Agrococcus, Arsenicicoccus, Arthrobacter, Brevibacterium, Citricoccus, Janibacter, Kocuria, Microbacterium, Microlunatus, Nocardioides, Nocardiopsis, Saccharopolyspora, Salinibacterium and Streptomyces. To our knowledge, this paper is the first report on the isolation of Microlunatus genus members from marine habitats. Of the 30 isolates, 11 strains exhibited antibacterial and/or antifungal activity, seven of which have activities against Bacillus subtilis and Candida albicans. All 30 strains have at least two biosynthetic genes, one-third of which possess more than four biosynthetic genes. This study demonstrates the significant diversity of Actinobacteria in the Chukchi Shelf sediment and their potential for producing biologically active compounds and novel material for genetic manipulation or combinatorial biosynthesis.Marine environments are a rich source of Actinobacteria and have the potential to produce a wide variety of biologically active secondary metabolites. In this study, we used four selective isolation media to culture Actinobacteria from the sediments collected from the Chukchi Shelf in the Arctic Ocean. A total of 73 actinobacterial strains were isolated. Based on repetitive DNA fingerprinting analysis, we selected 30 representatives for partial characterization according to their phylogenetic diversity, antimicrobial activities and secondary-metabolite biosynthesis genes. Results from the 16S rRNA gene sequence analysis indicated that the 30 strains could be sorted into 18 phylotypes belonging to 14 different genera:  Actinobacteria are Gram-positive bacteria with high guanosine-cytosine content in their DNA, ranging from 51% to >70% . The proOnly a few investigations on marine Actinobacteria have been performed in the Arctic marine environment. However, these studies have revealed a diversity of Actinobacteria in the Arctic marine sediments ,17,18,19Dispersion and differential centrifugation (DDC) was employed to isolate Actinobacteria from 10 samples of Chukchi Shelf marine sediments collected from 26 m to 69.6 m deep. After one week to four weeks, individual colonies were picked from the isolation media according to morphological characteristics and further isolated on fresh media as pure cultures. A total of 165 strains were isolated from the Chukchi Shelf marine sediments and grown on MISP2. Of all the strains, only 25 isolates formed powdery colonies with well-developed aerial hyphae. The remaining isolates formed white, yellow, orange, pink, red or brown pigmented colonies without aerial hyphae. The partial 16S rRNA gene sequence (~500 nt) analysis confirmed that 73 of the 165 isolates belonged to Actinobacteria. Repetitive DNA fingerprinting analysis was performed to dereplicate the actinobacterial isolates from the same sample. Then, from the 73 actinobacterial isolates, 30 representatives were selected for further investigation. Among the 30 representatives, six strains were obligate marine Actinobacteria that require seawater for growth, and nine strains grew better on the ISP2 media prepared with seawater than on those prepared with deionized water plus 3.2% NaCl  and on those prepared with deionized water . These rMicrolunatus, and was most closely related to the type strain of Microlunatus aurantiacus YIM 45721T isolated from a rhizosphere soil sample in Yunnan Province, China [Microlunatus genus members from a marine environment. These data indicated the considerable diversity of Actinobacteria within the Chukchi Shelf sediments.The nearly complete 16S rRNA gene sequences (1479 bp to 1506 bp) of the 30 representatives were PCR-amplified and sequenced. The results of the 16S rRNA gene sequences analysis are shown in e, China . To our Dietzia cercidiphylli was found to produce biosurfactants [Janibacter melonis [Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positive bacteria and possibly related to novel cyclic thiazolyl peptides [et al. found that Nocardiopsis dassonvillei subsp. dassonvillei could produce α-pyrones [Streptomyces albidoflavus was found to produce antifungal compounds, such as a non-polyene substance [etc. The 30 selected actinobacterial isolates obtained from the Chukchi Shelf sediments, which belong to 14 actinobacterial genera, were tested for antimicrobial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and the yeast, Candida albicans. The results are presented in Bacillus subtilis and yeast Candida albicans. Only one isolate, y146, and one isolate, y2, had activity against Gram-negative E. coli and Gram-positive bacteria Staphylococcus aureus, respectively. Furthermore, the data clearly show that strains that can produce antibiotic compounds belonging to three genera: Streptomyces, Nocardiopsis and Microlunatus.Actinobacteria are well known for their ability to produce secondary metabolites, many of which are active against pathogenic microorganisms. Some of the closest neighbors of our isolates were found to produce biologically active compounds. factants . Helquin melonis . Two strpeptides . Fu et a-pyrones . Streptoubstance , Antimycubstance , etc. ThphzE), dTDP-glucose-4, 6-dehydratase (dTGD), halogenase  and cytochrome P450 hydroxylase (CYP) genes. Several amplified gene fragments were cloned and sequenced, and subsequent analysis confirmed that these genes encode parts of the expected biosynthetic enzymes [phzE loci, six (20%) strains and four phylotypes; dTGD loci, 24 (80%) strains and 15 phylotypes; Halo loci, 12 (40%) strains and 11 phylotypes; and CYP loci, 10 (33.3%) strains and six phylotypes. These results suggested that the 6-deoxyhexoses glycosylation biosynthetic and type I PKS pathways were widespread in the Chukchi Shelf sediment-derived Actinobacteria. We successfully amplified secondary metabolite biosynthetic gene fragments from the genomes of these isolates with detectable antimicrobial activity. However, most of the non-active isolates have at least one kind of PKS I, PKS II, NRPS and phzE gene involved in the backbone biosynthesis of secondary metabolites and one of the dTGD and Halo genes encoding modification enzymes responsible for the introduction and generation of diversity and activity in several secondary metabolites [Janibacter, Salinibacterium and Arthrobacter genera without antimicrobial activity. We also sequenced the PCR products of the ketosynthase (KS) domain of PKS II from five Nocardiopsis strains genomes. The KS sequences from these strains displayed the amino acid sequence identities (67% to 69%) of the KS domain of fabF involved in the biosynthesis of pradimicin, an antifungal antibiotic from Actinomadura hibisca [Staphylococcus aureus (MRSA)-active antibiotic [Streptomyces, only a limited number of secondary metabolite biosynthesis gene clusters in rare Actinobacteria have been studied [A PCR-based screening for the presence of genes associated with biosynthetic pathways can rapidly predict the unknown strains that have the potential to produce secondary metabolites. Therefore, in parallel with antimicrobial activity testing, we attempted the PCR-based approach to detect type I polyketide synthase (PKS I), PKS II, nonribosomal peptide synthase (NRPS), aminodeoxyisochorismate synthase  nalidixic acid (25 μg/mL), nystatin (50 μg/mL) and cycloheximide (50 μg/mL) to inhibit the growth of Gram-negative bacteria and fungi. The isolation plates were incubated in the dark at 28 °C for 4 weeks. Colonies from various agar plates were selected based on differing colony morphologies. Isolates were obtained in pure culture after three successive transfers to fresh isolation media. Pure cultures were frozen in 20% glycerol at −80 °C for long-term storage.Repetitive DNA fingerprinting was performed on the actinobacterial isolates from the same sample. Genomic DNA was prepared using a bacterial genomic DNA kit , according to the supplied protocol from each isolate. PCR amplification was performed using the BOXA1R primer (5′-CTA CGG CAA GGC GAC GCT GAC G-3′) . The PCRThe dereplicated actinobacterial isolates were tested for the required seawater for growth. Frozen stored strains were inoculated onto the ISP2 agar medium prepared using seawater (MISP2). Once sufficient growth had occurred, a sterile wire loop was used to transfer cell material onto a new plate of the same medium prepared with seawater, a plate prepared with deionized water plus 3.2% NaCl and a plate prepared with deionized water. The plates were incubated at 28 °C for up to 4 weeks. Growth was monitored under a stereomicroscope at 64-fold magnification. If no growth was observed on the plate prepared with deionized water, that strain was considered to require seawater for growth.The 16S rRNA gene was amplified by PCR using primers 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) . The rea4·7H2O, 0.1 mg MnCl2·4H2O, 0.1 mg ZnSO4·7H2O, 15 g agar and 1 L sea water) [Staphylococcus aureus ATCC 29213 and Bacillus subtilis CMCC 63501, two Gram-negative bacterial strains, Escherichia coli ATCC 44102 and Pseudomonas aeruginosa ATCC 27853, and one yeast strain, Candida albicans ATCC 10231, were used as test strains. The antimicrobial activities were expressed as inhibition zones after 16 h of incubation at 28 °C.Actinobacterial isolates were transferred to the plates containing three different production media: PM3  . The plaTo amplify the genes for the KS domains of PKS I, the KS domains of PKS II, the adenylation domains of NRPS, the enzyme PhzE of the phenazine pathways, the enzyme dTGD of 6-deoxyhexoses glycosylation pathway, the enzyme Halo of halogenation pathway and the enzyme CYP in polyene polyketide biosynthesis, the degenerate primers as described previously were used . The comPKS I (50 μL): 2 μL template, 5 μL 10× buffer, 2.5 μL BSA (1 mg/mL), 2 μL dNTP (2.5 mM), 1 μL each primer (10 μM) and 2.5 U Taq DNA polymerase; 5 min at 95 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 60 °C and 2 min at 72 °C, followed by a 5-min extension at 72 °C.PKS II (50 μL): 2 μL template, 5 μL 10× buffer (Mg2+ free), 2 μL MgCl2 (25 mM), 4 μL BSA (1 mg/mL), 4 μL dNTP (2.5 mM), 5 μL each primer (10 μM) and 2.5 U Taq DNA polymerase; 5 min at 95 °C, followed by 40 cycles of 1 min at 95 °C, 1 min at 64 °C and 1.5 min at 72 °C, followed by a 15-min extension at 72 °C. NRPS (50 μL): 2 μL template, 5 μL 10× buffer, 2.5 μL BSA (1 mg/mL), 2 μL dNTP (2.5 mM), 1 μL each primer (25 μM) and 2.5 U Taq DNA polymerase; 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 2 min at 59 °C and 4 min at 72 °C, followed by a 10-min extension at 72 °C.phzE (25 μL): 1 μL template, 2.5 μL 10× buffer, 2 μL BSA (1 mg/mL), 2 μL dNTP (2.5 mM), 0.5 μL each primer (10 μM) and 1 U Taq DNA polymerase; 2 min at 94 °C, followed by 36 cycles of 1 min at 94 °C, 1 min at 54.7 °C and 2 min at 72 °C, followed by a 7-min extension at 72 °C. dTGD (20 μL): template 0.7 μL, 10× buffer 2.5 μL, BSA (1 mg/mL) 2 μL, dNTP (2.5 mM) 2 μL, each primer (10 μM) 1.5 μL and 3.5 U Taq DNA polymerase; 4 min at 98 °C, followed by 30 cycles of 40 s at 96 °C, 40 s at 50 °C and 1.5 min at 72 °C, followed by a 5-min extension at 72 °C. Halo (50 μL): template 2 μL, 10× buffer 5 μL, BSA (1 mg/mL) 2.5 μL, dNTP (2.5 mM) 2 μL, each primer (25 μM) 1 μL and 2.5 U Taq DNA polymerase; 3 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1.5 min at 58 °C and 1 min at 72 °C, followed by a 5-min extension at 72 °C.CYP (50 μL): 1 μL template, 5 μL 10× buffer (Mg2+ free), 2 μL MgCl2 (25 mM), 5 μL BSA (1 mg/mL), 4 μL dNTP (2.5 mM), 1 μL each primer (20 μM) and 2.5 U Taq DNA polymerase; 5 min at 96 °C, followed by 45 cycles of 1 min at 96 °C, 30 s at 60 °C and 45 s at 72 °C, followed by a 5-min extension at 72 °C.Bacillus subtilis and Candida albicans. All representatives have at least two biosynthetic genes, and one-third of these representatives possess more than four biosynthetic genes. The KS sequences from five Nocardiopsis strains with antimicrobial activity displayed the amino acid sequence identities (67% to 69%) of the KS domain of fabF involved in the biosynthesis of pradimicin or had 68% similarity with TamM, related to the biosynthesis of Tetarimycin A. This study demonstrates the significant diversity of Actinobacteria in the Chukchi Shelf sediment. The new strains might represent a valuable source of biologically active compounds with antimicrobial activity and genes for their biosynthesis.A total of 73 actinobacterial strains have been isolated from the Chukchi Shelf sediments in the Arctic Ocean. We used repetitive DNA fingerprinting analysis to select 30 representatives for further investigation. An unexpected variety of actinomycete genera were isolated. The 30 representatives were sorted into 18 phylotypes belonging to 14 different genera. Moreover, 25 of the 30 representatives are rare actinomycetes. We also screened them for biosynthetic genes and antimicrobial activities. Of the 30 isolates, 11 exhibited antibacterial and/or antifungal activity, seven of which have activities against


The results indicated that the single crystalline phase of (K0.5Na0.5)NbO3 was received in as-milled samples synthesized using optimized ball-to-powder mass ratio of 35 : 1 and at a milling speed of 600 rpm for 5 h. In the optimized as-milled samples, no remaining alkali carbonates that can provide the volatilizable potassium-containing species were found and (K0.5Na0.5)NbO3 nanopowders were readily obtained via the formation of an intermediate carbonato complex. This complex was mostly transformed into (K0.5Na0.5)NbO3 at temperature as low as 350°C and its existence was no longer detected at spectroscopic level when calcination temperature crossed over 700°C.The effects of ball milling parameters, namely, the ball-to-powder mass ratio and milling speed, on the synthesis of (K In addition, for the direct synthesis of (K0.5Na0.5)NbO3 as well as NaNbO3 [3 [Nowadays, with the tendency towards the sustainable and eco-friendly development, lead-containing compounds in general and commonly-used Pb Otoxicity , 2. For  density . To date density –11. Amon density , 10. By ed by us  for the s NaNbO3  or for t0.5Na0.5)NbO3 nanopowders by high-energy ball milling method using Na2CO3, K2CO3, and Nb2O5 as starting materials were described in detail in this paper. In addition, the thermal decomposition of the carbonato complex as an intermediate species during the reactive high-energy ball milling process was also investigated.The effects of ball-to-powder mass ratio and milling speed on the synthesis of (K0.5Na0.5)NbO3, all analytical grade K2CO3, Na2CO3, and Nb2O5, which were purchased from Aldrich (Germany), were dried at 200°C for 2 h prior to use in order to remove moisture. Stoichiometric mixtures of these raw materials were placed in a 125 mL stainless-steel vial of a Fritsch Pulverisette 6 planetary mill. The rotational speed was changed from 300 to 600 rpm. Different numbers of stainless-steel milling balls with diameters of 10 and 20 mm were used depending on the ball-to-powder weight ratio ranging from 25/1 to 40/1. For investigation of thermal decomposition of intermediate carbonate complex, as-milled samples synthesized at optimized conditions were calcined at temperatures ranging from 300 to 1000°C for 3 h. X-ray diffraction diagrams of obtained samples were recorded using Siemens D-5000 diffractometer  with CuKα radiation. For the determination of lattice parameters, Si  was used as an internal standard. The synthesized samples were also characterized by field-emission scanning electron microscopy , Fourier transform infra-red spectroscopy , and thermal analysis .For high-energy ball milling synthesis of NbO3 samples synthesized at rotational speed of 600 rpm for 5 h with different ball-to-powder weight ratios (BPRs) of 25/1, 35/1, 37/1, and 40/1 were shown in 2O5 (PDF card number 27-1003) only. It can be explained that the milling energy produced during the ball milling process was insufficient to activate Nb2O5 to react with alkali carbonates at atomic scale. Instead, this energy was mainly supplied for breaking down bulk Nb2O5 into nanocrystals and for the amorphization of alkali carbonates, similar to the case of NaNbO3 reported in [0.5Na0.5)NbO3 was obtained directly and uniquely without any other impurity. With the selected milling parameters, the generated milling energy was suggested to be enough for all reactants to react simultaneously and completely to form the desired (K0.5Na0.5)NbO3 crystalline phase. This alkali niobate phase was still detected together with the remaining Nb2O5 in the as-milled sample by increasing the ball-to-powder weight ratio to 37/1. When the BPR reached 40/1, however, only peaks of Nb2O5 crystalline phase existed in XRD diagram. Thus, the alkali niobate crystaline phase was only partially formed by selecting the ball-to-powder weight ratio of 37/1 that slightly differed from 35/1 and was completely absent when this value was large enough (40/1). It can be explained that when the ball-to-powder weight ratio crossed over 35/1, Nb2O5 was assumed to be activated, but K2CO3 and Na2CO3 were probably already well amorphized. As a result, these alkali carbonates undergo partially or did not undergo the mechanochemical reaction at all with niobium pentoxide to form alkali niobate phase. Another reason for the above-mentioned phenomenon is that the increased milling energy with the BPR might lead to the amorphization of milling mixture components by creating a more strain and a more defect concentration [0.5Na0.5)NbO3 samples synthesized at the rotational speed of 600 rpm for 5 h with different BPRs of 25/1, 35/1, 37/1, and 40/1 were shown in −1 belongs to symmetrical stretching mode of NbO6 octahedra. One can realize that the absorption band at 1467 cm−1 assigning to the asymmetrical C–O stretching vibration (ν3) of free CO32− was absent only for the sample synthesized mechanochemically with the BPR of 35/1. This implied that, with the selected preparative condition, no alkali carbonates were left in the milled product while were still detected in FTIR spectra of samples synthesized with other investigated BPRs. The damping of ν3 vibration was related to its splitting into three new bands at 1633, 1507, and 1338 cm−1. It is worth noting that the in-plane bending vibration of adsorbed water also contributed to the band at 1633 cm−1. For samples synthesized with the BPRs of 25/1, 37/1, and 40/1, only two bands at 1633 and 1338 cm−1 were detected, while all three bands occurred for the case with the BPR of 35/1. In addition, another new band belonging to the symmetrical C–O stretching vibration at 1055 cm−1 (ν1) that reported to be IR-inactive for the free CO32− of alkali carbonates was also found in all spectra [32− anion via the formation of an amorphous intermediate carbonato complex between CO32− and Nb5+ ions. These obtained results are in good agreement with those published previously [0.5Na0.5)NbO3 and were much lower than those obtained for as-milled samples synthesized with other investigated BPRs. It can be assumed that a larger amount of CO2 might be already released from reaction between alkali carbonates and niobium pentoxide to form carbonato complex during ball milling process and this led to the significant lowering in total weight loss. Thus, from above results, it is obvious that, with this optimized ball-to-powder, the volatilization of potassium-containing species produced from the decomposition of remained alkali carbonates during the calcination step that often required for mechanochemical-assisted solid-state reaction procedures can be avoided [X-ray diffraction (XRD) diagrams of an unmilled mixture containing Korted in . With thntration , 15. Thu spectra . The aboeviously . It can  avoided .2O5 (PDF card number 27-1003) was found in as-milled samples. By increasing the rotational speed to 500 rpm, (K0.5Na0.5)NbO3 phase occurred while diffraction peaks of Nb2O5 phase still presented. With the rotational speed of 600 rpm, there are only peaks of the crystalline phase of (K0.5Na0.5)NbO3 in XRD diagram. Thus, for the investigated milling time of 5 h, the milling energy that was generated during ball milling process increased with the rotational speed and finally became enough for all starting materials to be involved in the mechanochemical reaction simultaneously and stoichiometrically to obtain single crystalline phase of (K0.5Na0.5)NbO3 with the rotational speed of 600 rpm. When the milling time was increased to 10 h, the single crystalline phase of (K0.5Na0.5)NbO3 existed not only for the sample synthesized with the rotational speed of 600 rpm but also for that with 500 rpm NbO3 [ 500 rpm . For eac0.5)NbO3 , 18.0.5Na0.5)NbO3 was observed in XRD diagrams as shown in 0.5Na0.5)NbO3 phase only when heating temperature reached the value of 350°C. By increasing the calcination temperature, two bands at 1633 and 1338 cm−1 that are characteristics for this complex were detectable in FTIR spectra for samples calcined at 400 and 500°C NbO3 nanopowders were achieved with the average grain size of 40 nm for samples calcined at 700°C (0.5Na0.5)NbO3 nanopowders directly and at a large scale without the appearance of any remained alkali carbonates and hydrated carbonated phases.In order to study the thermal decomposition of intermediate carbonato complex, as-milled samples synthesized at optimized conditions were calcined at 300, 400, 500, 700, and 1000°C for 3 h. For all studied samples, only crystalline phase of (Knd 500°C . Its speat 700°C . From th0.5Na0.5)NbO3 nanopowders by reactive high-energy ball milling method using Na2CO3, K2CO3, and Nb2O5 as starting materials were systematically investigated. By selecting the rotational speed of 600 rpm with the ball-to-powder weight ratio of 35/1 after 5 h of milling time as optimized synthesis parameters, the volatilization of potassium-containing species can be avoided and (K0.5Na0.5)NbO3 nanopowders were readily obtained at a large scale via the formation of an intermediate carbonato complex. This complex was mostly transformed into (K0.5Na0.5)NbO3 at temperature as low as 350°C and the carbonato complex-free (K0.5Na0.5)NbO3 nanopowders were obtained with the average grain size of 40 nm for samples calcined at 700°C.The effects of the ball-to-powder mass ratio and milling speed on the synthesis of (K


Escherichia coli and an expression plasmid to isolate target-recognizing peptides. This display method is based on the formation, in bacteria, of complexes between a polyhistidine (His)-tagged peptide including random sequences and the peptide-encoding mRNA including an RNA aptamer against the His-tag. When this system was tested using a sequence encoding His-tagged green fluorescent protein that included an RNA aptamer against the His-tag, the collection of mRNA encoding the protein was dependent on the RNA aptamer. Using this display method and a synthetic library of surrogate single-chain variable fragments consisting of VpreB and Ig heavy-chain variable domains, it was possible to isolate clones that could specifically recognize a particular target (intelectin-1 or tumor necrosis factor-α). These clones were obtained as soluble proteins produced by E. coli, and the purified peptide clones recognizing intelectin-1 could be used as detectors for sandwich enzyme-linked immunosorbent assays. The Hishot display will be a useful method to add to the repertoire of display technologies.Display technologies are procedures used for isolating target-recognizing peptides without using immunized animals. In this study, we describe a new display method, named Hishot display, that uses  In addition, the peptides can be selected from high-variety libraries containing >1010 independent clones.Target-recognizing molecules are widely used in research, disease diagnosis, and clinical treatment. Among such molecules, monoclonal antibodies (mAbs) are one of the most common for molecular recognition because of their specificity, affinity, and versatility. Most mAbs are isolated via hybridoma technology, which requires B cells in antigen-immunized animals Escherichia coli and an expression plasmid. This display method uses the complex formed between a polyhistidine (His)-tagged peptide that contains random sequences and an mRNA that encodes the peptide with an RNA aptamer against the His-tag. With the Hishot display, the target-recognizing peptides were isolated as antibody fragments that could be obtained as soluble protein produced by E. coli and that bound the target specifically. The Hishot display is a useful method that should be added to the current repertoire of display technologies.These established display methods have many advantages, but they may not be able to identify every target-recognizing peptide. Therefore, in order to resolve the difficulties and ethics concerns associated with animal immunization, additional display methods that do not require the use of animals should be developed. In this study, we describe a new display method, named Hishot display, that is based on basic molecular biological procedures using NdeI and XbaI sites in a cold-shock expression vector, pCold IV  H) without complementarity-determining regions 3 (CDR3) was inserted into pCold IV with shot47; the resulting plasmid was named pHishot12. For the expression of the Fc-fused protein, we used an original mammalian expression vector, pHint, which has a human intelectin-1 signal peptide for secretion  (0.1 nmol), and reverse oligonucleotides (5′-CTAGTAGCGGCCGCTTATCTACCGCTGGAAACGGTAACCAGAGTACCCTGACCCCAGTAGTCGA-3′) (1 nmol) were mixed, and complementary strands were synthesized with Ex Taq (Takara Bio Inc.). The reaction conditions consisted of a denaturation step at 98°C for 30 s, followed by 5 cycles of 55°C for 1 min and 72°C for 1 min. The DNA was isolated by ethanol precipitation and treated with exonuclease I (Takara Bio Inc.) at 37°C for 18 h. Double-stranded DNA (dsDNA) was purified by phenol–chloroform extraction, digested with PstI and NotI, resolved by agarose gel electrophoresis using 3% NuSieve GTG agarose (Takara Bio Inc.), extracted with thermostable β-agarase , and purified by phenol extraction, phenol–chloroform extraction, and ethanol precipitation. The digested dsDNA (3 pmol) was inserted into PstI- and NotI-digested pHishot12 (0.3 pmol) using T4 DNA ligase (Takara Bio Inc.) in a ligation buffer (60 mM Tris buffer (pH 7.5) containing 60 mM MgCl2, 50 mM NaCl, 70 mM 2-mercaptoethanol, 1 mM ATP, 20 mM dithiothreitol, 10 mM spermidine, and 1 mg/mL bovine serum albumin (BSA)) at 14°C for 18 h. The plasmid was purified by phenol–chloroform extraction, precipitated with 10 µg of tRNA by ethanol precipitation, and transferred into E. coli DH5α electrocompetent cells (Takara Bio Inc.) by electroporation . The library (5–8×107 colony-forming units (CFU)) was used for the Hishot display. Sequencing of 48 clones from a library indicated that the insert rate was >97% (47/48 clones) and that none of the clones contained a stop codon in their random region.A plasmid library was made by inserting a synthetic CDR3 region containing random peptide sequences into pHishot12. Forward oligonucleotides, including 8 random codons  at 10°C with continuous shaking for 18 h. The following procedures were carried out at 4°C. The bacteria was centrifuged at 6,000× g for 10 min, washed with saline containing 10 mM ethylenediaminetetraacetic acid (EDTA), and incubated for 1 h with 20% sucrose (5 mL) containing 2 mg/mL lysozyme , 1 mM EDTA, and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5). The spheroid cells were washed gently with 20 mM HEPES-buffered saline (HBS) containing 0.1 mM magnesium acetate and then lysed with 4 mL of 20 mM HEPES (pH 7.5) containing 0.5% Triton-X100, 0.1 mM magnesium acetate, 100 µg/mL tRNA, 1 mg/mL BSA , 100 µg/mL rabbit IgG , 100 µg/mL human IgG , 100 µg/mL mouse IgG , 13 U/mL RNase inhibitor , 50 U/mL DNase I , and a protease inhibitor . The lysate was passed twice through a syringe with a 27-gauge needle and then diluted in 40 mL of HBS containing 0.5% Triton-X100 and 0.1 mM magnesium acetate. The lysate was then centrifuged at 14,000× g for 10 min, after which the supernatant was mixed for 90 min with 20 µL of Affi-Gel 10 gel  coupled with human intelectin-1 or human tumor necrosis factor (TNF)-α. The gels were precipitated by centrifugation at 3,000× g for 5 min, washed twice with 40 mL of HBS containing 0.5% Triton-X100 and 0.1 mM magnesium acetate, and incubated for 5 min with 150 µL of TRIzol reagent (Life Technologies). The eluate was filtered through an Ultrafree-MC GV 0.22 µm filter , and the RNA was purified according to the manufacturer's instructions and precipitated with 1 µL of Ethachinmate  in 50% isopropanol. The RNA was treated at 37°C for 30 min with RNase-free DNase  and purified by phenol–chloroform extraction and ethanol precipitation. The purified RNA clones were amplified by using a OneStep RT-PCR kit (Qiagen GmbH) in a 50 µL reaction mixture with 0.6 µM of primers (5′-CGGAAGACACGGCCGTTTATTACGCTG-3′ and 5′-CACTTAGCGGCCGCTTATCTACCGCTG-3′). The PCR conditions consisted of 20 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s. To obtain homologous dsDNA, excess primers (each 500 pmol) were added to the reaction mixture and incubated for 5 cycles at 55°C for 1 min and 72°C for 1 min following a prior denaturation at 98°C for 30 s. The dsDNA was collected by ethanol precipitation, treated with exonuclease I at 37°C for 18 h to digest excess primers, purified by phenol–chloroform extraction and ethanol precipitation, and solubilized in 10 µL of 10 mM Tris buffer (pH 7.0) containing 1 mM EDTA. The DNA (1 µL) was digested with PstI and NotI at 37°C for 7 h, purified by phenol–chloroform extraction and ethanol precipitation, and solubilized in 3 µL of 10 mM Tris buffer (pH 7.0) containing 1 mM EDTA. The purified DNA (1.5 µL) was inserted into PstI- and NotI-digested pHishot12 (0.3 pmol) using T4 DNA ligase in 50 µL of ligation buffer at 14°C for 18 h. The plasmid was purified by phenol–chloroform extraction, precipitated with 10 µg of tRNA by ethanol precipitation, and transferred into E. coli DH5α electrocompetent cells by electroporation (MicroPulser electroporator). The transformed E. coli was then used for the next round of Hishot display.600 reading reached 0.4–0.6. After incubation at 10°C for 30 min, the bacteria were cultured with 0.5 mM IPTG at 10°C for 18 h by continuous shaking. The cultures were centrifuged at 2,300× g for 20 min, and the supernatants were carefully removed by pipetting. The cells were lysed by freeze-thawing in 200 µL of 10 mM HEPES (pH 7.5) containing 0.5% Triton-X100, 1 mM EDTA, 13 U/mL benzonase nuclease , and 500 U/mL rLysozyme (Merck KGaA), and then 5 µL of 100 mM magnesium acetate was added. The mixture was then centrifuged at 2,300× g for 5 min, and the supernatants were screened by enzyme-linked immunosorbent assay (ELISA) as follows. Supernatants were diluted 3 times with 225 mM NaCl and incubated at 25°C for 2 h on an ELISA plate that had been precoated with 5 µg/mL of a target protein and blocked with 1% BSA. The plate was washed 3 times with Tris-buffered saline containing 0.1% Tween-20 and incubated at 25°C for 1 h with a horseradish peroxidase-conjugated anti-His-tag mAb  diluted 1∶3,000. The plate was washed 3 times with Tris-buffered saline containing 0.1% Tween-20 and incubated for 9 min with 1-Step Ultra TMB . The reaction was terminated by adding H2SO4, and the absorbance was then measured at 450 nm. Clones that bound nonspecifically to rabbit IgG were excluded.After 2 or 3 rounds of Hishot display, the clones were isolated by picking up colonies from an LB-agar plate and cultured in 500 µL of LB medium containing 100 µg/mL ampicillin in a 96-deep-well plate with continuous shaking until the ODE. coli DH5α cells transformed with the selected plasmid were cultured to an OD600 of 0.6 in 100 mL of LB medium containing 100 µg/mL ampicillin. Production of His-tagged surrogate scFv was then induced with 0.5 mM IPTG at 10°C for 24 h. The cells were collected by centrifugation at 6,000× g for 10 min, and lysed by freeze-thawing with 4 mL of 10 mM HEPES (pH 8.0) containing 0.5% Triton-X100, 1 mM EDTA, 13 U/mL benzonase nuclease, 500 U/mL rLysozyme, and a protease inhibitor (Complete Mini EDTA-free), and then 1 M magnesium acetate (20 µL) was added. After the lysate was incubated at 4°C for 30 min, the supernatant was collected by centrifugation at 14,000× g for 10 min and diluted with an equal volume of 10 mM HEPES (pH 8.0) containing 300 mM NaCl and 5 mM imidazole. The sample was applied to a Ni-NTA agarose column  equilibrated with HBS (pH 8.0) containing 20 mM imidazole. The column was washed with HBS (pH 8.0) containing 20 mM imidazole, and the His-tagged surrogate scFv was eluted with HBS (pH 8.0) containing 250 mM imidazole. The surrogate scFv was dialyzed against HBS, and its concentration was calculated from its coefficient of absorbance at 280 nm 5′-CGGAAGACACGGCCGTTTATTACGCTG-3′ and 5′-CTAGTAGCGGCCGCTGGAAACGGTAACCAGAGT-3′ (VIF) or 5′-AGGACACTGCAGTTTATTACTGTGCAGCGCGT-3′ and 5′-CTAGTAGCGGCCGCTGGAAACGGTAACCAGAGT-3′ (VISF). The PCR products were digested with PstI and NotI and inserted into VIF/pHint or VISF/pHint, respectively (The plasmid of the isolated clone was used as the template DNA. The random region of the clone was amplified by PCR using the following primers: ectively . The purHis-tagged peptides and shot47-tagged mRNA (Hishot complexes) can be formed in bacteria and then solubilized by bacterial lysis. After a target-binding Hishot complex has been isolated, its mRNA sequence can be amplified by RT-PCR and then cloned and sequenced to identify the target-binding peptide. We named this method the Hishot display as it uses Hishot complex.The RNA aptamer, shot47, binds strongly to the His-tag with a low picomolar dissociation constant H fragment at 3 different positions: pHishot1, in the 3′ UTR just after the stop codon and upstream of the terminator sequence; pHishot3, in the terminator sequence site in 3′ UTR; and pHishot4, in the 5′ UTR just after the transcription initiation site  with shot47 sequences in its 3′ untranslated region (UTR) was used . By affiion site . Althougion site , the higion site . Thereforesidues . Althougresidues , we wereresidues .H, the llama VHH fragment H through a peptide linker, (GGGGS)3. The surrogate scFv was more suitable than the other tested Ig fragments for obtaining a clone with good specificity and binding activity (data not shown). E. coli expression vector, pCold IV  at 4°C, they aggregated and precipitated from solution. To test the effect of fusion with the Fc region on storage stability, the clones were fused with the rabbit Fc region, and the Cys residues in VpreB and VH were reconstituted for formation of the disulfide bridge  most soluble peptides that can be produced by E. coli can be used as the base for target-recognizing peptides in a Hishot display, and the isolated peptide can, in principle, be largely purified; and (3) the method for selection of the target-recognizing peptide is not limited to panning, because the Hishot complex is small and stable; furthermore, it is possible to isolate clones of target-recognizing peptides efficiently, because many Hishot complexes can be formed and accumulated in bacteria.Hishot display has the following advantages, as follows: (1) it relies on basic molecular biological procedures using 8 CFU, which is comparable to that of B cells in mice On the other hand, one disadvantage is that there are fewer libraries available for Hishot display than that for phage or ribosome displays. However, a library with the titer of 10H and VL or a non-immunoglobulin protein Hishot display also has some additional problems that are still pending, which are difficulties common to all display methods. The reactivity of the isolated clone was strongly affected by the method of selection and screening. In particular, screening method affected the binding ability of clone, in that a clone that had a higher affinity in the target-coated ELISA did not bind specifically in the sandwich ELISA or with other methods for detection of target-binding (data not shown). Furthermore, the isolation efficiency for the target-binding clone was also influenced by various factors, such as the salt concentration of the buffer, the washing condition, and the procedure of selection . These problems might be caused by the inflexibility and instability of the base peptide containing random regions, rather than by any problems with the Hishot display. Although the surrogate scFv used in this study had relatively good specificity and binding activity, it was not able to isolate a strong binder that could work in multiple detection methods. On display methods that don't use animals, affinity maturation is often required to obtain a strong binder. The use of a carrier protein containing multiple random regions, such as a normal scFv containing both V26) is too large to screen in full, with the total weight of all peptides that could potentially be produced amounting to >300 kg. Since the number of clones that can be screened by Hishot or other display methods is <1/1013 only, it may be difficult to isolate specific clones reproducibly. Furthermore, many of the peptides in the selected pool that were not screened contained hydrophobic amino acids (data not shown). Although these homogeneous peptides would have been at a low concentrations overall in a lysate made from a pool of multiple clones, lysate from a single peptide clone would contain a high concentration of such peptides. In such cases, the peptides might aggregate with one another and become insoluble, therefore going undetected in the screening. To avoid possible aggregation and insolubilization of candidate peptides in the Hishot display, it would be necessary to surround the random peptide sequence with a hydrophilic carrier protein, such as a variable domain of the antibody. Proper selection of the hydrophilic carrier peptide may be important to ensure isolation of the target-recognizing peptides by Hishot display.Although target-binding peptides could be concentrated via the Hishot display by using random peptides , individFigure S1Sequences of genes inserted into the bacterial expression vector. The inserts are indicated as sequences between the Lac operator and terminator in pCold IV. The main restriction enzyme or shot47 sequences are underlined. The X in the amino acid sequences indicates an undetermined amino acid. V, N, or K in the DNA sequences indicates mixed bases as follows: V, C/G/A; N, T/C/A/G; K, T/G.(PDF)Click here for additional data file.Figure S2Sequences and structure of mammalian expression vectors. The structure of the pHint expression vector is shown as a schema. Sequences of the antibody gene in VIF/pHint and VISF/pHint are shown from the signal peptide of human intelectin-1 (hITLN1) to the stop codon. The main restriction enzyme or signal peptide sequences are underlined. The X in the amino acid sequences indicates an undetermined amino acid. V, N, or K of the DNA sequences indicates mixed bases as follows: V, C/G/A; N, T/C/A/G; K, T/G.(PDF)Click here for additional data file.


We hypothesized that higher doses would elicit more robust changes in functional and disease outcomes.We previously demonstrated that dietary vitamin D3 at 50xAI on functional outcomes  and disease severity , as well as disease onset, disease progression and lifespan in the transgenic G93A mouse model of ALS.To determine the effects of dietary vitamin Dad libitum with either an adequate  or high  vitamin D3 diet.Starting at age 25 d, 100 G93A mice  were provided HiD females consumed 9% less food corrected for body weight vs. AI females (P = 0.010). HiD mice had a 12% greater paw grip endurance over time between age 60–141 d (P = 0.015), and a 37% greater score during disease progression (P = 0.042) vs. AI mice. Although HiD females had a non-significant 31% greater CS prior to disease onset vs. AI females, they exhibited a significant 20% greater paw grip endurance AUC (P = 0.020) when corrected for clinical score.3 supplementation at 50x the adequate intake attenuated the decline in paw grip endurance, but did not influence age at disease onset, hindlimb paralysis or endpoint in the transgenic G93A mouse model of ALS. Furthermore, females may have reached the threshold for vitamin D3 toxicity as evidence by reduced food intake and greater disease severity prior to disease onset.Dietary D Amyotrophic lateral sclerosis  is a neurodegenerative/neuromuscular disease characterized by progressive degeneration of motor neurons in the central nervous system (CNS), resulting in muscle weakness followed by paralysis in vitro by 17% and 50% with 10 nM and 100 nM calcitriol, respectively) 3-1α-hydroxylase [1α(OH)ase] that converts the less active 25(OH)D3  into the fully active 1,25(OH)2D3  form of the vitamin  3 or its metabolites may attenuate multiple mechanisms involved in ALS pathology.Different dietary interventions have been studied in human and rodent models of ALS and are reviewed by Patel and Hamadeh 3 is the threat of toxicity as expressed by hypercalcemia 3 over 6 d allowed for 100% survival 3 wk post-injection, whereas 630 IU D3/d (∼27 IU D3/g b.wt./d) yielded 70% survival 3/d (∼9 IU D3/g b.wt./d) induced toxicity in male B10.PL mice 3 metabolites 3 and calcium showed no differences in body weights of supplemented mice compared to wild type controls at 12 wk, possibly due to the difference in murine strain 3 up to ∼9 IU D3/g b.wt./d, equivalent to ∼55 fold the adequate intake (AI), with a 0.5% calcium diet should be non-toxic in mice.The principal concern when supplementing with very high doses of vitamin D3 supplementation at 10x the AI attenuates the rapid disease-induced decline in paw grip endurance (7%) and motor performance (22%) vs. mice consuming the AI (1 IU/g feed) in the transgenic G93A mouse model of ALS 3 at 10x the AI does not affect these disease outcomes in this model. We wondered whether a higher dose of dietary vitamin D3 would elicit significant differences in disease outcomes, as well as more robust changes in functional outcomes. Hence, the objective of this study was to examine the effects of higher levels of vitamin D3 supplementation (50 IU/g feed) vs. the adequate intake (1 IU/g feed) on functional and disease outcomes, in the transgenic G93A mouse model of ALS.G93A mice transgenically overexpress the mutant human SOD1 gene and follow the same disease pattern as human ALS patients The experimental protocol in this study followed the guidelines of the Canadian Council of Animal Care and was approved by York University Animal Research Ethics Board (protocol # 2007-9). All necessary steps were taken to minimize suffering and distress to the mice in the study.3/g feed; Research Diet, New Brunswick, NJ). All animals were housed individually at age 25 d in a 12 h light/dark cycle.Male B6SJL-TgN(SOD1-G93A)1Gur hemizygous mice (No. 002726) were harem-bred with nonaffected female B6SJL control mice . The presence of the human-derived G93A transgene was confirmed using polymerase chain reaction (PCR) amplification of DNA extracted from ear tissue as outlined by Sigma-Aldrich . All breeding mice were housed 3 females per 1 male, and consumed Research Diet AIN-93G . At age 25 d, mice were housed in individual cages and divided into one of two groups: 1) adequate vitamin D3  and 2) high vitamin D3   between HiD and AI mice at age 113 d.One-hundred G93A mice  consumed a diet containing an adequate amount of vitamin Dick, NJ) . A subseWhen mice reached a CS of 3.0, food and calorie-free gel  were placed on the floor of the cage to fulfill the requirements of the ethics committee. The calorie-free gel contained synthetic polymers  and methanol (4.5% by weight). All measurements were conducted by two researchers who were blinded to the diets. The intra-tester coefficients of variation (CV) for researcher #1 were 1.8% for body condition, 0.0% for ability to move and 1.1% for clinical score, whereas for researcher #2 the intra-tester CV were 1.8% for body condition, 0.0% for ability to move and 1.2% for clinical score. The inter-researcher CV was 0.0% for body condition, 1.06% for ability to move and 0.96% for clinical score.Beginning at age 25 d, food intake and body weight were recorded twice per wk for all mice. When mice reached a clinical score of 3.0, body weight was recorded daily until endpoint.Beginning at age 60 d, BC was recorded twice per wk until mice reached a clinical score of 3.0, thereafter BC was recorded daily until endpoint. Body condition followed a 5-point scale: 5 =  obese mice, 4 =  over-conditioned mice , 3 =  well-conditioned mice , 2 =  under-conditioned mice , and 1 =  emaciated mice .Beginning at age 60 d, ATM was recorded twice per wk until mice reached a clinical score of 3.0, thereafter ATM was recorded daily until endpoint. Ability to move followed a 5-point scale: 4 =  normal mobility, 3 =  moving with limited use of the hindlimbs, 2 =  moving with the use of the forelimbs, 1 =  moving only for a short period with the use of the forelimbs, and 0 =  not moving.Beginning at age 60 d, PaGE was recorded 3 times every 10 d (measurements were separated by 2–3 days) until endpoint, using the modified hanging wire test Beginning at age 60 d, MP was recorded once every 10 d until endpoint using the rotarod test . Mice were placed on a rod  that rotated at a gradually increasing speed to 45 rpm over 60 s and remained at 45 rpm until the mouse could no longer stay on the rod. The rotarod apparatus was interfaced with a computer that initiated the test and recorded the competency score. Motion sensors located at the bottom of the rotarod chamber were activated when the mouse fell off the rod and, as a result, the computer ended the recording session. The test was performed in triplicate, with the highest score used for analysis.Beginning at age 60 d, CS was recorded daily until endpoint and followed an 8-point scale based on signs of weakness exhibited by the mice in order to establish disease severity: 0 =  no evidence of disease, 1 =  shaking or splaying of the hindlimbs when suspended by the tail (an indication of weakness in the hindlimbs), 1.5 =  weakness in one hindlimb (compensation for footdrop), 2 =  weakness in both hindlimbs , 2.5 =  extreme weakness in one hindlimb (inability to dorsiflex), 3 =  extreme weakness in both hindlimbs, 3.5 =  functional paralysis in one hindlimb, 4 =  functional paralysis in both hindlimbs, and 5 =  mouse cannot right itself within 20 s after being placed on its side  was used to determine significant sex differences over time in absolute food intake, food intake corrected for body weight, body weight, BC, ATM, PaGE, MP and CS. A two-way repeated measures ANOVA  was used to determine significant diet differences over time for BC, ATM, PaGE, MP and CS in three ways: 1) using data from the first day of testing (age 60 d) until the age at which the first group achieved a mean clinical score of 5 , 2) using data from the first day of testing (age 60 d) until the age at which the first group achieved a mean clinical score of 2  (i.e. prior to disease onset), and 3) using data from the age at which the first group achieved a mean clinical score of 2  until the age at which the first group achieved a mean clinical score of 5  (i.e. during disease progression). Statistical analysis using data for 60–105 d and 60–113 d with and without harvest mice were not different, therefore we reported results for 60–105 d without harvest mice. In order to achieve maximum statistical power, statistical analysis was performed including all mice when possible . When ANOVA indicated significance, a Newman-Keuls post-hoc test was used to determine the source of difference. A Student's t-test was used to determine significant differences in disease onset (CS2), hindlimb paralysis (CS4), endpoint (CS5), number of days between CS2 to CS5 (disease progression) and area under the curve (AUC) scores between the diets (within sex and when sexes were combined). Statistical analyses for AUC scores for BC, ATM, PaGE, MP and CS were conducted in three ways: 1) using data from first day of testing (age 60 d) until CS5 specific for each mouse, 2) using data from age 60 d until CS2 specific for each mouse (i.e. prior to disease onset), and 3) using data from CS2 until CS5 specific for each mouse (i.e. during disease progression). AUC scores for each mouse were corrected for the number of days between age 60 d and CS5 (first approach), between age 60 d and CS2 (second approach), or between CS2 and CS5 (third approach). Analysis of data was performed between age 60 d and CS5 to investigate the effect of the HiD diet over the entire study period, between age 60d and CS2 to investigate the effect of the HiD diet prior to disease onset, and between CS2 and CS5 to investigate the effect of the HiD diet during disease progression. A logrank test was used to determine differences in the rate at which the groups reached CS2, CS4 and CS5.A three-way repeated measures ANOVA  was also used to determine sex differences in the following correlations between BC, ATM, PaGE, and MP vs. CS; ATM, PaGE and MP vs. BC; PaGE and MP vs. ATM; as well as MP vs. PaGE. A two-way repeated measures ANOVA  was used for the above correlations to determine diet differences within sex or when sexes were combined. In addition, AUC for the above correlations were subjected to a two-way ANOVA (diet and sex) to determine sex differences, or a Student's t-test to determine diet differences within sex or when sexes were combined. Using data from mice followed to endpoint, correlations between the AUC for anthropometric (BC), functional  and disease  outcomes were conducted.tibialis anterior, quadriceps and brain weights. Body weight-adjusted tibialis anterior, quadriceps and brain weights were correlated with age at CS2 for each tissue harvest mouse. Group means for body weight-adjusted tibialis anterior, quadriceps and brain weights were also correlated with the group means for age at CS4, CS5 and disease progression for endpoint mice within the same diet and sex. A two-tailed test was used for all statistical comparisons for outcome measures except for BC, ATM, PaGE and MP where a one-tailed test was used, because based on the scientific literature we a priori hypothesized that high vitamin D3 would improve functional outcomes . Our lab has recently demonstrated that vitamin D3 supplementation at 10 fold the AI delays the decline in paw grip endurance and motor performance in the G93A mouse A Student's t-test was used to determine diet and sex differences in absolute and body weight-adjusted 3 intake of 3.37 IU/d (0.170 IU/g b.wt./d) (3 intake of 155.5 IU/d (8.11 IU/g b.wt./d). Males (3.33±0.05 g/d) consumed 8% more absolute food vs. females .Food intake was not significantly different between the diets . AI miceb.wt./d) . HiD micBody weight was not significantly different between the diets . AI miceBC was not significantly different between the diets . BetweenATM was not significantly different between the diets . BetweenBetween age 60–141 d, HiD mice had a 12% greater PaGE vs. AI mice, which was driven by HiD males having a 13% greater PaGE vs. AI males (P = 0.032) . Prior tMP was not significantly different between the diets or the sexes over time or for AUC . Over tiPrior to disease onset (age 60–105 d), HiD females had a non-significant 31% higher CS vs. AI females . In contBC, ATM and MP when corrected for CS, were not significantly different between the diets, although HiD males trended toward a 5% greater BC AUC vs. AI males (P = 0.065) . HoweverCS2 was not significantly different between the diets . BetweenNeither CS4 nor CS5 was significantly different between the diets . BetweenDisease progression was not significantly different between the diets or the sexes  and 6.tibialis anterior weights were not significantly different between the diets or the sexes . Body weight-adjusted tibialis anterior weights positively correlated with age at CS2 for both AI (P<0.001) and HiD (P = 0.090) mice (Body weight-adjusted he sexes . Between90) mice .quadriceps weights were not significantly different between the diets or the sexes . Body weight-adjusted quadriceps weights positively correlated with age at CS2 for AI mice (P<0.001), but not for HiD mice  is supported by our previous study in the same G93A mouse model of ALS 3 supplementation at 50 IU/g feed in the current study did not delay the age at disease onset and hindlimb paralysis, or prolong lifespan vs. AI mice, possibly due to tissue-specific effects of vitamin D in the CNS vs. skeletal muscle. A number of different interventions have demonstrated the effectiveness of calcitriol in the CNS Despite improvements in PaGE, vitamin DIn vivo, both a single oral dose of 400 IU vitamin D3 and intravenous injection of 0.4 µg calcidiol in vitamin D3-deficient rats significantly increased muscle leucine incorporation by 33% and 26% at 7 h and 4 h for vitamin D3 and calcidiol, respectively, compared to untreated controls 3 administration, demonstrating a direct role of calcidiol independent of calcitriol in muscle function in vitro rat epitrochlear muscle had greater leucine incorporation and ATP content in a medium containing 50 nmol/L calcidiol, but not 52,000 nmol/L vitamin D3 or 1.2 nmol/L calcitriol, vs. untreated muscle 3's action in skeletal muscle is conditional upon its conversion to calcidiol, and that calcidiol is the active vitamin D metabolite in skeletal muscle. Indeed, vitamin D3 supplementation rescues growth retardation observed in CYP27B1 knockout mice  Alternatively, evidence suggests that calcidiol possesses functions in muscle tissue independent of calcitriol. 3 toxicity as indicated by a significantly reduced food intake (absolute and corrected for body weight) and 31% greater disease severity prior to disease onset. Their vitamin D3 intake is equivalent to 40,582 IU/d for an 80 kg man and 39,049 IU/d for a 70 kg woman. The observed signs of toxicity in HiD females could be explained by sex differences in vitamin D metabolism. In B10.PL mice supplemented orally with 40 IU vitamin D3/d, females exhibited a ∼40% and ∼37% greater serum calcidiol at 38 d and 83 d vs. supplemented males, respectively, despite similar baseline serum calcidiol 3 and estrogen: estrogen enhances vitamin D function by increasing net calcitriol concentration via estrogen receptor-mediated down-regulation of CYP24A1 transcription. Estrogen increases vitamin D potency via the up-regulation of VDR, and, in turn, calcitriol enhances endogenous estrogen synthesis via VDR-mediated up-regulation of estrogen synthase In the current study, females consuming the HiD diet (50 IU/g feed) may have reached vitamin D3 toxicity, HiD females had greater PaGE compared to their AI female counterparts. Vitamin D3 toxicity may have been a result of high CNS calcitriol. In limited evidence, female mice consuming 40 IU vitamin D3/d (∼3.0 IU/g b.wt./d) had spinal cord calcitriol of ∼125 fmol/g 3/d (8.6 IU/g b.wt./d) likely resulting in supraphysiological levels of spinal cord calcitriol concentrations, inducing mild toxicity as evidenced by greater disease severity prior to disease onset and decreased food intake. However, considering that skeletal muscle is unable to perform the calcidiol-to-calcitriol conversion Taken together, the above data could explain the apparent paradox observed in the HiD G93A females in the current study: despite mild signs of vitamin D3 supplementation above the AI attenuates the decline in PaGE, but does not delay the age at disease onset, hindlimb paralysis or endpoint in the G93A mouse model of ALS.In closing, the current study confirms our previous observation that dietary vitamin DFigure S1Food intake, food intake corrected for body weight, body weight and body condition over time. A) Food intake (g), B) food intake corrected for body weight (mg/g b.wt.), C) body weight (g) for 54 adequate intake  and 46 high  vitamin D3 G93A mice, and D) body condition for 31 adequate intake  and 28 high  vitamin D3 G93A mice. A and B) HiD females consumed 10% less food (P = 0.008) and 9% less food corrected for body weight (P = 0.010) vs. AI females. C and D) There were no significant differences between the diets for body weight or body condition. Data are means ± SEM.(TIF)Click here for additional data file.Figure S2Relationship between functional outcomes and clinical score. A) Body condition vs. clinical score, B) ability to move vs. clinical score, C) paw grip endurance vs. clinical score and D) motor performance vs. clinical score for 31 adequate intake  and 28 high  vitamin D3 G93A mice. A) Corrected for clinical score, HiD males had a 5% greater body condition AUC vs. AI males (P = 0.065). B and D) Ability to move and motor performance vs. clinical score were not significantly different between the diets. C) Corrected for clinical score, HiD mice had a 13% greater paw grip endurance AUC vs. AI mice, mainly driven by a 20% greater AUC in HiD females vs. AI females. Data are means ± SEM.(TIFF)Click here for additional data file.Figure S3Relation between ability to move AUC and motor performance AUC vs. clinical score AUC. A) Ability to move AUC and B) motor performance AUC vs. clinical score AUC between CS2 – CS5 (during disease progression) for 31 adequate intake  and 28 high  vitamin D3 G93A mice. A) During disease progression, ability to move AUC negatively correlated with CS AUC for both AI  and HiD  mice. For AI mice: ATM AUCCS2 – CS5  =  (6.452±0.16) + [(−1.17±0.06) × (CS AUCCS2 – CS5)]. For HiD mice: ATM AUCCS2 – CS5  =  (6.06±0.18) + [(−1.02±0.07) × (CS AUCCS2 – CS5)]. B) During disease progression, motor performance AUC negatively correlated with CS AUC for both AI  and HiD  mice. For AI mice: MP AUCCS2 – CS5  =  (34.52±9.14) + [(−7.61±3.44) × (CS AUCCS2 – CS5)]. For HiD: MP AUCCS2 – CS5  =  (34.24±11.34) + [(−7.46±4.19) × (CS AUCCS2 – CS5)].(TIFF)Click here for additional data file.Figure S4Relation between paw grip endurance and clinical score area under the curve (AUC). A) Paw grip endurance AUC vs. clinical score AUC between age 60 d – CS5 for 18 adequate intake  and 15 high  vitamin D3 G93A male mice. B) Paw grip endurance AUC vs. clinical score AUC between CS2 – CS5 (during disease progression) for 13 adequate intake  and 13 high  vitamin D3 G93A female mice. A) Between age 60 d – CS5, HiD males  had an 18% greater PaGE AUC elevation (P = 0.082) vs. AI males . For AI males: PaGE AUC60 d – CS5  =  (126.4±33.64) + [(−8.33±23.84)) × (CS AUC60 d – CS5)]. For HiD males: PaGE AUC60 d – CS5  =  (128.6±30.68) + [(−0.1043±23.35)) × (CS AUC60 d – CS5)]. B) During disease progression, HiD females  had a 59% greater PaGE AUC elevation (P = 0.008) vs. AI females . For AI females: PaGE AUCCS2 – CS5  =  (88.79±72.00) + [(−14.98±26.84) × (CS AUCCS2 – CS5)]. For HiD females: PaGE AUCCS2 – CS5  =  (160.1±93.10) + [(−30.99±34.72) × (CS AUCCS2 – CS5)].(TIFF)Click here for additional data file.Figure S5Relation between body weight-adjusted tibialis anterior and quadriceps vs. disease onset (CS2). A) Tibialis anterior weight (mg/g b.wt.) vs. age at CS2 and B) quadriceps weight (mg/g b.wt.) vs. age at CS2 for 23 adequate intake  and 18 high  vitamin D3 G93A mice. A) Tibialis anterior weight (mg/g b.wt) positively correlated with age at CS2 for both AI  and HiD  mice. For AI mice: age at CS2 (d)  =  (62.47±7.67) + [(10.05±2.49) × )]. For HiD mice: age at CS2 (d)  =  (65.68±14.94) + [(8.48±4.70) × )]. B) Quadriceps weight (mg/g b.wt.) positively correlated with age at CS2 for AI  but not for HiD  mice. For AI mice: age at CS2 (d)  =  (63.74±7.36) + [(2.74±0.68) × (quadriceps weight (mg/g b.wt.))]. For HiD mice: age at CS2 (d)  =  (78.90±14.51) + [(1.27±1.35) × (quadriceps weight (mg/g b.wt.))].(TIFF)Click here for additional data file.Figure S6Group body weight-adjusted brain weights vs. group age at hindlimb paralysis (CS4) and endpoint (CS5). Group mean brain weights (mg/g b.wt.) for 23 adequate intake  and 18 high  vitamin D3 G93A mice vs. mean group age at A) CS4 and B) CS5 for 31 adequate intake  and 28 high  vitamin D3 G93A mice. A) Body weight-adjusted brain weights positively correlated with age at CS4 . Age at CS4 (d)  =  (94.55±3.90) + [(1.64±0.199) × (brain weights (mg/g b.wt.))]. B) A) Body weight-adjusted brain weights positively correlated with age at CS5 . Age at CS5 (d)  =  (95.47±4.75) + [(1.79±0.24) × (brain weights (mg/g b.wt.))]. Data are means ± SEM.(TIFF)Click here for additional data file.Results S1(DOC)Click here for additional data file.


In solid organ transplant (SOT) recipients it is unknown if natural infection with influenza confers protection from re-infection with the same strain during the next influenza season. The purpose of this study was to determine if infection with pandemic influenza A/H1N1 (pH1N1) resulted in a long-term immunologic response. Transplant recipients with microbiologically proven pH1N1 infection in 2009/2010 underwent humoral and cell-mediated immunity (CMI) testing for pH1N1 just prior to the next influenza season. Concurrent testing for A/Brisbane/59/2007 was done to rule-out cross-reacting antibody. We enrolled 22 adult transplant patients after pH1N1 infection. Follow up testing was done at a median of 7.4 months (range 5.8–15.4) after infection. After excluding those with cross-reactive antibody, 7/19 (36.8%) patients were seroprotected. Detectable pH1N1-specific CD4+ and CD8+ interferon-γ producing T-cells were found in 11/22 (50%) and 8/22 (36.4%) patients respectively. Humoral immunity had a significant correlation with a CD4 response. This is the first study in transplant patients to evaluate long-term humoral and cellular response after natural influenza infection. We show that a substantial proportion of SOT recipients with previous pH1N1 infection lack long-term humoral and cellular immune responses to pH1N1. These patients most likely are at risk for re-infection. Pandemic influenza A/H1N1 (pH1N1) caused widespread infection in 2009 and early 2010 creating a spectrum of disease in organ transplant recipients with a mortality rate of up to 7.8% It is also unknown if organ transplant recipients are able to produce similar humoral and cellular responses to pH1N1 infection compared to immunocompetent persons. Equally, it is unknown whether transplant recipients that recover from influenza infection retain a long-term humoral response or have a robust cellular response if rechallenged with the same viral subtype. Seasonal influenza vaccine responses in transplant recipients are known to be suboptimal. Monovalent pandemic vaccine responses in transplant recipients have been shown to be similarly low This study was approved by the institutional Ethics Review Board. All patients provided informed consent. Adult organ transplant recipients seen at the University of Alberta Hospital, Edmonton, were prospectively enrolled in the study if they had microbiologically proven pH1N1 during 2009–2010. All influenza A positive specimens were confirmed as pH1N1 by PCR. Serum and peripheral blood mononuclear cells (PBMCs) were collected from each patient prior to the onset of the next influenza season (2010–2011 season). Clinical information collected included demographic data, hospitalization due to the original pH1N1 infection, treatment of infection, and type of immunosuppression.2, counted and adjusted to a concentration of 1×106 cells per 400 µL. The samples were then stimulated for 24 hours with live pH1N1 virus  using a multiplicity of infection (MOI) of 0.5 and 1×106 cells per reaction. Anti-CD3 (10 µg/mL) was used as the positive control; media alone served as the negative control. Brefeldin A was added 2 hours after stimulation to halt further interferonγ (IFNγ) secretion. Using fluorescent monoclonal antibodies , the cells were surface stained for CD4 and CD8 with anti-human CD4 (PE-Cy7) and anti-human CD8 (APC-eFluor 780) respectively. After fixation, PBMCs were stained for intracellular interferonγ with PE. The percentage of pH1N1-specific CD4+ and CD8+ T cells producing interferonγ was determined using flow cytometry Serum and PBMCs were collected from transplant recipients with previous pH1N1 infection. Sera were stored at −80°C and underwent a hemagglutination inhibition assay (HAI) at the Ontario Agency for Health Protection and Promotion, Toronto, Ontario using a previously described method For the humoral response post-infection, seroprotection was defined as a strain-specific antibody titer ≥1∶40 Statistical analysis was performed using SPSS  and GraphPad Prism version 4.0. A univariate analysis was performed to determine variables affecting humoral and cellular responses. Correlations between humoral and cellular immunity were assessed using linear regression.During 2009–2010, 25 adult solid organ transplant recipients had microbiologically proven pH1N1 at our center. Three patients were lost to follow-up or did not provide consent for blood testing. Therefore, we were able to enroll 22 adult patients for follow-up blood prior to the 2010–2011 influenza season. Baseline characteristics of the cohort are shown in The median time from natural infection to assessment of humoral immune resonse was 7.4 months (range 5.8 to 15.4). Of the cohort, 10/22 (45.5%) patients met the criteria for long-term seroprotection to pH1N1 (≥1∶40 antibody titer). In 5/22 cases, patients also had cross-reactive antibody to A/Brisbane. In three cases, pH1N1 antibody titers did not exceed A/Brisbane titers by ≥4-fold; therefore, these results were considered indeterminate and patients were excluded from further analysis. Therefore, 7/19 (36.8%) patients were confirmed seroprotected for pH1N1. In these 7 patients, the median titer was 1∶160 (range 1∶40 to 1∶640). Patients hospitalized for pH1N1 were more likely to have a long-term seroresponse 4/5 (80%) than those not admitted to hospital 3/14 (21.4%); p = 0.038. Thoracic (heart and lung) transplant recipients had a trend towards a lack of long-term humoral response to pH1N1 infection compared to other SOT recipients . Shorter time to follow up of serologic measurement after infection was associated with a higher likelihood of seroprotection . For patients who were measured between 5–8 months post-infection, the seropositivity rate was 50% (6/12); for those measured between 8–12 months (seropositivity was 0% (0/5)) and for those between 13–15 months (seropositivity was 50% (1/2)). Factors that were not associated with a sustained humoral response included time from transplant, type of immunosuppression or calcineurin-inhibitor levels, early versus late antiviral treatment, presence or absence of lymphopenia and history of previous seasonal influenza vaccination. Of the three patients that were also immunized for pH1N1, only one had sustained seroprotection.Similar to the antibody assessment, the median time from infection to assessment of CMI was 7.4 months. All 22 patient PBMCs underwent flow cytometry analysis for pH1N1-specific IFNγ production from CD4+ and CD8+ T cells. The range of pH1N1 specific T cell frequencies is shown in Overall, 11/22 (50%) and 8/22 (36.4%) patients had a positive pH1N1-specific CD4+ and CD8+ T cell response respectively. A CD4+ IFNγ response was present in 3/7 (42.9%) of seroprotected patients but also 7/12 (58.3%) patients who had negative serology (p = NS). Similarly, a CD8+ IFNγ response was present in 6/7 (85.7%) of seroprotected patients and 9/12 (75%) of patients with negative serology (p = NS). Four of 19 patients (21.1%) were negative for all three immunological parameters. The three patients with indeterminate serology (i.e. cross-reactive antibody to A/Brisbane) had a CMI response in 1 of 3 patients for CD4 and 2 of 3 patients for CD8. Of the three patients that were immunized for pH1N1, only one had a persisting CD4+IFNγ and CD8+IFNγ response at the time of follow-up.2 = 0.428, p = 0.002 . No other correlations with immunosuppression were observed. There was no significant correlation between CD4+IFNγ or CD8+IFNγ frequency of T cell specific cells and the presence of lymphopenia, time from transplant to infection or the use of early antiviral therapy. In addition, hospitalization at the time of natural infection was not predictive of a T-cell response.The CD4+ T cell response had a significant association with pH1N1 antibody titer; R = 0.002 . TacroliWe determined the long-term humoral and cellular immune responses in 22 adult transplant patients who recovered from pH1N1 infection. We found that only approximately one-third of our cohort had a sustained humoral response. Cellular responses were present in a similar proportion of patients (50% CD4 and 36.4% CD8) and a significant proportion of patients had an absence of both humoral and cellular  responses (21.1%). Our results suggest that even after natural pH1N1 infection a substantial portion of transplant patients would be at risk for re-infection with the same strain in subsequent influenza seasons. To our knowledge, this is the first study examining the long-term responses to natural influenza infection in solid organ transplant recipients.Another study looked at humoral and cellular immunity to pH1N1 in patients following hematopoietic stem cell transplantation. Only short-term immunological responses were evaluated (within 12 weeks of infection); the authors found that only 6/11 (54.5%) patients had a humoral response compared to 100% of controls and that H1N1-specific T cells could be measured in only 2/8 (25%) patients compared to 4/4 in the control group et al. IL-8, IFNγ, IL-13, IL-10 were significantly higher in hospitalized patients vs. outpatients and controls. While both critical and non-critically hospitalized patients showed higher levels of IL-17 and TNFα than controls, only severe critically ill patients had significantly elevated levels of IL-17 and TNFα Cellular immune responses to pandemic H1N1 infection have previously been described in immunocompetent patients by Bermejo-Martin We also found that the ten heart and lung transplant recipients tended to have lower cellular responses to influenza infection compared with other types of transplant. This may reflect a higher net state of immunosuppression and is similar to that seen in influenza vaccine studies that suggest poor vaccine immunogenicity in lung transplant recipients compared to kidney recipients One limitation of our study is the small number of patients tested; to carry out similar studies in larger number of transplant patients with natural influenza infection, a multicenter approach would likely be needed. We did not do pre-infection testing or testing at earlier time points post-infection; however, similar to seroprevalence studies, we did serology for A/Brisbane to rule out cross-reactive H1N1 antibodies. We were also able to show diminished long-term response which is important when considering a revaccination strategy for subsequent annual vaccination. However, our findings represent an immune response to the infecting strain and not to significantly shifted influenza strains.Although the decline in humoral immunity shown in our study is similar to that shown by Wang et al. in the immunocompetent population, we believe this is of significant concern in the immunocompromised transplant patient. From previous literature, transplant patients are more likely to have a poor outcome from influenza infection and more likely to have greater tissue viral loads and prolonged shedding times


Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga  Chlamydomonas reinhardtii serves as a model organism for studying various aspects of cell biology BSA: 10 mg/ml Glycogen: 2.5 μg/μl Glycine: 1 MPhenol/chloroform/isoamylalcohol (25:24:1)Chloroform/isoamylalcohol (24:1)TE buffer containing 20 μg/μl boiled RNase AProteinase K: 10 mg/ml Na-acetate: 3 M NaCl: 5 M2x DNA loading dye: 15% Ficoll-400EDTA: 5 M (pH 8.0)Tris-HCl: 1 M (pH 8.0)Chlamydomonas cells in 400 ml TAP medium to a density of 4-8 × 106 cells/ml.1. Grow 9 cells in GSA tubes by centrifuging for 3 min at 3220 × g and 24°C [4°C if cells are not subjected to further treatments].2. Harvest 103. .4. Discard supernatant and resuspend cells.5. Add 10 ml of Crosslinking solution, mix gently for 10 min at 24°C .6. Add 1.25 ml of 1 M glycine, agitate for 5 min at 24°C (quenching).7. Centrifuge for 2 min at 3220 × g and 4°C, discard supernatant.8. Resuspend cells in 1 ml KH buffer and transfer them into a 2-ml microcentrifuge tube.9. Centrifuge for 2 min at 16, 100 × g and 4°C, discard supernatant.10. Add 400 μl Lysis buffer; after this step cells can be stored at -80°C for several months..11. Sonicate on ice with 55% output control, 60% duty cycle. To achieve an average DNA fragment size of ~200 bp, sonicate 20 times 20 sec with breaks of 20 sec between each sonication cycle (avoid frothing!). The sonicator tip needs to be as close as possible to the bottom of the tube.12. Transfer sonicated cell lysates to 15-ml Falcon tubes, add Lysis buffer to a total volume of 5 ml and mix carefully.13. Centrifuge for 2 min at 3220 × g and 4°C to remove cell debris.7cells). The input chromatin can be stored for several months at -80°C, but avoid multiple freeze/thaw cycles.14. Aliquot 50 × 100 μl .15. Take one 100- μl aliquot and add 400 μl of Lysis buffer and 50 μl of 5 M NaCl.16. Incubate over night at 65°C to reverse crosslinks.17. Extract twice with 500 μl phenol/chloroform/isoamylalcohol.18. Extract once with 500 μl chloroform/isoamylalcohol.19. Precipitate nucleic acids by adding 55 μl of 3 M Na-acetate (pH 5.2), 1 ml of 100% EtOH and incubating for 3 h at -20°C.20. Centrifuge for 15 min at 16, 100 × g and 4°C.21. Wash pellet with 800 μl of 70% EtOH (take care not to remove the tiny pellet).22. Centrifuge for 10 min at 16, 100 × g and 4°C.23. Dry pellet and resuspend it in 10 μl TE buffer containing 20 μg/μl RNase A.24. Incubate for 1 h at 37°C.25. Add 10 μl of 2 × DNA loading dye and separate sheared DNA on a 1.5% agarose gel. DNA fragments should have sizes of ~200 bp.26. Weigh-in 50 mg of Protein-A-Sepharose beads , resuspend beads in 1 ml ChIP-buffer and incubate them for 30 min at 4°C.27. Wash beads two times with 500 μl ChIP buffersoni (gives ~750 μl suspension with swollen beads).28. Discard supernatant and add 250 μl ChIP-buffer and 250 μl λDNAsoni solution and incubate for at least 30 min on ice).29. Prepare antibody solutions .31. Add 900 μl of ChIP buffer to each aliquot (from this step on consequently use stuffed tips to avoid DNA contaminations!).32. Centrifuge for 20 sec at 16, 100 × g and 4°C, and transfer supernatant to microcentrifuge tubes containing the prepared antibody solutions.33. Mix on a rotation wheel for 1 h at 4°C.34. Centrifuge for 20 sec at 16, 100 × g and 4°C, and transfer each supernatant to microcentrifuge tubes containing 60 μl sepharose beads.35. Mix on a rotation wheel for 2 h at 4°C.36. Centrifuge for 20 sec at 16, 100 × g and 4°C.37. Discard supernatant.38. Wash the beads once with 1 ml each of Washing buffer 1, 2 and 3, and twice with 1 ml TE buffer (use washing solutions that are stored on ice and centrifuge at 4°C).39. Elute precipitated chromatin with 250 μl Elution buffer for 15 min at 65°C.40. Repeat elution with another 250 μl Elution buffer and pool eluates.41. Add 50 μl of 5 M NaCl and incubate overnight at 65°C.42. To remove proteins in the precipitates add 10 μl 0.5 M EDTA (pH 8.0), 20 μl 1 M Tris-HCl (pH 8.0) and 2.1 μl proteinase K (10 mg/ml) and incubate for 1 h at 55°C.43. Extract once with 500 μl phenol/chloroform/isoamylalcohol.44. Extract once with 500 μl chloroform/isoamylalcohol.45. Precipitate DNA by adding 50 μl 3 M Na-acetate (pH 5.2), 2.5 μl glycogen (2.5 μg/μl) and 1 ml 100% EtOH and incubating for 3 h at -20°C.46. Centrifuge for 20 min at 16, 100 × g and 4°C.47. Wash DNA pellet with 500 μl 70% EtOH (take care not to remove the tiny pellet).48. Centrifuge for 15 min at 16, 100 g and 4°C.49. Dry DNA pellet in a clean bench, resuspend in 200 μl TE (pH 8.0).50. Use 5 μl (1:40) of the precipitate for qPCR analysis .2, 1 M betaine, 200 μM dNTPs, 2.5 U Taq polymerase (NEB), and 0.6 μM each of forward and reverse primers .Endpoint PCRs were carried out in 50- μl reactions containing 1 × Taq buffer (Fermentas), 5 μl chromatin immunoprecipitate, 1.5 mM MgClqPCR was performed using the StepOnePlus System (Applied Biosystems) and the Maxima SYBR Green kit from Fermentas. Each reaction contained the vendor's master mix, 200 nM of each primer  [As pointed out previously, the conditions used for the crosslinking of DNA with proteins are crucial for ChIP analyses and need to be optimized for each individual organism ,23,25. Ialdehyde . Followialdehyde -26 and 5otocols) .Chlamydomonas, we used as fixed parameters 2 × 107 cells as starting material, a crosslinking time of 10 min, and a DNA fragment size of 1000-3000 bp , or where the antibodies used are in large excess (e.g. when transcription factors or rare histone modifications are investigated). In the former case there should be a linear relationship between the amount of antibodies applied and antigen precipitated. In the latter case the amount of antigen precipitated under the respective condition should be constant and independent of the amount of antibodies used. To ensure that these premises are fulfilled it is important to titrate the antibodies used. Titration is also advisable to reduce costs, as commercially available antibodies against histones and histone modifications are expensive. Moreover, antibody titers may vary from batch to batch. Ideally, if the antigen is in excess, the amount of antibodies used should be adjusted such that robust signal intensities are obtained compared to a mock control.HSP70A, RBCS2 and CYC6 relative to input DNA was quantified by real-time PCR (qPCR). As shown in Figure CYC6 promoter, intermediate at RBCS2 and lowest at HSP70A, hence corroborating our earlier findings [To give an example for a proper antibody titration, we performed ChIP using different dilutions of antibodies directed against the unmodified C-terminus of histone H3 and employed affinity-purified antibodies against vesicle-inducing protein in plastids 2 (VIPP2) as mock control. The amount of precipitated chromatin from promoters findings .The advantages and disadvantages of different normalization approaches to handle qPCR data from ChIP experiments have been thoroughly discussed by Haring et al. . We will1. No matter whether antibodies against core histones, histone modifications or transcription factors are used for ChIP, the qPCR signals from ChIPs using specific antibodies need to be significantly stronger than those from ChIPs using mock controls. Of course, the investigated antigen must be present at the target site investigated. The choice of a proper control antibody depends on the nature of the specific antibody used. For example, if the specific antibody is affinity-purified (like that against the C-terminus of histone H3), the control antibody-targeting an unrelated antigen-should be affinity-purified as well (like that against VIPP2).2. Determine nucleosome occupancy and/or histone modifications at the locus of interest by normalizing qPCR signals from ChIPs with antibodies against a core histone and/or specific histone modifications with the qPCR signal from input chromatin (%IP). This eliminates variations in PCR efficiency for the respective locus and makes comparisons between different loci (and different PCR runs) possible.3. Express nucleosome occupancy and/or histone modifications determined for the locus of interest relative to that of a control locus known to contain nucleosomes and/or the histone modifications under investigation . As values for target and control loci are obtained from the same ChIP experiment, technical variations within that experiment are eliminated. This procedure also allows integrating biological replicates where for example antibody batches with varying efficiencies were used.4. As nucleosome occupancy may vary considerably between different loci , histone modifications generally need to be expressed relative to nucleosome occupancy.Chlamydomonas, it is important to keep in mind that the amino acid sequences of Chlamydomonas histones are not entirely conserved with those of mammals, against which many commercially available antibodies for the detection of histone modifications are directed. For instance, Chlamydomonas histone H3 contains a threonine instead of a serine at position 28 and the alanine at position 29 found in mammalian histone H3 is deleted in the Chlamydomonas protein. As a consequence, commercially available antibodies against methyl-H3K27 are not functional in Chlamydomonas [When investigating histone modifications in Chlamydomonas RBCS2 promoter by ~2- and ~6-fold, respectively, which correlated with a ~20% increase in nucleosome occupancy [Drosophila [Chlamydomonas HSP22F promoter occurred within the first minute after onset of heat stress, right after binding of HSF1 to the promoter [RBCS2 promoter during heat stress occurred as fast as HSF1-dependent remodelling at HSP22F. To address this question, we employed ChIP to monitor nucleosome occupancy and levels of H3 and H4 acetylation at the RBCS2 promoter during the first 10 min of heat stress.We have shown previously that heat stress caused reductions in levels of H3 and H4 acetylation at the ccupancy . Interesosophila . We coulpromoter . We wondCYC6 control promoter  qPCR signals from precipitates generated with specific antibodies were much stronger than those obtained from precipitates generated with mock antibodies (not shown). (2) qPCR signals obtained with antibodies against histone H3, di-acetylated H3 and tetra-acetylated H4 were first normalized to those gained with 10% input DNA and (3) subsequently normalized to the respective values obtained for the t stress ). (4) qPRBCS2 promoter was observed 2 min after onset of heat stress. Trends towards reduced levels of H3 acetylation and increased histone occupancy were also discernible after about 2 min of heat stress. Hence, HSF1-independent nucleosome remodeling at the RBCS2 promoter after heat stress is fast (within 2 min), but not as fast as HSF1-dependent nucleosome remodeling at the HSP22F promoter (within 1 min).As shown in Figure Chlamydomonas. We demonstrate the optimization of the most important parameters, which are the crosslinking conditions, the shearing of chromatin, the titration of antibodies and the normalization of results obtained by qPCR. Compared to earlier ChIP protocols described for Chlamydomonas [RBCS2 promoter rapidly decrease and nucleosome occupancy rapidly increases we provide an example for the suitability of our ChIP protocol for monitoring changes of chromatin structure in a minutes scale. Our protocol will prove useful for targeted and genome-wide studies of chromatin structure in Chlamydomonas and other eukaryotic microalgae.We describe here a detailed chromatin immunoprecipitation protocol for ydomonas ,6,9, ourydomonas . By demoThe authors declare that they have no competing interests.DS participated in the design of the study, carried out all experiments and drafted the manuscript. SS participated in the design of the study and helped with data evaluation. MS conceived and coordinated the study, and wrote the manuscript. All authors read and approved the final manuscript.


We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using  Epigenetic mechanisms are known to maintain long-lasting gene expression programs. These mechanisms involve several levels of regulation, including chemical modification of the DNA molecule by adding methyl groups at specific positions, often involving the dinucleotide sequence CpG . Such moSeveral lines of evidence are consistent with the hypothesis that epilepsy might be mediated by epigenetic processes -12. A pogria2, the gene coding the GluA2 subunit of the AMPA receptor. Evidence indicates that the presence of GluA2 subunit (encoded by gria2) in the heteromeric AMPA receptors impermeabilizes it to calcium Epigentek Bisulfite Kits (Qiagen) were used for bisulfite conversion of DNA as described in the manufacturer’s manual. 1µg of genomic DNA was used for a single-conversion reaction. Samples were prepared by performing nested PCR with one of the nested primers carrying a 5’ biotin modification. Primers used are listed in All primers designed against bisulfite-converted DNA using either MethPrimer online software  or pyrom® DNA SYBRGreen I master mix . Primers were purchased from IDT Technologies and are listed in gria2 mRNA expression, Taqman Multiplex real-time PCR was carried out using an ABI prism 7000 sequence detector , with 2 µL cDNA, 18 µm each primer, 5 µm probe, and Universal TaqMan 2 × PCR Mastermix (Applied Biosystems) to a final volume of 25 µL. All samples were run in triplicate. Primers and minor groove binder (MGB) TaqMan probes for gria2 (Rn00568514_m1) and gapdh (Rn017775763) were designed by Applied Biosystems (Assay-on-Demand) to avoid genomic amplification. The thermal cycling conditions used during the PCR were: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. gria2 mRNA levels were normalized to gapdh and the values were calculated relative to untreated controls.Mouse mRNA expression levels were quantified by real-time quantitative PCR on a Roche LightCycler® 480 Real-Time PCR System using LightCycler480Organotypic hippocampal slice cultures were fixed in 0.1 M phosphate buffer  with 4% paraformaldehyde overnight at 4 °C. Slices were then washed several times and dehydrated for 10 minutes in 30%, 50%, 70% ethanol and then stained with 0.5% cresyl violet. Further dehydration was performed in 90% and 100% ethanol and 45 minutes in 100% xylene. Nissl-stained slices were mounted using Permount mounting medium (Fisher Scientific) and imaged with an upright Zeiss Axioplan 2 microscope equipped with a 2.5x objective and a Zeiss Axiocam high-resolution color digital camera (Carl Zeiss AG).2, 2 mM MgCl2, 11.6 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM Glucose (pH 7.4). To determine excitability, whole-cell patch clamp recordings were performed in current-clamp mode from CA1 neurons using borosilicate glass pipettes . All recordings were made using an Axopatch 200A amplifier . Patch pipettes were filled with intracellular solution containing: 120 mM K-Gluconate, 1 mM EGTA, 10 mM HEPES, 5 mM Mg-ATP, 0.5 mM Na-GTP, 5mM NaCl, 5mM KCl, 10mM Phosphocreatine, and pH was adjusted to 7.3 with KOH. Cells that did not survive the entire recording were excluded from the final analysis. Spikes were detected offline and 3 or more spikes were grouped into bursts if the inter-spike interval was smaller than 600 ms.Slice cultures were inserted into a temperature-controlled chamber (30°C) mounted on an upright microscope  and continuously perfused with Tyrode solution containing: 137 mM NaCl, 2.7 mM KCl, 2.5mM CaClHippocampal slice cultures were tested for cell death. Briefly, slices were incubated with 5 µg/ml of propidium iodide (PI)  and imaged . Incubation of mouse hippocampus culture-slices in 15.7M KCl for 5 minutes was used to induce cell death in the KCl positive control slices.Hippocampal slice cultures were incubated with BrdU 0.5 µM for 3 days. Following the indicated treatments, slices were fixed at in 0.1 M phosphate buffer  with 4% paraformaldehyde overnight at 4 °C and washed extensively. The slices were then removed from their coverslips for BrdU staining. DNA was denatured using 50% formamide in 2 × SSC for 1.5 hour for 65 °C. After washing in 2 × SSC buffer, they were incubated at 37 °C in 2N HCL. The slices were then washed in 0.1 M PB and permeablized for 2 days at 4 °C in 0.1 M PB, 0.4% Triton X100 and 10% heat-inactivated horse serum. Primary antibodies  were applied overnight at 4 °C in 0.1 M PB + 0.4% Triton X-100 and 5% NHS. Secondary antibodies  were then added overnight at concentration of 1:300. Dentate gyrus region of the hippocampal slices were imaged on an upright confocal microscope .Nuclei were separated from mouse hippocampus slice using the protocol described by Matevossian et al., 2008  with thegria2 5’ region and promoter  Forward - TTT CTG CAG TTC AAG AGC AAT CCA CAG G; Reverse - TTT GGA TCC CTA TGA TGC AAG CAT AAT TCC; Probe region (antisense) Forward - TTT GGA TCC TTC AAG AGC AAT CCA CAG G; Reverse - TTT CTG CAG CTA TGA TGC AAG CAT AAT TCC according to the following cycle: 5min at 95°C followed by 40 cycles of 35 sec at 95°C, 35 sec at 56°C and 70sec/40sec (respectively) at 72°C. Amplification was followed by 10 min extension at 72°C. The PCR product was purified using Quickclean 5M PCR purification kit . PCR product containing the 5’ region and pCpGL-Basic plasmid (promoterless Luciferase reporter plasmid lacking any CpGs in its sequence), generously donated by Dr. Rehi’s lab, University hospital, Regensburg, Germany [Rat  Germany ; were diin vitro methylated using M.SssI CpG methyltransferase (New England Biolabs) in a 400µl reaction with the following ingredients: 10µg vector, 2µl SAM, 40µl Buffer, 3µl Enzyme. After 4 hours of incubation, 2µl of SAM and 1µl enzyme were added and the reaction mixture was incubated for additional 4 hours. Mock methylation followed the same protocol with exclusion of the M.SssI enzyme. Methylated plasmid was cleaned using Quickclean 5M PCR purification kit .The plasmid containing the 5’ region was gria2 promoter: Insert and vector were digested with BamHI and NcoI restriction enzymes  in 2xTango buffer, by incubation for 4 hours at 37c. Digestion products were cleaned using Quickclean 5M PCR purification kit . Vector and the gria2 promoter insert were ligated in a 1:2 molecule number ratio with T4 Ligase  based on the manufacturer recommendation. Incubation was conducted over-night at 22 °C. For the promoter plasmid, the promoter region PCR fragment was ligated under the same conditions to an empty pCpGL-Basic plasmid. After inserting the 'unmethylated' DNA, the ligated plasmids were purified from 1.0% agarose gel using Qiaquick Gel Extraction kit resulting in removal of excess unligated unmethylated DNA. The “patch” methylated constructs were transfected directly into SH-Sy5y neuroblastoma cells without cloning in bacterial cells to maintain the state of methylation of the patch. During cloning in bacterial cells the CpG methylation pattern is lost since E. coli does not harbor the DNA methyltransferase required to copy CpG methylation during replication.We then inserted an unmethylated PCR amplified fragment containing the 2, 37 °C). Cells were transfected using X-tremeGene HD  according to the manufacturer recommendations. In brief, 100,000 cells were platted on 6 well plates and grown to 80% confluency at the day of transfection. 1µg of vector was mixed with the transfection reagent (1:3 ratio) and added to the cell media. Cells were harvested 48 hour post transfection.SH-Sy5y human neuroblastoma cells  were grown in F12:DMEM 1:1 media  in controlled environment  and harvested using a cell scraper . Cells were spun down (5min at 1000rpm) and lysed in 30µl lysis buffer (25mM Tris-Phosphate (pH=7.8), 10% Glycerol, 1% Triton-X, 1mg/ml BSA, 2.5mM EDTA (pH=8.0) and 1x Complete-mini EDTA free protein inhibitors ) for 5 min on ice. The lysate was spun for 5 min at 13,000 rpm and the supernatant was removed for activity evaluation. 10µl of the lysate was added to each well with 100µl Luciferase assay substrate . The reactions were read using FluoStar Optime  and the results were normalized to the total protein concentration per sample.The protein concentration was measured using Bradford reaction assay  according to the manufacturer recommendations, and read using DU730 UV/Vis spectrophotometer .gria2 TSS we designed 5' forward primers from the 3’ region of the proposed regulatory region (“5’ region”) where a novel transcript would initiate if indeed this promoter is functional in vivo 5'-GGAATTATGCTTGCATCATAG and 3' reverse primer corresponding to the region of the known TSS 5'- AATATCAGCACCCTCCCAT . The PCR product was subjected to Sanger sequencing for confirmation of the sequence and its alignment to the DNA upstream of the known TSS using Multialin software [To test whether a 5' transcript is initiated upstream to the known software .gria2 5’ region and the 3 CpG sites in the mouse gria2 5’ region. The average DNA methylation value per animal was correlated with the number of convulsive seizures experienced, or number of recorded bursts in the slices, using Pearson’s correlation analysis. Significance between the different conditions in multiple-comparisons was calculated using the Student t-test with Holm-Bonferroni correction for multiple comparisons [For the DNA methylation results, we used Mann-Whitney U-test. For mRNA expression analysis, a Student’s t-test compared between treatment groups. To measure correlation between DNA methylation and either bursting or seizures, we first averaged the methylation levels at all 5 CpG sites in the rat parisons . All anaFigure S1NormFinder analysis of expression stability of several reference genes.Variability values calculated by NormFinder for 4 reference genes and a recommended combined reference. Note that all reference genes are below the acceptable variability cutoff previously defined by Wierschke et al. 2010 [al. 2010  as v<0.1(TIF)Click here for additional data file.Figure S2gria2 initiation site.Upstream An RT-PCR reaction was used to validate the existence of an alternate upstream gria2 TSS invivo. (A) Physical map of the rat gria2 promoter and 5’ region. CpG sites are marked by balloons and numbers indicate distance from previously known TSS. Primers spanning from the 3’ end of the gria2 5’ region (red arrow marked F) to the 5’ end of the known TSS (red arrow marked R) were used to amplify DNAse-treated rat hippocampus RNA. (B) PCR product was produced only after reverse-transcription (cDNA) and not when conducting the PCR reaction directly on the DNAse-treated RNA (RT -- ). (C) The PCR product was subjected to Sanger sequencing and aligned to the gria2 promoter, from the 5’ region to the previously reported TSS (C).(TIF)Click here for additional data file.Table S1PCR primers.(DOCX)Click here for additional data file.