Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis.The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga  Cyanidioschyzon merolae aurora kinase CmAUR regulates mitochondrial division by phosphorylating mitochondrial division ring components. Depletion of the evolutionarily conserved phosphorylation site on CmAUR targets causes defective mitochondrial divisions, suggesting an ancient origin of this regulation during endosymbiosis.Kato et al. show that red alga  The endosymbiosis of mitochondria is thought to be crucial for the evolution of eukaryotic cells3. Because the machinery regulating mitochondrial division, such as a mitochondrial division ring, is well conserved among eukaryotes, it can be predicted that the mechanism, which regulates mitochondrial division was acquired in the process of establishing this endosymbiosis5.Mitochondria are organelles that play important roles in energy production and several metabolic pathways in eukaryotic cells, and are thought to be derived from a sole alphaproteobacterium that invaded the protoeukaryoteDrp1, Opa1, and Fis1, control mitochondrial morphology in animal cells6. Drp1 forms a mitochondrial division ring at the mitochondrial division site. Mitochondrial division is induced by contraction, which is caused by a structural change in Drp1. Drp1 recruitment to mitochondria depends on an integral membrane protein, Fis1, which is located in the mitochondrial outer membrane6. In addition, the mitochondrial outer membrane protein Mff recruits Drp1 to mitochondria independently of Fis16. The dynamin-related protein Opa1, which is localized to the mitochondrial inner membrane, induces mitochondrial fusion6. In animal, yeast and plant cells, regulation of mitochondrial fragmentation in the G2/M phase by mitotic kinases is required for the proper distribution of mitochondria to daughter cells6. Previous studies using human cultured cells have revealed that mitochondrial proliferation occurs before mitosis, and that this phenomenon is controlled by phosphoregulation of Drp1 by cyclin-dependent kinase17. The mitochondrial localization of Drp1 is regulated by Aurora A via phosphorylation of RalA, which belongs to the Ras family, part of the small GTPase superfamily8. Phosphorylated RalA forms a protein complex with Drp1, RalBP1, Cyclin B, and CDK1, which is recruited to the mitochondrial outer membrane in a Mff-dependent manner8.Some nuclear-encoded genes derived from the ancestor host cell, such as Cyanidioschyzon merolae is an ultra-small (> 2 µm in diameter) unicellular red alga that inhabits hot and sulfate-rich springs. C. merolae has a simple cell structure, with a single mitochondrion and a single plastid10. The mitochondrion divides only once per cell cycle, during M phase11. These features would enable us to investigate the mitochondrial division precisely. In addition, sequencing of the C. merolae genome and the development of molecular genetic analysis methods have made it possible to investigate detailed molecular mechanisms in this organism16. Therefore, we assumed that C. merolae is the best model for investigating the molecular mechanisms of coordination between mitochondria and host cells.22. Although many other eukaryotes have two or three Aurora kinases, C. merolae has only one Aurora kinase gene (CmAUR) in its genome23. Previously, we revealed that a green fluorescent protein (GFP) fusion protein of CmAUR in C. merolae was localized to the spindle and the mitochondrion23. Localization to the mitochondrion was observed throughout the cycle, and CmAUR-GFP was especially accumulated at the mitochondrial division site in M phase, which is the only phase when mitochondrial division occurs in C. merolae23. Its localization to the mitochondrion was shown to be regulated by a kinesin-like, division-inducing protein (TOP)24. Given these findings, we presumed that CmAUR regulates mitochondrial division during M phase.Aurora kinases are members of a highly conserved mitotic kinase family, which controls mitotic spindle formation and chromosome segregationC. merolae. Our results indicate that Aurora kinase regulates mitochondrial division in G2/M phase through phosphorylation of the Drp1 ortholog CmDnm1. Our results also show that human Aurora kinases directly phosphorylate Drp1 in vitro. These results suggest the conservation of this mechanism among eukaryotes. The mechanism may have been acquired in common ancestral cells and may have contributed to the establishment of endosymbiosis.To test this hypothesis, here, we investigated whether regulation of mitochondrial division by a mitotic kinase occurs in primitive eukaryotic cells using C. merolae24, little is known about its role as an Aurora kinase, such as in mitotic progression through histone H3 Ser10 phosphorylation in mitosis25. Thus, we considered that it was important to test the Aurora kinase function of CmAUR. First, we conducted immunostaining to determine the cellular localization of CmAUR. Previously, we reported a CmAUR antibody and confirmed its specificity24. The immunostaining showed that CmAUR localized to the mitotic spindle and mitochondrion  in C. merolae. This mutant of CmAUR was designed by reference to previous reports of kinase-dead human Aurora A26 and B27, and we confirmed that recombinant CmAURK208R protein had no autophosphorylation activity -CmAUR strongly phosphorylated H3 Ser10 in vitro  antibody was able to detect self-phosphorylated CmAUR  but not Mda1 signals  in the mitochondrial replication process Fig. . The trases Fig. , 2. BecaC. merolae24. In co-stained M-phase C. merolae cells, CmAUR signals were partially co-localized with ProQ signals , which forms a ring around mitochondria and induces their division in eukaryotes such as mammalian, nematode, and yeast cells35. In these assays, CmAUR phosphorylated CmDnm1, Mda1, and TOP 33. We observed that overexpression of CmDnm1T139A/E and CmDnm1S726E caused mitochondrial division arrest. These results suggest that T139A/E and S726E function, respectively, in a dominant-negative mode. Interestingly, overexpression of CmDnm1 with T139 variations caused abnormal phenotypes such as mitochondrial distribution on one side of the cell -CmDnm1, sfGFP-CmDnm1C. merolae and human.Finally, we examined if recombinant human Aurora kinase could directly phosphorylate recombinant Drp1, which is a CmDnm1 orthologue found in human. Aurora A and Aurora B could phosphorylate Drp1 in vitro Fig. . This reC. merolae. Residues T139 and S726 of CmDnm1 were found to have functional impacts on mitochondrial division in C. merolae , Ef-Tu antiserum, Mda1 antiserum, α-tubulin antibody , phosphorylated histone H3 Ser10 antibody , and GFP antibody  were used at dilutions of 1:100, 1:20, 1:200, 1:100, 1:600, 1:125, and 1:100, respectively. The secondary antibodies, Alexa 488 and Alexa 546 , were used at a dilution of 1:1000. Images were acquired under an upright microscope  equipped with a DP72 digital camera (Olympus) or a GS3-U3-50S5M-C camera . The colocalization of the co-stained signals was quantified by EzColocalisation plugin in ImageJ software46.Immunostaining was performed as previously described24, phAUR , Aurora A (ab13824), and Aurora B (ab45145) (both from Abcam) were used at dilutions of 1:2000. As the secondary antibody, we used anti-rabbit IgG horseradish peroxidase (HRP)-linked species-specific whole antibody  and anti-mouse IgG (H + L) HRP conjugate  at a dilution of 1:10,000. Some membranes were stained with Coomassie Brilliant Blue or colloidal gold42 after luminescence reactions to check loading amounts. All original blotting images are shown in Supplementary Fig. Thiophosphate ester antibody (Abcam), His antibody (MBL) and phospho-histone H3 Ser10) antibody 05–598  were used as primary antibodies at dilutions of 1:5000, 1:2000, and 1:7500, respectively. Primary antibodies against CmAUR antibodym pH7.5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mm NaCl, 1 mm ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with complete protease inhibitor cocktail (Merck). After incubation for 90 minutes on ice, glass beads were added to the tube, and the tube was shaken using a bead beater (2600 rpm for 1 min). The supernatant of the extracts was used for western blotting.Synchronized cultured mitotic cells and normally cultured interphase cells were resuspended in lysis buffer  of CmAUR and the ORF–3′-UTR (140 bp) of Dnm1 were fused with the β-tubulin promoter of C. merolae (1955 bp) by polymerase chain reaction using PrimeSTAR HS . The PCR products were introduced into the multiple cloning sites of pTH2-PL and pMtGFP, respectively. Mutagenesis of CmAUR and Dnm1 was carried out by PCR with KOD plus-Neo polymerase . Primers are shown in Supplementary Table C. merolae as previously described15.To produce expression vectors for C. merolae transformants expressing CmDnm1 via a heat shock inducible promoter, linear vector vHS/bt3’ was amplified with primers T139HS(-1)R and T229btUTR(+ 1)  and dissolved in elution buffer . Recombinant GST-CmAUR was prepared with GSTrap HP as previously described43. Recombinant human Aurora A , Aurora B  and Drp1  proteins were purchased.Sequences encoding Dnm1, Mda1, and TOP were integrated into the multiple cloning site of pET28-His-TEV. The primers used for cloning are shown in Supplementary Table 43. When immunoblotting was used to detect phosphorylation, ATP was substituted by ATPγS to use the thiophosphate ester for detection. The substrates resolved in elution buffer were used at < 1/60th of the volume of the total reaction solution. The reaction volume was 25 µL. The reaction buffer contained 5 mm HEPES (pH7.5), 50 mm sucrose, 100 mm KCl, 0.1 mm CaCl2, 0.4 mm ethylene glycol tetraacetic acid, 1.2 mm ATPγS, and 20 mm MgCl2. After the reaction at 30 °C for 30 min, 1.5 μL of 50 mmp-Nitrobenzyl mesylate and 2.6 μL of 500 mm EDTA were added to the mixture.The CmAUR kinase assay was performed as previously describedm Tris-HCl, pH 6.8, 10 mm NaCl, 0.02 mm EDTA, 0.2 mm dithiothreitol (DTT), 0.14 mg/mL bovine serum albumin) at 37 °C for 20 min.Recombinantly expressed CmAUR was incubated with PP1 . Mammalian phAUR antibody was used to detect time-dependent dephosphorylation of CmAUR. Phosphatase assay was carried out in phosphatase buffer . The mixture was incubated at 37 °C for 30 min and then subjected to reductive alkylation treatment with iodoacetamide. Then, 50 mm ammonium bicarbonate was added and the urea concentration in the solution was diluted to 2 m. Finally, trypsin was added, and the mixture was incubated at 37 °C for 16 h. After the reaction, the peptide solution was desalted using a C18 Stage Tip. Phosphorylated peptides were fractionated and recovered from the peptide sample, and TiO2 resin was used for specific adsorption. The procedure was based on that recommended for the Titansphere Phos-TiO Kit . The eluted sample was dried under reduced pressure, and the residue was dissolved in a mixture of water, acetone, and trifluoroacetic acid (volume ratio 98:2:0.1) and then analyzed by LC-MS/MS. An Ultimate 3000 liquid chromatograph (Nippon Dionex K.K.) and Linear Trap Quadrupole (LTQ) Orbitrap Velos Mass Spectrometer (Thermo Fisher Scientific) were used for LC-MS/MS analysis. Acclaim PreMap100 (Nippon Dionex K.K.) was used as an analytical column. The composition of mobile phase A was water:acetonitrile:formic acid = 98:2:0.1 (volume ratio). The composition of mobile phase B was water:acetonitrile:formic acid = 5:95:0.1 (volume ratio). The gradient of acetonitrile was :  →  →  →  →  → . The MS scan range was m/z 350 to 1,200. The resolution was 30,000. MS/MS data were analyzed with Mascot Daemon ver. 2. 4. 0 (Matrix Science K.K.).CmDnm1 phosphorylated by CmAUR in vitro as described above was separated on 7.5% TGX gels, and the gels were stained with Bio-Safe Coomassie G-250 Stain. An LTQ-Orbitrap XL (Thermo Fisher Scientific) coupled with an EASY-nLC 1000 (Thermo Fisher Scientific) was used for nano-LC-MS/MS analyses as described previouslyt test and Welch’s t test were used for testing the difference in population means. Data sets whose P value of f test were larger than 0.05 were used Student’s t test, and one whose p value was < 0.05 were used Welch’s t test. For statistical analysis of count data based on mitochondrial morphology, we performed Pearson’s chi-squared test, and the residuals were calculated.Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Firmicutes being the most abundant phylum. The most prevalent bacterial groups were Streptococcus spp. (15/19 samples), Enterobacteriaceae (13/19) and Lactobacillus/Lactococcus/Pediococcus group (12/19). While the average total number of bacterial cells did not change significantly during storage, the prevalence of strict anaerobic Bacteroidetes increased threefold, from 3/19 to 9/19, while in 7 samples Clostridium clusters IV or XIVa became detectable after storage. Major HMO were not degraded. Lactate was present in 18/21 samples after storage (2.3–18.0 mM). Butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations ranging from 2.5 to 5.7 mM. We demonstrate enhanced prevalence and/or abundance of viable strict anaerobes from the Bacteroidetes and Clostridiales after 6-day anaerobic storage of human milk. Our data indicate that anaerobic cold storage did not markedly change total viable bacterial load, while HMO profiles were stable. Anaerobic cold storage of human milk for up to 6 days may be suitable for preserving milk quality for potential microbial transfer to the infant gut.Besides nutritional components, breast milk contains diverse microbes, which may be involved in colonization of the infant gut. Expressed milk is often stored for few days in the refrigerator. The aim of this study was to determine the abundance, prevalence and diversity of facultative and strict anaerobic bacteria using culture-dependent and -independent methods, and to determine changes in milk microbial and chemical composition during storage. Samples of mature breast milk from 21 women were collected 3–6 months post-partum and were analyzed fresh or after anaerobic storage for 6 days at 4°C. The cultivable bacterial population was analyzed using the most probable number (MPN) method or plate counts and different media. The abundance of major bacterial groups was determined using quantitative PCR and 16S rRNA gene sequencing. Lactose, lactate, short chain fatty acids (SCFA) and human milk oligosaccharides (HMO) were analyzed using chromatography techniques. Highest mean viable cell counts were obtained in yeast casitone fatty acids (YCFA) broth supplied with mucin (log 4.2 ± 1.8 cells/ml) and lactose (log 3.9 ± 1.4 cells/ml), or Columbia broth (log 3.0 ± 0.7 cells/ml). Mean total bacterial counts estimated by qPCR was 5.3 ± 0.6 log cells/ml, with  Breast milk harbors microbes that are potentially transferred to the neonate  and may Staphylococcus, Streptococcus, Lactococcus, Leuconostoc, Weissella, Enterococcus, Lactobacillus, Cutibacterium (formerly Propionibacterium) and Enterobacteriaceae . The most abundant secreted HMO include lacto-N-tetraose, lacto-N-neotetraose (LNnT), 2′fucosyllactose (2′FL), 3′fucosyllactose (3′FL), and the acidic HMO 3′sialyllactose (3′SL) and 6′sialyllactose (6′SL)  . Breast e (6′SL) . Beside e (6′SL) .Breast milk is suckled by the infant, or is expressed by mechanic or electric breast pumps and frequently stored aerobically at 4 or −20°C before feeding , which mA total of 21 breastfeeding women (27–40 years old) with infants aged 2.3–5.5 months were recruited in Switzerland. Exclusion criteria included the intake of antibiotics or probiotics within 1 month before the sampling day, chronic and acute diseases like fever, inflammation, diarrhea, and mastitis. Breast milk samples were collected from one breast lobe at one time point especially for this study. Written informed consent was obtained from the participants. The study protocol was approved by the Ethics Committee of the ETH Zurich (2018-N-14).2, 5% H2, 85% N2, Coy Laboratories, United States). Samples were aliquoted for the different analyses or storage assays, half of the aliquots were used for immediate cultivation or were centrifuged at 14,000 × g for 20 min at 4°C. Cell pellets and supernatants were frozen at −20°C until further analysis. Other aliquots were transferred to an anaerobic jar containing the Anaerocult A system (Merck), were stored at 4°C for 6 days and then processed as described above (stored samples). The pH of fresh and stored milk samples was determined using a pH meter .Prior collection, nipples and surroundings of one breast were cleaned using HibiScrub  to reduce bacteria load on the skin. Milk samples (approximately 10–15 mL) were collected in a sterile flask wearing gloves and using an electrical pump . The first 3–5 ml were discarded. After expression, samples were immediately placed in an anaerobic jar  containing the Anaerocult A system  and transported to an anaerobic chamber  was added when the temperature of the medium was approximately 50°C. Columbia broth was transferred to Hungate tubes flushed with CO2, tubes were sealed, and autoclaved. Yeast extract, casitone and fatty acid (YCFA) medium is used to cultivate gut microbes that depend on the presence of volatile fatty acids for growth  was transferred to Hungate tubes flushed with CO2, and tubes were sealed and autoclaved. Reinforced clostridial broth  and Bacteroides Mineral Salt Broth , which is a general medium used for the cultivation of fastidious bacteria was produced aerobically (Col_ae) according to the instructions of the supplier, or anaerobically (Col_an) to cultivate facultative anaerobes and strict anaerobes. Therefore, the Columbia powder was mixed with distilled water, boiled for 15 min, cooled down and flushed with COr growth . We adapr growth . YCFA waoth BMS;  were preoth BMS; , with 1.ium spp. .2HPO4, 240 mg/l KH2PO4; pH 7.4) in tenfold dilution series. For each dilution, 180 μl of medium were supplied in microtiter plate  to which 20 μl of diluted sample were added. Microtiter plates were covered with a lid, and incubated at 37°C for 48 h inside the anaerobic chamber. For determination of MPN of aerobically grown cells, microtiter plates were prepared outside the anaerobic chamber and were incubated aerobically at 37°C for 48 h. Before and after incubation the optical density at 590 nm (OD590) was determined . An increase in OD590 of ≥0.1 compared to the corresponding baseline was considered growth, and was confirmed visually. Based on these OD590 data, the MPN was calculated using the Excel Spread Sheet provided by the Food and Drug Administration . Following the OD590 measurement, 5 replicates of the 10–1 dilution were pooled and centrifuged at 13,000 × g for 15 min. Supernatants and pellets were kept at −20°C until further analysis. To determine the cultivable lactic acid bacteria and bifidobacteria, 100 or 200 μl of fresh or stored milk and ten-fold dilutions were spread on modified mMRS or mWC agar plates inside the anaerobic chamber in duplicates, and were incubated anaerobically for 2–4 days at 37°C.Cultivable cells were assessed in freshly expressed and stored breast milk using the most probable number (MPN) technique. A ten-replicate design  adapted To identify colonies from agar plates, DNA was isolated from liquid culture as described below, and the 16S rRNA gene was amplified using PCR and primers F8 (5′-AGA GTT TGA TCM TGG CTC-3′), and 1391R (5′-GAC GGG CGG TGT GTR CA-3′), both 10 μM, Microsynth, Switzerland). Amplicons were sent for Sanger sequencing to Eurofins Genomics (Germany). The resulting sequences were aligned against the 16S rRNA gene database provided by NCBI using blastN.Cell pellets obtained from fresh and stored breast milk samples and from bacterial cultures (1 ml) were thawed and the DNA extraction was done using Fast DNA SpinKit for Soil which includes a bead beating step . DNA concentration was determined using QuBit and the dsDNA HS Assay Kit (Thermo Fisher), and ranged from 90 pg/ul to 39.3 ng/μl, whereas three samples were below detection limit (<0.5 pg/μl) . DNA samFirmicutes, Bacteroidetes, Lactobacillus/Leuconostoc/Pediococcus spp.  and Veillonella spp., Clostridium cluster IV (both correction factor 4) and XIVa (correction factor 6)  .Libraries covering the V3 region of the 16S rRNA gene were prepared using a two-step PCR approach. In the first reaction, 100 ng DNA was used as template and was amplified with 12 μl AccuPrime SupermixII , 0.5 μl of with barcoded primers NXt_338F (5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG ACW CCT ACG GGW GGC AGC AG-3′) and NXt_518R  and water to a final volume of 25 μl. Thermocycling was conducted as follows: initial denaturation at 95°C for 2 min, 25 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s and extension at 68°C for 30 s followed by final extension at 68°C for 4 min.To add adapters and indexes, a second PCR was performed using 12 μl of AccuPrime Pfx Fusion mix (Thermo Fisher), 2 μl of corresponding P5 and P7 primer , 2 μl of the PCR product from the first PCR and nuclease-free water to a final volume of 25 μl. The PCR temperature profile was as follows: 98°C for 1 min, followed by 12 cycles of: 95°C for 15 s, 55°C for 20s and 72°C for 20 s and a final extension step at 72°C for 5 min. The amplified fragments were purified using AMPure XP beads  according to manufacturer’s manual. Prior to pooling, libraries were quantified using a Qubit fluorometer (Thermo Fisher) and were mixed in approximately equal concentrations.Due to low concentration of DNA in most milk samples , low effn = 12) yielded 584,096 reads , sequencing of milk samples (n = 6) yielded 148,590 reads .Sequencing was carried out on an Illumina MiSeq at the Genetic Diversity Center of ETH Zurich. Preparation of the library used the V2 2 × 250 bp paired end NextTera chemistry supplemented with 30% of PhiX. Raw sequencing reads were processed by merging the paired reads using USEARCH (v8.1.1756) with to a minimum length of 100 bp setting the expected error threshold to 1 and the minimum overlapping to 15 bp. Raw sequencing reads were filtered using PRINSEQ-lite (v0.20.4). Sequences that passed quality filtering were clustered into OUTs at 97% identity level using UPARSE (usearch v8.0.1623). Representative sequences were aligned using PyNAST (QIIME-1.8.0) and taxonomically assigned using UTAX (usearch v8.1.1756). Sequencing of MPN samples . Detection limits were 1 mM for lactose, 0.9 mM for lactate, and 0.5 mM for formate, succinate, acetate, propionate and butyrate. A representative chromatogram is shown in The supernatants from fresh and stored milk, and from randomly selected MPN milk samples were subjected to high performance liquid chromatography  with refractive index detection (HPLC-RI) using an Aminex HPX-87H column . Thawed supernatants (40 μl) were, without additional extraction procedure, eluted at 40°C with 10 mM HN-neotetraose, N-acetylneuraminic acid supplied by Glycom A/S (Denmark) as external standards.HMO profiles before and after milk storage were qualitatively analyzed using high pH anion exchange chromatography with amperometric detection (HPAEC-PAD) on a Dionex IC3000 equipped with a CarbopacPA20 column (Thermo Fisher) and an electrochemical detector with a gold electrode. HMO were analysed directly from milk supernatants without further extraction procedure. Water (A), 200 mM NaOH (B) and 1 M Na-acetate (C) were used as solvents at a flow rate of 0.25 ml/min with the following gradient: 0 min 30.4% B, 1.3% C, 22 min 30.4% B, 11.34% C, followed by washing and regeneration. Main HMO were identified using 2′FL, 3′FL, 3′SL, 6′SL, lacto-t-test or Kruskal Wallis One Way Analysis of Variance of Ranks with Dunn’s method for pairwise comparison (SigmaPlot 13), respectively. Differences between cell counts in fresh and stored samples determined using qPCR were evaluated using the paired two-tailed t-test. The degree of correlations between parameters and their significance was tested using IBM SPSS Statistics 20.0 . Significance was set at p < 0.05.Differences in log viable counts between fresh and stored samples, and between cultivation media were tested using paired two-tailed PRJNA557136.16S rRNA gene sequences are available at ncbi’s sra archive under bioproject accession number Enterococcus and Streptococcus spp., and Cutibacterium acnes. No growth was observed on mWC agar plates used to detect bifidobacteria.The viable cell counts in fresh and cold stored breast milk were assessed using a cultivation-based approach combining MPN technique and cultivation on agar plates with different semi- and non-selective media. Significant higher mean cell counts in fresh breast milk were obtained in YCFA containing either mucin or lactose , or aerobic (log 3.2 ± 0.7 cells/ml) and anaerobic (log 3.0 ± 0.7 cells/ml) Columbia medium compared to the other media tested . The addp < 0.05, Δlog 0.6 ± 0.8 cells/ml) compared to fresh milk samples, but no significant change was measured for the other cultivation conditions .During storage, mean cell counts significantly increased in YCFAwo , log MPN were significantly higher for stored samples tested in anaerobic Columbia Broth  and in YCFA + Lact . No group difference was detected for other media and for fresh samples in all media. Age of the infants negatively correlated with fresh milk bacterial cell counts in RC , and mMRS  and YCFA + Lact , and positively with bacterial load in Col_an (r: 0.64 p = 0.034) of stored samples. The age of the mother was negatively correlated with log MPN after cold storage in RC medium .After grouping data for infants according to age , formation of acetate (42.4 ± 8.7 mM), lactate (11.9 ± 1.4 mM) and propionate (2.3 ± 0.4) was detected in 5/6 samples. In YCFA + Muc in 6/12 samples from day 0, lactate (7.9–68.2 mM), acetate (3.4–13.2 mM) and formate (3.1–7.3 mM) were formed, propionate was detected in one sample (12.1 mM). In two samples, this metabolic profile differed at day 6 with lactate utilization (−5.7 to −6.9 mM) observed while the other three samples showed reduced lactate and/or acetate formation. In four fresh milk samples, lactate was used (−6.0 to −11.2 mM) and acetate was formed (6.2–19.3 mM) with little difference between day 0 and day 6. For one sample, metabolic activity (lactate formation and acetate utilization) was only observed at day 0, while for one sample there was little metabolic activity at both sampling days.Supernatants of selected samples grown in YCFAwo, and YCFA + Muc were analyzed by HPLC-RI to investigate the formation of SCFA. In YCFAwo  correlated positively with MPN of the sample (p < 0.05). The Shannon index, which equally weighs richness and evenness, ranged from 0.01 to 0.67, while Chao1 indices ranged from 10 to 42.We also determined the 16S rRNA gene composition of selected MPN samples in overnight cultures of the −1 dilutions with total cell counts ranging from log 2.8–7.3 cells/mL . Unclassn = 19) were analyzed by qPCR to estimate cell counts of total bacteria and of selected bacterial groups representing the predominant phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria in fresh and stored milk, using correction for multiple 16S rRNA gene copies per cell. Fresh and stored milk samples contained an average of total bacteria of log 5.4 ± 0.5 cells/ml (Breast milk samples (cells/ml . Only licells/ml .Firmicutes contributing the majority of cells (85%) in all samples. Streptococcus spp., Lactobacillus/Leuconostoc/Pediococcus spp. and Veillonella spp. were present in 79, 63, and 53% of the fresh samples, respectively  were detected in 68 and 58% of the samples before and after storage, respectively, at low abundance. Propionibacterium/Cutibacterium spp. was detected in 5 and 16% of the samples before and after storage, respectively, and Bifidobacterium 16S rRNA genes (both phylum Actinobacteria) could not be amplified in any sample. After storage, the prevalence of Bacteroides/Prevotella/Porphyromonas group was threefold increased from 16 to 47%  and Proteobacteria , and at lower relative abundance Actinobacteria (Bifidobacteriaceae).To investigate bacterial diversity, we tested 16S rRNA gene amplicon sequencing. As the standard protocol did not yield and PCR products, we employed a nested PCR approach. Visible bands after the initial and the 1st PCR were obtained for samples that contained >2 ng/μL DNA, or >log 6 cells/ml, which corresponded to only six samples in total. However, this approach yielded a high proportion (13–70%) of unclassified reads even at phylum level  indicatir: −0.529, p = 0.016) but was not correlated with the lactose concentration change. Free butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations in the range from 2.5 to 5.7 mM. Acetate, propionate or formate were not present in detectable amounts in any sample.Breast milk samples were analyzed by HPLC-RI to determine the concentrations of lactose, SCFA and lactate. The average lactose content in fresh milk samples was not impacted by milk storage, with 161.8–235.8 mM and 156.3–227.6 mM in fresh and stored milk, respectively . LactoseN-acetylneuraminic acid), and no accumulation of glucose or galactose, indicating that at least the most prevalent and abundant HMO were not degraded during storage.HMO profiles were generated from fresh and stored breast milk samples using HPAEC-PAD . The majHuman breast milk banks usually store samples aerobically at −20°C to minimize or prevent biological and chemical degradation, after batchwise pasteurization is performed . These tn = 55) (n = 12) from preterm infants (n = 7)  , between infants  and <log (n = 7) . In agre (n = 7) . We supp (n = 7) . Bacteri6–7 cells/ml of milk after correcting for multiple 16S rRNA gene copies per cell  viable strict anaerobes were present in breast milk albeit at low abundance, and that (2) anaerobic storage of breast milk at 4°C promoted the growth of some strict anaerobes.Culture independent 16S rRNA gene analysis frequently  but not Bacillaceae were a major viable bacterial population recovered in MPN samples in agreement with Firmicutes being the major phylum present in breast milk according to qPCR analysis. The presence of Bacillus spp. in breast milk was observed before ((Unclassified) d before .S. epidermis, Streptococcus spp. and C. acnes from mMRS agar plates which belong to the natural skin microbiota . The participants provided their written informed consent to participate in this study.MV, CL, and CS conceived the study. EV and AG conducted the experiments and run analysis. EV, AG, and CS analyzed the data. EV and CS wrote the manuscript with the support of all authors. MV was employed by the company Medela A/S Switzerland. The remaining 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.


Our results suggest developmental hypoxia improves cardiomyocyte anoxia-tolerance of juvenile turtles, which is supported by enhanced myofilament Ca2+-sensitivity and a superior ability to suppress ROS production. Maintenance of low ROS levels during anoxia might limit oxidative damage and a greater sensitivity to Ca2+ could provide a mechanism to maintain contractile force. Our study suggests developmental hypoxia has long-lasting effects on turtle cardiomyocyte function, which might prime their physiology for exploiting hypoxic environments.For some species of ectothermic vertebrates, early exposure to hypoxia during embryonic development improves hypoxia-tolerance later in life. However, the cellular mechanisms underlying this phenomenon are largely unknown. Given that hypoxic survival is critically dependent on the maintenance of cardiac function, we tested the hypothesis that developmental hypoxia alters cardiomyocyte physiology in a manner that protects the heart from hypoxic stress. To test this hypothesis, we studied the common snapping turtle, which routinely experiences chronic developmental hypoxia and exploits hypoxic environments in adulthood. We isolated cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (21% O Often, 2 i, pHi and ROS, respectively, at room temperature. [Ca2+]i and pHi were measured simultaneously, by co-loading cardiomyocytes with Fura-2  and BCECF . ROS were measured separately by loading cardiomyocytes with DHE . Following the loading protocol, cells were resuspended in fresh isolation solution for 15–20 min to allow de-esterification. Cardiomyocytes were then placed in the recording chamber (at room temperature), perfused with normoxic saline and stimulated to contract at a frequency of 0.2 Hz, which is within the normal physiological range of heart-rates in common snapping turtles  ([Ca2+]e), from 1 to 5 mM , while simultaneously measuring relaxed cell length, according to a protocol by Wisløff et al. i), normalized Δ[Ca2+]i, diastolic [Ca2+]i, systolic [Ca2+]i, pHi, ROS production, cardiomyocyte shortening efficiency and times to rise and half-decay. For the GLM analyses, the between-group factor was developmental O2 (N21 versus H10), while time and treatment (normoxia and anoxia) were the within-group factors. A further GLM was used, followed by Sidak post hoc tests, to reveal the effect of the developmental O2 and experimental period  on cardiomyocyte shortening (the dependent variable), while controlling for Δ[Ca2+]i (the covariate). Finally, a mixed-effects, repeated-measures GLM, followed by Sidak post hoc tests, was used to reveal the effect of developmental O2 on myofilament Ca2+-sensitivity, with relaxed cardiomyocyte length as the dependent variable, while controlling for extracellular [Ca2+] (the covariate). Additionally, regressions were used to fit lines through the data. The Δ[Ca2+]i was calculated by subtracting the value of the diastolic [Ca2+]i from the systolic [Ca2+]i, and ROS values were normalized to the 5-min time-point and described as relative changes in production. All data are presented as means ±s.e.m. and considered significantly different, when p ≤ 0.05.Statistical analyses and graph plotting were carried out using S3.2 and treatment (anoxia and reoxygenation) on multiple aspects of juvenile turtle cardiomyocyte structure and function. The results are described below, and statistical support for the findings  are given in electronic supplementary material, tables S2–S7.The mixed-effects, repeated-measures GLMs revealed significant effects of developmental O(a)While the N21 and H10 turtles did not differ in body or heart mass , there w(b)2 on Δ[Ca2+]i and cell-shortening efficiency; a significant effect of treatment on cell shortening, Δ[Ca2+]i, pHi, ROS production, normalized Δ[Ca2+]i, systolic [Ca2+]i, time to rise and time to half-decay; a significant interaction between normoxia and developmental O2 on the Δ[Ca2+]i and cell-shortening efficiency; and a significant interaction between anoxia and developmental O2 on cell shortening, pHi, ROS production and cell-shortening efficiency. See electronic supplementary material, tables S3–S5, for full details of these statistical interactions.The mixed-effects, repeated-measures GLMs revealed a significant effect of developmental O(c)i of N21 and H10 cardiomyocytes were similar , but the average intracellular Ca2+ transient (Δ[Ca2+]i) was more than twice the size in N21 versus H10 cardiomyocytes (b); this meant the efficiency of shortening (% cell shortening as a function of Δ[Ca2+]i) was significantly greater in the H10 cohort (f). The large difference between Δ[Ca2+]i values can be explained by lower average N21 versus H10 diastolic [Ca2+]i levels, but similar average systolic [Ca2+]i; nevertheless, the differences in diastolic [Ca2+]i did not reach levels of statistical significance . Lastly, relative intracellular ROS production did not change during the control period (e).Under control conditions, cell shortening and pH similar a,d, but myocytes b; this m0 cohort f. The laificance c,d. Lastl period e.Figure(d)2 rapidly dropped inside the chamber to undetectable levels (within approx. 54 s), and N21 and H10 contraction declined in association with a reduction in Δ[Ca2+]i, pHi and ROS . However, while N21 cell shortening remained depressed throughout the anoxic period, H10 cell shortening recovered to control levels, after 15 min of anoxia (a), despite a significantly larger acidosis in the H10 cells (d) and a similar proportional decline in Δ[Ca2+]i to the N21 cohort (c). Therefore, the efficiency of cell shortening during anoxia tended to be higher in the H10 versus N21 cardiomyocytes; this effect was only statistically significant at the 25- and 30-min time-points (f). In both cell groups, the reduction in the Δ[Ca2+]i amplitude (b) was caused by a progressive increase in diastolic [Ca2+]i and a decrease in systolic [Ca2+]i, although these values did not reach levels of statistical significance . Anoxia also slowed the times to rise and half-decay of both cohorts .At the onset of anoxic perfusion, O and ROS a,b,d,e. f anoxia a, despit10 cells d and a s1 cohort c. Therefe-points f. In botmplitude b was cauificance c,d. Anox cohorts . It shou10 cells e.Table (e)2 was restored (approx. 63 s), N21 and H10 cardiomyocyte shortening recovered to pre-anoxic levels (a), in association with the restoration of pHi, ROS production , [Ca2+]i and the times to rise and half-decay 2+ transients, suggested that the H10 cohort has greater myofilament Ca2+-sensitivity. Therefore, we investigated the correlation between cardiomyocyte contraction and Δ[Ca2+]i . As expected, regression lines fitted to the data revealed that shortening is positively correlated with Δ[Ca2+]i, during all three experimental periods, with the exception of N21 cells in anoxia. However, when controlling for Δ[Ca2+]i, the extent of the shortening was greater in H10 cardiomyocytes, regardless of the experimental period . Furthermore, relaxed cardiomyocyte length decreased by a greater proportion in the H10 cohort, at the range of external Ca2+ concentrations tested (c). Collectively, our data suggest that the myofilaments of H10 cardiomyocytes have a higher sensitivity to Ca2+ than N21 cells. See electronic supplementary material, tables S6 and S7, for full details of these statistical interactions.The observation that H10 cardiomyocyte shortening was either equal to, or larger, than N21 cells, despite smaller CaΔ[Ca2+]i a,b. As el period a,b. Furts tested c. Collec4.f), higher myofilament Ca2+-sensitivity (figure 2e).Numerous studies across different vertebrate taxa show that developmental hypoxia alters cardiac morphology and function ,13,26–33figure 2sitivity  and a su(a)Previous work has shown developmental hypoxia is associated with cardiac enlargement in embryonic snapping turtles , but thi2+]i), compared with N21 cardiomyocytes (b). Differences in cardiomyocyte Δ[Ca2+]i can be explained by numerous factors, including Ca2+-influx pathways (e.g. L-type Ca2+ channel or the sarcoplasmic reticulum), efflux pathways (e.g. the Na+/Ca2+-exchanger) or buffering capacity . While we did not investigate these Ca2+-cycling mechanisms here, we found that H10 cardiomyocytes could still develop the same level of shortening as the N21 cells, despite the smaller Δ[Ca2+]i. Our findings suggest that these differences can partly be explained by improved myofilament Ca2+-sensitivity in the H10 cohort . For example, Ca2+-cycling is a major consumer of ATP, on account of the high energetic demand of Ca2+-removal pathways, such as the sarcoplasmic reticulum Ca2+-ATPase and the Na+/Ca2+ exchanger, which is indirectly dependent on the Na+/K+-ATPase i, which reduces myofilament crossbridge formation, and a profound intracellular acidosis, brought on by a respiratory and a metabolic acidosis, which reduces myofilament Ca2+-sensitivity [2+]i and a lower pHi than N21 cardiomyocytes. Evidently, prior exposure to developmental hypoxia improved cardiomyocyte anoxia-tolerance in juvenile turtles.N21 and H10 cardiomyocyte shortening was dramatically impaired within the first 5 min of the anoxic challenge. Consistent with this finding, c output ,41. Our sitivity . HoweverDanio rerio) exposed to hypoxia produce offspring that are more hypoxia-tolerant i remained relatively stable during anoxia, the mechanism underlying the rebound in cell shortening of H10 cardiomyocytes must be Ca2+-independent . Consistent with the hypothesis that developmental hypoxia enhances myofilament Ca2+-sensitivity, cell shortening in N21 and H10 cardiomyocytes was similar within the first 5 min of anoxia, despite the lower intracellular pH of the H10 cohort. However, the time course of the rebound in H10 cell shortening suggests separate mechanisms were activated to recover contractile function. One possibility is the suppression of ROS production. Low O2 tensions are associated with excessive ROS production, which is known to reduce cardiac force production, by directly modifying sarcomere proteins and reducing myofilament Ca2+-sensitivity i and ROS production all recovered to intermediate or pre-anoxic levels within the first 5 min of reoxygenation, despite the persistent increases in diastolic and systolic [Ca2+]i. These results suggest the turtle heart can withstand reoxygenation without apparent cellular injury, irrespective of developmental O2 tension.Although anoxia can produce extensive tissue damage in mammals, the greatest risk to cell survival occurs after reoxygenation, when the mitochondria produce a surge of ROS that can trigger apoptotic and necrotic cell death ,52. By c5.2 deprivation throughout their lifetime [2+-sensitivity and a superior ability to suppress ROS production. From an ecological perspective, these modifications might be beneficial to turtles and possibly other ectotherms, when they exploit hypoxic environments.North American freshwater turtles experience prolonged periods of Olifetime ,9. We ha


Here, we use targeted RNA capture (‘RNA CaptureSeq’) in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer’s and Parkinson’s disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain  Over the past decade, genome-wide association studies (GWAS) have facilitated a multitude of discoveries in human genetics, identifying variants associated with a number of complex phenotypes and diseases . This incis-regulatory DNA sequences such as enhancers or silencers (cis- and trans-acting non-coding (regulatory) RNAs, many of which may not yet be documented in public databases. Indeed, recent studies have indicated that human transcriptome annotations are far from complete, particularly in relation to long non-coding RNAs (lncRNAs)  .XIST, NEAT1, HOTAIR), many have been shown to play unexpectedly diverse roles in epigenetic signaling and gene regulation including genetic imprinting, shaping chromosome conformation, forming subcellular organelles  long-read cDNA sequencing in order to achieve an unprecedented level of sensitivity and resolution. While two recent studies have coupled CaptureSeq with Pacific Biosciences (PacBio) sequencing , the preUsing the NHGRI GWAS database , we idenin silico chromosome (chrIS)   . Overall) (chrIS . We reta) (chrIS . In para)  . This waR2 = 0.851, slope = 0.822) and non-captured  sequins alike  contained a total of 939,558 splice junctions. In comparison, we detected a total of 234,138 uniquely mapped junctions in the matched Illumina data. We found that 164,183 of these junctions were shared between the two technologies; that is, 17.5% of junctions detected using ONT were validated by Illumina, and 70.1% of junctions detected by Illumina were validated by ONT . Of the R2_26_1, with a 4,375 nt read that covered all 18 of its exons . Another notable example was a 3,473 nt read which mapped to R1_24_1 , spanning 33 out of 36 exons. Most synthetic isoforms < 2.5 kb were fully sequenced with single ONT reads, allowing complex, alternatively spliced synthetic loci to be unambiguously deconvoluted (chrIS (mean = 762 nt) and hg38 (mean = 937 nt)  .chrIS annotation, at the base-, exon-, intron-, transcript- and gene-levels. When considering the raw read alignments, we observed high sensitivity across these features, but large numbers of false-positive events led to poor precision scores (chrIS junctions (true-positives) and unannotated chrIS junctions , finding that ONT performed poorly in comparison to Illumina (1 to cluster together ONT reads (mapQ = 60) having similar exon/intron structures, and then create a consensus of the clusters by calculating the median of exon boundaries from all transcripts in the cluster . After clustering, we observed marked improvements in precision with only minor drops in sensitivity, validating that the approach was effective in processing raw ONT reads (We used the gffcompare tool  to assesn scores . This isn scores . We compIllumina . We usedNT reads . After bNT reads .n = 279), finding that there was significantly less variation between bases for ONT (median CV = 0.0339) than Illumina sequencing (median CV = 0.172) .Using sequins, we observed that ONT sequencing had a slight length bias, with synthetic transcripts at around the ∼1 kb size over-represented and transcripts at either end of the size spectrum under-represented. This effect was seen for both captured  and non-= 0.172) . This diLong-read sequencing provides accurate full-length isoform structures, but suffers from a relatively high sequencing error rate that leads to spurious splice junction detection . ConversNext, we developed a comprehensive pipeline to retain only high-confidence transcripts that overlapped targeted haplotype blocks . We filtered out all transcripts that met any of the following criteria: (i) no overlap with any capture probe, (ii) contained < 3 exons; (iii) contained an intron > 1 Mb, (iv) contained a non-canonical splice junction ; (v) had any of the following gffcompare  classifiWe compared our hybrid transcriptome to the GENCODE (v29) annotation using gffcompare . Only 2,The combined resolution of CaptureSeq with ONT long-read sequencing enabled us to identify 109 novel intergenic transcripts overlapping GWAS SNPs associated with neuropsychiatric traits. Only two of these transcripts were predicted to have protein-coding potential; the remaining 107 are therefore classed as putative lncRNAs . We inveFor example, we discovered a novel intergenic locus overlapping a GWAS haplotype block associated with ‘smoking behavior’ (rs1847461)  on chromRIMS2 gene, which encodes a protein that interacts with various synaptic proteins that are important for normal neurotransmitter release   Figure . This SN release . We idenvel ORFs . The firNRGN gene on chromosome 11  , locatedosome 11 . NRGN enan brain . We idenchrIS as a proof-of-principle to verify that ONT sequencing could reliably detect such events. For example, the sequin locus R1_22 comprises two alternatively spliced isoforms that contain a distant mutually associated pair (dMAP) of exons  of exons    on chrom9556711) . This ex9556711) . This coThis study has successfully employed RNA CaptureSeq to reveal the rich transcriptional diversity hidden within non-coding regions of the genome that have previously been associated by GWAS with neuropsychiatric functions. In doing so, we have vastly expanded existing transcriptome annotations of these regions, creating an expression atlas of thousands of novel isoforms in human brain. Many of these assembled isoforms show preliminary evidence of functional roles, including highly conserved exons and promoter regions, TSSs supported by CAGE peaks and epigenetic marks, and novel predicted ORFs.The improved sensitivity of CaptureSeq allowed us to assemble 109 novel intergenic transcripts in regions of the genome that were previously thought to be transcriptionally silent. As a class, these transcripts resemble annotated lncRNAs, as judged by their protein-coding probability scores, relatively low overall expression and evolutionary conservation compared to protein-coding genes, which is typical of regulatory sequences . While oin vivo, leading to the identification of hundreds of lncRNAs that have an effect on cell growth  has hindered their functional characterization. Promisingly, several technologies for probing lncRNA functions and mechanisms have begun to emerge, including ChIRP-seq (for assaying DNA/protein binding partners) and SHAPE-seq (for RNA structure) . Furtherl growth . Mechanil growth . Indeed,l growth , providiper se, or whether it is merely the act of their transcription that is indicative of some underlying function  can act as a comprehensive ‘ground truth’ reference against which FP and FN findings with RNA-seq can be evaluated. Because chrIS emulates the features of a real human chromosome, we would expect similar rates of TP and FP events for human transcripts. Notably, this type of analysis is not possible with previous spike-in controls . Since GWAS simply associates traits with regions of the genome that are in LD, the causative SNP could be anywhere within the region of LD. In order to ensure the causative SNP is included, it is necessary to capture the full haplotype block associated with the SNP reported by GWAS. As such, we used SNAP  and repeat elements (RepeatMasker). Probes were designed in accordance with previous guidelines . This re2O, with the eluate being placed back onto the columns for an additional run in order to ensure maximum recovery and concentration.Brain tissue from three de-identified, neurologically healthy males of European ancestry was supplied by the NSW Brain Tissue Resource Centre (BTRC) under Project ID 0379 and ethics approval number HC15878. Donors were aged between 61 and 64, had cardiac causes of death, with post-mortem intervals of brain collection between 17 and 41 h. From each brain, 3 mg of tissue was dissected by the BTRC from four regions: prefrontal cortex (PFC), primary visual cortex (VCx), caudate, and superior colliculus (SupCol). RNA was extracted from samples using the protocol for QIAGEN’s miRNeasy kit: 700 μL QIAzol was added per sample on ice, which were then transferred to 2 mL tubes containing a single 5 mm ball bearing and lysed on the Tissue Lyser II for 2 min at 20 Hz. Upon completion of the cycle, adapters were rotated and shaken for an additional 2 min at 20 Hz. Once lysed, the samples were purified according to the miRNeasy mini kit protocol instructions, including the on-column DNAse treatment. Elution was in 15 μL nuclease-free H°C for 5 min and immediately cooled on ice. Four μl of First Strand buffer and 2 μl 100 mM DTT  were added to the samples, which were mixed by tapping, briefly spun by microfuge and incubated at 42°C for 2 min. One μl of SuperScript II Reverse Transcriptase was added to the 19 μl reaction mixes, which were mixed by tapping, briefly spun by microfuge and incubated with the following thermocycler conditions: RT reaction at 50°C for 50 min; denaturation at 70°C for 15 min; and holding indefinitely at 4°C. The second strand synthesis reaction mix was assembled as follows : 20 μl of first strand cDNA, 10 μl buffer, 5 μl enzyme mix, and 45 μl nuclease-free water. Reactions were incubated with the following thermocycler conditions: second strand synthesis at 16°C for 1 h; and holding indefinitely at 4°C. Resulting cDNA was purified by a 10 min incubation with 144 μl (1.8X) Agencourt AMPure XP beads , 2 × 200 μl washes with 80% ethanol for 30 s each, 5 min air drying, and elution in 52 μl nuclease-free water.RNA CaptureSeq was performed as previously described , with thFollowing reverse transcription, the four samples had an average fragment length of 1500 bp with the following concentrations: 6.99 ng/μl (SupCol), 5.56 ng/μl (PFC), 5.5 ng/μl (VCx), and 6.5 ng/μl (caudate) according to a genomic DNA screentape analysis . The samples were each end-prepped and dA-tailed using the NEBNext Ultra II End Repair/dA-Tailing Module (E7595) as follows: 45 μl DNA, 7 μl Ultra II End-prep reaction buffer, 3 μl Ultra end-prep enzyme mix, and 5 μl nuclease-free water. Each reaction was incubated at 20°C for 30 min followed by 65°C for 30 min. Following end-prep, the samples were purified using AMPure beads (A63882) at a 1:1 ratio with incubation at room temperature for 5 min, two 140 μl washes with 70% ethanol, and elution for 2 min in 31 μl nuclease-free water. The PCA adapters from the ONT ligation sequencing kit 1D (SQK-LSK108) were ligated onto the end-prepped samples as follows: 30 μl of the end-prepped DNA, 20 μl PCR adapters (PCA), and 50 μl Blunt/TA ligase master mix (NEB) were combined and incubated at room temperature for 10 min. The adapted DNA was purified using Agencourt AMPure Beads as described above. The DNA was then eluted in nuclease-free water. Next, each sample was amplified in a 100 μl reaction using NEB Long-Amp Taq (M0323) as follows: 1X LongAmp Taq reaction buffer, 300 μM dNTPs, 2 μl PRM primers, and 48 μl DNA from the previous step. The reaction for each sample was divided into two 50 μl reactions and amplified with the following thermocycler conditions: an initial denaturation at 95°C for 1 min; 18 cycles of 95°C for 15 s, 62°C for 15 s, and 65°C for 2 min; a final extension at 65°C for 10 min; and a 4°C hold. Following amplification, the samples were purified using a 1:1 dilution of AMPure beads as described above.°C for 10 min. The 10.5 μl contents were briefly spun by microfuge and immediately transferred to a pre-warmed 4.5 μl aliquot of the NimbleGen SeqCap EZ Capture probe in a 0.2 ml PCR tube housed in a thermocycler set to 47°C (lid 57°C). Following overnight incubation (∼18 h), 100 μl M-270 Streptavidin Dynabeads  per sample were washed twice with 200 μl 1x wash buffer , once with 100 μl, and then placed in the thermocycler set to 47°C. Incubated samples (15 μl) were transferred immediately to the beads for 45 min. Non-target DNA was removed through washing the beads with buffers of the SeqCap EZ Hybridization and Wash Kit  as per manufacturer’s instructions with the following modifications: pipette-mixing was replaced by inversion-mixing and brief centrifugation. Beads were finally resuspended in 48 μl nuclease-free water. The following PCR reaction was set up: 48 μl sample; 2 μl PRM primers; 50 μl LongAmp Taq 2x master mix . The reaction mixes were split into 50 μl aliquots and amplified with the following thermocycler conditions: initial denaturation at 95°C for 3 min; 22 cycles of 95°C for 15 s, 62°C for 15 s, and 65°C for 10 min; a final extension at 65°C for 10 min; and holding indefinitely at 4°C. Resulting DNA was purified by a 10 min incubation with 70 μl (0.7X) Agencourt AMPure XP beads , 2 × 200 μl washes with 80% ethanol for 30 s each, 3 min air drying, and elution in 52 μl nuclease-free water. Sample concentrations were determined by Qubit as: 43.5 ng/μl (SupCol), 48.6 ng/μl (PFC), 30.5 ng/μl (VCx), 38.2 ng/μl (caudate).Following ligation of the PCR adapters, the 4 samples had the following concentrations: 36.0 ng/μl (SupCol), 25.2 ng/μl (PFC), 30.2 ng/μl (VCx), and 24.6 ng/μl (caudate) according to genomic DNA screentape analysis . To prepare the hybridization reaction, 1 μg of each of the samples was combined with 5 μg Cot-1 DNA  and 1 μl of 1mM blocking oligo (5′ AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAA 3′) and dried down in a vacuum concentrator for 1 h. The dry contents were resuspended by addition of 7.5 μl hybridization buffer and 3 μl hybridization component A , mixed by tapping, briefly spun by microfuge, and denatured at 95The captured samples were barcoded using ONT 1D native barcoding genomic DNA kit (EXP-NBD103), and the library prep was performed using the 1D genomic DNA by ligation kit and protocol for the PromethION sequencer (SQK-LSK109). The preparation was performed according to manufacturer recommendations with some modifications. Briefly, each sample underwent end-prep in a 60 μl reaction containing the product from capture, 7 μl Ultra II End-prep reaction buffer, and 3 μl Ultra end-prep enzyme mix. The resulting DNA samples were purified with AMPure beads at a ratio of 1:1 as above. After end-prep, 120 ng of each sample was barcoded by ligation of a barcoding adaptor in a 50 μl reaction containing 2.5 μl of the respective barcode and 25 μl NEB Blunt/TA ligase master mix (M0367). The reaction was incubated at room temperature for 10 min and the resulting product was purified with AMPure beads at t a 1:1 dilution as above. Then, 100 ng of each sample was combined into a final reaction to ligate on the sequencing adaptor in a 100 μl reaction containing 400 ng total combined DNA, 25 μl ligation buffer, 10 μl NEBNext Quick T4 DNA ligase, and 5 μl adapter mix. The reaction was incubated for 10 min at room temperature and then purified with AMPure beads at a.55 ratio, to preserve the smaller cDNA fragments, at room temperature for 5 min. The DNA bound to the beads were then washed with and resuspended in 250 μl short fragment buffer and rebound to the magnets twice before being eluted from the beads for 10 min in 25 μl of elution buffer. A PRO002 PromethION flow cell was primed and loaded with the resulting 41.04 ng of captured and barcoded cDNA according to the manufacturer’s recommendation. The LSK109 sequencing protocol was executed, and the sequencing ran for 52 h.2. Demultiplexing base-called reads was performed using Porechop (v0.2.3)3 with the enforced barcode detection parameter. Demultiplexed ONT reads were mapped to a combined index comprising the human genome (hg38) and in silico chromosome (chrIS)   (v1.8.5). Quality control of reads was undertaken using ONT ‘fastq deconcatenate’ (v0.6.2) and POREquality (chrIS)  using mi (chrIS) . The folv0.10.6) , with thSamples were spiked with RNA sequins  Mix A; , using t5. Reads were aligned using STAR (v2.5.3a)  (Adapters were trimmed from reads using CutAdapt  (v1.8.1)v2.5.3a)  to a comv 1.3.3) , withoutchrIS annotation and our hybrid transcriptome as a reference. For ONT samples, we used the following non-default parameters: –fldMean 1000 –fldSD 100 –libtype U. For Illumina samples, we used –libtype ISR. Salmon outputs expression measurements in TPM.We compared quantitative gene expression measurements for ONT and Illumina using Salmon (v0.11.3) , providiUniquely mapped reads were isolated from BAM files using samtools (v1.6)  ‘view’  ‘bamtobe10 scale). We then carried out simple linear regression of captured and non-captured sequins separately. The LoD was defined as the spike-in transcript of lowest abundance that was detected in more than one sample. To estimate the enrichment provided by capture, we compared the measured abundance (TPM) of captured vs. non-captured standards at each of the shared concentration points. At each shared point, we divided the average TPM for captured standards by the average TPM for non-captured standards, and then averaged all of these to obtain an overall enrichment. CV was calculated by dividing the standard deviation (SD) of each spike-in by its mean TPM across all four samples. To assess quantitative accuracy at the gene-level, we first counted the number of reads mapping uniquely to annotated sequin loci with featureCounts (v1.6.3)  of each sequin isoform against its input concentration (log(v1.6.3)  (using t(v1.6.3)  for eachn = 279) using BEDtools (v2.25.0) (t-test (two-tailed).To assess the uniformity of sequencing coverage, we calculated the read coverage of every base in every sequin exon targeted for capture (v2.25.0)  ‘coverag6.To generate a hybrid transcriptome that leverages both long- and short-read data, we used the FLAIR tool (v1.2) . First, We then undertook a number of steps in order to produce a high-confidence set of multi-exonic transcripts overlapping GWAS haplotype blocks associated with neuropsychiatric functions. First, we filtered our transcripts using gffread  with theOur set of filtered transcripts was split into unique introns and internal exons using in-house Perl scripts . We compared these feature counts with the current version of GENCODE (v29), three previous GENCODE releases  and MiTranscriptome (v2) . Only muhttps://sourceforge.net/projects/rna-cpat/files/v1.2.2/prebuilt_model/. For putative coding transcripts (coding probability ≥ 0.364), open reading frames (ORFs) were predicted and annotated using TransDecoder (v5.3.0)  , using t(v5.3.0) . Protein(v5.3.0)  and UniP(v5.3.0)  database(v5.3.0) ; the ave(v5.3.0) . For botWe retrieved promoter coordinates from our transcripts using BEDtools ‘flank’ (with –l 1 –r 0 –s options). We downloaded the latest dataset of cap analysis of gene expression (CAGE) robust peaks (hg38) from the FANTOM5 consortium . We usedWe used our previously developed scripts , 2018 toGenomic alignments were visualized using the UCSC genome browser  and IGV Statistical analyses and plotting were carried out using Prism (v7) and R (v3.5.0).This study was carried out in accordance with the recommendations of the Australian NHMRC National Statement on Ethical Conduct in Human Research and the NSW Health Sydney Local Health District Human Research Ethics Committee, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the NSW Health Sydney Local Health District Human Research Ethics Committee.JM, SB, GH, and CL conceived the study and experimental design. SB, SH, NB, and TM designed the RNA capture panel. SH and SB carried out bioinformatics analysis. MS supervised ONT sequencing experiments, bioinformatics analyses, and manuscript preparation. JB and KB carried out RNA capture and library preparation for ONT long-read sequencing. DK carried out RNA capture and library preparation for Illumina short-read sequencing. SC processed ONT sequencing data. HT carried out long-range exon coordination analysis. SH and JM wrote the manuscript, with advice from all authors.MS has received travel and accommodation expenses to speak at Oxford Nanopore Technologies conferences. TM is an inventor on a patent (PCT/AU2015/050797) relating to sequin spike-in controls. The remaining 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.


This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques. The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2 The AMPK is one of the major sensors of energy status in mammalian cells and as such plays an essential role in the regulation of cellular homeostasis, metabolism, cell growth, differentiation, apoptosis, and autophagy . The e anemia , as a coe anemia , AMPKα2-tolerant ,6. The stolerant .The AMPK also has an important role in regulating vascular function, with distinct roles reported in endothelial cells, vascular smooth muscle cells, and immune cells (for review see ). While ∆MC littermates. Experiments were performed under basal conditions, as well as in response to hypoxia  to ensure AMPK activation. The wild-type and AMPK α2∆MC samples clustered according to the genotype  2B (∆MC than in wild-type cells (Among the proteins significantly upregulated in monocytes from AMPKα2(MAT) 2B A the cat(MAT) 2B B and pro(MAT) 2B C, and fi(MAT) 2B D. Duringpe cells E. AMPKα2pe cells F,G.∆MC mice. The methylation of the CpG island, which overlaps the 5′untranslated region and the first exon, in the Vegfa gene, was higher in macrophages from AMPKα2∆MC than from wild-type mice  islands play a major role in chromatin compaction and consequently gene silencing ,20. Giveype mice A, a phenype mice B. Similarophages C, and corophages D and prorophages E. Functiype mice F. ∆MC mice under basal conditions  and IFN-γ -γ for 4 h (M1 polarization) or interleukin (IL)-4 and IL-13 for 16 h (M2 polarization). Differences in the expression of IL-1β, resistin like beta  and chitinase-like 3 (YM1) were evident in macrophages from wild-type and AMPKα2nditions A,B. Whilnd IFN-γ C. M2 pol∆MC mice were crossed onto the apolipoprotein E–deficient (ApoE-/-) background to generate ApoExα2∆MC mice and fed a western diet for four months. The western diet elicited the deposition of atherosclerotic plaques in the aortic root as well as in the aortae of ApoExα2fl/fl mice . Immunohistochemistry of the aortic root after four months of a western diet, however, revealed a clear decrease in macrophage infiltration (Mac2 positive regions) in plaques from the ApoExα2∆MC mice C. This c∆MC mice D and an ∆MC mice . Howeveranalyses . It fail∆MC mice also developed smaller plaques than carotid arteries from ApoExα2ff/fl mice . Despit∆MC mice, as well as the decrease in atherosclerotic plaque formation. The downregulation of the AMPKα2, but not the AMPKα1 subunit was previously reported to attenuate neutrophil isocitrate dehydrogenase expression and the production of α-ketoglutarate . As a co-/- background) with increased atherosclerotic calcification [Few studies have assessed the consequences of AMPKα1 versus AMPKα2 deletion in a head-to-head manner. Recently, a direct comparison of the consequences of the global deletion of AMPKα1 and AMPKα2 on collateral remodeling and arteriogenesis revealed that the loss of both proteins attenuated the recovery of blood flow after ischemia . The confication , but decfication . The lat∆MC mice was distinct. While the studies focused on the link to methylation, it is clear that other mechanisms can underlie the regulation of many of the proteins found to be differentially regulated. Indeed, in addition to its effects on the maturation of a spectrum of microRNAs [Taken together, the present study revealed that protein expression in bone marrow-derived monocytes from wild-type and AMPKα2icroRNAs , and traicroRNAs , the resicroRNAs . IL-4, IL-13 and IFN-γ were from PeproTech . The antibody against MAT2B was from GeneTex/Biozol , anti-DNMT1 was from Novus Biologicals/Bio-Techne . The antibodies against MMP9 , β-actin  were from Sigma/Merck . Unless otherwise mentioned, all other materials were from Sigma/Merck or Applichem .-/- mice were purchased from Charles River . Floxed AMPKα2 mice were kindly provided by Benoit Viollet  [Lysm promoter (B6.129P2-Lyz2tm1(cre)Ifo/J; Jackson Laboratories) to generate mice lacking the α2 subunit specifically in myeloid cells, as described [∆MC mice were crossed onto the ApoE-/- background to generate ApoExα2∆MC mice. For the feeding experiments and the partial carotid ligation ApoExα2fl/flxCre-/- (ApoExα2fl/fl) and LysM Cre+/- ApoExα2∆MC) as controls were used. All animals were housed in conditions that conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication No. 85-23). Both the University Animal Care Committee and the Federal Authority for Animal Research at the Regierungspräsidium Darmstadt  approved the study protocol (#F28/34). For the isolation of organs, mice were killed using 4% isoflurane in air and subsequent exsanguination or decapitation.ApoE France)  and bredescribed . The resfl/fl and ApoExα2∆MC mice underwent partial ligation of the left carotid artery (LCA) as previously described [ApoExα2escribed , whereasfl/fl and ApoExα2∆MC mice were fed a western-type diet , for 2 or 4 months. After this time, the mice were killed and the vessels were excised, cleaned from connective tissue and either used for dissection to single cells for FACs analysis of infiltrated immune cells or for histological and immunohistochemistry experiments. The excised arteries were pinned on agarose plates, opened and stained with Oil red O after fixation Six-week-old ApoExα2To section the aortic root, hearts were excised and fixed overnight in Histofix  and embedded in Tissue-Tek OCT. Using the cryostat, the aortic roots in the range of the aortic valve were cut into 10 µm sections. Prior to staining, the sections were washed with phosphate buffered saline (PBS) and blocked with 0.3% bovine serum albumin, 5% horse serum (Fisher Scientific) and 0.1% Triton X-100 for 1 h at room temperature. The antibodies against Mac2 , MMP9  and Collagen IV  were diluted in PBS and incubated at 4°C overnight. After washing four times with PBS, the secondary antibodies , or anti-goat Alexa Fluor 568  were incubated with FITC conjugated smooth muscle actin antibody . After washing with PBS the sections were embedded in mounting medium: 1 mmol/L dithiothreitol, 50% Glycerin, and 1 µg/mL 4’,6-diamidino-2-phenylindol (DAPI), and the fluorescence was detected using a LSM780 (Zeiss). Picrosirius red and Masson Trichome stainings were performed on cryosections washed with PBS. For picrosirius red, slides were incubated for 1 h in 0.1% picrosirius Red solution  in a saturated aqueous solution of picric acid (Sigma/Merck). Sections were destained with 0.05% acetic acid and mounted with Entellan (Sigma/Merck). Manson Trichome staining was performed using a kit according to the manufacturers (Sigma/Merck) protocol. Total RNA was extracted using TriReagent (Sigma-Aldrich/Merck) and concentration was determined by OD260nm . For the generation of cDNA total RNA (200 ng) was reverse transcribed using the SuperScript III  and random hexamer primers according to the manufacturer’s protocol. The amount of mRNA was quantified using the cycle threshold (cT) value using a SYBR green master mix  with intron spanning primers in a Mic qPCR system . CT values obtained were converted into relative amounts based on a standard curve obtained from serial dilutions of a positive control and mRNA levels were normalized to 18S rRNA. Primers for PCR were ordered from Biospring , sequences are listed in Vegfa and Mmp9 was assessed by qPCR using the primers listed in DNA was isolated using the DNAeasy Blood and Tissue kit from Qiagen . 2 µg of genomic DNA was sheared in ice-cold water (15/15 rounds high/low shear of 10 s on/off cycles) using the Bioruptor plus from Diagenode  to achieve fragments of 200–1000 bp in length. The immunoprecipitation of methylated DNA was performed using the abcam kit  according to the manufacturer’s instructions. In brief, wells were preincubated with an anti-methyl cytosine antibody, following the removal of unbound antibody sheared genomic DNA was added and incubated for 2 h at room temperature. Following several washing steps, the samples were treated with proteinase K and enriched methylated DNA was purified using silica columns. The methylation status of the CpG islands in Cells were lysed in Triton X-100 buffer and detergent-soluble proteins were solubilized in SDS-PAGE sample buffer, separated by SDS-PAGE and subjected to Western blotting as described . ProteinMice were sacrificed by 4% isoflurane in air, the heart was punctuated and NaCl solution (15 mL 0.9%) containing 5 U/mL heparin was perfused. The carotid arteries or the aortas were excised and cleaned from connective tissue. To obtain single cell suspensions from the entire aortas or carotid arteries the vessels were microdissected and digested with 125 U/mL collagenase type XI, 60 U/mL hyaluronidase type I-s, 60 U/mL DNase1, and 450 U/mL collagenase type I in PBS containing 20 mmol/L HEPES at 37 °C for 1 h . Cell suMurine bone marrow cells were obtained from 12 weeks old mice. Suspended cells were seeded on tissue culture dishes in RPMI 1640 , and after 30 min the medium was changed to RPMI with 10% FCS. For differentiation to macrophages cells were in the presence of granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor  for 6 days. For the polarization to M1 macrophages the cells were treated for 4 h with LPS (0.1 ng/mL) IFN-γ (1 ng/mL), for the polarization to M2 macrophages IL-4 and IL-14 (each 1 ng/mL) was applied for 16 h. Pellets of isolated monocytes were solubilized in 10% SDS, 150 mmol/L NaCl, 100 mmol/L Tris/HCl pH 7.6, 100 mmol/L dithiothreitol. Samples were sonicated for 5 s and heated at 95 °C for 5 min to facilitate protein solubilization. Samples were then incubated at 56 °C for 30 min and centrifuged to remove insoluble material. Total protein (100 µg per sample) was diluted by adding 200 µL, 8 mol/L urea, 50 mmol/L Tris/HCl, pH 8.5 and loaded onto spin filters with a 30 kDa cut off , and prepared as described . Proteinm/z with a resolution of 70,000, and an automatic gain control value of 3 × 106 total ion counts with a maximal ion injection time of 160 ms. Only higher charged ions (2+) were selected for MS/MS scans with a resolution of 17,500, an isolation window of 2 m/z and an automatic gain control value set to 105 ions with a maximal ion injection time of 150 ms. Selected ions were excluded in a time frame of 30 s following fragmentation event. Fullscan data were acquired in profile and fragments in centroid mode by Xcalibur software .LC/MS was performed using a Q Exactive Plus  equipped with an ultra-high-performance LC unit  and a Nanospray Flex Ion-Source (Thermo Scientific). Peptides were loaded on a C18 reversed-phase precolumn followed by separation on a 2.4 μm reprosil C18 resin  in-house packed picotip emitter tip  using a gradient from eluent A  to 30% eluent B  for 110 min followed by a second gradient to 60% eluent B for 45 min with a flow rate 300 nL/minute. MS data were recorded by data dependent acquisition using the Top10 method to select the most abundant precursor ions in positive mode for higher-energy collisional dissociation fragmentation. The lock mass option was enabled to ensure high mass accuracy during many following runs. The full MS scan range was 300 to 2000 http://141.61.102.17/maxquant_doku/doku.php?id=start, accessed on: 30 March 2015) proteomics software [http://www.perseus-framework.org/, accessed on: 27 March 2015; MPI for Biochemistry, Martinsried, Germany) [t-tests were used. Xcalibur raw files were analyzed using the Max Quant  . Identifm/z 385.1/136.1, 385.1/88.0; SAM: m/z 399.1/250.1, 399.1/136.1). Calibration curves were performed with authentic standards. The intensity of the measured metabolite was normalized to SAM-d3 internal standard. Analyst 1.6.2 and MultiQuant 3.0 , were used for data acquisition and analysis, respectively.The quantification of SAM and SAH was performed by LC-MS/MS. Cells were scraped in acidic methanol  and snap frozen. The supernatant was used for reversed-phase LC separation using a Waters Acquity HSS T3 column  at 35 °C, operated by an Agilent 1290 Infinity pump system . The LC mobile phase was A) 100% miliQ water with 0.1% formic acid and B) 100% methanol with 0.1% formic acid. Starting condition for the separation was 98% solvent A held for 2 min, followed by a 0.5 min gradient from 60% A to 5% A, hold 55 for 0.5 min, followed by an equilibration step to 98% A, making 6 min total LC run time. The flow rate was 330 μL/minute, injection volume 5 μL. The QTrap 5500 mass spectrometer  was operated using positive mode electrospray ionisation using the following parameter: CUR 30 psi, CAD medium, IS 4000 V, TEM 350 °C, GS1 15 psi, GS2 20 psi. Two MRM transitions were used for each metabolite, one serving as quantifier one as qualifier, respectively (SAH: p < 0.05 were considered statistically significant. Data are expressed as mean ± SEM. Statistical evaluation was performed using Student’s t test for unpaired data, one-way ANOVA followed by a Bonferroni t test or ANOVA for repeated measures where appropriate. Values of