Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. MEFs were converted into chemically induced neurones by four compounds including ISX9, Forskolin, CHIR99021, and I-BET151 . A BET fin vitro and in vivo . Re. Re67]. Pdx1, Ngn3, and MafA [in vivo, proper differentiation from pluripotent stem cells requires many kinds of culture media, growth factors, and small molecules at each step of the differentiation [The number of patients suffering from diabetes mellitus has been rapidly expanding all over the world, but the fundamental treatment is difficult without transplantation of islet or functional β cells. So the development of induced pancreatic β cells from other somatic cells is one of the most attractive strategies to treat diabetes. A number of studies have reported that functional β cells were transdifferentiated from pancreatic exocrine cells, pancreatic ductal cells, intestinal crypt cells, stomach cells, and hepatocytes by forced expression of β cell lineage-specific transcription factors such as and MafA . These sntiation ,78.In consideration of these background factors, chemical compound-based direct conversion of human dermal fibroblasts into functional β cells is challenging. In practice, such chemical conversion into β cells has not yet been reported. Sheng Ding and colleagues demonstrated that transient expression of the reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc or P53 shRNA converted mouse and human fibroblasts into an intermediate ,80. ThenIns2 and Pdx1 [Pdx1 gene in both primary human islets and the ductal cells. 5′-Azadeoxycytidine (AZA), a DNA methyltransferase inhibitor, induced Ngn3 expression in PANC-1 cells and promoted an endocrine differentiation in the cells [The high-throughput screening of small molecules from several groups have identified candidate compounds for chemical compound-based pancreatic reprogramming. BRD7389, an inhibitor of ribosomal S6 kinase (RSK), changed the cell shape of mouse pancreatic α cells to a more β cell-like one and increased the expression of β cell-specific genes, and Pdx1 . BRD7552and Pdx1 . BRD7552he cells . Harminehe cells . More rehe cells .Challenges to converting either differentiated endoderm cells or fibroblasts into the progenitor cells have been noted by several groups. Wang et al.  reportedCdh1, Sox17, FoxoA2, and HNF4α. ciEPCs could be differentiated into hepatocyte-like cells in the specialized medium including chemical compounds and growth factors, DAPT, Dexamethasone, A83-01, hepatic growth factor (HGF), and oncostatin M (OSM). The hepatocyte-like cells differentiated from ciEPCs exhibited an expression pattern similar to the one of iPS cells-derived hepatocytes. ciEPCs were also differentiated into pancreatic progenitor cells expressing Pdx1; however, insulin expression was not confirmed. It is also a promising strategy to prepare clinically applicable hepatocytes and pancreatic β cells if human adult fibroblasts are successfully converted into the endoderm progenitor cells as well. Similar combinations of the chemicals and the protocols might be useful for performing the conversion of human fibroblasts into ciEPCs.Recently MEFs were chemically converted into endoderm progenitor cells by two sets of combinations of chemical compounds and growth factors . MEFs we. [Katsuda et al.  successf. . A diffeMSCs are multipotent somatic stem cells and can be differentiated into osteocytes, chondrocytes, and adipocytes. MSCs are generally isolated from the tissues such as bone marrow, adipose tissue, and umbilical cord blood. MSCs also have immunomodulatory effects to suppress immune response and participate in tissue repair by secreting multiple cytokines . Human dIn 2013, Hou et al.  first reSall4 and Lin28a, which makes the pluripotent state more accessible. The transition via XEN-like cells in the reprogramming process is unique for chemical-based, but not observed in transcription factor-based, reprogramming. In addition, the effects of several new compounds such as AM580, EPZ004777, SGC0946, and 5-Aza-dC on the chemical reprogramming were investigated. The retinoic acid receptor agonist and epigenetic modulators in combination with VC6TF increased the efficiency up to 1000-fold greater than that described in their previous reports. Not only MEFs but also neonatal dermal fibroblasts and adult lung fibroblasts were chemically reprogrammed, suggesting a broad availability of the chemical cocktail for the reprogramming. Further studies are required to reveal in more detail the molecular mechanism by which each chemical compound facilitates the chemical reprogramming through regulation of targetted signaling pathways and epigenetic modifications. The identification of an intermediate state is an important step to further improve the experimental protocol and the combination of chemical compounds for rapid and better reprogramming to a pluripotent state that resembles that of ES cells.Approximately 2 years later, the same group showed that extraembryonic endoderm (XEN)-like cells were an intermediate in the process of CiPSC generation . The XENMoreover, their group also successfully converted mouse neural stem cells and small intestinal epithelial cells into CiPSCs . SimilarCiPSCs might have higher developmental potentials than iPSCs induced by the reprogramming transcription factors ,93,95. CBecause human somatic cells have not been reprogrammed into CiPSCs by chemical compounds only, the generation will have a great impact on this field. Several differences of signaling pathways between mouse and human pluripotent stem cells have been reported ,98. One in vivo ones.In most cases, the combination of chemical compounds required for each direct conversion were empirically determined, and the detailed molecular mechanisms of the direct conversions remain largely unknown. However, identification of the chemical compounds provides great insights into how signaling pathways regulate the direct conversion. In addition, we need to understand how multiple signaling pathways could synergistically change one cell fate to another. A better understanding of the reprogramming process is obviously helpful to improve the efficiency and to perform complete epigenetic reprogramming into various cell types which are more similar to the A number of common chemicals are frequently used in the direct conversions from both mouse and human fibroblasts. CHIR99021 is one of the most frequently used compounds in various conversions into neurones, neural stem cells, cardiomyocytes, endoderm progenitor cells, and iPS cells, suggesting that activation of WNT signaling pathway by inhibiting GSK3 might potentially increase plasticity for cell fate changes in fibroblasts. On the other hand, our report indicated that CHIR99021 severely inhibited the brown adipogenic reprogramming, which suggests that a cell type-specific regulation of signaling pathways is required for chemical direct reprogramming . TGFβ siSox2 gene and activated the expression [A few articles have reported a possible molecular mechanism underlying chemical direct reprogramming. A study on neural stem cell reprogramming suggested that downstream effectors, Elk1 and Gli2, in MAPK and SHH signaling pathways, respectively, were both directly bound to the promoter of pression . In humapression . In addiOne of the characteristics in chemically induced cells is low tumorigenicity, although whether it is really lower than that for the cells converted by transcription factors remains unknown. More careful studies will be required to evaluate the tumorigenicity in chemically induced cells by transplantation into animal models. The treatment with chemical compounds likely changes broad gene expression via downstream transcription factors of the related signaling pathways, which might also lead to an irreversible disruption of proper epigenetic states. It might increase the risk for tumorigenesis although this should be dependent on the properties of chemicals, concentration, incubation time, and so on.Compared with gene expression regulated by specific transcription factors, chemical compounds do not always activate expression of a specific set of transcription factors required for the cell fate conversion and have an influence on a wide range of gene expression and epigenetic modifications. Therefore, to more specifically and selectively regulate signaling pathways and histone-modifying enzymes, the strategy of chemical biology is required to identify novel compounds from amongst a tremendous array of chemicals. Such novel compounds might improve on the direct reprogramming efficiency, and lead to the conversions into more kinds of cell types that have not yet been reported.This review provides an overview of recent advances in chemical compound-based direct reprogramming. This field has rapidly developed based upon numerous studies on transcription factor-based direct reprogramming. The combinations of chemical compounds and the cell type-specific medium including growth factors will likely enable more cell fate conversions of somatic cells in the future. The use of chemical compounds instead of forced expression of transcription factors resolves several barriers which have been raised in the use of iPS cells and cells directly reprogrammed by transcription factors. Such small molecule-based direct conversions in a chemically defined medium will robustly facilitate the cell-based study on transplantation therapy, disease modeling, drug screening, and personalized medicine .Further studies are required to more fully manipulate cell fates by chemical compounds only. In addition, most reported protocols for chemical compound-based direct reprogramming are performed and repeated by the same groups. To increase the availability in other laboratories as well as hospitals, the same or similar experiments need to be actively repeated by independent groups. Particularly, the chemical conversion of human fibroblasts into endoderm lineage cells such as hepatocytes and pancreatic β cells is clinically anticipated. The chemical compound-based strategy could become one of the main sources for cell transplantation therapy in the near future.


Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5 against Salmonella Typhimurium. Non-fermented and fermented media containing 5.6% WPI were fractionated at a 3 kDa cut-off and the filtrate was analyzed by mass spectrometry. The non-fermented WPI medium contained 109 milk derived peptides, which originated from β-casein (52), αs1-casein (22), αs2-casein (10), κ-casein (8), and β-lactoglobulin (17). Most of these peptides were not found in the fermented media, except for 14 peptides from β-casein and one peptide from αs2-casein. Database searches confirmed that 39 out of the 109 peptides had established physiological functions, including angiotensin-converting-enzyme (ACE) inhibitory, antioxidant, antimicrobial, or immunomodulating activity. A total of 75 peptides were found in the LH-2 cell free spent medium (CFSM): 54 from β-casein, 14 from k-casein, 4 from β-lactoglobulin and 3 from αs2-casein. From these peptides, 19 have previously been associated with several categories of bioactivity. For La-5 CFSM, a total of 15 peptides were sequenced: 8 from β-casein, 5 from αs1-casein, 2 from β-lactoglobulin. Only 5 of these have previously been reported as having bioactivity. Many of the peptides remaining in the fermented medium would contain low-affinity residues for oligopeptide binding proteins and higher resistance to peptidase hydrolysis. These properties of the sequenced peptides could explain their accumulation after fermentation despite the active proteolytic enzymes of LH-2 and La-5 strains. Down-regulated expression of hilA and ssrB genes in S. Typhimurium was observed in the presence of La-5 and LH-2 CFSM. Downregulation was not observed for the Salmonella oppA mutant strain exposed to the same CFSM used to treat the S. Typhimurium DT104 wild-type strain. This result suggests the importance of peptide transport by S. Typhimurium for down regulation of virulence genes in Salmonella.Peptides in the 3-kDa ultrafiltrate of fermented whey protein isolate (WPI) medium could be responsible for the antivirulence activity of  Salmonella enterica subsp. enterica serovar Typhimurium is considered a major foodborne pathogen with public health and economic concerns. This foodborne pathogen has developed resistance against a broad range of antibiotics  controls the production and activity of the Type III secretion system which enables the membrane ruffling process, in which the epithelial cell cytoskeleton is rearranged to engulf the Salmonella into cytoplasmic vacuoles  is the result of the balance between proteolytic activity and peptide consumption . The propeptides .S. Typhimurium. oppA gene expression in S. Typhimurium is modulated by nitrogen compounds and regulates the expression of other genes such as Lrp (leucine-responsive regulatory protein) or CodY  is one of the most abundant periplasmic proteins in  or CodY . Mutatiopeptides .oppA gene expression and virulence gene expression in Salmonella in the presence of antivirulence peptides was investigated using a S. Typhimurium oppA mutant strain.In this study, the proteolytic and peptidolytic activities of LH-2 and La-5 strains in Whey Protein Isolate (WPI) are explored through genome sequencing and biochemical assays on substrates. Peptide profile analysis by mass spectrometry was conducted to understand the potential factors that lead to accumulation of antivirulence peptides in the growth media. Furthermore, the correlation between Salmonella Typhimurium oppA mutant, which was provided by Prof. Eric Brown, McMaster University. Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5 probiotic strains were grown under anaerobic conditions at 37°C for 48 h on De Man, Rogosa, Sharpe broth . Salmonella Typhimurium hilA::lux and Salmonella Typhimurium ssrB::lux were grown on Luria-Bertani broth  supplemented with 50 μg/ml of ampicillin (Amp). Both constructs were grown aerobically overnight on a shaking incubator at 37°C. S. Typhimurium DT104 and S. Typhimurium oppA mutant strains were grown under the same condition as S. Typhimurium constructs, but without Amp. Solid media preparation for all strains were carried out under the same conditions but with addition of 15 g/l of agar and incubated under the same conditions.All bacterial strains used in this study were obtained from the Canadian Research Institute for Food Safety (CRIFS) except for the L. helveticus LH-2 and L. acidophilus La-5, respectively. The media were filter sterilized through 0.45 μm pore-size filters . An overnight culture of each strain in MRS broth was washed and inoculated at 5% (v/v) into WPI based media and incubated anaerobically at 37°C for 48 h. Following growth, bacterial cells were removed by centrifugation at 15,000 × g for 30 min at 4°C. The supernatant was filtered through a 0.20 μm pore-size filter (Corning) to obtain sterile CFSM which was subsequently freeze-dried  for 72 h and stored at −80°C.Whey Protein Isolate (WPI)  was dissolved at 5.6% (v/v) in sterile sugar solution. Glucose or sucrose  at 0.5% (v/v) were added for S. Typhimurium hilA::lux and ssrB::lux (luxCDABE operon from Xenorhabdus luminescens was isolated and cloned with an Amp resistance gene into a plasmid (pSB377), which was further fused with the hilA and ssrB promoter regions, separately. The expression of the lux genes is controlled by these promoter regions, so that light is emitted when the gene is expressed. Each overnight culture of the constructs was diluted 1:100 with fresh LB broth with and without supplementation of 10% (v/v) of neutralized (pH 7) CFSM from L. helveticus LH-2 and L. acidophilus La-5, which had been reconstituted in sterile deionized water at 10-fold concentration compared to the initial medium. The samples were incubated aerobically at 37°C with shaking for 3 h. Luminescence was measured with the FB 12 luminometer . The results are presented as Relative Light Units (RLU)/OD600 nm.For initial screening of the antivirulence effects of CFSM, bioluminescent reporter strains srB::lux  were useS. Typhimurium DT104 and S. Typhimurium oppA mutant following 3 h growth in fresh LB broth with and without 10% neutralized (pH 7) CFSM with shaking at 37°C, 2-ml samples were centrifugated at 5,000 × g for 4 min at RT. The supernatant was discarded and cells were mixed with 1 ml of RNA Protect reagent  and incubated for 5 min at RT. Cells were collected again by centrifugation at 5,000 × g for 10 min at RT. Cell pellets were suspended in 200 μl of Tris-EDTA buffer, pH 8.0 , 60 μl of 20 mg/ml lysozyme (Fisher Scientific) and 20 μl of proteinase K (Qiagen). The suspensions were incubated at 37°C for 1 h with shaking at 450 rpm. RNeasy Plus Mini Kit (Qiagen) was used to extract RNA from all samples following the manufacturer's instructions. DNA was eliminated by using RNase-Free DNase Set (Qiagen). In brief, 40 μl of total RNA was incubated for 10 min at RT with 2.5 μl DNase I stock solution, 10 μl RDD buffer in a total volume of 100 μl. RNA purification and concentration were performed with RNeasy MinElute Cleanup kit (Qiagen) and solubilized in 30 μl molecular-grade water. The quantity of quality RNA was determined by measuring the absorbance at 260 and 280 nm using a NanoDrop 1000 spectrophotometer . RNA integrity was verified by gel electrophoresis.RNA was extracted from The same procedures were used for a mixture of the following synthetic peptides  (Synpeptide) at a concentration of 0.2 mg/ml for each peptide.The purified RNA was used for reverse transcription by using high-capacity cDNA reverse transcription kit . RNA (1 μg) was mixed with 2 μl of 10× RT buffer, 2 μl of 10× random hexamer primers, 0.8 μl of 25× dNTP (100 mM), 1 μl of Multiscribe reverse transcriptase (50 U/ml) in a total volume of 20 μl. A no reverse transcription control was included to confirm the absence of contaminating DNA. The synthesis of cDNA was conducted using a Mastercycler Gradient Thermocycler  under the following settings: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min and a holding step at 4°C. The cDNA was stored at −20°C until use.TM 7 Real-Time PCR System  and PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) were used for RT-qPCR. The primers (E = 10 (−1/slope). The PCR was performed in a total volume of 20 μl; 10 μl of PowerUp™ SYBR™ Green Master Mix, 1.6 μl of forward primer (5 μM), 1.6 μl of reverse primer (5 μM), 5 μl of 1/10 diluted cDNA and 1.8 μl of molecular-grade water with the final primer concentration of 400 nM for all the genes except 16S  and rpoD (sigma factor). For these two genes, a final primer concentration of 200 nM was used with reaction mixture, 10 μl of SYBR® Select Master Mix, 0.8 μl of forward primer (5 μM), 0.8 μl of reverse primer (5 μM), 5 μl of 1/10 diluted cDNA and 3.4 μl of molecular-grade water. Each PCR was performed in triplicate in the 96 well plates . PCR conditions were as follows: UDG activation at 50°C for 2 min and Dual-Lock™ DNA polymerase activation at 95°C for 2 min, followed by 40 repeated cycles of denaturation, annealing and amplification, at 95°C for 15 s, 54°C for 30 s and 72°C for 45 s. Subsequently, a default dissociation curve  was performed in the instrument and specific amplicon was verified by a single melting-temperature peak. The transcript levels were normalized to the geometric average of expression for all housekeeping genes for each sample  according to the manufacturer's instructions. DNA was eluted in 10 mM Tris-HCl (pH 8.0). The concentration and the purity of the purified DNA was measured at 260 and 280 nm using a Nanodrop 1000 spectrophotometer. The integrity of the DNA was verified by agarose (1%) gel electrophoresis. Extracted DNA samples were stored at −20°C. Library preparation and sequencing were performed at the Plateforme d'Analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes . In short, the libraries were prepared using 500 ng of mechanically fragmented DNA by a Covaris M220 (Covaris) using the NEBNext Ultra II kit (New England Biolabs). TruSeq HT adapters (Illumina) were ligated instead of NEBNext adaptors. The libraries were checked for quality using Bioanalyzer and quantified with Picogreen. The libraries were sequenced on a fraction of a MiSeq run  following the manufacturer's instructions. De novo assembly of the reads was performed using CLC Bio Genomic Workbench version 10.1  at the Genomics Facility of the Advanced Analysis Centre, University of Guelph. Both the assembled reads and the de novo assembled contigs were BLAST searched (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparative genomic analysis of the proteolytic system was conducted using other related strains L. helveticus CNRZ 32  for L. helveticus LH-2 and L. acidophilus NCFM  for L. acidophilus La-5 using the NCBI microbial genome database and Blast alignment tools.Genomic DNA was extracted from L. helveticus LH-2 and L. acidophilus La-5 were grown anaerobically in WPI—sugar based medium supplemented with 10 mM CaCl2.2H2O at 37°C for 24 h. The cells were harvested by centrifugation at 15,000 × g for 10 min at 4°C and washed twice with 10 mM CaCl2-saline solution. The supernatant designated as extracellular extract (EE) was used for the measurement of cell lysis rate while the cells were re-suspended in Tris buffer  to an optical density at 600 nm (OD600) of approximately 10 and were used for CEP and aminopeptidase enzyme assays.−1cm−1. One unit of protease activity was defined as 1 nmol of p-nitroanilide released per minute. The specific protease activity was calculated as one unit of protease from 1 mg of cell protein. The protein content was estimated using the method of Bradford  as described previously . The reaBradford . Bovine 600 of approximately 10 were used for preparation of intracellular extracts (IE) according to Pescuma et al. (g at 4°C for 5 min) and the supernatant fluid was designated as the intracellular enzymatic extract (IE) which was maintained on ice and immediately used for enzymatic assays. Intracellular aminopeptidase activity was assayed with the chromogenic substrate Lys-ρNa (Sigma) as previously described . The filtrate was freeze dried for 72 h. A control sample of filter sterilized WPI CFSM was included. The freeze-dried filtrate of 30 ml CFSM was reconstituted in 300 μl Milli-Q water and peptides were identified on an Agilent 1200 HPLC liquid chromatograph interfaced with an Agilent UHD 6530 Q-Tof mass spectrometer at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph  and subjected to post hoc test with a P-value of ≤ 0.05 considered significant.All experiments were carried out three independent times with triplicates of each sample. Means and standard deviations were analyzed using ANOVA followed by Tukey's S. Typhimurium hilA::lux and ssrB::lux were used for initial screening of the antivirulence effects of CFSM obtained from WPI fermented with L. helveticus LH-2 and L. acidophilus La-5. CFSM from both strains reduced luminescence of the S. Typhimurium hilA::lux while only La-5 could decrease ssrB::lux reporter luminescence; indicating down-regulation of the hilA and ssrB genes  than LH-2 CFSM . The expression of hilA and ssrB genes in S. Typhimurium oppA mutant strain was −2.23 and −1.61, respectively, in the presence of La-5 CFSM while in the presence of LH-2 CFSM, expression of both genes was −0.92 and −0.77, respectively . The relative expression of the same genes in the S. Typhimurium oppA mutant strain in the presence of the same peptide mixture was −1.60 and −1.67, respectively  with the absence of prtH, prtH2 and prtM genes   .L. helveticus LH-2 for oligopeptide transport elements and peptidases shows the presence of these proteolytic components with high identity (97–100%) (L. acidophilus La-5 genome codes for oligopeptide transport elements and peptidases of L. acidophilus NCFM with high identity (99–100%), except for oppB2 (33% identity)  . The L. dentity) .L. helveticus LH-2 and L. acidophilus La-5 were measured based on the in vitro hydrolysis of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The optimal temperature for the proteinase activity of LH-2 strain was 45°C with enzyme activity 3.60 while the maximum CEP activity of La-5 was 0.25 ± 0.01 at 40°C  analysis of unfermented WPI and WPI fermented with LH-2 and La-5 showed that peptides are almost all fragments of the main milk proteins  . Out of 2-casein . Most pe2-casein –9.L. helveticus LH-2 CFSM, 19 of these peptides have previously been associated with several categories of bioactivity . For L. acidophilus La-5 CFSM, a total of 15 peptides were sequenced, 5 of these peptides have previously been reported with ACE-inhibitory, antioxidant, antimicrobial and anti-caries activities. Some of these peptides have multifunctional properties that can modulate two or more physiological processes  and these signals affect the growth, metabolism and virulence of bacteria  and PrtM (maturase) in L. acidophilus La-5 confirmed that La-5 can digest large proteins extracellularly and produce small peptides. Genay et al.  is not different from other previously reported L. helveticus strains (40–50°C) (L. acidophilus strains (45–50°C) previously reported  –104. Thereported .L. helveticus LH2 CEP activity in WPI was lower than the CEP activity of other L. helveticus strains previously reported in skim milk  after ultrafiltration of milk during the WPI production process. The presence of these endogenous peptides in the growth media could explain the lower CEP activities of LH-2 and La-5 in WPI compared with other related strains in milk-based growth media. The CEP activity levels of lactobacilli depend on the strain, nature and quantity of peptides present in the growth media  supplemented with different nitrogen sources  in WPI fermented by LH-2 and La-5 strains indicates some similarity in the affinity of the proteinases of these strains for certain cleavage sites.The presence of endogenous peptides in unfermented WPI indicates the activity of endogenous milk proteases which are mainly plasmin, elastase and cathepsin D, B, and G . PlasminD, and G , 113. DaD, and G  concludeAccording to peptide analysis data, most of the identified peptides are casein derived, even though whey was used. The presence of casein derived peptides indicates that these proteins were exposed to proteolytic cleavage during processing . The thrL. helveticus 1198 while Sadat-Mekmene et al. . The lack of the prtH CEP gene in the L. helveticus LH-2 genome does not explain the absence of αs1-casein derived peptides in LH-2 CFSM, as some of these are present in the control medium. Strain LHC2, which has a similar combination of CEP homologs (PrtH3/PrtH4), does produce peptides from αs1-casein within a 3-h time scale  of identified peptides in the WPI and WPI fermented with LH-2 and La-5, respectively, were derived from β-casein. Thus, it can be concluded that the endogenous and microbial proteases of LH-2 and La-5 may preferentially attack β-casein. These findings are consistent with previously studies on bovine casein and kefir . The dege et al.  concludeme scale  reported strains  while κ- strains . β-lacto strains . LH-2 anL. acidophilus strains tested was 6.5 with a temperature optimum of 45–50°C, which might explain the lower number or diversity of peptides produced by La-5 at a lower pH and temperature (pH 4.5–5.2 at 37°C). The La-5 genome codes for only one CEP when compared with LH-2, which could result in a lower number or diversity of released peptides accumulating in the growth medium. Even though La-5 produces fewer types of peptides, those that are produced have higher antivirulence activity compare to those produced by LH-2, which depends on the nature and structure of the peptides. None of the peptides found in WPI fermented by La-5 are similar to those found in previous studies of the same strain grown in skim milk  of these strains, thus remaining in the spent medium if they are not transported into the cell. In Salmonella, the antivirulence activity of milk protein-derived peptides is related to the presence of the oppA gene. The undigested and fermented WPI by LH-2 and La-5 strains could be considered as a possible source of natural and functional ingredients, which may be used to increase the biological activity of food products. Further studies are required to explore the antivirulence ability of individual synthetic peptides with the same sequence at different concentrations compared to the antivirulence activity of fermented WPI.https://doi.org/10.5683/SP2/43M6GX].The datasets generated for this study can be found in the the University of Guelph Research Data Repository [EA carried out the experiments, interpreted the results, and wrote the first draft of the manuscript. SN participated in interpretation and presentation of the peptide results. SA-E, AE-L, and ES provided help for interpretation of the results. GL was involved in the design of the study, interpretation of the results, and writing 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.


Previous data showed that the GTPase-interacting components, Gic1 and Gic2, control cell polarity through its binding with Cdc42 and PtdInsP2 in the plasma membrane in budding yeast. However, whether the Gic proteins regulate polarized exocytosis is unknown.Cell polarity refers to spatial difference in morphology, structure, and function within different parts of a single cell, which plays important roles in a wide range of cellular processes. In eukaryotic cells, the small GTPase Cdc42 and phosphatidylinositol 4,5-bisphosphate Pgic1Δgic2Δ are synthetically sick with sec3ΔN. We demonstrated that Gic1 and Gic2 are required for polarized exocytosis in a yeast strain harboring the N-terminal domain deletion of Sec3, which is also known as an effector of Cdc42 GTPase. Gic proteins are required for polarized localization of exocyst, growth, and efficient secretion in sec3∆N mutant. In addition, we found that the N-terminal domain of both Gic2 and Sec3 share the similar binding sites of Cdc42. Surprisingly, not all the Sec3/Gic binding deficient cdc42 mutants displayed defects of growth and secretion, indicating that disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects in cdc42 mutants.In this study, we found that Gic2 co-immunoprecipitates with the exocyst complex, suggesting Gic proteins may be involved in exocytosis. Although we could not show the direct interaction between Gic2 and exocyst, we found 2, the regulation of Gic protein and Sec3 on polarized secretion may also be controlled by PtdInsP2. Further experiments need to be performed to test this hypothesis. Our findings provide important clues for understanding the molecular mechanism of cell polarity establishment in eukaryotic cells.We conclude that Gic1/2 and Sec3 act in parallel to regulate polarized post-Golgi secretion, but this regulation is not solely controlled by their upstream factor Cdc42. Considering that N-terminal domain of Gic2 and Sec3 can bind to both Cdc42 and PtdInsPThe online version of this article (10.1186/s13578-019-0295-x) contains supplementary material, which is available to authorized users. It is essential for many processes such as cell growth, differentiation, and morphogenesis, which rely on successful implementation of polarized secretion –4. Previmembrane , 15–21. membrane –25. Cdc4membrane , 8. Disrmembrane , 17, 18.2 via a polybasic region [sec3∆N exo70-38 double mutant can be rescued by adding the N terminus of Gic2 to sec3∆N [The GTPase-interacting components Gic1 and Gic2, also called Gic proteins, share 39% identity and 54% similarity in protein sequence, and play critical role for establishment of cell polarity. They are involved in initiation of budding and cellular polarization through interacting with Cdc42 via the Cdc42/Rac-interactive binding (CRIB) domain and with PIPc region , 26–30. o sec3∆N , it is ncdc42 mutants. Our data suggests that Gic proteins may regulate polarized secretion by the coordination of both Cdc42 and PtdInsP2.In this study, we find that Gic2 physically interacts with exocyst complex, and Gic proteins are required for polarized exocytosis in a mutant strain harboring the deletion of Sec3N-terminal domain. But the disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects and growth inhibition in 2 via a polybasic region [sec3∆N exo70-38 double mutant can be rescued by adding the N terminus of Gic2 to sec3∆N [Because both Sec3 and Gic proteins bind to Cdc42 via the Cdc42/Rac-interactive binding (CRIB) domain and with PIPc region , 29, 30,o sec3∆N , we spec2 binding, but is mostly independent of actin [sec3∆N mutant protein was not able to bind to Cdc42 and PIP2 in vitro, but the exocyst subunits were polarized to bud tip in this mutant strain [sec3∆N, gic1∆ gic2∆ and gic1∆ gic2∆ sec3∆N. As shown in Fig. gic1∆ gic2∆ and sec3∆N, which is similar to that in wild type cells. In gic1∆ gic2∆ sec3∆N triple mutant cells, however, most exocyst proteins were either depolarized or diffused inside the cells. We also examined the actin pattern in these mutant strains, and found that actin organization is mostly polarized in these mutants at 25 °C Pof actin –28, 30. t strain . We examsec3∆N mutant, we speculate that Gic2 and its a paralog, Gic1, may function in parallel with Sec3 for post-Golgi secretion, and a strain with deletion of Gic1 and Gic2 would have synthetic growth effect with sec3∆N mutant. To test this hypothesis, we deleted GIC1 and GIC2 either alone or in a sec3∆N mutant background yeast strain. As shown in Fig. gic1∆ gic2∆ or sec3∆N alone at 25 °C, 32 °C, 35 °C and 37 °C. This result suggests that Gic1 and Gic2 may be involved in regulating exocyst complex in a pathway parallel to Sec3.Because Gic proteins are required for polarized localization of exocyst in sec3∆N and gic1Δ gic2Δ double mutant at 25 °C, and a small amount of Bgl2 was accumulated in the sec3∆N gic1Δ gic2Δ triple mutant. After shifting to 37 °C for 2 h, although the sec3∆N and gic1Δ gic2Δ double mutants still did not accumulated Bgl2 protein inside cytosol, the sec3∆N gic1Δ gic2Δ triple mutant showed a pronounced amount of Bgl2 accumulation. As a control, the sec10-2 cells did not accumulate Bgl2 at 25 °C, but accumulate Bgl2 at 37 °C. These results indicate that combining sec3∆N with gic1Δ gic2Δ greatly aggravates the Bgl2 secretion defects.To determine whether these mutations cause secretion defects, we have analyzed the secretion of two cargoes, the cell wall endo-glucanase Bgl2 and the periplasmic enzyme invertase, which are well-characterized markers for the two major classes of post-Golgi vesicles that deliver proteins to the cell surface . As showsec3∆N, gic1Δ gic2Δ double mutant, and sec3∆N gic1Δ gic2Δ triple mutant. As shown in Fig. sec3∆N or gic1Δ gic2Δ mutant cells secreted > 85% of the total invertase outside of cytosol. However, only about 62% of invertase in sec3∆N gic1Δgic2Δ triple mutant cells was secreted outside at 25 °C. After a 2-h shift to 37 °C, the triple mutant cells only secreted about 30% of the total invertase.We also examined the secretion of the periplasmic enzyme invertase in the sec3∆N, gic1Δ gic2Δ double mutant, and sec3∆N gic1Δ gic2Δ triple mutant, we performed thin-section electron microscopy on these mutants. As shown in Fig. sec3∆N and gic1Δ gic2Δ double mutant, whereas the sec3∆N gic1Δ gic2Δ triple mutant accumulated about 62.9 ± 9 vesicles/section vesicles. These results clearly show that Sec3 and Gic1/2 function at parallel to regulate exocytosis.To examine whether exocytic vesicles are accumulated in Since Gic proteins and Sec3 are downstream effectors of Cdc42, we want to know whether the regulation of Gic proteins and Sec3 on polarized exocytosis is under the control of Cdc42.Saccharomyces cerevisiae share a high degree of sequence similarity. The crystal structure of the N-terminus of Sec3 (“Sec3N”) alone and the Sec3N–Rho1 complex were resolved [cdc42 mutants were made in this study to confirm its binding to Sec3: cdc42-301 (V36A), cdc42-302 (V36A F37A), cdc42-303 (Q61A), cdc42-304 (Y64A) and cdc42-305 (Y64A R66A). Our in vitro binding assay showed that these cdc42 mutants were defective in binding to GST-Sec3N (a.a.71–241) P2, and these associations are important for polarized cell growth [cdc42 mutants was performed using GST-tagged Gic2N (amino acids 1–155), which contains its Cdc42-interacting domain [cdc42-302, the binding of GST-Gic2N to the other four cdc42 mutants decreased dramatically. These results suggest that the binding sites of Cdc42 in Gic2 is very similar to Sec3.The N-terminus of Gic2 contains the classic Cdc42/Rac1 interactive binding (CRIB) domain , 27. Beco sec3∆N , we testl growth . The in g domain . As show PtdIns4,P2, and tSaccharomyces cerevisiae under control of the endogenous Cdc42 promoter. The growth of the mutant cells was examined on YPD plates at 25 °C or 37 °C Pgic1Δgic2Δ double mutant is synthetically sick with sec3ΔN single mutant and regulates cell gowth and exocytic secretion. Gic proteins regulate polarized secretion by targeting the tethering complex to bud tip of yeast cells P2 binding region, and the interaction of Sec3N-terminus with both Cdc42 and PtdInsP2 are important for Sec3 function. Yamashita et al. [sec3 mutants deficient in the binding of either Rho1 or PtdInsP2 can still be localized to the bud tip even in the presence of Latrunculin B, which disrupts the actin microfilament cytoskeleton, while the sec3 mutants deficient in the binding of both Rho1 and PtdInsP2 are almost completely depolarized in the presence of Latrunculin B. These results suggest that both Rho GTPase and PtdInsP2 can act as receptors for the exocyst complex in a complementary fashion. Gic proteins are considered to be targeted to the bud tip and plays an important role in early bud formation in yeast. The GTP-bound Cdc42 interacts with Gic2 through the Cdc42/Rac interactive binding domain located at the N terminus of Gic2 and activates Gic2 during bud emergence [2 in the plasma membrane. This physical interaction is also necessary for the polarized localization of Gic2 protein to the bud tip and is important for the function of Gic2 in cell polarity. These facts indicate that PtdInsP2 and Cdc42 act in concert to regulate polarized localization and function of Gic2 during polarized cell growth in the budding yeast. Based on these previous reports and the results shown in our study, we speculate that either the Cdc42–Sec3/Gic interaction or PtdInsP2–Sec3/Gic interaction can targeting the exocyst complex to the polarized growth sites, which explains the result that three Sec3-binding deficient cdc42 mutants did not show growth defect in Fig. cdc42 mutants can not bind to Sec3 and/or Gic2.Zhang et al. , 10 founa et al.  also foumergence , 27. Orlmergence  have idecdc42 mutants may also be defective in interaction with other effectors. It is possible that the interaction of Cdc42 with other effectors are also destroyed in the two cdc42 mutants, cdc42-303 and cdc42-305. But it is unlikely that the ATP loading capacity of these mutants is the main reason for the binding defect because no interaction was detected between cdc42 mutants and Sec3, and neither GDP nor GTP affected the binding of Sec3 to all five mutants . Further experiments are needed to explore the molecular mechanism of Gic proteins interacting with exocyst complex.We found that Gic2 is physically associated with exocyst complex. It is likely that its paralog, Gic1, may also interacts with this protein complex. However, it is unclear whether Gic proteins bind directly to one of the exocyst subunits, or bind to another protein which interacts with exocyst complex. Another exocyst subunit, Exo70, binds to both Cdc42 and PtdInsPecretion , 18, 21.600 units of early log phase cells were collected in a 1.5-ml centrifuge tube and washed once with 0.5 ml of distilled water. 240 μl PEG 3350 (50% wt/vol), 36 μl 1.0 M LiAc, 5 μl 10 mg/ml sonicated salmon sperm DNA (Agilent Technologies), and 0.1–10 μg DNA were added and the tube was vortexed until the cell pellet had been completely mixed. The cells were then shocked in a water bath at 42 °C for 20–25 min. 2–200 μl of the transformation mix was plated onto Petri dish plates containing solid synthetic complete medium. The plates were incubated at 25 °C for 2–4 days to recover transformants.Standard methods were used for yeast growth and genetic manipulations . All strpRS314-CDC42 plasmid [pRS316-HA-CDC42 plasmid balancer. The strains used in this study are listed in the strain table. Standard methods were used for yeast media and genetic manipulations.All mutagenesis was performed based on the  plasmid  using th plasmid  and the SEC5, SEC8, EXO70 and EXO84 by Green Fluorescence Protein (GFP) was performed as previously described [Chromosomal tagging of escribed . Cells wescribed . F-actin2, and 1 mM CaCl2). The cells were spheroplasted and fixed with 1% glutaraldehyde  at 4 °C overnight. The spheroplasts were washed in 0.1 M cacodylate buffer and were post-fixed twice with ice-cold 0.5% OsO4 and 0.8% potassium for 10 min each. After dehydration and embedding in Spurr’s epoxy resin , thin sections were cut and transferred onto 600 mesh uncoated copper grids  and were post-stained with uranyl acetate and lead citrate. Cells were observed on a transmission electron microscope  at 100,000× magnification.Cells were grown at 25 °C in YPD media to log phase, and were then processed for thin section EM analysis using a Jeol-1010 transmission electron microscope. Cells for EM were collected by vacuum filtration using a 0.45 µm nitrocellulose membrane and were fixed for 1 h at room temperature in fixation buffer  at 25 °C. NaN3 and NaF were added directly to the culture at a final concentration of 10 mM each. 10 OD600 units of cells were collected, washed with cell wash buffer . The cells were resuspended in 300 µl of spheroplast solution buffer  and incubated at 37 °C in a water bath for 30 min. The spheroplasts were pelleted gently by centrifuge at 2000 rpm for 5 min at 4 °C. 100 µl of supernatant was carefully transferred to a new tube and mixed with 20 µl of 6× SDS loading buffer . This is the external pool. The remaining supernatant was removed and the pellet (spheroplasts) was washed once with 1 ml of spheroplast wash buffer  to remove residue external pool. The spheroplasts were dissolved in 300 µl of lysate buffer  by leaving on ice for 10 min. The cell debris was removed after the sample was centrifuged at 13,000 rpm for 5 min at 4 °C. 100 µl of supernatant (lysates) was transferred to a new tube and mixed with 20 µl of 6× SDS loading buffer. This is the internal pool. The internal pool and external pool samples were boiled at 95 °C for 5 min, loaded into 12% SDSPAGE gel. Bgl2 was detected by Western blotting with an anti-Bgl2 rabbit polyclonal antibody (1:2000). For temperature-sensitive mutants, the cells were grown at 25 °C or shifted to 37 °C for 90 min before being processed for the Bgl2 assay.Analyses of the secretion of Bgl2 and invertase were carried out as previously described . 20 ml o600 is 0.6–1.0) at 25 °C. 1 OD cells were transferred to a new tube and washed with 1 ml of ice cold 1 mM NaN3. The cells were then resuspended in 1 ml YP plus glucose medium  and incubated at 25 °C for 1 h to induce invertase expression. After 1 h of incubation, the cells were collected and washed once with 1 ml 10 mM NaN3. The cells were resuspended in 1 ml 10 mM NaN3 and kept on ice. The external invertase was measured directly on the whole intact cells, whereas the internal invertase was measured after preparation of lysates. 0.5 ml of cells were removed and mixed with 0.5 ml of 2× spheroplast cocktail mix . The cells were incubated in water bath at 37 °C for 45 min. The spheroplasts were collected and the supernatant was removed carefully without disturbing the pellet. The spheroplasts were dissolved at 4 °C in 0.5 ml 0.5% Triton X-100. The invertase assay was performed in 13 × 100 mm tubes. 20 µl of sample was placed in the tube and 80 µl of 50 mM NaAc, pH 5.1, was added. Then 25 µl of 0.5 M sucrose was added and the tube was incubated at 37 °C for 30 min. 150 µl of 0.2 M K2HPO4 was added and the tube was placed on ice to stop the reaction. The sample was boiled for 3 min and put on ice. 1 ml of assay mix was added  and the sample was incubated at 37 °C for 30 min. 1 ml of 6 N HCl was added into the tube and the value of A540 was measured by the Spectrophotometer . For temperature-sensitive mutants, the cells were grown at 25 °C or shifted to 37 °C for 90 min and then were grown in low-glucose medium (0.1% glucose) at the same temperature for 1 h.Invertase secretion was examined as described previously . 20 ml ocdc42 were expressed as His6-tagged fusion protein (His6-Cdc42 or His6-cdc42). 20 μg Sec3 (a.a.71–241) or Gic2 (a.a.1–155) and 10 μg Cdc42 or cdc42 mutant were used for in vitro binding assay as previously described [Amino-terminus of Sec3 (a.a.71–241) and Gic2 (a.a.1–155) were expressed as GST fusion proteins. Cdc42 and g at 4 °C, and the supernatant was further clarified by centrifugation at 100,000g at 4 °C. The published Hot-SDS protein extraction protocol was used with slight modifications [Yeast extracts were prepared using glass beads as described previously . Yeast cications . In brieFor immunoprecipitation of GFP-tagged Sec5, total proteins were extracted by using the glass beads method . A rabbiAdditional file 1: Figure S1. Polarization of actin in gic1∆gic2∆sec3∆N mutant. Cells were grown to log phase at 25°C, and then fixed. Actin was stained by Alexa Fluor 488-conjugated phalloidin. Scale bar, 5μm. (B) Quantification of cells with polarized actin in each mutant. 90 cells were counted for each group (n=3). Error bars represent standard deviations. The representative results from three independent experiments are shown.Additional file 2: Figure S2. Structural integrity of the exocyst complex in the gic1∆gic2∆sec3∆N triple mutant. The exocyst component Sec5 in wild-type and mutant strains was GFP-tagged by chromosomal integration. The cells grown to log phase were incubated at 25°C or shifted to 37°C for 2 h. The exocyst was immuno-isolated from the wild type and mutant cells by anti-GFP antibody, and individual exocyst components were detected by Western blot. At both 25°C and 37°C, the exocyst components were coprecipitated with Sec5-GFP. Our result suggests that the assembly of the exocyst complex is largely unaffected in the gic1∆gic2∆sec3∆N triple mutant. The representative results from three independent experiments are shown.Additional file 3: Figure S3. The cdc42 mutants were not able to bind to Sec3N in vitro with either GDP or GTP in the binding buffer. GST fusion proteins containing (a.a.71–241) of Sec3 was purified and conjugated to glutathione Sepharose. Cdc42, cdc42-301, cdc42-302, cdc42-303, cdc42-304 and cdc42-305 were expressed as a Hisx6 fusion and purified from bacteria. The in vitro binding assay was performed using GST-Sec3 and Hisx6-Cdc42 in the presence of GTPγS or GDP. The Hisx6-Cdc42 fusion protein bound to the GST-Sec3N Sepharose was detected by Western blotting with anti-Hisx6 antibody (bottom). Equal amounts of Sec3 fusion proteins on beads were used in the binding assay . Equal amounts of cdc42 wild type and mutants were used . The representative results from three different experiments are shown. The representative results from three independent experiments are shown.Additional file 4: Table S1. Yeast strains and genotypes. Table S2. Plasmids used in this study.


Each pancreas was perfused in situ with histidine-tryptophan-ketoglutarate (HTK) or with University of Wisconsin solution and placed into a transport container. Temperature of the grafts was maintained at 4 ± 2 °C during transport to our hospital and MR scanning. A 1.5 T clinical scanner was used for the measurements. Single-voxel PRESS spectra were acquired using transmit–receiver head coil.2–)n , and tissue water . Average total fat, and intracellular lipids of NAPC concentrations were 79.2 ± 100.8 (range 2.4–304.4), and 2.9 ± 1.2 mmol/kg ww, respectively.Relaxation times were measured for lipid (–CH1H-MRS is a useful tool for the estimation of pancreas graft lipid concentrations. Total pancreatic fat and especially content of intracellular lipids of NAPC are valuable measures for inspection of graft quality prior to transplantation or islet of Langerhans isolation.We have shown that  The pancreas plays an important role in the synthesis and secretion of digestive enzymes and metabolism-regulating hormones. The majority of pancreas volume contains exocrine glands that are dedicated to producing enzymes which help to digest proteins, fats, and carbohydrates (sugars). Approximately 2% of the pancreas mass consists of different hormone-producing endocrine cells which clump together into small clusters called islets of Langerhans. The hormones made by alpha and beta cells in the islets produce glucagon and insulin, respectively. These two hormones regulate the sugar levels in the blood and cells.Pancreas transplantation is the treatment of choice for patients whose pancreas does not make enough, or sometimes any, insulin. These patients suffer type 1 diabetes, or insulin-dependent type 2 diabetes. A relatively new and minimally invasive therapy is transplantation of the islets of Langerhans , 4.2 .Relaxation times ies Fig.  by a Lev2O per 1 g wet weight tissue  where A and B are constants. T1 temperature coefficient decreased from dT1/dT = 9.5 ± 0.16 ms/°C to 5.35 ± 0.08 ms/°C in the interval from 65 to 25 °C. Linear dependence (0.9 ± 0.03 ms/°C) was found between T2 and temperature. These T1, T2 dependences and the temperature difference ~ 33 °C between in vivo and cold storage explain the shorter relaxation times.In vivo estimation of pancreatic water relaxation times e et al. . Our shoeous fat , 29. Thieous fat  reportedIn this study, we found the pancreatic fat content (f/w) to be very similar, to those reported for healthy subjects , 12, 13.1H-MRS is unable to discriminate between intracellular lipids of acinar and the islet of Langerhans cells.Pancreatic intracellular lipid accumulation is currently of considerable interest. It was shown that an increased content of cytosolic lipid droplets in islets of Langerhans leads to decreased glucose-stimulated insulin secretion , 33. ThiThe main limitation of this study is the small number of grafts and age of donors. It should be noted that the grafts were considered unsuitable for transplantation mainly due to advanced age. A further limitation is the fact that increased pancreatic fat content hinders detection of intracellular lipids of NAPC. The usability of the measured relaxation times is limited to the grafts perfused by HTK or UW solution and to the temperature of cold storage (4 ± 2 °C).1H-MRS is a useful tool for quantification of pancreas graft lipid concentrations using water as the internal concentration reference. Total pancreatic fat and especially content of intracellular lipids of NAPC are valuable measures for inspection of graft quality prior to transplantation or islet of Langerhans isolation.The present results suggest that


Alzheimer’s disease (AD) is a progressive neurodegenerative disease. The study of blood-based biomarkers has lasted for a long time in AD, because it supports the concept that peripheral changes are involved in AD pathology. But it is still unclear how peripheral blood is involved in the temporal characteristic molecular mechanisms of AD from aging to mild cognitive impairment (MCI) and which cells are responsible for the molecular mechanisms. The main purpose of our study is to gain a systematic and comprehensive understanding of temporal characteristic networks of peripheral blood in AD using whole blood samples with 329 case-control samples, including 104 normal elderly control subjects (CTL), 80 MCI who are susceptible to AD, and 145 AD, by the weighted gene co-expression network analysis (WGCNA). The module-trait relationships were constructed and module preservation was validated with independent datasets GSE63061, GSE97760, GSE18309, GSE29378, GSE28146, and GSE29652. Our results indicate that the down-regulated protein modification and ubiquitin degradation systems, and the up-regulated insulin resistance both play a major role in MCI, while the up-regulated inflammatory cascade dominates in AD, which is mainly mediated by monocytes, macrophages. Although there is mixed activation of M1 and M2 macrophages in all stages of AD, the immune neutral state or M2 polarization may predominate in MCI, and M1 polarization may predominate in AD. Moreover, we found that TRPV2, NDUFV1, ATF4, HSPA8, STAT3 and LUC7L3 may mediate the pathological changes in MCI, while SIRPA, LAMP-2, NDUFB5, HSPA8, STAT3 and FPR2 may mediate the conversion from MCI-AD or the pathological changes in AD, which provide a basis for the treatment based on the peripheral blood system. In addition, we also found that the combined diagnosis based on a panel of genes from the red, blue, and brown modules have a moderate diagnostic effect on distinguishing MCI and AD from CTL, suggesting that those panels of genes may be used for detection of MCI and prediction of this conversion from MCI to AD. Our research emphasizes that pathological changes, based on temporal characteristics of peripheral blood, provide a theoretical basis for targeted peripheral treatment based on appropriate times and identified several diagnostic markers. Alzheimer’s disease (AD), the most common type of dementia, affects more than 50 million people worldwide Hodson, . More imAlthough accurate diagnosis has been achieved by novel CSF biomarkers , a bioinformatics analysis method, has been proven to effectively detect the complex module-trait relationships , 80 MCI who are susceptible to AD, and 145 AD. Then, we identified seven modules associated with different stages of AD and module preservation for each of the seven modules was validated with independent datasets. Next, we tested the overlap between the module and cell signature gene lists using GeneOverlap and performed functional enrichment analysis. Finally, we obtained the hub genes of modules using cytohubba plugin in Cytoscape and performed receiver operating characteristic curve analysis to detect its diagnostic power.The human whole blood mRNA expression dataset GSE63060 provided by AddNeuroMed Cohort, which is a large cross-European prospective biomarker study, was downloaded from the Gene Expression Omnibus (GEO) database . GO terms and KEGG pathways  suggest that RNA metabolism and inflammatory signaling pathway were down-regulated in both microglia and peripheral blood monocytes and macrophages, while ubiquitin-mediated proteolysis and protein modification process were down-regulated in peripheral blood monocytes and macrophages. The black module with high preservation in peripheral whole blood or monocyte datasets indicates that ribonucleoprotein complex biogenesis and RNA processing were up-regulated in regulatory T cells and Th1 cells. It is worth emphasizing that those modules are almost simultaneously involved in M1 and M2 macrophages or regulatory T cells and Th1 cells, which suggests that mixed activation of the peripheral blood immune system may exist in MCI. In these modules, we found that a part of the hub genes may be involved in the pathology, development and diagnosis of AD. For example, Enoyl-CoA hydratase 1 (ECH1), a member of the hydratase/isomerase superfamily, was proven to be a promising marker for early diagnosis of AD  cell type specific genes are based on reference datasets, and do not completely reflect cell type proportions. Changes in monocytes can occur in AD compared to controls, and this change within the same cell type will be lost or incorrectly interpreted using the currently used approach of assigning cell specificity to particular genes; and (2) since this is a cross-sectional study rather than a longitudinal study, the above results cannot fully represent the pathological changes from CTL-MCI-AD, and further experimental verification is needed.HL and RT: study design. RT: literature search, data analysis and article writing. HL: article revision. Both authors approved the final version of the article.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.


N-acetylglutamate synthase deficiency  is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD. NAG is an essential allosteric activator of carbamylphosphate synthetase 1 , which catalyzes the initial step of ureagenesis – ATP-dependent synthesis of carbamylphosphate from ammonia and bicarbonate2. Primary NAGS deficiency  results from defects in NAGS gene and protein leading to decreased abundance or absence of NAG, decreased or absent CPS1 activity, reduced or absent urea synthesis and hyperammonemia4. Hyperammonemia is the principal biochemical symptom of NAGSD which manifests clinically with nausea, vomiting, cognitive changes, seizures and, in severe cases, coma and death; other biochemical symptoms include high plasma glutamine and low or absent plasma citrulline4. Additionally, NAGSD can arise secondary to defects in organic and fatty acid metabolism4.N-acetylglutamate synthase  is a urea cycle enzyme that catalyzes formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme ANAGS gene is located on chromosome 17 within band 17q21.31; NAGS spans approximately 8.5 kb and has seven exons that encode a 1605 bp open reading frame, starting at chr17:42,082,032 (GRCh37/hg19), six introns, a promoter, and an enhancer located about 3 kb upstream of the transcription start sites9. The NAGS promoter directs transcription of the NAGS gene from multiple transcription start sites located between 20 and 120 bp upstream of the NAGS translation initiation codon9. The NAGS promoter controls transcription of the NAGS gene through binding of specificity protein 1 (Sp1) and cAMP response element binding (CREB) transcription factors9. The NAGS enhancer binds hepatic nuclear factor 1 (HNF1) and nuclear factor Y (NF-Y) transcription factors and is responsible for liver-specific expression of the NAGS gene9.Human 10. Majority of patients with NAGSD are homozygous for the disease causing NAGS allele11. Most NAGSD causing sequence variants are within NAGS coding region or affect splicing of the NAGS mRNA15; a sequence variant in the NAGS enhancer (NM_153006.1:c.-3064 C > A) caused NAGSD by reduced NAGS expression due to decreased binding of HNF1 to its binding site8. Quick and accurate diagnosis of NAGSD is essential because it is the only urea cycle disorder that can be completely treated with a single drug, i.e. N-carbamylglutamate (NCG), a chemical analog of NAG that binds to and activates CPS117 to completely restore ureagenesis in patients with NAGSD18. NAGSD can be diagnosed by sequencing of the NAGS gene and the diagnosis is supported by the markedly enhanced ureagenesis in response to treatment with NCG8.Primary NAGSD is an autosomal recessive disorder that affects approximately 1 in 2,000,000 peopleNAGS enhancer that occurred independently in two families whose members had NAGSD, totaling to three independent families with NAGS enhancer variants with apparent clinical consequences. This novel variant is pathogenic because it is associated with recurrent episodes of hyperammonemia (and epilepsy) that were promptly resolved upon treatment with NCG and caused reduced expression in a functional assay consisting of a reporter gene in cultured cells.We describe a novel sequence variant in the All methods were performed in accordance with the relevant guidelines and regulations of the participating institutions. Mutation analysis was conducted with the approval of the Institutional Review Boards of the Erasmus Medical Center of the Sophia Children’s Hospital and the University Hospital of the University of Padua. Informed consent was obtained either from all study subjects or their parents/guardians.The female subject is the only child of non-consanguineous parents. Mother is from Russia while father is Dutch. Pregnancy, delivery and neonatal period were normal. She was breastfed until the age of 3 months. She had a normal development, smiling, first words at 6 months and walking at 18 months. At the age of 2–3 years, she refrained from milk and dairy products, fish and meat.3 18.6 mmol/L, BE −7), elevated lactate 4.0 mmol/L (ref. 1.0–1.8) and ammonia . Amino acids in plasma (prior to treatment) showed increased alanine 1043 µmol/L (ref. 176–480), normal glutamine 482 µmol/L (ref. 400–720), and decreased citrulline 10 µmol/L (ref. 17–50) and arginine 17 µmol/L (ref. 32–128); isoleucine, leucine and valine were also decreased. Urinary orotic acid was normal as was blood acylcarnitine profile. She was started on a high caloric drip, without protein but did not improve clinically and was therefore transferred to the Pediatric Intensive Care Unit (PICU). The patient suffered a similar period with abnormal behavior a month prior to this event, but the cause of her strange behavior remained unclear.A female patient, 3 years and 9 months old, was referred because of encephalopathy . Her CT scan indicated diffuse bilateral obliteration of peripheral cerebral spinal fluid, hypodensity of white matter with loss of white-gray demarcation, and diffuse hypodense aspect of the white matter as a result of global cerebral ischemia. Four days prior, she had been restless, drowsy/lethargic, and repeating incomprehensible sentences. She seemed tired, but would get up in the night. With increasing loss of consciousness, she was admitted to the hospital. An EEG was suspicious for focal epilepsy. She was treated with diazepam and valproate. Brain MRI, taken after treatment, was normal. Laboratory investigations revealed compensated metabolic acidosis , she was started on i.v. arginine . Ammonia levels decreased from maximum 462 µmol/L to 103 µmol/L, but increased again after 3 days to 194 µmol/L. Therefore, nitrogen scavengers were increased to 400 mg/kg/d and NCG started simultaneously (5 × 200 mg/d). With this treatment and after reintroduction of protein at final dose of 1 g/kg/d, ammonia levels normalized and remained normal.Based on initial results, ornithine transcarbamylase (OTC) deficiency was suspected and NCG treatment was stopped. Two weeks later, another episode of hyperammonemia occurred which resolved immediately after reintroduction of NCG as the only treatment. This fast response to NCG together with the self-imposed protein restriction suggested NAGSD.NAGS and CPS1 coding exons, including the flanking intronic regions, did not reveal any sequence variants. Liver enzyme activity analyses were all normal: NAGS 94 µIE/mg protein (ref. >34), NAGS after arginine stimulation 197 µIE/mg protein (ref. >144), CPS1 26.7 mIE/mg protein (ref. >12), and OTC 489 mIE/mg protein (ref. >160).Routine DNA analysis of NAGS enhancer region was identified  of chromosome 17  which remained elevated despite continuous i.v. glucose fluids. Due to persistent hyperammonemia, treatment with sodium benzoate was initiated. A complete metabolic work-up including the plasma amino acid profile showed high glutamine (895 µmol/L), alanine (719 µmol/L) while urinary organic acids and blood acylcarnitine profile were normal. No orotic acid was detected in the urine collected during acute hyperammonemic episode. Subsequently, an allopurinol loading test was performed showing neither orotic acid nor orotidine. Screening of the gene panel for UCD, which includes Three months after this episode, she presented again with a similar psychotic picture that required hospitalization. Metabolic investigations revealed hyperammonemia (236 µmol/L), elevated glutamine in plasma (855 µmol/L) and in the CSF . These data, consistent with a proximal urea cycle defect, including NAGSD, suggested to change therapy from sodium benzoate to NCG at an initial dose of 100 mg/kg/d. Shortly after this change, ammonia normalized and the child regained her normal activity.th percentile and the neurological and developmental assessments were normal. Despite the negative results for NAGS mutation(s) in the coding region and splice sites we decided to continue the therapy with NCG. After mutation analysis of patient 1 revealed the NM_153006.2:c.-3026C > T variant in the NAGS enhancer we revisited the previous sequencing results in this patient and found that she is also homozygous for the c.-3026C > T sequence variant  and the conservation scores for each position were downloaded from the UCSC Genome Browser (https://genome.ucsc.edu/). Sequences of NAGS enhancers from 30 mammals  according to manufacturer’s instructions. Presence of the enhancer mutation was confirmed by DNA sequencing of the resulting plasmid.Expression plasmid p4.23hE_Mut, which harbors 2. Cells were plated in the 24-well plates at 1.5 × 105 cells/well; 24 hr. later cultured cells reached 90–95% confluency. Cells were transfected with Lipofectamine 3000 reagent (Life Technologies) mixed with 0.5 µg of plasmid DNA. The ratio of experimental plasmids, which express firefly luciferase luc2, and pGL4.74, which expresses Renilla reniformis luciferase and was used as a transfection efficiency control, was 1000:1.Human HepG2 hepatoma cells (ATCC) were cultured in complete EMEM medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 5% penicillin/streptomycin (Life Technologies); cells were cultured at 37 °C in atmosphere containing 5% CORenilla luciferase to correct for differences in transfection efficiency, and to firefly luciferase activity from the p4.23hE plasmid harboring wild-type NAGS enhancer. Results are average of three independent experiments, each carried out in triplicate. Values were expressed and mean ± SEM and analyzed using Student t-test.Cells were harvested 24 hr. after transfection and used for reporter gene assay with the Dual Luciferase Reporter Assay System (Promega) and Berthold Centro 960 luminometer according to the manufacturer’s instructions. Firefly luciferase activities were normalized to activity of NAGS gene regulatory region encompasses approximately 4 kb; it harbors several regions of high conservation in mammalian genomes that cluster in the NAGS promoter, immediately upstream of NAGS exon 1, and in the NAGS enhancer, which is located about 3 kb upstream of the NAGS translation initiation codon . ENCODE project revealed that human NAGS enhancer also binds transcription factor NF1C in HepG2 cells .Human don Fig. . Human Nver Fig.  and8,9. lls Fig.  and22.FiNAGS exons to include the NAGS gene regulatory regions.All three patients in this study presented with hyperammonemia and a metabolic profile suggestive for a proximal urea cycle disorder. Treatment with NCG led to normalization of plasma ammonia in patients 1 and 2 while withdrawal of this treatment resulted in recurrence of hyperammonemia in both patients. The third patient in this study markedly increased dietary protein intake after initiation of treatment with NCG. Therefore, genetic investigations were expanded beyond the routine sequencing of the coding NAGS regulatory region in all three patients revealed a C to T change at position chr17: 42,079,006 (GRCh37/hg19 human genome assembly), which is 3026 bp upstream of the NAGS translation initiation codon, within intron1 of PYY, and within the Nuclear Factor IC (NFIC) transcription factor binding site 25 and Phylo-P26 scores for the base pair affected by the c.-3026C > T variant are 4.51  and 2.749 , respectively, indicating evolutionary constraint at this position while the PhastCons score26 of 0.999  indicates that the c.-3026C is part of a conserved element. The CADD (Combined Annotation-Dependent Depletion) scaled C-score, which includes the previous scores in a weighted model, for the c.-3026C > T is 19.36 indicating that this variant is among 1–2% of the most deleterious variants in the human genome27, and MutationTaster228 classified this variant as disease causing. Additionally, c.-3026C > T variant was classified as deleterious by each of the five variant prediction tools  that are combined into PredictSNP2 platform29. Because computational prediction of the effects of sequence variants are not always accurate we carried out functional testing in cultured cells.Sequencing of the ite Fig. . This seNAGS gene and NAGSD in three patients. The C to T change corresponding to the c.-3026C > T variant was engineered into p4.23Enh construct8 and resulting construct p4.23hE_Mut was transfected into HepG2 cells. The p4.32Enh construct, harboring wild-type NAGS enhancer, was used as a positive control and p4.23, which lacks NAGS enhancer, was a negative control. The relative firefly luciferase activity was approximately 20% lower in HepG2 cells transfected with the p4.23hE-Mut construct than in cells transfected with the construct harboring wild-type NAGS enhancer  binding site, which has been identified using chromatin immunoprecipitation followed by sequencing carried out as part of the ENCODE project31. Although NFIC transcription factor is a DNA-binding protein that interacts with transcription machinery32, the presence of its binding site within an enhancer is not unusual as many enhancers have been shown to interact with transcription machinery when regulating expression of their target genes33. The high conservation scores of the base pair affected by the c.-3026C > T variant indicate that it resides in a conserved element, and suggests evolutionary constraint in vertebrates and importance for the NAGS enhancer function. This is reflected in computational predictions that c.-3026C > T variant is deleterious (PredictSNP2) and disease causing (MutationTaster2). Because computational predictions of the functional effects of sequence variants do not always agree with the expert opinion34, we carried out functional testing with reporter gene constructs, which revealed that the c.-3026C > T variant could affect expression of the NAGS gene. Taken together clinical, biochemical and molecular evidence strongly suggest that the c.-3026C > T variant is pathogenic and caused NAGSD in three patients experiencing physiological stress. As a consequence, and including the previous single case, we recommend adding sequencing of the NAGS enhancer region both in direct NAGS gene analysis using Sanger sequencing and when next generation approaches are performed.The c.-3026C > T variant maps in the vicinity of the NF-Y transcription factor binding siteFigureS1 and TableS1Dataset 1


According to multivariate logistic regression analysis, the variability of the renin concentration was associated with NTG (p = 0.006). In conclusion, the systemic concentration and variability of renin levels were elevated in NTG patients. An altered renin concentration could represent a difference in RAAS function in NTG patients.The purpose of this study was to investigate the function of the renin–angiotensin–aldosterone system (RAAS) in normal tension glaucoma (NTG) patients by measuring the level of renin and angiotensin II (AngII) in the plasma. Twenty-four patients with NTG and 38 control subjects were included in this study. Renin and AngII were measured in the blood samples of all subjects by enzyme-linked immunosorbent assay (ELISA). No significant differences were found in the complete blood count, fasting glucose, low-density lipoprotein (LDL), and high-sensitivity C-reactive protein (hs-CRP) levels between the control and NTG groups. The systemic concentration and variability of the renin concentration in the blood was significantly higher in the NTG group ( Glaucoma is characterized by the progressive death of retinal ganglion cells (RGCs) and the associated visual field (VF) defect. Elevated intraocular pressure (IOP) is traditionally known to be the main cause of glaucoma . HoweverGlaucoma patients with normal IOP, known as normal tension glaucoma (NTG), have distinct clinical features associated with vascular dysregulation. According to several studies, orthostatic hypotension, systemic arterial hypotension, Raynaud’s phenomenon, and migraines are common in NTG patients ,5,6. AccThe renin–angiotensin–aldosterone system (RAAS) is important for regulating hemodynamic stability and fluid volume in the body . When hyThe purpose of this study was to investigate the systemic hemodynamic features associated with RAAS function in patients with NTG. To the best of our knowledge, no study has been conducted measuring renin and AngII levels in the blood of NTG patients. We measured the concentration of renin and AngII, as well as the complete blood count, fasting glucose, total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in the blood of the study subjects. The results of the plasma concentrations of renin and AngII, including the variability, were compared to those of normal controls.This study was performed according to the tenets of the Declaration of Helsinki and was approved by the Institutional Review and Ethics Board of Incheon St. Mary’s Hospital, South Korea. All subjects of this single-center, case–control study provided written, informed consent. Normal controls were recruited from a routine ocular examination group, and patients with NTG were recruited from the glaucoma clinic of Incheon St. Mary’s Hospital. Patients were excluded if they met any of the following criteria: (1) a history of non-glaucomatous optic neuropathy; (2) a history of eye trauma or surgery, except for uncomplicated cataract extraction; (3) pathologic myopia ; (4) other retinal diseases, including diabetic retinopathy and retinal vascular diseases such as vascular occlusion or uveitis.All subjects underwent comprehensive ophthalmic examinations, including best-corrected visual acuity (BCVA), refraction, Goldmann applanation tonometry, slit-lamp examination, gonioscopy, and dilated fundus bimicroscopy. Fundus photography, stereoscopic photography of the optic disc , and perimetry with the Swedish Interactive Threshold Algorithm (SITA) 24-2 test on a Humphrey Field Analyzer were performed to diagnose NTG. The diagnostic criteria for NTG were as follows: (1) open angle by gonioscopy; (2) IOP <22 mmHg; (3) glaucomatous-appearing optic disc morphology corresponding to glaucomatous VF defects on SITA 24-2 results, as follows: a glaucomatous-appearing optic disc accompanied by increased cup-disc ratio (CDR) of more than 0.7, a difference in vertical CDR of more than 0.2 between both eyes, retinal nerve fiber layer (RNFL) defects, or neural rim thinning. We defined glaucomatous VF defects as meeting two of the following three criteria: (1) a cluster of 3 points with a probability lower than 5% on the pattern deviation map in at least one hemifield and including at least 1 point with a probability lower than 1% or a cluster of 2 points with a probability lower than 1%; (2) glaucoma hemifield test results outside normal limits; (3) a pattern standard deviation outside 95% of the normal limits. VF defects were confirmed by at least two consecutive and reliable tests . All NTG patients had anti-glaucomatic eyedrops and showed stable disease status without VF progression for three years.BP was measured after the study subjects had rested in a sitting position for 5 min. Trained clinicians took measurements of two consecutive systolic and diastolic BPs, and the average value was used. For hypertension patients, the type of hypertensive medication was identified, and subjects with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) were excluded, as these medications could interfere with systemic renin and angiotensin II levels.After fasting for 12 h and resting for 30 min, blood samples for the biochemical analysis were obtained by standard venipuncture. Blood was collected into tubes containing EDTA as an anticoagulant for plasma preparation. As soon as a blood sample was collected, the tube was frozen at −20 °C, and repeated freeze–thaw cycles were avoided. Frozen samples were thawed before conducting ELISA of renin  and EIA of AngII  using commercially available kits and according to the manufacturer’s instructions. In brief, standards, samples, and controls were added to each well of a microplate pre-coated with a secondary antibody specific for renin and AngII. After incubation and washing the plate, a primary antibody was added, and the plate was again incubated and washed. Subsequently, a chromogenic substrate was added to each well for the development of color, and the intensity was measured by a microplate reader. All measurements were assayed in duplicate and the mean values were calculated. In addition, the standard deviations (SDs) of the measurements were calculated to assess the variability of renin and AngII.t-tests were used to compare the continuous variables between the control and NTG groups. Chi-square tests were used to compare the categorical variables between the two groups. To identify the factors associated with NTG, logistic regression analyses were performed. Variables with p-values <0.1 in the univariate analyses were included in the multivariate analysis. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each variable. All statistical analyses were performed with SPSS version 24.0 . p < 0.05 was considered statistically significant.All data are expressed as mean ± standard deviation. Independent This study included 24 patients with NTG and 38 controls as study subjects. p = 0.027) and HDL was lower (p = 0.036) in the NTG patients.The laboratory findings were comparable between the two groups, as shown in p = 0.005; SD: 17.79 ± 14.29 vs. 49.45 ± 49.41, p = 0.005). The mean concentration and SD of AngII were higher in the NTG group but not statistically significant . According to The mean concentration and SD of renin and AngII from each group are reported in p = 0.033, 0.003, and 0.002, respectively). In the multivariate analysis, including the variables with p-values ≤0.10 from the univariate analyses, only the SD of renin was significantly associated with NTG (p = 0.006).The factors related to NTG from the clinical and laboratory findings were evaluated using logistic regression analyses . In the The systemic features representing hemodynamic instability include systemic arterial hypotension, orthostatic hypotension, and a nocturnal BP dip. These features have been reported as commonly found characteristics in NTG patients ,13,14. AWhen systemic hypotension occurs in the body, the RAAS starts to regulate the blood volume and systemic vascular resistance . The RAAIn the present study, we found that the systemic mean concentration and variability of renin was higher in the NTG patients. Additionally, the SD of the renin concentration was a meaningful factor associated with NTG. AngII was thought to be elevated in response to an increase in renin; however, there was no statistically significant difference between the NTG and control groups. In short, this study identified a link between systemic RAAS functioning and NTG through the measurement of the systemic concentration of renin and AngII. RAAS function showed a different pattern in the NTG patients when compared to normal controls.According to our previous study, the systemic concentration of endothelin-1 (ET-1) and macrophage chemoattractant protein-1 (MCP-1) are elevated in NTG patients . ET-1 isIn healthy eyes, retinal blood flow is mediated by endothelial cells because there is no autonomic nervous system innervation . AngII, In addition, most RAAS components and receptors were found in the retinal tissue. The local RAAS from tissue, including the retina, has been reported as a system that maintains the homeostasis of tissue . ProreniIn this study, total cholesterol and HDL were different between the two groups. The NTG group had worse lipid profiles in terms of dyslipidemia. However, lipid profiles were not independently associated with NTG in the regression analyses. Indeed, there is controversy regarding the relationship between dyslipidemia and glaucoma. Wang et al. suggested that hyperlipidemia is related to an increased risk of glaucoma and IOP elevation . It has The other limitations of this study should be mentioned. First, clinical features associated with vascular dysregulation, such as optic disc hemorrhage, migraine, and Raynaud’s phenomenon, were not included in our analysis. Since normal controls were recruited from routine medical examinations, information on their hemodynamic features was not available. Several studies have already reported the correlation of vascular dysregulation with optic disc hemorrhage , migrainIn conclusion, NTG patients showed elevation and fluctuation of systemic renin concentration. The difference in RAAS function in NTG patients may be associated with diverse features of vascular dysregulation. Further studies investigating the relationship between RAAS and NTG could suggest a novel approach for disease modulation.