Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.Navigation of fast migrating cells such as amoeba  Migratory cells that move by crawling do so by extending and retracting their plasma membrane. When and where these events take place determine the cell shape, and this is directly linked to the movement patterns. Understanding how the highly plastic and interconvertible morphologies appear from their underlying dynamics remains a challenge partly because their inherent complexity makes quantitatively comparison against the outputs of mathematical models difficult. To this end, we employed machine-learning based classification to extract features that characterize the basic migrating morphologies. The obtained features were then used to compare real cell data with outputs of a conceptual model that we introduced which describes coupling via feedback between local protrusive dynamics and polarity. The feature mapping showed that the model successfully recapitulates the shape dynamics that were not covered by previous related models and also hints at the critical parameters underlying state transitions. The ability of the present approach to compare model outputs with real cell data systematically and objectively is important as it allows outputs of future mathematical models to be quantitatively tested in an accessible and common reference frame. Dictyostelium and migrating immune cells are versatile in their patterns of movement that range from random exploratory movements with frequent turns to more persistent migration in a straight path. Dictyostelium cells exhibit both random migration , [A0 = 78.83 [μm2] (~ 5 μm radius circle), protrusive force by actin polymerization awW = 0.8–4.0 [pN/μm] (for W ~ 1)  [’ so that the time scale of the simulated cell trajectories match with that of real data as described in the later section. The area constraint M = 0.5 pN/μm3 and the size of the boundary layer ε = 1.0 [μm] were set close to those in the earlier studies [The equation describes the dynamics of a continuous state variable ϕ2(1−ϕ)2 . The sec A0. The . The thi.0 [pN], , cell arr W ~ 1) ,73. Sinc[pN/μm2] , which w studies ,70.U and V describe an excitable reaction network for transient protrusive dynamics, and the third equation for W describes polarization dynamics, respectively  [V by a positive-feedback described in the first term. Increase in V is then slowed down due to depletion in U, and the system eventually recovers the original resting state.The ‘ideal cell’ that can take close to all of the basic phases of morphology features that we have examined  should cectively ; S1 Texttivation ,70,74. H∫dr|∇ϕ|2,τ′∂(ϕV)∂+ϕN,τ′∂(ϕW)∂W governed by Dictyostelium [W and its reciprocal state W* is conserved (Wtot = const.) [W* diffuses much faster than W and thus can be approximated as uniform in space. Hence we obtainThe expanding membrane region is determined by ostelium . The fir const.)  and thatW regions due to stalling of a transitory wave front—a property known as “wave pinning” [Fprot = aWW in W is high, and the rest of the domain with high Wtot /A0 -W as a result contracts due to area conservation imposed by the second term in W only specifies where the protrusions and retraction take place and does not assume the origin of their driving force. The variable W can thus be interpreted as an abstract representation of bistable signals such associated with the leading edge of polarize cells as Rac-GTP, or alternatively, Wtot/A0-W with the reciprocal spatial profile to indirectly represent signals that regulate rear contraction such as RhoA-GTP. A recent study has shown that Rac-GTP accumulates at the front of migrating neutrophils regardless of whether it is F-actin filled or blebbing [W positively regulates the positive-feedback amplification of V  were chosen for detail analysis. Parameters  that define the kinetics of U, V were fixed to those used earlier [F. Because grid-search in 8 dimensional parameter space was unattainable due to heavy computational load, we adopted parameter search around manually selected reference parameter sets. First, based on preliminary simulations performed with various parameters, a single parameter set R1 was chosen which gave rise to polarized cell morphology; i.e. elongated shape in the moving direction. Around the reference set R1, the following parameters were varied: aw and Wtot which appeared to affect the elongation of cell in the moving direction,χU and DW related to the split of the leading edge, and γ that seems to affect the appearance of new pseudopods. We performed a grid search around R1 in 2-dimension  for χU = 0 and χU = 50  for χU = 0 and 50 , , and  at both χU = 0 and 50 , and the closest simulations (N = 44 from 2 timeseries). HL-60 (N = 68 from 5 timeseries), and the closest simulations (N = 77 from 3 timeseries).For both experiments and simulation data, events of pseudopod formation were detected manually by eye as either ’de novo’, ’Y-split’ or ‘one-way-split’ based on . New pseS1 Text1. Details of our mathematical model. 2. Feature extraction from the intermediate-layer. 3. Analysis based on commonly used intuitive features. 4. Details of related mathematical models.(DOCX)Click here for additional data file.S1 FigF for Dictyostelium (agg)(C), HL60 (D) and fish keratocyte (E).  Orientation dependency for the oval shape (L = 0.75L0 (F) and L = L0 (G). (H-J) Mapping of circles and polygons with various aspect ratios (x-axis: y-axis) 1:3, 2:5, 1:2, 2:3, 1:1, 3:2, 2:1, 5:2 and 3:1. Circles, squares and rhombuses (H), triangles, pentagons and star shapes in the upright (I) and inverted (J) orientation. (K) Orientation dependency of a complex shape with multiple edges. The values of accuracy (A) and loss (B) during training (blue) and validation (red) of the deep convolutional neural networks. (C-E) Representative time series of the feature vector al shape  L = 0.75(TIF)Click here for additional data file.S2 Figh1 (A) and h2 (B). The first feature h1 and the second feature h2 is the degree of elongation parallel and orthogonal to the front-tail axis direction. Those values were extracted using the normalized mask image. (C) Definition of h3. The third feature h3 is circularity of a cell mask. (D) PC1-PC2 diagram obtained from each snapshots using hand-crafted features (left). The same diagram obtained using DNN-based features was also shown for comparison (right). The aggregation-stage Dictyostelium, HL-60, and fish keratocyte were shown as dark red, dark red, and yellow colors. Definition of (TIF)Click here for additional data file.S3 FigDictyostelium prespore cell-type). Cell-to-cell variation (A) and a representative temporal variation of a single cell (B). Blue circled regions represent 95% confidence eclipses for the mean of all combined timeseries (dotted) and the mean of individual cells (filled).  Mapping of mouse T cells in the PC1-PC2 space. Filled rhombuses: T helper 1 (C) and regulatory T cell (D). Circled regions in the background indicate the reference data in Dictyostelium dark green (dark green), HL60 (dark red) and fish keratocyte (yellow)). Mapping of cell shape with anterior-posterior elongation without lateral pseudopods ((TIF)Click here for additional data file.S4 FigV, green U. (D) Time series of the local curvature of the boundary (left panel) and local elongation (right panel) for (C). (E-G) The 1-variable scheme (E) and its representative morphology dynamics . Blue indicates the high W region. (H) Time series of the local curvature at the boundary (let panel) and local elongation (right panel) for (G). (I) Feature mapping of the polar morphology in 1-variable scheme. Time average (solid circles) and time samples (filled). Right panels in colored frames indicate representative snapshots. See Table O in (A-D) The 2-variable scheme (A) and its morphology dynamics (B-D). Representative simulations showing traveling patches (B) and lamellipodium-like protrusions (C). Color overlay; red (TIF)Click here for additional data file.S5 FigχU = 0), R1’ , R2 (upper right panels) and R2’ (lower right panels). Each square in the grid represents a sampled parameter condition.  Representative simulation time series for parameters R1, R2’ and (1)-(8) in (A). (D) Feature scores  in the (γ-aW) plane around R1. (E) Feature scores  in the (kW1-μ) plane around R2’. See Table O in (A) Two-dimensional grids around manually selected reference parameters R1 Click here for additional data file.S6 Figγ, ρ, DW, χU of the simulated morphology  and the feature values . See Table O in Dependency on (TIF)Click here for additional data file.S7 Figθ = 1.4, σ = 0.075) (A). Mapping in PC1-PC2 plane in Score-K rank1 simulation (B).  The same analyses at . Shape and orientation fluctuations observed under the default noise size ((TIF)Click here for additional data file.S8 FigDictyostelium (agg) (green +), HL-60 (dark red +), keratocyte (yellow +), Dictyostelium (veg) (magenta inverted triangles), Dicytostelium racE- strain (red squares) and Nocodazole-treated HL-60 (blue triangles). (B) Mapping of model simulations (grey circles), the top-ranking simulations based on the feature vector F (dark green circle for Dictyostelium (agg), magenta circle for Dictyostelium (veg)) and those based on 256-dimensional features (light green circle for Dictyostelium (agg), pink circle for Dictyoste the lium (veg)).  Time-series of simulations with the highest feature similarity to Dictyostelium (aggregation-stage) (C), vegetative Dicyostelium (D). See Table O in (A) PCA of the 256 dimensional intermediate layer. Microscopy dataset of (TIF)Click here for additional data file.S9 Figet al., 2019 [ The model by Bhattacharya et al . The PC1l., 2019 . PC1-PC2l., 2019  (A). Locl., 2019  (B).(TIF)Click here for additional data file.S10 FigV, green U. (B) Feature mapping: present model (triangle), the PIP2-modulated cell (Video 4 in [V at the cell edge taken from the simulation in (A). Arrows indicate bifurcating V-shaped fronts. (D) Kymographs of the local curvature (top panels) and the protrusion size (bottom panels) along the cell boundary; for the real cell data (Video 4 in [(A) A representative simulation result of a fan-shaped cell with a fluctuating front. Color overlay; red deo 4 in ) (circledeo 4 in ) (cross)deo 4 in ) (left),deo 4 in ) (middle(TIF)Click here for additional data file.S11 FigDictyostelium (agg) data (left), Score-D(agg) rank1 simulations in the Moreno et al. (middle) and our model (right). (C) Mapping of individual snapshots of HL-60 data (left), Score-H rank1 simulations in the Moreno et al. (middle) and our model (right). (D) Proportion of de novo pseudopod formation in real cell data and the rank1 simualtions; Dictyostelium (agg) (left lanes) and HL-60 (right lanes).(A) Mapping of simulated cell morphology from 105 parameter sets of the model (blue circle). The results from our model (black circle) shown for comparison. (B) Mapping of individual snapshots of the (TIF)Click here for additional data file.S1 MovieDictyostelium (agg), HL60 and fish keratocyte (from left to right).Timelapse sequence of representative training data images; (MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.S4 Movie(MP4)Click here for additional data file.S5 Movie(MP4)Click here for additional data file.S6 Movie(MP4)Click here for additional data file.S7 Movie(MP4)Click here for additional data file.S8 Movie(MP4)Click here for additional data file.S9 Movie(MP4)Click here for additional data file.S10 Movie(MP4)Click here for additional data file.S11 Movie(MP4)Click here for additional data file.


S-adenosylmethionine (SAM), the principal biological methyl donor. Glycine N-methyltransferase (GNMT) further utilizes SAM for sarcosine formation, thus it regulates the ratio of SAM:S-adenosylhomocysteine (SAH). (2) Methods: by analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that breast cancer patients with higher MAT2A had worse survival rate (p = 0.0057). Protein expression pattern of MAT1AA, MAT2A and GNMT were investigated in the tissue microarray in our own cohort (n = 252) by immunohistochemistry. MAT2A C/N expression ratio and cell invasion activity were further investigated in a panel of breast cancer cell lines. (3) Results: GNMT and MAT1A were detected in the cytoplasm, whereas MAT2A showed both cytoplasmic and nuclear immunoreactivity. Neither GNMT nor MAT1A protein expression was associated with patient survival rate in our cohort. Kaplan–Meier survival curves showed that a higher cytoplasmic/nuclear (C/N) MAT2A protein expression ratio correlated with poor overall survival . Accordingly, a MAT2A C/N expression ratio ≥ 1.0 was determined as an independent risk factor by Cox regression analysis . In vitro studies found that breast cancer cell lines with a higher MAT2A C/N ratio were more invasive. (4) Conclusions: the subcellular localization of MAT2A may affect its functions, and elevated MAT2A C/N ratio in breast cancer cells is associated with increased invasiveness. MAT2A C/N expression ratio determined by IHC staining could serve as a novel independent prognostic marker for breast cancer.(1) Background: methionine cycle is not only essential for cancer cell proliferation but is also critical for metabolic reprogramming, a cancer hallmark. Hepatic and extrahepatic tissues methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A that catalyze the formation of  In the US, the cancer incidence had been stable in women and declined by approximately 2% per year in men (2006 to 2015). The cancer death rate decreased annually by 1.4% and 1.8% (2007 to 2016), respectively . The breBreast cancer usually displays frequent intra- and inter-tumor heterogeneity presenting genetic and non-genetic alterations that often promote the progression of cancer cells ,6,7. AltS-adenosylmethionine (SAM) through the transfer of adenosine from ATP to the methionine sulfur that is catalyzed by methionine adenosyl transferases (MATs). Mammals have three distinct forms of MAT , encoded by two distinct genes (MAT1A and MAT2A). Among the MATs, MAT1A is mainly expressed in the liver, whereas MAT2A and MAT2B are widely expressed in non-parenchymal cells of the liver and extrahepatic tissues [MAT genes plays a significant role in the development of gastrointestinal cancers including hepatocellular carcinoma, cholangiocarcinoma, tumors from colon, gastric, and pancreas tissues [MAT2A and β regulatory subunit encoded by MAT2B. Hepatocellular carcinoma (HCC) is characterized by the low expression of the liver-specific MAT1A gene that encodes the SAM synthesizing isozymes MATI/III; and the high expression of the widely MAT2A that encodes the MATII isozyme and high expression of MAT2B that encodes a β-subunit without catalytic action, but it can regulate MATII enzymatic activity [Folate-mediated one-carbon metabolism is essential for methylation status  and nucl tissues . Accumul tissues  as well activity .MAT genes beyond the liver cancer development. In human colorectal cancer cell lines, inhibition of MAT2A and MAT2B by SAM or miR-34a/b expression inhibited tumor migration and invasion in vitro [MAT2A and MAT2B [MAT2A and MAT2B could be important targets for inhibiting cancer metastasis.Wang et al. recently demonstrated that methionine cycle flux can specifically modulate the epigenetic state of cancer cells and drives tumor initiation . Studiesin vitro . The tumnd MAT2B . These sMAT2B has been found to be correlated with worse relapse-free survival in triple-negative breast cancer (TNBC) [MAT2A/MAT2B confers growth and survival advantage to cancerous cells and enhancing tumor migration [MAT genes in tumorigenesis can help develop potential and effective strategies for cancer treatment and chemoprevention. Direct inhibition of MAT2B suppressed cell growth and migration and induced apoptosis in breast cancer cell MDA-MB-231 and MDA-MB-468 [MAT2A was upregulated in tamoxifen-resistant-MCF-7 cells [MAT genes could be potential therapeutic intervention for TNBC and the role of MATs in human breast cancer needs further investigation.A higher level of r (TNBC) . Inductiigration ; hence, A-MB-468 . The bas-7 cells . These sMAT2A to synthesize SAM and maintain their epigenome. The combination of methionine depletion and MAT2A inhibition has been used to suppress SAM biosynthesis and eradicate CD44hi/C24low cancer stem cell population. Methionine depletion induced MAT2A mRNA and protein that sensitized cancer stem cells to MAT2A inhibition by siRNAs or cycloleucine. The combination of dietary methionine restriction and cycloleucine was effective in suppressing primary and lung metastatic tumor burden in a murine TNBC model. SAM biosynthesis is a unique target for drug-resistant cancer stem cells [Many transformed cells and embryonic stem cells are dependent on em cells .Glycine N-methyltransferase (GNMT) catalyzes the transfer of a methyl group from SAM to S-adenosylhomocysteine (SAH), which is subsequently converted to the amino acid homocysteine by removal of the adenosine base ,23. We pGNMT knockdown results in an inhibition of proliferation, and induces G1 arrest and apoptosis in prostate cancer cell lines. Hence GNMT may play an important role in promoting prostate cancer cell growth via the regulation of apoptosis, and serve as a marker of malignant progression and poor prognosis of prostate cancer [GNMT may have distinct roles in different types of cancers. GNMT is commonly diminished in human liver cancers and is undetectable in cancer cell lines. GNMT nuclear localization was associated with induction of apoptosis that is independent of its catalytic activity or folate binding . Overexpe cancer .Expressions of sarcosine metabolism-related proteins including GNMT varied according to subtype of breast cancer . Tissue Cancer cells within tumors are heterogeneous and dynamic. Proteome-wide mass spectrometry profiling revealed that MAT2A is among the cell cycle-dependent translocating proteins. Further analyses indicated that MAT2A may translocate to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication . This stGNMT, MAT1A and MAT2A mRNA levels in breast carcinoma using RNA-seq data from the TCGA/GTEx datasets within GEPIA2, and further validated the findings in our own breast cancer cohort by immunohistochemistry. GNMT, MAT1A, MAT2A-biomarker IHC panel was compared with the clinical survival record in breast cancer patients, to examine the accuracy of IHC-based methods for identifying clinical prognosis.In the present study, we explored the clinical significance of methionine cycle genes, including GNMT, MAT1A and MAT2A mRNA expression of the methionine cycle genes were verified by the GEPIA web tool   . mRNA exsurvival A,B. A dibe lower C. Howeveanalyses D. The cop = 0.004, p < 0.001, p < 0.001, The nucleus translocation of MAT2A has been proposed to enable epigenetic histone methylation maintenance during DNA replication in vitro . The prep = 0.050, p = 0.050, IHC staining of tissue microarray discovered that a higher cytoplasmic/nuclear (C/N) ratio of MAT2A protein was observed in more  patients aged above 65 (p < 0.001). Patients aged 65 and above were associated with a significant increase in 5 years mortality rate compared with those aged below 65  1.186–6.472) . Taken tP53 and DNA Damage Regulated 1 (PDRG1) encoded protein PDRG1 has been reported as an interaction target of methionine adenosyltransferases in the control of the nuclear methylation status [PDRG1 mRNA expression and breast cancer patient survival , which activates both sites, can increase MAT2A expression in a dose- and time-dependent manner [MAT2A in breast cancer, and there could be a link between inflammation and MAT2A expression during breast cancer progression. Previously, we discovered in vitro and in vivo evidence that low-dose anti-inflammatory DMARD methotrexate inhibits MAT genes, proteins, and enzyme activity [The cause of increased gulation . Nucleart manner . TNF-α lt manner . Hence, activity  and thusMAT2A overexpression facilitates mouse hepatic fibrosis through the regulation of intracellular SAM concentration [MAT2A may modulate human disease pathogenesis via SAM supply. Carbon tetrachloride-induced ntration . Transfontration , suggestntration . Furtherntration . In vitrntration . MAT2A pntration .Pdrg1 gene expression is not related to breast cancer survival. No association was observed between overall survival and mRNA expression levels of PDRG1  that leads to reduced DNA methylation. Increased PDRG1 expression is detected in acute liver injury and hepatoma cells, together with decreased MAT1A expression and nuclear accumulation of MATα1. Silencing of PDRG1 in hepatoma cells downregulates genes associated with tumor progression according to GO pathway analysis. Yeast two hybrid and rat liver library revealed that onco-protein PDRG1 is an interacting target of MATs . These dof PDRG1 .GNMT was downregulated in breast tumor tissues compared with normal breast tissues. GNMT has been proposed to be a novel tumor suppressor in cellular defense against DNA damage . ConversMAT2A to synthesize SAM and maintain their epigenome. Higher level of MAT2B has been found to be correlated with worse relapse-free survival in the TNBC [MAT2B encodes a β-subunit without catalytic action, but it can regulate MATII enzymatic activity [MAT2A/MAT2B favors tumor growth and survival and also enhances tumor migration [MAT2B suppressed cell growth and migration and induced apoptosis in breast cancer cell MDA-MB-231 and MDA-MB-468 [Many transformed cells rely on the TNBC . MAT2B eactivity . Inductiigration . Direct A-MB-468 . Future GNMT, MAT1A and MAT2A mRNA in breast tumor and breast normal tissues was calculated using ANOVA. The Kaplan–Meier survival analysis of GNMT, MAT1A, and MAT2A mRNA expression was performed on the BRCA RNA-seq data of the TCGA/GTEx datasets available within GEPIA2, by autoselecting the median values between the lower and upper quartiles into high and low expression. More information can be found at http://gepia2.cancer-pku.cn/#index (accessed on 25 October 2020) [The expression analysis of er 2020) .Contralateral primary breast tumor and adjacent normal breast tissues of 252 breast cancer patients receiving surgical resection were acquired from Changhua Show Chwan Memorial Hospital from March 2011 to January 2017. Computed tomography (CT) was applied for diagnosis in the 265 breast cancer patients prior to surgery. The diagnosis parameters and clinical outcomes were recruited until patient death or loss to follow-up. The age of all patients was between 29 and 95 years (mean ± SD = 54.88 ± 12.32). Clinical parameters and survival data were recorded from the cancer registry system of Changhua Show Chwan Memorial Hospital. Survival data was annotated to be the following time from the date of primary surgery to the date of death. During this survey, 30 patients died and 42 patients exhibited tumor metastasis, with the metastasis sites, including skin, abdomen, pleura, bone, lung, liver, chest wall, breast, and lymph node. The median overall survival of all breast cancer patients was 48 months. This project was approved by the Ethics Committee of the Institutional Review Board of Show Chwan Memorial Hospital .For each patient, representative tissue cores of the BC tumor section as well the adjacent normal section were carefully collected and made into tissue microarray. Immunohistochemistry (IHC) staining was used to evaluate GNMT, MAT1A, and MAT2A protein expression. The GNMT antibody  was purchased from Proteintech Group, Inc. . MAT1A antibody  was purchased from Novus Biologicals, LLC, Inc. ., and MAT2A antibody  was purchased from GeneTex, Inc. . IHC evaluation and protocol were used to obtain score have been descripted previously ,46. The 2 at 37 °C.The human breast cancer cells were purchased from the American Type Culture Collection . The human breast cancer MCF7, Hs578T, MDA-MB-231, and BT549 cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS . T47D and BT549 were grown in RPMI 1640  with 10% FBS. Cells were maintained in a humidified incubator with 5% COg for 10 min at 4 °C and then lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer  with 100 µL protease inhibitor cocktail . Equal amounts of protein (30 µg) were separated by SDS-PAGE (10% gel) and subsequently transferred to a polyvinylidene difluoride membrane. Subsequent to blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies, including anti-MAT2A , anti-β-actin , anti-α-tubulin , anti-Histone H3 , followed by incubation at room temperature for 2 h with HRP-conjugated polyclonal secondary antibody . All Western blots were visualized using the enhanced plus chemiluminescence assay kit , according to the manufacturer’s protocol. Protein expression levels in cells were quantified by ImageJ software .The cells were harvested using a curet and centrifuged at 1000× 4 cells per chamber) in serum-free medium were seeded in the upper chamber, and 10% fetal bovine serum (FBS)  was used as a chemoattractant in the bottom well. After incubation for 24 h at 37 °C, the non-invasive cells on the upper surface of the membrane were removed with a cotton swab, and the invasive cells on the bottom side were fixed in 100% methanol at room temperature for 5 min, stained with 1% crystal violet at room temperature for 10 min and counted using a microscope  under ×200 magnification with five fields of view per cells.Cell invasion was investigated using Matrigel invasion chambers with a pore size of 8 μm . Briefly, MCF7, Hs578T, MDA-MB-231, and BT549 cells  was used for all statistical analyses.The association between GNMT, MAT1A, and MAT2A protein expression and the clinical and pathological parameters was calculated using Chi-square and paired-sample t-tests, and survival curves were plotted using the Kaplan–Meier method and compared using log-rank test. Cox’s proportional hazards regression model was used to analyze the association between the variables and survival data. The present study demonstrated a novel strategy that used the MAT2A C/N ratio rather than the MAT2A expression for breast cancer prognosis. Furthermore, it is noteworthy that high C/N ratio (>1) of MAT2A protein expression was present in more than 50% of the breast cancer specimens in our cohort. In vitro studies found that breast cancer cell lines with a higher MAT2A C/N ratio were more invasive. MAT2A C/N expression ratio determined by IHC staining could serve as a novel independent prognostic marker for breast cancer. The modulation of MAT2A subcellular localization and function may serve as a potential novel therapeutic strategy for breast cancer.


All data were analyzed with non-parametric statistics. Patients with CD had similar to healthy controls 25(OH)D3 levels (p > 0.05) and higher 25(OH)D3/24,25(OH)2D3 ratios (p < 0.05) throughout the study. They also had lower baseline free 25(OH)D levels (p < 0.05) despite similar DBP levels (p > 0.05) and lower albumin levels (p < 0.05); 24-h urinary free cortisol showed significant correlation with baseline 25(OH)D3/24,25(OH)2D3 ratio . The increase in 25(OH)D3 after cholecalciferol intake was similar in obese and non-obese states and lacked correlation with BMI (p > 0.05) among patients with CD, as opposed to the control group. Overall, patients with CD have a consistently higher 25(OH)D3/24,25(OH)2D3 ratio, which is indicative of a decrease in 24-hydroxylase activity. This altered activity of the principal vitamin D catabolism might influence the effectiveness of cholecalciferol treatment. The observed difference in baseline free 25(OH)D levels is not entirely clear and requires further study.In this study we aimed to assess vitamin D metabolism in patients with Cushing’s disease (CD) compared to healthy individuals in the setting of bolus cholecalciferol treatment. The study group included 30 adults with active CD and the control group included 30 apparently healthy adults with similar age, sex and BMI. All participants received a single dose  of cholecalciferol aqueous solution orally. Laboratory assessments including serum vitamin D metabolites (25(OH)D Cushing’s disease (CD) is one of the disorders associated with endogenous hypercortisolism and is caused by adrenocorticotropic hormone (ACTH) hyperproduction originating from pituitary adenoma . Skeleta3, or cholecalciferol) and its active metabolites  are universally considered as the essential components of the osteoporosis management  unless otherwise specified. Mann-Whitney U-test and Fisher’s exact two-tailed test were used for comparisons between two groups. Friedman ANOVA was performed to evaluate changes in indices throughout the study and pairwise comparisons using Wilcoxon test with adjustment for multiple comparisons (Bonferroni) were also made if the Friedman ANOVA was significant. Spearman rank correlation method was used to obtain correlation coefficients among indices. A p > 0.05). Both groups consisted predominantly of young and middle-aged women and the majority of patients were overweight or moderately obese (p < 0.05).The groups were similar in terms of age, sex and BMI (ly obese . PatientThe features of the underlying disease course in the study group are listed in p > 0.05) and although there was a slight difference in alcohol consumption (p < 0.05), the absolute values were minor in both groups . They also had higher levels of urine phosphorus-creatinine ratio (p < 0.05). The rest of the studied biochemical parameters did not show significant difference between the groups (p > 0.05). 3 patients (10%) from the study group and 5 patients (17%) from the control group had secondary hyperparathyroidism, one patient with CD (3%) was diagnosed with mild primary hyperparathyroidism.Patients with CD had several alterations in biochemical parameters, in particular, lower baseline serum creatinine and albumin levels, while magnesium levels were higher than in the control group , with only two patients (7%) from the study group and one patient (3%) from the control group having sufficient vitamin D levels, according to the Endocrine Society and the Russian Association of Endocrinologists guidelines , whereas the levels of 3-epi-25(OH)D3 and 24,25(OH)2D3 were lower in CD patients. Further calculation of 25(OH)D3/24,25(OH)2D3 and 25(OH)D3/1,25(OH)2D3 ratios corresponded to the observed levels of metabolites: 25(OH)D3/24,25(OH)2D3 ratio was higher in the study group (p < 0.05) assuming lower 24-hydroxylase activity and 25(OH)D3/1,25(OH)2D3 ratio was equal between the groups (p > 0.05). As for the assessment of vitamin D metabolism, unexpectedly the levels of 25(OH)D30 ng/mL ,42). Thep < 0.05) and the levels of DBP did not differ between the groups (p > 0.05). Although calculated free 25(OH)D showed prominent positive correlation with the measured free 25(OH)D in both groups , the association appeared to be weaker in the study group. In the control group, DBP levels correlated with both measured and calculated 25(OH)D levels , while in patients with CD there was no association with measured free 25(OH)D levels .Levels of free 25(OH)D were lower in CD patients (p < 0.05) and urine calcium-creatinine ratio  among assessed biochemical parameters, and only with 25(OH)D3/24,25(OH)2D3 ratio among the parameters of vitamin D metabolism .Correlation with 24-h UFC in CD patients was observed for serum albumin level  from the control group completed the study. p = 0.006) and a tendency to an increase in the urine phosphorus-creatinine ratio by Day 7 (p = 0.02). Patients from the control group showed a clinically insignificant increase in serum creatinine levels by Day 1 (p = 0.002) and a non-significant trend towards an increase in serum total and albumin-adjusted calcium (p = 0.01 for both measurements). No change in PTH levels was observed in patients with CD during the follow-up (p > 0.05), while in the control group there was a tendency for PTH to decrease by Day 3 (p = 0.02). There were no new cases of hypercalcemia in both groups during the follow-up. One patient from the study group and one patient from the control group had persistently increased urine calcium-creatinine ratio throughout the study. Four patients from the study group (13%) and none from the control group developed hypercalciuria during the follow-up, however these patients had no clinical manifestations during the observation period.The observed baseline differences in biochemical parameters mostly preserved during the follow-up. In the study group there was an increase in serum phosphorus levels by Day 1 (3 levels (≥30 ng/mL). Levels of 25(OH)D3 continued to increase by Day 3 in both groups (p < 0.001), after which tended to decrease in the study group (p = 0.01) and remained stable in the control group (p = 0.65). The increase in 25(OH)D3 after cholecalciferol intake was equal between the groups . In the presence of obesity, Δ25(OH)D3 was higher in the CD patients than in the control group , while in non-obese patients no difference was observed (p > 0.05).By Day 7, 25 patients (83%) from the study group and 22 patients (79%) reached sufficient 25(OH)D3 , while in the control group it was significantly lower in obese patients . BMI showed significant correlation with Δ25(OH)D3 only in the control group , while in CD patients there was no such association  D > 0.05) .2D3 levels increased in CD patients by Day 1 and were stable during the follow-up in the control group. The rest of the studied parameters of vitamin D metabolism changed in a similar way between groups: 3-epi-25(OH)D3 levels increased until the Day 3, after which they decreased by the Day 7; 24,25(OH)2D3 levels showed more graduate elevation throughout the follow-up. In both groups 25(OH)D3/24,25(OH)2D3 ratios increased by Day 1, after which they decreased by Day 7, and 25(OH)D3/1,25(OH)2D3 ratios increased by Day 1, after which they remained stable. DBP levels didn’t change and free 25(OH)D levels showed an increase in both groups during the follow-up. The levels of 25(OH)D2 did not exceed 0.5 ng/mL in all examined individuals throughout the study. Among assessed parameters of vitamin D metabolism, higher 25(OH)D3/24,25(OH)2D3 ratios in the study group was the only difference between the groups which remained significant throughout the observation period (p < 0.05)  < 0.05) . 3 levels and its response to the therapeutic dose of cholecalciferol in patients with CD as compared to healthy individuals. We observed no difference in baseline 25(OH)D3 assessed by UPLC-MS/MS between groups. Similar to our data were obtained in most studies conducted specifically in the state of endogenous hypercortisolism in humans [3 by UPLC-MS/MS, since the initial difference between the groups revealed by ELISA data with lower total 25(OH)D levels in the study group was not replicated by UPLC-MS/MS. It should be noted that our ELISA method did not participate in an external quality control program at the time of the study unlike UPLC-MS/MS; furthermore, a lower analytical performance was previously described for this technique with tendency for low specificity and lower measurement results [The main goal of our study was to evaluate the 25(OH)Dn humans ,15 and dn humans . The stun humans , while in humans . Somewha results . 3/24,25(OH)2D3 ratio in CD patients, both initially and during the observation after the intake of the cholecalciferol loading dose, indicating consistently reduced activity of 24-hydroxylase, the main enzyme of vitamin D catabolism. Earlier clinical and experimental studies also suggested altered activity of enzymes of vitamin D metabolism in hypercortisolism. However, these studies were heterogeneous and aimed predominantly at studying the activity of 1α-hydroxylase [2D3 levels in rats [2D3 levels [2D3 levels before and after hypophysectomy as well as compared to control dogs [2D3 in patients with Cushing’s syndrome [3/24,25(OH)2D3 ratio, which has recently emerged as a new tool for vitamin D status assessment [3 in obese and non-obese state and lacked correlation between Δ25(OH)D3 and BMI, as opposed to the control group. Moreover, the increase in 25(OH)D3 in obese patients from the control group was lower not only than in non-obese controls, but also than in obese patients with CD. When assessing other parameters of vitamin D metabolism, the most significant finding was the higher 25(OH)Droxylase ,11,12,14 in rats , while isyndrome , which isyndrome , liver asyndrome  and in rsyndrome . Howeversessment ,47. GiveAnother intriguing finding was lower levels of free 25(OH)D observed in patients with CD despite similar DBP levels and lower albumin levels, which, on the contrary, allows one to expect higher values of free 25(OH)D. Considering the weaker correlation between the measured and calculated free 25(OH)D in patients with CD, as well as the lack of correlation of the measured 25(OH)D with the main transport protein, an altered affinity of DBP might be suspected. One possible explanation is protein glycosylation as a consequence of diabetes mellitus, which was present in half of the patients ,48,49. AThe obtained discrepancies in the biochemical parameters characterizing calcium and phosphorus metabolism were generally consistent with the data of early studies discussed in the introduction ,27,28,29Our research is distinguished by a number of important strengths: a prospective design, substantial sample of patients with CD, accounting for social and behavioral factors affecting vitamin levels D, comprehensive spectrum of vitamin D metabolism parameters investigated and participation in an external quality control program for vitamin D metabolites measurement.p = 0.07 for both parameters). Also, the study lacked a study group of patients with remission of CD to test the hypotheses put forward, however, this is a promising direction for further research.Nevertheless, the study also had several limitations: the amount of dietary vitamin D and phosphorus, as well as possible differences in DBP affinity to vitamin D metabolites due to genetic isoforms of DBP  or other3/24,25(OH)2D3 ratio than healthy controls, which is indicative of a decrease in 24-hydroxylase activity. This altered activity of the principal vitamin D catabolism might influence the effectiveness of cholecalciferol treatment. There is also a lack of clarity regarding the lower levels of free 25(OH)D observed in patients with CD, which require further study. To test the proposed hypotheses and to develop specialized clinical guidelines for these patients, longer-term randomized clinical trials are needed.We report that patients with endogenous ACTH-dependent hypercortisolism of pituitary origin have a consistently higher 25(OH)D


It plays an important role in many physiological and pathological processes. Recent studies indicate that H2S can improve myocardial I/R injury, however, its mechanism is not fully understood, especially the involved signal pathways. In this review, we summarize the related researches about the role of the signaling pathways involved in the protective effects of exogenous H2S on myocardial I/R injury, so as to provide theoretical reference for the future in-depth researches.Ischemia/reperfusion (I/R) injury refers to the functional and structural changes in the process of blood flow recovery after ischemia. In addition to ischemia, the blood flow recovery can also lead to very harmful damage, such as the obvious cell swelling and the irreversible cell necrosis. I/R injury is related with many diseases, including myocardial I/R injury. Myocardial I/R injury refers to the aggravation of ischemic myocardial tissue injury due to sudden disorder of blood circulation. Although there are many studies on myocardial I/R injury, the exact mechanism is not fully understood. Hydrogen sulfide (H Ischemia/reperfusion (I/R) injury is used to describe the functional and structural damages which become apparent when the blood flow is restored after a period of ischemia. In addition to the ischemia, the recovery of blood flow can leads to potentially very harmful effects, such as the cell necrosis, the notable cell swelling, and the uneven blood flow of all parts of the recovered tissues . I/R inj2S), which has the smell of rotten eggs, is a toxic, colorless and corrosive gas. Structurally, it is a sulfur analog of water molecules and can be oxidized to elemental sulfur, sulfate (SO42–), sulfur dioxide (SO2), and thiosulfate (S2O3–)  and carbon monoxide (CO), is the intracellular signal transduction molecules. It has been found that the low concentration of H2S plays an vital role in the physiological process (2S: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) . The sulfur of 3-MP was catalyzes by 3-MST to convert into H2S  . H2S wasdogenous . In rece process . Three ee (3MST) . CBS catinto H2S . It has l system . In rece2S) postconditioning attenuated the rat myocardial I/R injury by improving systemic hemodynamics, reducing myocardial infarct size and inhibiting cardiomyocyte apoptosis. While AG-490, a JAK2 inhibitor, abolished the cardioprotective effect of exogenous H2S. The in depth researches showed that NaHS postconditioning increased the expression of p-STAT3 and bcl-2, and decreased bax expression in the rat heart with I/R injury, while AG-490 counteracted these changes. Therefore, it can be deduced that H2S postconditioning protected the rat hearts against I/R injury through JAK2/STAT3 signaling pathway  signaling pathway is activated by a variety of interferons and cytokines, and widely involved in the tumor signal transduction. JAK is activated by the bingding of cytokines to its specific receptors of the cell membrane, which induces STAT phosphorylation. The phosphorylated STAT binds to the specific DNA elements and promotes the gene transcription . The JAK pathway . Rapamyc channel , suggest channel , therefooxidants . Therefo2S improves diabetes-accelerated atherosclerosis by suppressing oxidative stress via Keap1 sulfhydrylation at Cys151 to activate Nrf2 signaling  in the heart of diabetic mice were reduced. The levels of free H2S and sulfane sulfur also were notably decreased in the heart and the blood of diabetic mice. Treatment with H2S in the form of sodium sulfide (Na2S) 24 h before myocardial ischemia (Na2S precondition) or 7 days before myocardial ischemia (Na2S 7d precondition) could notably reduce rat diabetic myocardial I/R injury by decreasing infarct size. Moreover, the myocardial protective effect of Na2S 7d precondition was better than that of Na2S PC. Pretreatment with Na2S decreased the oxidative stress and the apoptosis induced by myocardial I/R injury through reducing lipid peroxidation levels and cleaved caspase-3, respectively, suggesting that Na2S improve rat myocardial I/R injury by inhibiting oxidative stress and the apoptosis. The mechanism researches revealed that Na2S pretreatment activated Nrf2 signaling in rat diabetic myocardium with I/R injury, while diabetes impaired Nrf2 signaling. NQO1 and HO-1 are important antioxidants. Na2S 7d PC increased the expression of NQO1 and heme oxygenase-1 (HO-1) by promoting Nrf2 binding to the promoter of NQO1 and HO-1. Bach1, a known HO-1 transcription inhibitor, which impeded the Nrf2 binding to HO-1 promoter. The in depth researches showed that Na2S 7d PC could upregulate HO-1 expression by promoting the ERK1/2-dependent removal of Bach1 from the nucleus. Collectively, exogenous H2S improved diabetic myocardial I/R injury by upregulating the expression of NQO1 and HO-1 through activating Nrf2 signaling pathway in an ERK-dependent manner, which needs to be further confirmed with the inhibitor of Nrf2 signaling pathway -like 2] is a transcription factor and is inhibited by interacting with the redox sensitive protein Kelch-like ECH-associated protein 1 (Keap1). Nrf2 is the main regulator of a group of antioxidant response elements containing cell protective genes induced in the stress response. It has been reported that Hignaling . H2S alsap1-Nrf2 . Extraceap1-Nrf2 . It has pathways . So far, pathway . At pres+-dependent protein deacetylase, which deacetylates downstream peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) to promote its activity  levels, and decreasing malondialdehyde (MDA) level. Exogenous H2S also upregulated the expression of SIRT1 and PGC-1α in the rat heart with I/R injury. EX-527, a selective SIRT1 inhibitor, reversed the above changes induced by H2S, suggesting that SIRT1/PGC-1α signaling pathway mediated the protective effect of exogenous H2S on myocardial I/R injury , FOXO family transcription factors, and ubiquitination can regulate SIRT1 activity  is a highly conserved NADactivity . The SIRactivity . There aactivity , howeverR injury . The entR injury , which m pathway . Studiesactivity . Therefo2S, suggesting that exogenous H2S activated PI3K/SGK1/GSK3β signaling pathway in cardiomyocyte with H/R injury. The H/R treatment of rat cardiomyocytes reduced cell viability, and aggravated cell injury by increasing LDH releasing, which were reversed by exogenous H2S. Further researches showed that autophagy was notably increased in cardiomyocytes exposed to H/R, which was reversed by exogenous H2S. The inhibition of PI3K with LY294002 (a PI3K inhibitor) or knocking down SGK1 with SGK1 siRNA promoted autophagy and inhibited the anti-autophagy, and cardioprotective effects of exogenous H2S. While blocking GSK3β by tws119 (a GSK3β inhibitor) has the opposite effect. Collectively, it can be induced that exogenous H2S alleviated myocardial I/R injury by inhibiting autophagy through activating PI3K/SGK1/GSK3β signaling pathway  is a group of plasma membrane associated lipid kinases, which consists of three subunits . It is i pathway . The rel2S. The above suggested that exogenous H2S notably improved myocardial I/R injury. The in-depth studies showed that in the cardiomyocytes with I/R jury, exogenous H2S also increased the endogenous H2S level and induced the activity of CSE, SOD and GSH-Px, inhibited the activity of SOD, and reduced the level of phosphorylated JNK2. This indicated that exogenous H2S may alleviate myocardial I/R injury through antioxidant and JNK signaling pathway  is a member of the mitogen activated protein kinase (MAPK) family and regulate the cell responses to a variety of exogenous and endogenous damages, including reactive oxygen species (ROS), radiation, DNA damage, bacterial antigens, heat, and inflammatory cytokines. In particular, the JNK signaling regulates many important physiological processes including metabolism and tissue homeostasis, cell damage repair and cell death/survival, and affects the life span of organisms . The JNK pathway , which n pathway , so whet2S on myocardial I/R injury as follows: (1) exogenous H2S postconditioning improves the rat myocardial I/R injury through JAK2/STAT3 signaling pathway; (2) exogenous H2S protected diabetic mouse myocardial I/R injury through activating Nrf2 signaling pathway in an ERK-dependent manner; (3) exogenous H2S alleviates myocardial I/R injury in rats by activating SIRT1/PGC-1α signaling pathway; (4) exogenous H2S alleviated myocardial I/R injury by inhibiting autophagy through activating PI3K/SGK1/GSK3β signaling pathway; and (5) exogenous H2S alleviates myocardial I/R injury through inhibiting JNK signaling pathway (In this review, we summerized the signaling pathways involved in the protective effect of exogenous H pathway .2S in the process of myocardial I/R injury remains to be further elucidated. For example, in addition to the signal pathways mentioned in this manuscript, is there any other signal pathway involved in the above effects? Can high concentration of H2S aggravate myocardial I/R injury through the specific signaling pathways? In addition, the studies indicate that the cardioprotective effect of H2S is related to gender. Estrogen can regulates the production and release of H2S in cardiovascular cells to increase cell proliferation, cell migration, and vasodilation, which exert its cardiovascular protective effects (2S can improve myocardial inflammation in diabetic mice by inhibiting NLRP3 inflammasome (2S can improve myocardial I/R injury by inhibiting NLRP3 inflammasome and the related signaling pathways deserve further study.Hydrogen sulfide is now regarded as the third kind of signal gas transmitter after NO and CO. It has a wide range of physiological and pathophysiological functions, including vasodilation, induction of angiogenesis, regulation of inflammatory response, regulation of glucose homeostasis, and regulation of neuronal activity. However, its role has not been fully studied. The mechanism of H effects . The aboammasome , I/R canammasome , so whet2S in the process of myocardial I/R injury, the use of H2S donor in the treatment of myocardial ischemia-reperfusion injury will become a very promising therapeutic strategy.With the in-depth study of the signal pathway of HHW devised, wrote, and funded with the manuscript. SL wrote and funded with the manuscript. XL drew the figures. SZ and HL wrote the manuscript. 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.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.


PR8NA tetramer (NAtet) could provide complete homologous protection against A/PR8 (H1N1) virus infection in mice, while the protection of H1N1PR8NA monomer (NAmono) was moderate. Higher levels of NA-reactive binding and inhibition antibodies and less weight loss were observed in the H1N1PR8NAtet-vaccinated group. Similarly, H5N1VNNAtet immunization exhibited a preferable heterologous protection than H5N1VNNAmono, but neither H7N9SHNAtet nor H7N9SHNAmono vaccination showed heterosubtypic protection. We also compared the effect of three adjuvants, aluminum, 3′3′-cGAMP (cGAMP), and Poly(I:C), on the humoral response and protective efficacy induced by H1N1PR8NAtet. H1N1PR8NAtet protein adjuvanted with aluminum was observed to exhibited better capacity in inducing NA-specific humoral immunity and preventing weight loss than with cGAMP or Poly(I:C). In conclusion, our data demonstrate that tetrameric NA with natural conformation is required to induce protective anti-NA immunity. The NA tetramer could provide homologous protection and subtype-specific cross-protection. In addition, the aluminum adjuvant is preferable in recombinant NA protein vaccination.Influenza neuraminidase (NA) is able to induce cross-subtype immunity and is considered as a promising target for the development of universal influenza vaccines. However, commercial influenza vaccines only induced low NA-specific immune responses due to the low amounts and the denatured conformation of NA proteins in current inactivated or split influenza vaccines. Here we investigated the protective efficacy of recombinant tetrameric and monomeric NA proteins to determine whether the conformation contributed to induce protective immunity. We found that H1N1 The seasonal epidemics and less frequently global pandemics of influenza viruses result in high morbidity and mortality every year. Vaccination is the most effective way to prevent influenza circulation. The currently licensed vaccines, including inactivated and attenuated influenza vaccines, induce protection mainly by eliciting neutralizing antibodies (NAbs) against the major glycoprotein, hemagglutinin (HA), of influenza virus . VaccineNeuraminidase (NA) protein is the second most abundant glycoprotein on the membrane of influenza virion. The native influenza NA protein is a tetramer with enzymatic activity  that canPR8NAtet tetramer.In this paper, we investigated the factors that potentially affect the immune response and protective efficacy induced by NA protein in BALB/c mouse models. We compared the difference of the protective efficacy between tetrameric and monomeric NA proteins and investigated the humoral responses induced by tetrameric and monomeric NA proteins. We also assessed the influence of adjuvants on the humoral response and protective efficacy induced by H1N1Madin Darby canine kidney (MDCK) and human embryonic kidney 293 (HEK293T) cells were obtained from the American Type Culture Collection and grown in complete high-glucose Dulbecco’s modified Eagle’s medium  supplemented with antibiotics  and 10% fetal bovine serum . Expi293F (Thermo Fisher Scientific) cells were grown in SMM 293-TII expression medium .50) of viruses were calculated by the Reed and Munch method.The influenza A virus (IAV) used in this study was the mouse-adapted strain A/Puerto Rico/8/1934 (H1N1). The IAV was propagated in MDCK cells in serum-free DMEM media in the presence of 1 μg/ml TPCK-trypsin (Sigma-Aldrich). The median tissue culture infective dose and median lethal dose  beads . Briefly, the NA ectodomains with additional N-terminal Igκ-light chain secretion sequence, followed by a hex-histidine tag (HHHHHH), a human vasodilator stimulating phosphoprotein (VASP) tetramerization domain (SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRG), and a thrombin cleavage site (SLVPRGSPSRS) were constructed into eukaryotic expressing plasmid pcDNA3.1 to express NA tetramer (NAtet). The monomeric NA (NAmono) proteins were constructed in the same way as NAtet but without the VASP domain. Recombinant NA proteins were purified from the supernatant of transiently transfected Expi293F cells by Ni-NTA beads. The concentration of proteins was adjusted to 1 mg/ml with phosphate-buffered saline (PBS) and frozen at −80°C.Recombinant NA proteins derived from A/Puerto Rico/8/1934 (H1N1), A/Shanghai/37T/2009 (H1N1), A/Hong Kong/16/68 (H3N2), A/Vietnam/1204 (H5N1), and A/Shanghai/4664T/2013 (H7N9)—referred to as H1N1Recombinant NA proteins were then analyzed by Western blotting. Briefly, 2 μg of NA were mixed with 4× SDS-loading buffer containing 10% β-mercaptoethanol. The samples were heated for 10 min at 98°C and were afterward loaded on an SDS gradient gel . The gel was run for 100 min at 120 V, and Western blotting transfer was performed. Following the transfer onto the nitrocellulose membrane, the membrane was blocked with TBS with 0.1% Tween 20 (TBS-T) containing 5% milk powder for 2 h at room temperature (RT). Then, mouse anti-HIS primary antibody (Younuoke Biotechnology Inc.) was added for 12 h at 4°C . The membrane was washed three times with TBS-T after 12 h, and a secondary goat anti-mouse horse radish peroxidase (HRP) antibody (Jackson Immuno Research) was added for 1 h at RT . The membrane was then washed three times with TBS-T before it was visualized using Pierce Chemiluminescence (ECL) Western Blotting Substrate (as per the instructions of the manufacturer) on Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).The extent of tetramerization and/or multimerization was investigated by cross-linking of NA with glutaraldehyde (Sigma-Aldrich). Briefly, 5 μg of NA was diluted in 25 μl of PBS in the presence of 0.3 mM of glutaraldehyde cross-linker. The mixture was incubated at RT for 5 min, and then glutaraldehyde was quenched by adding 1 M Tris–HCl buffer (pH 8.0) to a final concentration of 50 mM. Afterward, the protein samples were loaded on 4–20% SDS gradient gel. The gel was run for 60 min at 180 V and confirmed by Coomassie staining.The enzymatic activity of recombinant NA proteins was determined by the cleavage of two specific substrates of NA, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA)  and fetu2, pH 6.5) were incubated with 20 mM MUNANA (Sigma-Aldrich) at 37°C for 40 min. The reaction was stopped by adding a stopping buffer , and the degree of fluorescence was detected by EnSight Multimode plate reader (PerkinElmer).For the MUNANA-based enzymatic activity assay, twofold gradient diluted recombinant NA proteins that range from 0.0015625 to 1.6 μg/ml in morpholine ethanesulfonic acid (MES) buffer  values were read at 450 nm on a Multiskan FC plate reader (Thermo Fisher Scientific).For the fetuin-based enzyme-linked lectin assay (ELLA), MaxiSorp Nunc-immuno 96-well plates were coated with 100 μl of fetuin (Sigma-Aldrich) at a concentration of 50 μg/ml and refrigerated at 4°C overnight. The plates were blocked with 200 μl blocking buffer (PBS containing 1% FBS and 5% dry milk) for 1 h at RT and washed six times with PBS-T solution. Following the blocking, 50 μl of the sample diluent [Dulbecco’s phosphate buffered saline (DPBS) with 1% bovine serum albumin and 0.5% Tween 20] and 50 μl of serially diluted recombinant NA protein or purified virus were then added to the fetuin-coated plates and mixed well. The plates were then incubated for 14–16 h at 37°C before being washed six times with PBS-T. After the extensive wash, the NA enzymatic activity was detected by horse-radish peroxidase-labeled peanut agglutinin (Sigma Aldrich) and developed with the 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich). The reaction was stopped by the addition of 2 N HFemale BALB/c mice (6–8 weeks old) from the Laboratory Animal Center of Shanghai Public Health Clinical Center (SHPHCC) were used for all animal experiments. The protocols were reviewed and approved by the Ethics Committee of SHPHCC .PR8NAtet, H5N1VNNAtet, H7N9SHNAtet, H1N1PR8NAmono, H5N1VNNAmono, and H7N9SHNAmono, respectively. The antigens were diluted in 100 μl PBS and mixed with aluminum adjuvant (1:1). The mice were immunized with PBS as control. To investigate the influence of adjuvants on the immunogenicity of NA tetramers, the mice were intraperitoneally immunized with 20 μg H1N1PR8NAtet protein mixed with aluminum (i.p.), 3′3′-cGAMP  and Poly(I:C) (i.p.), respectively. Aluminum is a strong inducer of Th2 responses  immunized with 20 μg H1N1esponses . cGAMP iesponses . The TLResponses . These t50 of A/Puerto Rico/8/34 (H1N1) virus 2 weeks after the final immunization to evaluate the protective efficacy. The weight loss and survival rates of mice were monitored for 14 days after the challenge.Sera were collected 1 week after the final immunization to detect NA-specific antibodies. The mice were intranasally challenged with 5 LD2CO3, 40 mM NaHCO3, and pH 9.6) at 4°C overnight. To detect the NA-specific antibodies, the respective immunogen to sera of immunized mice was coated. To detect the cross-binding activity of mice sera, the tetrameric NA protein of H1N1PR8NAtet was coated. The plates were washed three times with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 1% FBS and 5% dry milk) for 1 h at RT. The sera were fourfold serially diluted starting at 1:100 in disruption buffer . Then, 50 μl of diluted serum was added to each well and incubated for 1 h at RT. After three times of washing with PBS-T, bound antibodies were detected by horseradish peroxidase-labeled goat-anti mouse IgG antibody (Jackson Immuno Research) and substrate ABTS (Thermo Fisher Scientific). The OD was measured at 405 nm on a Multiskan FC plate reader (Thermo Fisher Scientific). The isotypes of NA-specific antibodies were determined by ELISA with Mouse Monoclonal Antibody Isotyping Reagents (Sigma-Aldrich) following the instructions of the manufacturer. The antibody titers were defined as the highest sera dilution at which the OD values were twice of those by the control sera.The NA-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA). To explore the immunogenicity of tetrameric and monomeric NA proteins, the sera samples which were collected 1 week after the final immunization were tested for NA-reactive antibodies by ELISA, including NA-specific binding antibodies to the respective immunogen, cross-subtype binding antibodies against NA of A/PR8 (H1N1), and NAI antibodies. The subtypes of NA-specific antibodies were also analyzed. Briefly, MaxiSorp Nunc-immuno 96-well plates (Thermo Fisher Scientific) were coated with 2 μg/ml NA proteins (100 μl/well) in carb/bicarb coating buffer  virus was added to each well of the serially diluted serum plate, and the plates were incubated at 37°C for 1 h. The amount of split A/PR8 (H1N1) virus used in the NAI assay corresponded to 90% of the maximum signal. After incubation, 100 μl of the serum/virus mixture was added on the blocked fetuin-coated plates and incubated for 16–18 h at 37°C. The remainder of the NAI assay was performed as ELLA as described above. The values of test wells obtained from the plate reader were divided by the average value for virus-only control wells and then multiplied by a factor of 100 to obtain the NA activity. Percent inhibition was calculated by subtracting the NA activity from 100.P-value < 0.05 was considered significant. All the statistical analyses were performed by GraphPad Prism, version 8.00 .The survival rate was compared by log-rank test. Antibody titers, viral titers, and body weights among groups were compared by one- or two-way ANOVA. A The NA tetramers and monomers were expressed using Expi293F cells and purified by Ni-NTA. The proteins were analyzed by Western blotting ,B and crPR8NAtet or H1N1PR8NAmono significantly protected the mice against the lethal dose of the A/PR8 (H1N1) influenza challenge , with only 37.5% (3/8) of H5N1VNNAmono-immunized mice surviving. No protection was observed in mice that were immunized with either H7N9SHNAtet or H7N9SHNAmono proteins (pdm09NA induced moderate cross-protection against A/PR8 (H1N1) virus infection while H3N2HKNA not  virus. As shown in proteins ,F, indicHKNA not .VNNAtet and H5N1VNNAmono  than the six survivors that were immunized with H1N1PR8NAmono. In contrast, the NAI activity was barely detectable in sera from two deceased mice in this group -adjuvant, but both adjuvants induced a similar level of NAI antibodies , on the protective efficacy and humoral responses induced by the H1N1tibodies . These rSHNAtet tetramer could not provide heterosubtypic protection against A/PR8 (H1N1) virus infection. This finding is supported by the report that vaccination with the recombinant NA protein of A/PR8 (H1N1) could provide complete homologous protection against A/PR8 (H1N1) virus infection but not heterosubtypic protection against H3N2 virus  virus infection without provoking cross-reactive NAI antibodies, which probably depended on the differences among the epitopes around the active enzyme sites (VNNAtet induced high titers of subtype-specific binding antibodies to the NA protein of A/PR8 (H1N1) virus. Those cross-binding antibodies may potentially contribute to partial cross-protection. Further study is required to clarify whether the cross-protection was correlated with the NA-binding antibodies.It was suggested that the protection induced by the NA protein was mediated by NAI antibodies . In thisme sites . HoweverIn addition, adjuvant is also an important consideration in NA protein-based vaccine design since we found that it worked on the immunity induced by the NA proteins. In contrast, we found that the mice of aluminum and cGAMP groups all survived the lethal influenza virus challenge, although the NAI antibody level of the aluminum group was slightly higher. We speculate that it may refer to the property of adjuvants or immune methods (i.p. vs. i.d.), but this still remains to be further determined. In short, adjuvant aluminum might be preferred for NA protein-based vaccination.In conclusion, our data suggests that tetrameric NA provides better homologous protection against influenza virus infection, and it could also confer preferable subtype-specific cross-protection. NA-reactive binding and inhibition antibodies are related to protection. Furthermore, aluminum adjuvant is preferential in vaccination of recombinant NA protein than cGAMP and Poly(I:C). We hope that this information could be useful for influenza vaccine formulation and administration.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Ethics Committee of Shanghai Public Health Clinical Center.JH conceived and designed the experiments and supervised the project. XD, ML, and QZ expressed and purified the recombinant NA proteins. XD performed the MUNANA-based enzymatic activity assay and ELISA and ELLA assays. XD and QW performed the mice experiments. XD, JH, and FW analyzed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.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.


CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes. Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) bacterial adaptive immune systems capture and store viral sequences in the genome for expression as guide RNAs that direct Cas proteins to disrupt viral infection  or dCas9 (pRH227) on the pC194 backbone  with ΦNM4γ4 targeting spacers of different lengths were built by inserting the spacers in pBD184 (S. aureus RN4220 as previously described elsewhere (S. aureus (in vivo) experiments are mentioned in Construction of the plasmids containing the backbone  were desbackbone  by ampli10-Cas9-Cas1-Cas2-Csn2 as reported earlier (Escherichia coli BL21 Rosetta (DE3) cells. Cultures were induced for 16 h with isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were lysed by sonication. The cell lysate was first purified by Ni-NTA affinity chromatography. The eluates were dialyzed and further subjected to ion-exchange chromatography by using Heparin columns. For ion-exchange chromatography, elution was performed using a concentration gradient with Buffer A  and Buffer B . For binding and molecular weight measurements, the elution peak from the Heparin column containing all four proteins was mixed with a spacer and sgRNA of choice and incubated at 4The co-expression of the supercomplex was done using a construct containing His earlier .2, pH 7.4) and heating at 95°C for 5 min followed by slow cooling to room temperature. A list of all different prespacers used in this study have been shown in in vitro processing assays and for selection of full-site integration products are:All DNA oligonucleotides used in this study were synthesized by integrated DNA technologies. Prespacers were prepared by annealing the complementary strands in hybridization buffer , UV (260 nm) and light scattering signals at 90° were collected. The eluting fractions were analyzed to assess the molecular weights of the components. Bio-SEC software (Agilent) was used for data analysis and molecular mass calculations.2 5mM, pH 7.4). The reactions were quenched with 25 mM EDTA and Proteinase K. Loading buffer containing bromophenol blue (BPB) was added to the reactions and heated at 80For prespacer processing experiments, Cas1-Cas2 integrase or the supercomplex with a complementary sgRNA/non-complementary sgRNA/sgRNA scaffold were incubated together at a concentration of 0.5 μM on ice for 1 h. The respective protein mixtures (Cas1 and Cas2) or the ribonucleoprotein complexes (supercomplex or Cas9) was incubated with 1 μM annealed prespacer substrates for 60 min at 37°C in processing buffer  and eluted with 6 μl water. The purified products were subjected to a gap-filling reaction  in which 3′ ends were extended by a non-displacing DNA polymerase and nicks were ligated: 6 μl purified acquisition reaction, 2 μl Taq DNA ligase , 1 μl T4 DNA Polymerase , dNTP Solution Mix , 2 μl 10x Taq DNA Ligase buffer (NEB), 6.5 μl water. Gap-filling reactions were again purified using the Zymo Research kit as described above. A Golden Gate-compatible chloramphenicol cassette was generated by PCR using a forward primer:Prespacer substrates were designed to facilitate Golden Gate-mediated insertion of a chloramphenicol selection cassette into pCRISPR (containing an Ampicillin cassette) only upon full-site integration of the prespacer by Cas1-Cas2 integrase or the supercomplex. Specifically, two abutted and inverted BbsI restriction sites were encoded in the center of each prespacer substrate, with overhangs designed to be complementary to the BbsI-generated overhangs at the edges of the chloramphenicol selection cassette. In total, the BbsI recognition and cleavage sites occupied the central 24 bp of each prespacer. GGCCGAAGACGCAGATCTTATATCGTATGGGGCTGACTTCAGGTTGATCGGGCACGTAAGAGGTTCCand a reverse primer:GGCCGAAGACGCGAAACTTATATCGTATGGGGCTGACTTCAGGACCAATAAAAAACGCCCGGCencoding BbsI sites, and this amplicon was purified with the E.Z.N.A Cycle Pure Kit (Omega Biotek). The purified gap-filled products and chloramphenicol selection cassette were subjected to Golden Gate cloning using a standard BbsI assembly protocol.2O, and 3 μl was electroporated into 25 μl E. coli strain E. cloni 10G Elite electrocompetent cells (Lucigen). Electroporated cells were recovered in 975 μl pre-warmed Lucigen recovery medium for 1 h at 37 C, serially diluted in LB media, spot-plated (5 μl per spot) on LB agar containing 100 μg/ml carbenicillin and 25 μg/ml chloramphenicol and incubated overnight at 37 C. Individual colonies were picked into 1 ml TB medium containing carbenicillin and chloramphenicol in 96 deep well blocks (Costar) and grown in a shaking incubator (Multitron) overnight at 37°C and 750 rpm. Plasmids were purified and Sanger sequenced with primers binding inside the chloramphenicol resistance cassette and oriented outward toward the flanking CRISPR array:Golden Gate reactions were purified using the Clean and Concentrate 5 kit as described above, eluted in 6 μl HGGCCCGTCTCACTTTCATTGCCATACGAAATTCCGGATGAGCand GGCCCGTCTCGAAAGACGGTGAGCTGGTGATATGG.For each colony, the forward and reverse Sanger sequencing reads were concatenated and annotated using a reference feature database derived from known features on pCRISPR. Colonies were excluded from analysis if Sanger sequencing traces were low quality or uninterpretable; if the spacer was integrated at a site other than the edge of a direct repeat; if the integrated spacer was flanked by nonconsecutive spacers from the original CRISPR array; or, in rare circumstances, if mutations or indels in the prespacer ends were observed (likely stemming from oligonucleotide synthesis errors).2 and 10 μg/ml of chloramphenicol. The cultures were then grown for 1 h and 15 min and normalized to OD600 = 0.3. About 150 μl of each culture was added to three wells of a 96-well plate. When appropriate, bacteriophage was added directly to the well at a MOI of 1. Bacterial growth was monitored over time in a microplate reader (TECAN Infinite 200 PRO) by recording the OD600 of each well every 10 min.Overnight cultures were diluted 1:100 in 5 ml of BHI with 5 mM CaCl2 and 10 μg/ml of chloramphenicol was plated on BHI plates. About 3 μl of 10-fold serial dilutions of phage were plated on the lawn of cells once the agar had solidified. The next day, photos of the plates were taken using a plate imager (FluorChem HD2).About 100 μl of an overnight culture mixed with melted 50% heart infusion agar with 5 mM CaCl2 and grown for 1 h and 15 min. Then, the optical density (OD600) was recorded, and all the cultures were diluted to OD600 = 0.3. The cultures were infected with ΦNM4γ4 at a MOI of 100 for 30 min. Next, the cells were pelleted at 10 000 RPM for 3 min and flash frozen in liquid nitrogen. The frozen pellets were stored at -80°C until plasmid extraction for next-generation sequencing.Spacer acquisition was performed as previously described  with sliPlasmid from frozen RN4220 pellets were obtained using a modified QIAprep Spin Miniprep Kit protocol described elsewhere . PCR ampUsing a custom Python script, the spacer sequences aligning perfectly to ΦNM4γ4′s genome was extracted and the number of reads and the length for each spacer were recorded. The number of reads for each spacer sequence was normalized to account for PCR biased as previously described . For eacS. pyogenes Cas1, Cas2, Csn2, Cas9 complexed with a guide RNA, we aimed to determine its role in the acquisition of new spacers into the CRISPR array   were challenged with ΦNM4γ4 phage   and harbas9 gene  showed nas9 gene . This ise Figure . Deep see Figure . These r in vivo ,15, implin vitro assays from the type II-A system focused on the final step of the integration pathway ) and the spacer sequence.Previous studies using  pathway ,23 withoi Figure . Acquisii Figure . CRISPR i Figure  and the i Figure . Notablyi Figure . No suchSince Cas1 and Cas2 alone did not exhibit DNA trimming activity in our assays, and since Csn2 has no known enzymatic function , we testStreptococcus pyogenes Cas9 contains two endonuclease domains (RuvC and HNH) that together generate DNA double-strand breaks by cleaving opposite strands of the DNA helix  or a H840A (HNH-inactivating) Cas9 variant   ,29. We tes Cas9) . Under tes Cas9) . Surprises Cas9) . In the es Cas9) . Togethex Figure . In ordex Figure . The resx Figure , but thix Figure .S. pyogenes Cas9 can convert DNA fragments with short 3′-overhangs into properly trimmed substrates for integration into the CRISPR array (Figure Genomic loci with high propensity toward stalled replication forks or double-stranded breaks are sources of spacers generated in bacteria . Double-y Figure .S. pyogenes, the Cas9-integrase supercomplex binds to prespacers, trims them to a size of ∼30 bp and catalyzes integration into the CRISPR array. Cas9 is a precise RNA-guided endonuclease for generating double-stranded DNA-breaks during CRISPR-based adaptive immunity (Our results suggest that in the type II-A CRISPR-Cas system of immunity ,27,28. Tin vitro need to be understood and explored further. Csn2 has been reported to act as an interface for the interaction of Cas9 with Cas1-Cas2 for supercomplex assembly (in vitro. Our experiments indicate that while the supercomplex can form in the absence of a prespacer DNA, it is most stable when both sgRNA and a prespacer DNA are present. Since prespacer trimming is mediated by both Cas9 alone as well as an intact supercomplex, it can be argued that prespacer trimming and adaptation can be mediated by a sequential interaction of Cas9 and the adaptation module respectively. However, the trimming activity of Cas9 within the supercomplex is enhanced, indicating that association of Cas9 with Cas1-Cas2-Csn2 accelerates the kinetics and/or extent of prespacer processing.Complex formation between Cas1, Cas2, Csn2 and Cas9 from the type II-A system has been reported previously ,31, but assembly ,31. Addiassembly , indicatE. coli, DnaQ exonucleases have been shown to perform prespacer processing, with the canonical sized spacers being integrated by Cas1-Cas2 at the leader-repeat junction in the correct orientation (S. pyogenes, Cas9 is involved in prespacer trimming but the precise mechanisms underlying PAM specificity of prespacer processing and directional integration of spacer into the CRISPR array remain to be explored. Sequencing analysis from full-site integration experiments showed that spacers integrated by the supercomplex are always trimmed to 30 bp but the integration is neither specific to the leader-repeat junction nor are the spacers always integrated in the physiologically relevant orientation. The supercomplex might restrict site-specific interaction of Cas1-Cas2 with the leader-repeat junction and points toward a kinetic timeline where the dissociation of Cas9 and Csn2 from the supercomplex would make the Cas1-Cas2 integrase available for site-specific integration. The dissociation of Cas9 from the supercomplex would leave behind a Cas1-Cas2-Csn2 complex, the structure of which has recently been reported (While the mechanism of prespacer generation in type II-A CRISPR-Cas systems is not well understood, the machinery responsible for prespacer trimming has been reported for a few other CRISPR-Cas systems. In the type I-E system from entation . In the entation ,34. In treported . In the reported . This sereported .Since the nuclease activity of Cas9 is dispensable for acquiring canonical-sized spacers upon phage-infection, we believe that other cellular nucleases in addition to Cas9 can mediate prespacer processing. Cas9′s capacity for prespacer trimming may enable horizontal gene transfer of CRISPR-Cas systems into hosts with diverse genetic backgrounds  by avoidThe raw deep sequencing data collected for this study is available under the bioproject ID PRJNA670708.gkab123_Supplemental_FileClick here for additional data file.


Variability in the 25-hydroxyvitamin D [25(OH)D] response to prenatal and postpartum vitamin D supplementation is an important consideration for establishing vitamin D deficiency prevention regimens.We aimed to examine interindividual variation in maternal and infant 25(OH)D following maternal vitamin D supplementation.n = 1300) received a prenatal cholecalciferol (vitamin D-3) dose of 0, 4200, 16,800, or 28,000 IU/wk from 17 to 24 wk of gestation followed by placebo to 6 mo postpartum. A fifth group received 28,000 IU cholecalciferol/wk both prenatally and postpartum. In a subset of participants, associations of 25(OH)D with hypothesized explanatory factors were estimated in women at delivery (n = 655) and 6 mo postpartum (n = 566), and in their infants at birth (n = 502) and 6 mo of age (n = 215). Base models included initial 25(OH)D and supplemental vitamin D dose. Multivariable models were extended to include other individual characteristics and specimen-related factors. The model coefficient of determination (R2) was used to express the percentage of total variance explained.In a randomized trial of maternal vitamin D supplementation , healthy pregnant women . Additional characteristics, including BMI, contributed negligibly to remaining variance (<5% increase in R2). Variance in neonatal 25(OH)D was explained mostly by maternal delivery 25(OH)D and prenatal vitamin D intake (R2 = 82%). Variance in 25(OH)D in later infancy could only partly be explained by numerous biological, sociodemographic, and laboratory-related characteristics, including feeding practices (R2 = 43%).Supplemental vitamin D intake and initial 25(OH)D accounted for the majority of variance in maternal 25(OH)D at delivery and postpartum (RPresupplementation 25(OH)D and vitamin D supplemental dose are the major determinants of the response to maternal prenatal vitamin D intake. Vitamin D dosing regimens to prevent maternal and infant vitamin D deficiency should take into consideration the mean 25(OH)D concentration of the target population. The high prevalence of vitamin D deficiency in pregnant women and newborns has been recognized as a global issue, with variability within and across WHO world regions . It is wSubstantial heterogeneity in the 25(OH)D response to vitamin D intake has been observed in nonpregnant adults , 9, yet n = 1300), having an uncomplicated singleton pregnancy, were enrolled at 17–24 wk of gestation and randomly assigned to 1 of 5 equally sized trial arms, comprising a prenatal:postpartum regimen of 0:0 (placebo); 4200:0; 16,800:0; 28,000:0; or 28,000:28,000 IU cholecalciferol (vitamin D-3) per week until 26 wk postpartum. In addition to the intervention dose, all participants were provided with standard iron–folic acid supplements as per usual care, and calcium supplementation  to mitigate effects of low dietary calcium intakes. Ethical approval was obtained from the Research Ethics Committees of the Hospital for Sick Children in Toronto (REB1000039072) and the International Centre for Diarrhoeal Disease Research, Bangladesh (PR-13055). Written informed consent was provided by all women prior to commencing the trial. The trial was registered prospectively at clinicaltrials.gov (ID: NCT01924013).This secondary analysis used data from participants of the Maternal Vitamin D for Infant Growth Trial of maternal prenatal and postpartum vitamin D supplementation in Dhaka, Bangladesh , 13. BriTo permit consideration of basal 25(OH)D in our modeling approach as a determinant of the vitamin D intake–25(OH)D response , 15, matPlacebo and vitamin D-3 tablets were manufactured by the Toronto Institute of Pharmaceutical Technology. All tablets were identical in appearance, color, and taste. For each lot of tablets, manufacturer verification of the vitamin D content was confirmed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) to a composition range within 10% of the labeled dose. Participant adherence was calculated by dividing the number of tablets consumed by the total number of assigned tablets. Average weekly supplemental vitamin D intake  was estimated from the measured tablet composition and individual participant adherence.General health and sociodemographic information was collected at enrollment (17–24 wk of gestation) through interviewer-administered questionnaires. Asset index, as a proxy for socioeconomic status, was determined by ownership of household items, using principal components analysis . Habitua3, 3-epi-25-hydroxyvitamin D3, and 25-hydroxyvitamin D2 [25(OH)D2] was achieved. National Institute of Standards and Technology quality control materials (SRM 972a) and DEQAS standards  were used throughout the analysis. Mean inter- and intra-assay CVs for 25(OH)D3 were 7.0% and 4.9%, respectively. The lower limit of quantification (LLoQ) for 25(OH)D3 was 1.25 nmol/L . The LLoQ for 25(OH)D2 was 1.25 nmol/L; however, because 25(OH)D2 concentrations were undetectable or negligible in this cohort D was performed at the Analytical Facility for Bioactive Molecules , which participated in the Vitamin D External Quality Assessment Scheme  throughout the study period. Circulating 25(OH)D was measured in women , umbilical cord blood, and infants (at 6 mo of age) by high-performance LC-MS/MS, using standardized methods that have been described in detail elsewhere . Chromats cohort , only coactivity . For simAdditional maternal and infant metabolites, including circulating C-reactive protein (CRP), ferritin , maternal serum folate and retinol, and infant serum creatinine were measured using commercially available kits . Inter- 1) days since last dose was defined as the number of days between blood collection and the last administered intervention dose; 2) time of day of blood collection was categorized as morning (00:00 to 11:59), afternoon (12:00 to 16:59), or evening (17:00 to 23:59); 3) assay drift was represented by the number of months between completion of the first 25(OH)D assay and the assay concerning the 25(OH)D measurement of interest; and 4), acknowledging the high stability of 25(OH)D D introduced through collection and handling of blood samples, we derived several sample-related variables:  25(OH)D . We calcCRP and ferritin distributions were right-skewed and were therefore natural log-transformed to approximate normality. Pairwise correlations among variables were assessed using Pearson and Spearman correlation coefficients, and bivariate relations were examined using scatter plots with locally weighted regression. Biomarker concentrations below the LLoQ were assigned objective values equal to or half the LLoQ . Data we1) maternal enrollment–delivery; 2) maternal delivery–6 mo postpartum; 3) maternal delivery–neonatal , and 4) neonatal–6 mo of age. Regression models were constructed with 25(OH)D as the (end point) outcome variable, measured at 4 corresponding  time points: 1) maternal 25(OH)D at delivery; 2) maternal 25(OH)D at 6 mo postpartum; 3) neonatal 25(OH)D; and 4) infant 25(OH)D at 6 mo of age. As a reflection of basal status, initial 25(OH)D was represented by the measurement at the preceding time point . For the maternal delivery and 6-mo postpartum end points, initial 25(OH)D was defined as enrollment and delivery 25(OH)D, respectively. For neonatal and infant 6-mo 25(OH)D, initial concentrations were defined as maternal delivery and neonatal 25(OH)D, respectively. We assumed that initial 25(OH)D accounted for nonsupplemental sources of vitamin D from cutaneous production and dietary intake, which was expected to be low relative to the supplemental doses provided (To identify factors associated with the 25(OH)D response to supplementation, we defined 4 intervals based on paired time points at which 25(OH)D was measured in maternal-infant pairs: provided .Supplemental Table 2). For each interval and for each candidate factor, we first used a simple linear regression model (Model A) to estimate the unadjusted association of the hypothesized factor with 25(OH)D. We extended the model to adjust for initial 25(OH)D and calculated vitamin D dose per week (Model B), which enabled an assessment of the interindividual variation in end-of-interval 25(OH)D explained by the factor of interest, but which was not already explained in the base model. A parsimonious multivariable model (Model C) included initial 25(OH)D, calculated vitamin D dose per week, and any other covariate with a P value < 0.1 in its respective Model A and/or B, as well as selected variables that were a priori hypothesized to affect vitamin D status . Finally, for each interval, a multivariable model (Model D) consisted of all noncollinear covariates listed in r ≥0.6 was used to define collinearity between pairs of continuous variables, and prompted consideration for removal from multivariable models. Gestational age at enrollment was tightly correlated with the duration of supplementation, as was maternal weight with BMI at each time point, and therefore only duration of supplementation and enrollment or habitual BMI remained in the final models.For each interval, a base model included initial 25(OH)D and the calculated vitamin D dose per week as the only predictor variables. We then considered a range of other covariates that were hypothesized to be associated with 25(OH)D . For eacP < 0.1). Irrespective of statistical significance, a decision was made a priori to include interaction terms for BMI and feeding practices in models concerning maternal delivery or neonatal 25(OH)D, and infant 6-mo 25(OH)D, respectively, based on known attributes of vitamin D metabolism , the goodness-of-fit for the base model, Model C, and Model D were compared using Akaike information criterion and partial F-tests, where asticity , 30. AnaOf 655 women with 25(OH)D measured at delivery, 471 (72%) had a complete dataset  for inclusion in multivariable models. Complete data were available for 541 of 566 (96%) women at 6 mo postpartum, 329 of 502 (66%) infants at birth, and 174 of 215 (81%) infants at 6 mo of age . Addition of other covariates (Models C and D) yielded a statistically significant improvement in model fit , but with small increases in the proportion of variance explained D (base model) collectively explained a high proportion of total variance in maternal 25(OH)D at delivery and at 6 mo postpartum  . Addition = 651; P = 0.31 for the interaction term). There was some evidence for attenuation of the response to supplementation at higher initial 25(OH)D, but effects were minor such that none of the interactions between vitamin D status classifications and calculated dose per week reached statistical significance (P = 0.71) . Although there was considerable interindividual variation in response within each intervention group, few women  achieved a delivery 25(OH)D <50% of the predicted group mean D, with a major inflection point between intakes of 4200 IU/wk and 16,800 IU/wk . InitialSupplemental Figure 3). A similar interaction was found between maternal height and intervention dose in their effects on postpartum 25(OH)D  had a potent effect on maternal 25(OH)D at 6 mo . There w 25(OH)D . Other m 25(OH)D  and 3. O 25(OH)D . Materna 25(OH)D . The impMaternal 25(OH)D at delivery strongly correlated with neonatal concentrations D at 6 mo of age . Of the Determination of safe and effective strategies for prevention of micronutrient deficiencies is reliant on an understanding of interindividual variation in the response to supplementation and dietary intake . DespiteDespite recent efforts to bridge knowledge gaps surrounding dietary vitamin D requirements, interindividual variability in the dose–response relation of 25(OH)D to vitamin D intake remains a relatively understudied consideration in the estimation of requirements . BetweenThe present analysis extends the previous work by Moon and colleagues , 40, who2) compared with the previous trial populations.Consistent with previous intervention trials, including pregnant , pediatrA substantial proportion of variance in 25(OH)D remained unexplained for infants at 6 mo of age. As dietary diversity increases with the introduction of complementary foods, and sun exposure behavior influences cutaneous vitamin D synthesis, the difficulty of capturing direct sources of variation in 25(OH)D increases with advancing age. We found that maternal postpartum supplementation attenuated the difference in 25(OH)D between breastfed and formula-fed infants, results that corroborate previous findings that high-dose maternal vitamin D supplementation can increase breast milk antirachitic activity, in turn improving vitamin D status of the breastfed infant . Unlike CYP24A1), folate could promote a higher or sustained 25(OH)D concentration as the rate of catabolism declines D data, have long been raised as concerns for the reporting of 25(OH)D and other biomarker data . In the The well-characterized population and dose-ranging design are particular strengths of this study, permitting exploration of several potential contributors to interindividual variability in 25(OH)D across a range of administered doses. Unlike previous trials , 58, theThese findings support the expectation that the biochemical response of most pregnant women to a given dose of supplemental vitamin D can be reliably predicted, assuming good adherence. Given substantial between-population variation in average 25(OH)D during pregnancy , mean manxab265_Supplemental_FileClick here for additional data file.