ,108,109.The good news shared by such trustworthy bodies as the European Food Safety Authority (EFSA), the UK EVM, the IOM, and WHO, which was also supported by clinical and nonclinical data, clearly states that using MK-7 as a dietary supplement according to the recommended dosages is safe ,111,112.