Desert ecosystems, spanning a third of the earth’s surface, harbor remarkably diverse microbial life despite having a low potential for photosynthesis. In this work, we reveal that atmospheric hydrogen serves as a major previously overlooked energy source for a large proportion of desert bacteria. We show that both chemoheterotrophic and photoautotrophic bacteria have the potential to oxidize hydrogen across deserts sampled across four continents. Whereas hydrogen oxidation was slow in native dry deserts, it increased by three orders of magnitude together with photosynthesis following hydration. This study revealed that continual harvesting of atmospheric energy sources may be a major way that desert communities adapt to long periods of water and energy deprivation, with significant ecological and biogeochemical ramifications. 2), to conserve energy and fix carbon independently of photosynthesis. In this study, we investigated whether atmospheric H2 oxidation occurs in four nonpolar desert soils and compared this process to photosynthesis. To do so, we first profiled the distribution, expression, and activities of hydrogenases and photosystems in surface soils collected from the South Australian desert over a simulated hydration-desiccation cycle. Hydrogenase-encoding sequences were abundant in the metagenomes and metatranscriptomes and were detected in actinobacterial, acidobacterial, and cyanobacterial metagenome-assembled genomes. Native dry soil samples mediated H2 oxidation, but rates increased 950-fold following wetting. Oxygenic and anoxygenic phototrophs were also detected in the community but at lower abundances. Hydration significantly stimulated rates of photosynthetic carbon fixation and, to a lesser extent, dark carbon assimilation. Hydrogenase genes were also widespread in samples from three other climatically distinct deserts, the Namib, Gobi, and Mojave, and atmospheric H2 oxidation was also greatly stimulated by hydration at these sites. Together, these findings highlight that H2 is an important, hitherto-overlooked energy source supporting bacterial communities in desert soils. Contrary to our previous hypotheses, however, H2 oxidation occurs simultaneously rather than alternately with photosynthesis in such ecosystems and may even be mediated by some photoautotrophs.How the diverse bacterial communities inhabiting desert soils maintain energy and carbon needs is much debated. Traditionally, most bacteria are thought to persist by using organic carbon synthesized by photoautotrophs following transient hydration events. Recent studies focused on Antarctic desert soils have revealed, however, that some bacteria use atmospheric trace gases, such as hydrogen (HIMPORTANCE Desert ecosystems, spanning a third of the earth’s surface, harbor remarkably diverse microbial life despite having a low potential for photosynthesis. In this work, we reveal that atmospheric hydrogen serves as a major previously overlooked energy source for a large proportion of desert bacteria. We show that both chemoheterotrophic and photoautotrophic bacteria have the potential to oxidize hydrogen across deserts sampled across four continents. Whereas hydrogen oxidation was slow in native dry deserts, it increased by three orders of magnitude together with photosynthesis following hydration. This study revealed that continual harvesting of atmospheric energy sources may be a major way that desert communities adapt to long periods of water and energy deprivation, with significant ecological and biogeochemical ramifications.Author Video: An author video summary of this article is available. Actinobacteriota, Acidobacteriota, and Proteobacteria  to oxidize atmospheric H2. Activity studies have shown that electrons derived from H2 oxidation can support aerobic respiration and, for some cells capable of the Calvin-Benson-Bassham cycle, carbon dioxide fixation , has a low activation energy, and yields a large amount of free energy when oxidized , Proteobacteria (17%), and Chloroflexota (10%) , especially Nostoc, Microcoleus, and Tychonema taxa  were collected from a sparsely vegetated arid desert in South Australia see . In commta (10%) . Cyanobaema taxa , all of ema taxa –64. Metaeroidota . However10.1128/mSystems.01131-20.7TABLE S1Table S1, XLSX file, 0.01 MB.Soil physicochemical properties of the Australian desert soil microcosms at each sampling point. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.8TABLE S2Table S2, XLSX file, 0.02 MB.Sequencing statistics for the metagenomes and metatranscriptomes and taxonomic information and metabolic classification of the 39 metagenome-assembled genomes (MAGs). Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.9TABLE S3Table S3, XLSX file, 0.2 MB.Bacterial and archaeal community composition of Australian desert soil microcosms and other desert samples based on metagenomic searches using GraftM. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the c sulfide dehydrogenases [16%]), including MAGs affiliated with Actinobacteriota, Proteobacteria, and other abundant phyla , Actinobacteriota (type IE), and Proteobacteria  (Crenarchaeota), reverse tricarboxylic acid cycle (MAGs of nitrite-oxidizing Nitrospirota), and 3-hydroxypropionate cycle (short reads matching Chloroflexota reference sequences)  . Also dequences)  and S4. quences) , 37, thi10.1128/mSystems.01131-20.10TABLE S4Table S4, XLSX file, 2.8 MB.Metabolic marker gene abundance in metagenomic and metatranscriptomic short reads and sequences of their hits in assemblies and MAGs. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 oxidation in this ecosystem, we classified sequences encoding hydrogenase catalytic subunits using the recently expanded HydDB scheme , Proteobacteria (group 1d), and Cyanobacteria (group 2a)  . The lowenous H2 , 67, 68,enous H2 . Concordypothrix . While Hll known , 70, to  strains , 72. How strains , it is aA microcosm experiment was performed to investigate how community and metabolic composition, gene expression, and biogeochemical activities of the sampled Australian soils  varied d10.1128/mSystems.01131-20.1FIG S1FIG S1, TIF file, 1.1 MB.Geographical details of the samples studied. (a) Map showing location of four study sites colored by Koppen-Geiger Climate Classification. (b) Photograph of the exact sampling site used for the microcosm experiments near Roxby Downs, South Australia. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.2FIG S2FIG S2, TIF file, 2.3 MB.Overview of study design and setup of the Australian desert soil microcosms for the control samples (a) and water treatment samples (b). Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.3FIG S3FIG S3, TIF file, 0.4 MB.Temperature and humidity profiles of the Australian desert soil microcosms. Temperature and humidity were monitored by iButtons embedded in each microcosm, with measurements taken every 20 min over a 44-day period. The traces show the average hydration and temperature across the five independent control microcosms and five independent treatment microcosms. The three wetting time points are depicted by the green dotted lines, and the four sampling points are depicted by red dashed lines. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Nostocales) and anoxygenic photosynthesis , concomitant with a decrease of similar magnitude of two actinobacterial orders . This trend was reversed as soils returned to a desiccated state at the end of the experiment  than for controls (C2 and C3), whereas the abundance of Actinobacteriota-associated group 1l [NiFe]-hydrogenase and type IE RuBisCO decreased to an even greater extent  Estimated richness based on Chao1 index. (b) Taxon diversity based on Shannon index. Download FIG S4, TIF file, 0.3 MB.Alpha diversity of Australian desert soil microcosms. Values are based on the single-copy ribosomal marker gene Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.5FIG S5FIG S5, PDF file, 0.1 MB.Phylogenetic tree of amino acid sequences of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalytic subunits. Sequences are shown of binned (red) and unbinned assembled (blue) sequences retrieved from the metagenomes alongside representative reference sequences (gray). The RuBisCO subgroup associated with each reference sequence is shown, with type IB associated with photoautotrophs and the retrieved type IA, IC, ID, and IE being associated with chemoautotrophs. Trees were constructed by the neighbor-joining method, gaps were treated with partial deletion, and bootstrapping used 200 replicates. Download Copyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01131-20.6FIG S62 oxidation. Trees were constructed by the neighbor-joining method, gaps were treated with partial deletion, and bootstrapping used 200 replicates. Download FIG S6, PDF file, 0.02 MB.Phylogenetic tree of amino acid sequences of group 1 and 2 [NiFe]-hydrogenase catalytic subunits. Sequences are shown of binned (red) and unbinned assembled (blue) sequences retrieved from the metagenomes alongside representative reference sequences (gray). The hydrogenase subgroup associated with each reference sequence is shown, with group 1h, 1l, 2a, and 1f [NiFe]-hydrogenases previously shown to mediate atmospheric HCopyright © 2020 Jordaan et al.2020Jordaan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 and carbon monoxide. While these findings lend support for continual energy harvesting from trace gases, fewer community members expressed genes to harvest light energy . As expected, the levels of cyanobacterial photosystem I (5.1 TPM), photosystem II (4.1 TPM), and RuBisCO (2.2 TPM) were also low  and compared them to the metatranscriptomes of composite dry control microcosms (C2). The most expressed metabolic genes surveyed in the dry condition were the standard complexes of the respiratory chain  followed by group 1h and 1l [NiFe]-hydrogenases (144 TPM), formate dehydrogenase , and carbon monoxide dehydrogenase  . The higalso low , in linealso low , suggestalso low .Cyanobacteria, together with a 1.7-fold decrease in chemosynthetic RuBisCO lineages   to survive in these soils. Remarkably, hydration caused a significant 950-fold increase (P < 0.0001) in the rate of H2 oxidation of the treatment microcosms at the second and third sampling points (k = 0.87 ± 0.19 s−1) compared to the control microcosms (2 to subatmospheric levels down to the quantification limit (61 ppbv) within 3 h  , and oxive state , 37. How reasons . These r reasons , 76, unl reasons . Insteadcrocosms . The comthin 3 h . This suetection . A highl14CO2) to compare the rates of chemosynthetic and photosynthetic carbon fixation during the microcosm experiment. CO2 incorporation occurred at low but detectable rates in the native dry soils compared to the heat-killed controls (0.089 ± 0.011 pmol g−1 h−1), though it was not significantly affected by the availability of light and/or H2  in dark CO2 assimilation rates for the treatment microcosms at the second and third sampling points (1.30 ± 0.99 pmol g−1 h−1) (−1 h−1)  in CO2 fixation compared to the dark condition for the treatment microcosms, but not the controls   compared(−1 h−1) . Light icontrols . In linecontrols  and 2, tcontrols , H2 suppommunity  and tranommunity , though Cyanobacteria were scarce (0 to 0.3%)  and occurred to subatmospheric levels  that hydrogen-oxidizing bacteria are abundant community members that are strongly stimulated by hydration, demonstrating the generalizability of our core conclusions.To test whether these findings were generalizable to deserts in other continents, we sampled arid soils from the climatically distinct Namib, Gobi, and Mojave deserts. Metagenomic profiling showed that the community composition of these soils was relatively similar to those of the Australian microcosms at the phylum and order levels . Howeverto 0.3%) . As for to 0.3%) . Particuto 0.3%) . Activitry soils  but was c levels . This su2, are important for the endurance of microbial communities in desert environments. We previously postulated that the relative importance of photoautotrophs  and trace gas oxidizers  would shift depending on hydration levels  or consume exogenous atmospheric H2 . In addition, further studies are required to gain a more detailed understanding of carbon fixation and energy acquisition strategies at the population and cellular levels.In accord with conventional ecological theory, our study highlights that  regions , 37, 39.2 oxidation occurred at low rates in dry soils and was greatly stimulated by hydration. This suggests that low moisture content greatly inhibits atmospheric H2 oxidation, though likely not as much as other processes such as photosynthetic carbon fixation. It seems improbable that the low rates of H2 oxidation measured under dry conditions are sufficient to sustain the large community of H2-oxidizing bacteria detected by metagenomics. However, given reports of extremely low energy requirements of dormant bacteria  in sustaining them. It also remains to be explained why, despite harboring a similar relative abundance of predicted H2-oxidizing cells, atmospheric H2 oxidation in dry native samples occurred at much lower rates in the four desert soils described here than in previously reported Antarctic samples . In order to limit sampling bias and to ensure sampling homogeneity, the site was divided into three 10-m2 sections (located ∼120 m apart) and further subdivided into three 1-m2 subplots. Nine surface soil samples (0 to 10 cm) were aseptically collected and transferred into sterile 25-liter plastic containers. To create a representative sample for the complete site, the nine individual subsamples were pooled and homogenized by sieving through sterilized 2-mm grid size sieves. Samples were transported to the laboratory and stored at ambient temperature until microcosm setup 1 week after collection. Samples were also separately collected from three other arid deserts : a gravel plain (0 to 10 cm) from the Namib Desert, Namibia , a soil surface (0 to 2 cm) from the Gobi Desert, China , and a loess pediment (0 to 10 cm) from the Mojave Desert, Nevada . In all cases, samples were aseptically collected and transferred into zipper storage bags before transport to Monash University’s quarantine approved facilities.Soils for the microcosm experiments were collected from an arid site  on the outskirts of Roxby Downs, South Australia, on 31 October and 1 November 2018. As depicted in the photograph in  deserts : a grave−1) and moderate (∼3 mm h−1) rainfall events in the sampled region and was predetermined through a trial experiment to prevent excess drainage of water and nutrients from soil microcosms. Microcosms were then left to dry for 31 days to simulate a desiccation event. Microcosms were destructively sampled at four points: day 9 , day 11 , day 13 , and day 42 . At each sampling point, the contents of five control microcosms and five treatment microcosms were used for downstream soil physicochemical analysis, nucleic acid extraction, gas chromatography analysis, and carbon fixation assays.The Australian desert samples were subjected to a microcosm experiment that simulated a hydration-desiccation cycle. A total of 40 microcosms  were constructed . These cPhysicochemical analysis of Australian soil samples was conducted at the Environmental Analysis Laboratory (EAL), Southern Cross University, Australia, in accordance with ISO/IEC 17025 standard procedures. Composite soil samples were created for each sampling point and treatment group prior to analysis . PhysicoMetagenomic DNA was extracted from 40 soil samples collected during the Australian desert soil microcosm experiment (5 replicate microcosms × 2 treatment groups × 4 sampling points). DNA was extracted using 0.25 g of soil with the DNeasy PowerSoil kit (Qiagen) according to the manufacturer’s instructions. DNA purity and concentration were evaluated on an ND-1000 spectrophotometer (Thermo Fisher Scientific). DNA extracts for all soil replicates were pooled (equimolar basis) to generate a composite sample for each combination of sampling points and treatment group . Samples were stored at –80°C. Community genomic DNA (gDNA) samples were subjected to library preparation using the Nextera Flex DNA library prep kit followed by paired-end 150-bp sequencing on the Illumina HiSeq2500 platform at CD Genomics (New York). This yielded an average of 27.3 million paired reads per sample, as detailed in 2PO4 (pH 6.5) to remove excess salts. The pellets were mixed with 0.5 g of glass beads  and resuspended in 1 ml of lysis buffer  and 1 ml of TRIzol reagent . Samples were vortexed at 3,200 rpm for 1 min at room temperature, followed by chloroform (200 μl) and isopropanol (500 μl) washes. RNA was precipitated overnight at –20°C using 20 μl of 3 M sodium acetate and 800 μl of 100% (vol/vol) ethanol. RNA was collected by centrifugation at 14,000 × g for 15 min at 4°C. Pellets were air dried at room temperature and resuspended in 50 μl of TE buffer . RNA extracts were treated with TURBO DNase , and the absence of DNA was confirmed by quantitative PCR (qPCR) of the 16S rRNA gene using the Platinum SYBR Green qPCR SuperMix-UDG kit . RNA purity and concentration were evaluated on an ND-1000 spectrophotometer (Thermo Fisher Scientific). The extracted RNA was stored at –80°C before further analysis. Prior to mRNA enrichment, DNase-treated RNA for all soil replicas were pooled (equimolar basis) to generate a composite sample each for the control microcosms (one pooled C2 sample) and treatment microcosms (one pooled T2 sample). Composite RNA samples were applied to the subtractive hybridization method using the MicrobeExpress kit  to remove rRNA from the mRNA. Purified mRNA was eluted in 25 μl of TE buffer and reverse transcribed to cDNA using the SuperScript III first-strand synthesis system kit  following the manufacturer’s protocol. Community cDNA samples were subjected to library preparation using the Nextera Flex DNA library prep kit, followed by paired-end 150-bp sequencing on the Illumina HiSeq2500 platform at CD Genomics (New York). Sequencing yielded 41.2 million and 29.0 million paired reads for the treatment (T2) and control (C2) samples, respectively  was used to trim Illumina adapter sequences, remove reads aligning to the PhiX genome, and trim low-quality bases , discarding reads less than 50 bp in length after trimming. Metatranscriptomic data were additionally filtered with BBDuk to remove reads corresponding to rRNA  by aligning to a database of single-copy ribosomal proteins. SingleM OTUs of the ribosomal protein gene rplB were clustered at 97% identity and used for downstream analysis. Shannon index  and Chao1 (richness estimated) were calculated from the rplB OTU table in R v3.6.0 with the phyloseq v1.28.0 . Genes were predicted from the assembled contigs using Prodigal v2.6.3  -p meta cd-hit- was usedTo obtain sufficient sequences for metagenomic binning, four samples representing the desert soils in their native dry and wet states  were subjected to additional deep metagenomic sequencing. They were sequenced on a 2 × 150-bp NextSeq500 run at the Australian Centre for Ecogenomics, yielding an average of 86.1 million paired reads per sample. For each of the four samples, raw reads sequenced from the two runs were pooled to produce one single cometagenome. Raw reads derived from the four cometagenomes and other four single-run metagenomes  were quality controlled by clipping off primers and adapters and then filtering out artifacts and low-quality reads using the Read_QC module within the metaWRAP pipeline . The eig2 oxidation was measured through monitoring headspace H2 mixing ratios over time of sterilized 120-ml serum vials incubated with 4 g of desert soil. For the Australian desert samples, 4-g soil samples were collected from each microcosm at each sampling point (T1 to T4 and C1 to C4) and transferred into serum vials. For the Namib, Gobi, and Mojave desert samples, 4-g quantities of either native dry soils or experimentally wetted soils were transferred into the serum vials; soils were wetted by adding approximately 4 g of soil to an empty sterilized microcosm vessel with a mesh base, pouring 4 ml of Milli-Q water, and allowing covered vessels to drain at ambient temperature for 24 h. Serum vials were sealed with 0.1 M NaOH-treated butyl rubber stoppers, flushed with pressurized air, and injected with H2  to achieve a final headspace mixing ratio of ∼10 ppmv. Headspace gas (2 ml) was sampled using a gas-tight syringe and measured as described previously (2 oxidation was measured in both live samples and heat-killed controls (autoclaved at 121°C and 15 lb/in2 for 60 min). All sampling needles and syringes were flushed with helium (99.999% pure) prior to penetration of the vial septum to avoid interference from air and contamination from the previous sample. For long-term measurements, vials were supplemented with helium gas as necessary to allow headspace sampling to continue, with dilution factors accounted for when calculating H2 mixing ratios. First-order rate constants (k) for H2 oxidation were estimated using the exponential nonlinear regression function in GraphPad Prism 8.4.3.Heviously  on a gas14C-labeled CO2. Gaseous 14CO2 (1% [vol/vol]) was generated by mixing 15 μl of radiolabeled sodium bicarbonate solution  with equal volumes of 10% HCl solution in a sealed 5-ml glass vial and incubating it for 2 h at room temperature. Samples (0.5 g) of either live soil or heat-killed controls (autoclaved at 121°C and 15 lb/in2 for 60 min) were collected from each microcosm at each sampling point (T1 to T4 and C1 to C4). They were transferred to sterile 5-ml glass vials sealed with rubber septa. Each vial was injected with 14CO2 (1% [vol/vol]) gas to achieve a final initial headspace concentration of 400 ppmv, which otherwise comprised ambient air. The samples were incubated for 24 h at 28°C in technical quadruplicate under four conditions: (i) light plus 14CO2, (ii) light plus 14CO2 + 100 ppmv of H2, (iii) dark plus 14CO2, and (iv) dark plus 14CO2 plus 100 ppmv of H2. Samples incubated under dark conditions were covered with aluminum foil prior to incubation, while those incubated under light conditions were incubated under a light box. Unfixed 14CO2 was removed by transferring soils to 10-ml scintillation vials and suspending them in 2 ml of 10% acetic acid in ethanol. Samples were left to dry in a chemical fume hood at ambient temperature for 48 h. Approximately 10 ml of scintillation cocktail (EcoLume) was added and 14C counts were measured with a liquid scintillation counter  operating at 93% efficiency. Background luminescence and chemiluminescence were corrected through internal calibration standards and by including an additional background vial. Background scintillation counts were subtracted from sample counts prior to calculations. The amount of 14CO2 fixed per sample was quantified as the mass of 14CO2 per mass of soil per time unit (picomoles per gram per day).Chemosynthetic and photosynthetic carbon fixation was measured for the Australian soil microcosm samples by tracing incorporation of PRJNA647683.All metagenomes, metatranscriptomes, and metagenome-assembled genomes are available at the Sequence Read Archive under BioProject accession number


Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium.Capsular polysaccharide is a key factor underlying the virulence of  Streptococcus pneumoniae, a Gram-positive human pathogen, causes a series of serious diseases in humans. SPD_1495 from S. pneumoniae is annotated as a hypothetical ABC sugar-binding protein in the NCBI database, but there are few reports on detailed biological functions of SPD_1495. To fully study the influence of SPD_1495 on bacterial virulence in S. pneumoniae, we constructed a deletion mutant (D39Δspd1495) and an overexpressing strain (D39spd1495+). Comparative analysis of iTRAQ-based quantitative proteomic data of the wild-type D39 strain (D39-WT) and D39Δspd1495 showed that several differentially expressed proteins that participate in capsular polysaccharide synthesis, such as Cps2M, Cps2C, Cps2L, Cps2T, Cps2E, and Cps2D, were markedly upregulated in D39Δspd1495. Subsequent transmission electron microscopy and uronic acid detection assay confirmed that capsular polysaccharide synthesis was enhanced in D39Δspd1495 compared to that in D39-WT. Moreover, knockout of spd1495 resulted in increased capsular polysaccharide synthesis, as well as increased bacterial virulence, as confirmed by the animal study. Through a coimmunoprecipitation assay, surface plasmon resonance, and electrophoretic mobility shift assay, we found that SPD_1495 negatively regulated cps promoter expression by interacting with phosphorylated ComE, a negative transcriptional regulator for capsular polysaccharide formation. Overall, this study suggested that SPD_1495 negatively regulates capsular polysaccharide formation and thereby enhances bacterial virulence in the host. These findings also provide valuable insights into understanding the biology of this clinically important bacterium.IMPORTANCE Capsular polysaccharide is a key factor underlying the virulence of Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium. Streptococcus pneumoniae is a major pathogenic bacterium that causes various serious diseases in humans worldwide and may lead to high morbidity and mortality, especially among young children and elderly , l-rhamnose (Rha), l-rhamnose, l-rhamnose, glucose, and glucuronic acid (GlcUA), and all the CPSs, except for serotypes 3 and 37, are synthesized in the Wzx/Wzy-dependent pathway , arabinose, xylose, glucose, fructose, lactose, sucrose, and maltose as the only sugar sources in C+Y medium promoted bacterial growth. The results showed that the addition of Neu5Ac, arabinose, and xylose as the only sugar sources in the culture medium almost rescued the growth of both the WT and mutant strains. The supplementation of glucose, fructose, lactose, sucrose, and maltose as the only sugar sources in C+Y medium can recover cell survival at different levels. Compared to that of D39-WT, D39Δspd1495 showed obvious hysteresis of growth, especially when grown in C+Y medium supplemented with fructose and sucrose, whereas D39spd1495+ exhibited a marginal increase in growth. These results indicated that SPD_1495 may be a sugar-binding protein in S. pneumoniae and improve sugar utility of bacteria, which is consistent with the annotation in the NCBI database, but its detailed biological function was not reported.Since SPD_1495 is annotated as an ABC sugar-binding protein in the NCBI database, the utility of sugar source of D39-WT and D39Δ strains . WithoutS. pneumoniae, we used iTRAQ-based proteomics to identify differentially expressed proteins between D39-WT and D39Δspd1495. A strong correlation (r2 = 0.91374)   indicate0.91374) . More th0.91374) , and the10.1128/mSystems.00025-20.1TABLE S1Table S1, PDF file, 0.02 MB.Detailed information regarding 54 differentially expressed proteins. Download Copyright © 2020 Zheng et al.2020Zheng et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P value among the three pathways. Following deletion of the spd1495 gene, 34 proteins, including Cps2C, Cps2K, Cps2I, Cps2T, Cps2E and Cps2D etc., which are associated with CPS synthesis were obviously upregulated  analysis  pathway enrichment analysis revealed that the differentially expressed proteins were enriched in three biological processes, namely, polyketide sugar unit biosynthesis, streptomycin biosynthesis, and acarbose and validamycin biosynthesis  and E. Iegulated . For exaynthesis . Furtherynthesis  and G shanalysis . MoreovePH, etc. . The 20 S. pneumoniae, transmission electron microscopy (TEM) was used to detect the thickness of the CPS. The images of TEM showed that the thickness of CPS decreased in the following order: D39Δspd1495 > D39-WT > D39spd1495+. This indicates that SPD_1495 could negatively regulate CPS formation  tag affinity chromatography. There was a band of ∼45 kDa, corresponding to the calculated molecular weight of SPD_1495 in SDS-PAGE, the Western blotting with SPD_1495 antibody confirmed the expression of SPD_1495  results revealed that SPD_1495 cannot directly interact with cps operon  was expressed and purified to check its interaction with SPD_1495 by surface plasmon resonance (SPR). The result of SPR studies also showed that ComED58E can interact with SPD_1495 directly in vitro  assay was performed to identify the proteins interacting with SPD_1495. Compared to that of IgG in the control group, SPD_1495 specifically captured some proteins of molecular weight between 20 and 37 kDa . These pin vitro , and theps probe , indicatΔspd1495 . MoreoveStreptococcus. A database search showed that several highly homologous proteins of SPD_1495, for example, B7692_06535, YESO_2, and SPAR10_0271 are also annotated as sugar ABC transporter substrate-binding proteins. The detailed biological function of this protein family has not been reported. This is the preliminary report of the function of SPD_1495 in S. pneumoniae CPS synthesis. Hence, the regulation of capsule formation and bacterial virulence of SPD_1495 observed in the present study may represent a novel biological function of this cluster of proteins.To assess the conservation of SPD_1495 in bacteria, amino acid sequence alignment and the phylogenic tree construction were carried out  and B. TS. pneumoniae is one of the key virulence factors required for effective infection host  with 0.5% yeast extract (Oxoid) or on Columbia agar  containing 5% sheep blood  at 37°C in a 5% CO2 incubator . Escherichia coli DH5ɑ and BL21  were both cultured in Luria-Bertani (LB) medium at 37°C in an incubator shaker . All strains were stored at –80°C in 15% glycerol. Erythromycin  at 0.25 mg/ml, chloramphenicol  at 4 mg/ml, or ampicillin  at 100 μg/ml was added to the medium when needed for selective culture. All of the bacterial strains and plasmids used are listed in The spd1495 , was replaced by an antibiotic resistance cassette gene (erm). The reconstructed region, including erm (829 bp), upstream (817 bp) and downstream (624 bp) of spd1495 was ligated by LFH-PCR using primers P3 to P8. The products of LFH-PCR were transferred into strain D39-WT, and the positive transformant was selected by Erm-containing Columbia sheep blood agar plate. D39Δspd1495 strains were stocked after seven sequential passages in the THY medium with 0.25 μg/ml Erm. To construct spd1495-overexpressing strains, the recombinant plasmid pIB169-spd1495 was constructed by inserting the spd1495 gene  into pIB169 plasmid using a ClonExpress II one-step cloning kit . The recombinant plasmid pIB169-spd1495 containing an Cm resistance cassette was then transferred into the D39-WT strain. The transformants were screened in Cm-containing Columbia sheep blood agar plate. Finally, all of the mutant strains, including D39Δspd1495 and D39spd1495+, were confirmed by Western blotting (Long-flanking-homology PCR (LFH-PCR) was used to construct strain D39Δspd1495 , 30, andblotting .spd1495 gene was amplified from S. pneumoniae D39 genomic DNA  and then transferred into E. coli BL21. For SPD_1495 expression, BL21/pGEX-4T-1-spd1495 was cultured in LB medium with 100 μg/ml Amp in a shaking incubator at 37°C. When the optical density at 600 nm (OD600) reached 0.6 to 0.8, protein expression was induced by adding 0.5 mM isopropyl-β-d-thiogalactopyranoside  with a further 5 h of incubation. The bacteria were harvested by centrifugation at 8,000 × g for 10 min at 4°C and washed with 0.01 M phosphate-buffered saline (PBS) three times. The harvested cells were suspended in 0.01 M PBS and lysed by sonication. GST-SPD_1495 fusion protein was isolated by glutathione-Sepharose 4B  and digested with thrombin in 0.01 M PBS buffer according to the manufacturer’s instructions to remove the GST tag. The purified SPD_1495 was then used as an antigen for antibody preparation . The BALB/c mice  were immunized with 100 μg of GST-SPD_1495 by subcutaneous injections. After 2 weeks, secondary immunization was performed. A booster dose was administered intravenously to enhance immunity. The spleen cells of the immunized mice were then fused with hybridoma cells and diluted in Dulbecco modified Eagle medium. Hybridomas were tested by indirect enzyme-linked immunosorbent assay . The isotype of each monoclonal antibody (MAb) was determined. Ascites were produced in BALB/c mice, and then MAbs were purified and biotinylated  membrane . Anti-SPD_1495 antibody was incubated with polyvinylidene difluoride membrane at 4°C, and horseradish peroxidase-conjugated goat anti-mouse was used as a secondary antibody. The results were visualized with Clarity Western ECL Substrate  and captured using ImageMaster 2D Platinum 6.0 . Meanwhile, SDS-PAGE gels stained with Coomassie brilliant blue G250 were used as a loading control.2 incubator. The OD600 was continuously measured every hour by UV-visible spectroscopy  for 12 h, and the data were analyzed by Prism 6.0 .The D39-WT and mutant strains were cultured in C+Y medium (no sugar) with Neu5Ac, arabinose, xylose, glucose, fructose, lactose, sucrose, and maltose as only sugar source, respectively, at equivalent inoculation doses at 37°C in a 5% COspd1495 strains in the exponential growth phase, digested with trypsin  at 37°C for 16 h, and lyophilized. Then, a iTRAQ reagent multiplex kit  was used to label peptide samples according to the manufacturer’s protocol. D39-WT and D39Δspd1495 strains were labeled with 114 and 116 isobaric tags for 1 h at room temperature, respectively. The labeled peptides from the two groups were mixed in equal proportions and dried. High-performance liquid chromatography  with a TechMate C18-ST column  was used to separate the labeled peptides. In brief, an iTRAQ-labeled peptide mixture was eluted with a gradient of 2 to 80% acetonitrile in 20 mM ammonium formate (pH 10.0) for 65 min at a flow rate of 0.8 ml/min. The peptide elution was monitored at 214 nm. The eluted peptides were collected every minute, pooled into six fractions, and then lyophilized. MS analyses were performed with a TripleTOF 5600 (AB Sciex) mass spectrometer. The acquired raw data files (wiff) of each fraction were combined to search against the protein database of S. pneumoniae D39, and ProteinPilot software 4.5 (AB Sciex) was used to identify and quantify the proteins. The parameters used for mass spectrometry and quantitative analysis were set as follows: sample type, iTRAQ 4plex (peptide labeled); cysteine alkylation, iodoacetic acid; digestion, trypsin; instrument, Triple-TOF 5600; ID focus, biological modifications; database, S. pneumoniae D39_fasta; search effort, thorough; and detected protein threshold [unused ProtScore (Conf)], >1.30 (95.0%)  , 35. The (95.0%) . The finKEGG was used for the pathway enrichment analysis of 54 differentially expressed proteins in the “Wu Kong” platform , 38. Prospd1495, and D39spd1495+ strains in the exponential growth phase were harvested by centrifugation at 10,000 × g for 5 min at 4°C and washed with 0.01 M PBS for three times. Then, all cell pellets were fixed with 2.5% glutaraldehyde fixative and postfixed in 1% osmium tetroxide for 2 h at 4°C. Subsequently, 2% uranyl acetate was added to dye the cell pellets for 2 h at room temperature. After the dyeing step, the sample was dehydrated with 50% acetone for 15 min, 70% acetone for 15 min, 90% acetone for 15 min, and twice in 100% acetone for 20 min. All samples were then fixed in epoxy resin and left to polymerize at 37°C for 24 h, 45°C for 24 h, and 60°C for 48 h. Each sample was cut into approximately 50- to 80-nm thin slices and then dyed with 3% uranyl acetate and lead citrate. Finally, the prepared samples were detected using TEM .The D39-WT, D39Δspd1495, and D39spd1495+ strains in the exponential growth phase were harvested by centrifugation at 8,000 × g for 10 min at 4°C and washed with 0.01 M PBS three times. Next, the bacteria were resuspended the in Tris-HCl/MgSO4 buffer  and centrifuged at 8,000 × g for 5 min at 4°C to remove the supernatant and then resuspended with 500 μl of Tris-HCl/MgSO4 buffer again. Next, 200 μl of bacterial solution and 1.2 ml of 12.5 mM tetraborate solution were mixed and heated at 100°C for 5 min. All of the samples were placed in ice water, and 20 μl of 15% m-hydroxybiphenyl solution was added to each sample, followed by thorough mixing. Meanwhile, the same volume 0.5% NaOH was added in control group, Finally, the concentration of each sample was detected in UV-visible spectroscopy reader at 520 nm  according to the manufacturer’s protocol following treatment with 100 mg/ml lysozyme. The RNA concentration was determined by using a NanoDrop 2000 UV-VIS spectrophotometer (Thermo Fisher Scientific). A 1-μg mRNA sample was reverse transcribed using a transScript one-step gDNA removal and cDNA synthesis SuperMix kit for qPCR  according to the manufacturer’s instructions. RT-qPCR was performed using the StepOne system  with the TransStart Tip Green qPCR supermix kit (TransGen Biotech); 16S RNA was used as an internal control. The cycle threshold (TC) value was recorded, and the relative quantification of gene expression was calculated using the 2–ΔΔCT method ; animal experiments were approved by the Ethics Committee for Animal Experiments of Jinan University. The 6- to 8-week-old female BALB/c mice were cared for according to the institutional guidelines for animal care under standard conditions.spd1495, and D39spd1495+ mutants were grown in THY medium and harvested in the exponential growth phase. The bacteria were resuspended in 0.01 M PBS and adjusted to the concentration of 1 × 107 CFU/50 μl. Then, 50-μl portions of the D39-WT, D39Δspd1495, and D39spd1495+ strains were injected into 6- to 8-week-old female BALB/c mice (n = 8) via tail vein injection. The survival of the mice was recorded every 12 h for 4 days.For mouse infections, D39-WT, D39Δg for 10 min at 4°C and washed three times with 0.01 M PBS. D39-WT was resuspended in fresh cell lysates  until the cell lysates were fully dissolved. The supernatant was collected by centrifugation at 10,000 × g for 10 min at 4°C and then incubated with 40 μl of protein A/G-agarose  and 5 μl of anti-mouse IgG for 1 h at 4°C, respectively, to remove the nonspecific binding protein, which can bind to IgG, followed by centrifugation at 2,500 × g for 5 min at 4°C. Protein A/G-agarose was added, and the supernatants were incubated again. After centrifugation, the concentrations of supernatants were measured by a BCA protein assay kit. SPD_1495 antibody (2 μg) and IgG (2 μg) were added to the supernatants, followed by incubation for 16 h at 4°C, respectively. Then, 40 μl of protein A/G-agarose was added to bind the proteins at 4°C for 6 h, which were collected by centrifugation at 2,500 × g for 5 min at 4°C and then resuspended in 800-μl portions of lysates. The isolated proteins were separated by SDS-PAGE, was stained by AgNO3, and then digested and identified by mass spectrometry .D39-WT in the exponential growth phase was harvested by centrifugation at 10,000 × D58E in 0.01 M PBS running buffer were injected sequentially into the chamber at different concentrations . The interaction of SPD_1495 with ComE was detected by using Open-SPR  at 25°C. The binding time and disassociation time were both 300 s, the flow rate was 20 μl/s, and the chip was regenerated with hydrochloric acid (pH 2.5). A one-to-one diffusion-corrected model was fitted to the wavelength shifts corresponding to the various protein concentrations. The data were retrieved and analyzed by using TraceDrawer software.For SPR analysis, SPD_1495 protein (30 μg/ml) was fixed on a NanoGold-COOH sensor chip by capture coupling, and then recombinant ComE and ComEcps gene cluster was amplified by PCR . Proteins of interest  and 100 ng of labeled probe were incubated at 37°C for 30 min in 20 μl of reaction buffer, including 1×binding buffer, 1 μg of poly(dI-dC), 2.5% glycerol, 0.05% NP-40, and 5 mM MgCl2; the cps without binding protein was used as a negative control. After incubation, electrophoresis in 6% native Tris-borate-EDTA polyacrylamide gels was performed at 100 V for 60 min to analyze the binding reactions. The polyacrylamide gels were transferred to nylon membranes at 380 mA for 40 min. The membranes were cross-linked for 10 min using a UV lamp equipped with 254-nm bulbs, and subsequently biotin-labeled DNA was detected by chemiluminescence according to supplier’s instructions (Thermo Fisher Scientific).For EMSA, the 218-bp upstream promoter region of the S. pneumoniae D39 was used to query the NCBI database using the protein BLAST tool. The multiple sequence alignment and cluster analysis of the high-scoring proteins were performed by the software package Clustal-X 2.1. MEGA5.05 was applied to construct the protein evolutionary tree.The amino acid sequence of SPD_1495 from t tests, and are expressed as means ± the standard deviations (SD). Statistical analysis was conducted using Prism 6.0 . Results were considered significant at P < 0.05.Data were analyzed by two-tailed, unpaired Student http://www.ebi.ac.uk/pride; username, reviewer70538@ebi.ac.uk; password, x5Zecn7h).The raw proteomic data and search results have been deposited to the ProteomeXchange Consortium via the PRIDE  partner


Scanning electron microscopy evaluated morphological alterations of erythrocytes. Western blotting assessed caspases. PGE2 was measured by ELISA. Adhesion of erythrocytes on endothelial cells was evaluated by gravity adherence assay. Results: HNE in the concentration range between 10–100 µM induces eryptosis, morphological alterations correlated to caspase-3 activation, and increased Ca++ levels. The process is not mediated by redox-dependent mechanisms; rather, it strongly depends on PGE2 and ceramide. Interestingly, HNE induces significant increase of erythrocytes adhesion to endothelial cells (ECs) that are in turn dysfunctionated as evident by overexpression of ICAM-1. Conclusions: Our results unveil a new physiopathological role for HNE, provide mechanistic details of the HNE-induced eryptosis, and suggest a novel mechanism through which HNE could exert pro-inflammatory effects.Background: Eryptosis is a physiological, apoptosis-like death of injured erythrocytes crucial to prevent premature haemolysis and the pathological sequalae generated by cell-free haemoglobin. When dysregulated, the process is associated to several inflammatory-based pathologies. 4-Hydroxy-trans-2-nonenal (HNE) is an endogenous signalling molecule at physiological levels and, at higher concentrations, is involved in the pathogenesis of several inflammatory-based diseases. This work evaluated whether HNE could induce eryptosis in human erythrocytes. Methods: Measurements of phosphatidylserine, cell volume, intracellular oxidants, Ca Hallmarramblase . This prUtterly distinct from the erythrocytic senescence, eryptosis ostensibly serves the same purpose as apoptosis, i.e., the disposal of defective cells without breaking cell membrane integrity and releasing eventual pro-inflammatory, cytosolic material to the extracellular environment. Eryptosis can be, therefore, envisaged as a preemptive measure of the body to reduce premature haemolysis of injured RBC, thus preventing the detrimental increase of cell-free haemoglobin in circulation .Excessive eryptosis, however, has been implicated in a number of pathological conditions such as anaemia, renal and hepatic failure, sepsis, and malignancy ,4,5,6. M++ levels play a key role within the eryptotic process. It has, indeed, been shown that several proeryptotic stimuli can activate phospholipase A2 (PLA2) that releases arachidonic acid (AA), leading to prostaglandin endoperoxide synthase (PGHS) activation. The resulting increase of PGE2 levels then activates and opens nonselective cationic membrane (NSCM) channels, leading to an increment of Ca++ endocellular levels that in turn activates several molecular targets including Ca++-dependent K+ channels. The resulting membrane hyperpolarization increases the electrical driving force for Cl− exit, and the resulting cellular loss of KCl, with osmotically obliged water efflux, determines RBC shrinkage [++ influx, together with platelet activating factor (PAF), activates membrane sphingomyelinase (SMase) responsible for the increase of ceramide levels that plays a key role in the apoptotic process [++ and ceramide are able to respectively activate scramblase and to inhibit flippases, involved in PS externalization. Finally, increase of Ca++ levels also activate cysteine-calpain endopeptidases responsible for the membrane blebbing that increases the adhesion capacity of RBC [Eryptosis is triggered by several pathophysiological cell stressors and orchestrated by a complex machinery of lipids, membrane proteins, ion channels, and cellular enzymes. In this regard, the increase of endocellular Cahrinkage . On the  process ,4. Indeey of RBC .trans-2-nonenal (HNE) is an α,β-unsaturated aldehyde endogenously generated by the radical-mediated peroxidation of ω-6 polyunsaturated fatty acids. Its reactive carbonyl moieties, together with the conjugated double bonds, generate two electrophilic centres (the carbonyl carbon atom and the β-carbon one), both of which can condense with suitable nucleophilic moieties of biological targets, such as proteins, DNA, and phospholipids [4-Hydroxy-holipids ,9. Thankholipids  and its holipids ,15,16,17holipids ,18. On tholipids ,20,21. Aholipids . CovalenIn light of the mechanisms orchestrating the eryptotic process, strongly regarding membrane structure and function and considering the key role of HNE in membrane alterations supporting inflammatory-based pathologies , we hereUnless otherwise specified, all reagents and chemicals were from Sigma-Aldrich  and of the highest purity grade available.g at 4 °C over Ficoll  gradient. RBCs (0.4% haematocrit) were incubated at 37 °C, 5% CO2, and 95% humidity in Ringer solution containing (mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)/NaOH, 5 Glucose, and 1 CaCl2 at pH 7.4 for the indicated time.Blood was collected from healthy volunteers  with informed consent, and RBCs were immediately isolated by 20 min centrifugation at 2000× v/v) ethanol (EtOH) concentration. Preliminary experiments showed that, under these conditions, EtOH did not have any effect on cells and therefore control RBCs were incubated with EtOH.RBCs were treated for 24 h with HNE and were added in Ringer’s solution at a final 0.1%  diluted in EtOH.In selected experiments, RBCs either were incubated in Ringer’s solution in the absence of CaClThe recruitment of the healthy volunteers was conducted within the project “Discovery of molecular and genetic/epigenetic signatures underlying resistance to age-related diseases and comorbidities (DESIGN)” Bando 2015 Prot 20157ATSLF, funded by the Italian Ministry of Education, University, and Research. The study protocol was approved by the Ethic Committee of Palermo University Hospital  The study was conducted in accordance with the Declaration of Helsinki and its amendments.6 cells/mL with a binding buffer according to the manufacturer’s instructions . In experiments designed to evaluate PS externalization, cell suspension (100 μL) was incubated with 5 μL Annexin V-FITC at room temperature in the dark for 15 min. Samples of at least 1 × 104 cells were then analysed by flow cytometry (FACS) for both FSC and Annexin V-fluorescence intensity , with an Epics XL™ flow cytometer, using Expo32 software, version 1.1C .RBCs were washed once in Ringer’s solution, pH 7.4, and adjusted at 1.0 × 105 cells, 30 min before the end of the treatment, in the dark. Cells were collected by centrifugation , washed, resuspended in PBS, and analysed as above reported.Level of Reactive Oxygen and Nitrogen Species (RONS) was monitored by measuring the fluorescence changes resulting from oxidation of dichloro-dihydro-fluorescein diacetate (DCF-DA). Briefly, 10 μM fluorescence probe was added to 1.0 × 10++ was monitored by measuring the fluorescence changes of the cell-permeable dye fluo-3 AM resulting from its binding with Ca++. Briefly, 10 μM fluorescence probe was added to 1.0 × 105 cells 30 min before the end of the treatment in the dark. Cells were collected by centrifugation , washed, resuspended in PBS, and analysed as above reported.Levels of intracellular Ca5 cells were incubated with 1 µg of a mouse antihuman, ceramide monoclonal antibody  in PBS containing 0.1% bovine serum albumin (BSA) for 1 h at 37 °C. Cells were then washed twice with PBS-BSA and incubated for 30 min with 20 µL of a goat anti-mouse, polyclonal, fluorescein isothiocyanate-conjugated, secondary antibody  diluted 1/50 in PBS-BSA. Cells were then collected by centrifugation , washed, resuspended in PBS, and analysed as above reported.Level of ceramide was monitored by flow cytometry as follows. Briefly, 24 h after the treatment, 1.0 × 10g for 10 min to precipitate the cells. The absorbance of the supernatant was then evaluated at 408 nm . Similarly, a volume of the incubation mixture was treated with 10 volumes of 5 mM sodium phosphate buffer, pH 7.4 (hypotonic PBS), and briefly exposed to an ultrasonic bath. The absorbance value at 408 nm was taken as 100% haemolysis.After 24 h treatment, a volume (0.2 mL) of the incubation mixture was diluted with 10 volumes of PBS and centrifuged at 1000× 2, 2 mM PMSF, 0.5 mM DTT, and 2 mg/mL Lysozyme) with protease inhibitor cocktail , and sonicated for 60 s on ice with Labsonic LBS1-10 . After centrifugation at 40,000× g for 1 h at 4 °C, supernatant was collected. Protein concentration of each sample was determined by using Bradford protein assay reagent . Protein samples (50 μg/line) were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane. Immunoblots were blocked overnight at 4 °C with 5% skimmed milk, followed by incubation with a 1:1000 dilution of mouse antihuman, caspase-3 monoclonal antibody  for 1 h at room temperature. Blots were washed two times with Tween 20/Tris-buffered saline and incubated with a 1:2000 dilution of rabbit anti-mouse, horseradish peroxidase-conjugated anti-IgG antibody  for 1 h at room temperature. Blots were then washed five times with Tween 20/Tris-buffered saline and then developed by enhanced chemiluminescence .RBCs were washed twice with PBS, resuspended in lysis buffer : each step was repeated twice for 10 min. After the last passage in 100% ethanol, the samples were treated with HMDS (hexamethyldisilazane) and left for 24 h in solution until complete evaporation. Finally, samples were grafted onto the supports for visualization by means of silver paste and subsequently metallized with gold and observed using a scanning electron microscope . At each step, the samples were centrifuged at 1000× Intracellular GSH/GSSG levels were measured by employing a glutathione colorimetric assay kit according to manufacturer’s instruction .2 released (pg/1 × 108 cells) was quantified using a PGE2 Enzyme Immunoassay Kit  in accordance with the manufacturer’s protocol. Briefly, after treatment, cells were pelleted by centrifugation at 4 °C, for 5 min at 450× g. Samples of the supernatant were diluted at 1:2.5 with assay buffer. Then, a 100 µL sample, a 50 µL alkaline phosphatase PGE2 conjugate, and a 50 µL monoclonal anti-PGE2 EIA antibody were applied to a goat anti-mouse IgG-containing microtiter plate and incubated at room temperature for 2 h. After washing, 200 µL of p-nitrophenyl phosphate substrate solution was added and incubated at room temperature for 45 min. Finally, optical density at 405 nm was measured on a microplate reader. PGE2 concentrations in the samples were calculated from a PGE2 standard curve (25–5000 pg/mL) that was run in parallel.After incubation, as indicated above, PGE2 flasks with endothelial cell basal medium MV2 (PromoCell) containing 100 pg/mL of heparin, 10% foetal bovine serum (GIBCO), 120 U/mL penicillin/streptomycin, and 30 pg/mL of endothelial cell growth supplement. At 85% confluence, cells were subcultured in 96-well, flat-bottom plates. Cellular monolayers were co-incubated with HNE-stimulated RBCs (25 or 50 µM) or control RBCs for 48 h. After treatment, the adherence of RBCs to HUVEC was measured by the gravity adherence assay as previously reported [Human Umbilical Vein ECs (HUVEC) were purchased from PromoCell, Milan, Italy and used for up to four additional passages. Cells were grown in 75-cmreported .2. Aliquots (0.1 to 0.5 × 106 cells in 20 μL) were incubated at 4 °C with 2 μg/well of a mouse antihuman ICAM-1 monoclonal antibody FITC-conjugated . After 1 h at 4 °C, cells were washed and analysed by FACS.After 24 h co-incubation with RBCs, HUVECs were washed with phosphate buffered saline (PBS), harvested with cell dissociation medium, and diluted in a washing buffer containing 0.1% bovine serum albumin and 1mM CaClp < 0.05 level.Results are expressed as mean ± SD of six independent experiments and conducted in triplicates. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Fisher’s correction for multiple comparisons using Prism version 8 . In all cases, significance was accepted if the null hypothesis was rejected at the p < 0.05, HNE, instead of a mere by-product of oxidative stress, is capable of modifying nucleophilic residues and, depending on its concentration, can either orchestrate cell signalling events or modify target proteins, thus dysregulating functional cellular processes such as proliferation and apoptosis ,30,31,32p < 0.05, In order to determine whether the HNE-mediated eryptosis was also associated to necrotic events, we next evaluated if and to what extent the aldehyde was able to induce haemolysis. To this end, RBCs were incubated for 24 h in the absence (control) or in the presence of HNE in the range between 25 and 50 µM and Hb release was measured. With respect to control cells, HNE induced a significant increase of haemoglobin release only at 50 µM with a value increasing from 3.8% ± 0.3% to 7.6% ± 0.4% . Our results suggest that HNE-induced eryptosis is accompanied with an apparent increase of RBC volume and differs from those reported in literature for other eryptotic models. It has indeed been demonstrated that RBCs, either stimulated with other eryptotic agents or isolated from patients suffering from sepsis or cardiometabolic or blood disorders, are characterised by a reduction of cell volume [As before-mentioned, eryptosis is well known to induce evident morphological variations characterised by cell volume reduction and membrane blebbing. With the aim to investigate and characterise the HNE-induced morphological alterations in RBCs, we assessed FSC variations and performed scanning electron microscopy evaluations in both control and HNE-treated RBCs. As shown in l volume ,4,6.In order to further investigate these discrepancies, we next employed a SEM approach and analysed the morphological alteration in eryptotic RBCs. By analyzing in details the complex cytological framework, primary echinocytes and polyhedrocytes were detected and identified B,C. ThesNotwithstanding that RBC is devoid of nucleus, mitochondria, and other organelles, it does have a molecular machinery, saved during the differentiation from erythroid progenitors, with functional caspases. Activation of these effectors is a key step in the eryptosis induced by either specific xenobiotic or physiopathological conditions ,5,6,43. p < 0.05, In light of the morphological changes triggered by HNE in eryptotic RBCs, we next evaluated the involvement of caspase-3 in the HNE-induced eryptosis. To this end, RBCs were incubated for 24 h in the absence (control) or in the presence of HNE in the range between 25 and 50 µM and caspase-3 cleavage was evaluated. With respect to control RBCs, stimulation with HNE at 25 and 50 µM significantly modified caspase-3 levels that increased from a control value of 0.12 ± 0.01 Arbitrary Units (AU) to 1.45 ± 0.2 and 1.73 ± 0.1 AU respectively. Conversely, procaspase-3 levels decreased from 1.13 ± 0.04 AU to 0.94 ± 0.05 and 0.70 ± 0.05 AU  or in the presence of HNE in the range between 25 and 50 µM and Ca++ endocellular levels were evaluated and expressed as % of positive cells. As shown in ++ with values rising from a control value of 1.8% ± 0.2% to 20.5% ± 0.4% and 41.5% ± 0.4% with 25 and 50 µM HNE, respectively (p < 0.05).As abovementioned, a wealth of studies confirmed a strict correlation between Calization ,4,6. In ++ in the eryptotic process, RBCs were incubated with HNE either in the absence or in the presence of Ca++ and PS externalization was evaluated. As expected, absence of Ca++ resulted in a significant reduction of PS externalization, induced by HNE at 25 or 50 µM, by 68% ± 1% and 72% ± 1% respectively  or in the presence of HNE between 25 and 50 µM and both endocellular RONS and GSH/GSSG levels evaluated. As shown in RBCs are considered “reporter cells” for the oxidative state since they can be particularly vulnerable to RONS due to their constant exposure to by-products generated by autoxidation of haemoglobin . Moreovelization . In ligh++-dependent, HNE-induced eryptosis. Other mechanisms, related to NSCM activation, could therefore underlie the process.Notwithstanding the role of oxidative stress in eryptosis induced by other agents ,3,52 and2 are deeply involved in the eryptotic signalling induced by several stimuli, through the opening of NSCM channels that leads to an increase of cytosolic Ca++ levels. Along these lines, we therefore evaluated whether the Ca++ influx in HNE-treated RBCs was dependent by activation of PGHS. To this end, RBCs were preincubated either in the absence or in the presence of 50 µM ASA and stimulated with HNE in the range between 25 and 50 µM, and then, both endocellular Ca++ levels and PS externalization were evaluated. As shown in ++ release was reduced by −71% ± 2% and −69% ± 1%, respectively.PGHS and PGE2 production. To this end, RBC were incubated for 24 h in the absence (control) or in the presence of HNE in the range between 25 and 50 µM and PGE2 release was evaluated. When compared to control cells, treatment of RBCs with HNE significantly modified endocellular PGE2 levels that increased from a control value of 5.1 ± 0.1 to 10.1 ± 0.3 and 14.6 ± 0.3 pg/108 RBCs with 25 and 50 µM HNE, respectively  and accompanied by the resulting increase of PGE2 levels. While ruling out a direct interaction of HNE with NSCM channels, our data also implied an HNE-induced synthesis of PGE2 in the RBCs. The aldehyde, therefore, seems to share with other proeryptotic stimuli a common PGE2-dependent mechanism to promote an increase of Ca++ levels [As a whole, these results demonstrate that the HNE-induced eryptosis is mediated by PGHS activation  or in the presence of HNE in the range between 25 and 50 µM and then ceramide levels were assessed as % positive cells. Interestingly, treatment of RBCs with HNE significantly modified the percentage of RBCs containing ceramide that increased from a control value of 5.4 ± 0.1 to 20.5 ± 0.3 and 44.3 ± 0.3 with 25 and 50 µM HNE respectively .In line with this, we finally investigated to what extent HNE-induced eryptosis would increase RBC adherence to ECs. To this end, confluent HUVEC were co-cultured for 48 h either with control RBCs or with HNE-stimulated RBCs in the range between 25 and 50 µM. Adherence of RBCs to ECs was evaluated from the ratio of Hb of lysed HUVEC to Hb of RBCs applied to well. As shown in RBC adherence to ECs is a physiopathological process eventually leading to endothelial activation and dysfunction, i.e., overexpression of endothelial cell-surface adhesion molecules, such as ICAM-1 . Inducedp < 0.05).In order to assess whether HNE-induced eryptosis could have also induced EC dysfunction, we finally assessed ICAM-1 levels in ECs co-incubated for 24 h with either control RBCs or with HNE-stimulated RBCs in the range between 25 and 50 µM. As shown in Relevantly, our results demonstrate that HNE-induced eryptosis could foster adhesion of eryptotic RBCs to endothelium and, importantly, lead to its dysfunction. Several pathological conditions, characterized by high plasma levels of HNE, are associated with vascular complications. Our experimental evidences suggest that eryptotic RBCs could contribute to vascular dysfunctions in these diseases.2 (In this work, we unveiled for the first time a new physiopathological role for HNE, i.e., its ability to exert eryptotic effects. Mechanistic details of the process pointed out a crucial role for lipid-derived signalling molecules such as ceramide and PGE2 . SEM ana


Salmonella enterica serovar Enteritidis ODA 99‐30581‐13 is a relatively heat-resistant strain isolated from shell eggs. The strain has a 4,777,965-bp genome sequence (52.1% GC content) that was predicted to encode 4,455 proteins, including heat stress response proteins and stress response regulators; these may be involved in its heat resistance. Salmonella enterica serovar Enteritidis ODA 99‐30581‐13 is a relatively heat-resistant strain isolated from shell eggs. The strain has a 4,777,965-bp genome sequence (52.1% GC content) that was predicted to encode 4,455 proteins, including heat stress response proteins and stress response regulators; these may be involved in its heat resistance. Salmonella enterica serovar Enteritidis has caused numerous salmonellosis outbreaks linked to egg consumption  using a QIAamp DNA minikit  according to the manufacturer’s protocol. The purity and concentration of the gDNA were evaluated by the NanoVue Plus spectrophotometer . DNA libraries of the gDNA were prepared using the Nextera DNA Flex library preparation kit  and barcoded using the Nextera DNA CD index library indexing kit (Illumina) according to the manufacturer’s instructions. The DNA libraries were quantified using the Qubit fluorimeter , and their sizes were measured using the 2100 Bioanalyzer system . The genome was sequenced using the Illumina MiSeq platform . The platform generated 478.5 Mb of data, including 1,910,230 raw reads  with 214.3× coverage and a quality score of 37, calculated by FastQC v0.11.9 software , which were ordered against the reference genome of Salmonella enterica serovar Typhimurium LT2 by progressiveMauve v2.4.0 , followed by culturing in tryptic soy broth (BD); incubation in both transfers was done at 37°C for 24 h. Genomic DNA (gDNA) was isolated from 1 ml of the overnight culture 109 CFU/ml Salmonella Enteritidis ODA 99‐30581‐13 genome sequence showed that it has 4,777,965 bp  and encodes 4,455 proteins, 75 tRNAs, 15 rRNAs, and 15 noncoding RNAs (ncRNAs). Strain ODA 99‐30581‐13 belongs to sequence type 11 and serotype Enteritidis, as determined by MLST v2.0  and heat stress response regulators (rpoS and rpoH) , which promotes heat resistance specific to Salmonella Enteritidis (Analysis of the LST v2.0 , respecteritidis , was encSalmonella Enteritidis ODA 99‐30581‐13 was deposited at GenBank under accession number JACTGY000000000.1, BioProject accession number PRJNA659787, BioSample accession number SAMN15925966, and SRA accession number SRX9029726.The genome sequence of


TM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs . In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrep Over the last two decades, extracellular vesicles (EVs) have been recognized as a critical component of intercellular communication in both physiological and pathological conditions . InteresSeveral sophisticated models based on multivariate parameters of embryo scoring have been designed to predict pregnancy rates in humans after in vitro fertilization and to optimize the selection of embryos in specific subgroups of patients . AlthougEmbryonic secretions might predict the true developmental capacity of an embryo. The embryo culture medium could, therefore, reveal important quality aspects of the embryo that could assist the selection of the most competent embryos . RecentlTM), can be utilized to remove contaminating impurities such as non-specific argonaute proteins, and high-density lipoproteins [The separation of EVs still represents a critical step due to several reasons: first, the methods to isolate EVs are currently highly diverse, and second, depending on which separation method is employed, the results can be considerably different, even when starting from the same sample . To dateproteins . Apart fOur aim is to assess the suitability of different methods to separate EVs with maximum specificity and efficiency from embryo-conditioned medium. We evaluate the impact of three separation methods , OptiPrep™ density gradient ultracentrifugation (ODG), and size-exclusion chromatography (SEC)) on yield, purity, size, and morphology of EVs secreted by bovine embryos.A total of 1500 presumed zygotes were cultured in ultracentrifuged SOF + ITS + BSA to attain 3 mL of conditioned medium on Day 8. For each EV separation method, 1 mL of conditioned medium was used. A schematic representation of the DU, ODG, and SEC isolation protocols used in this study is shown in 10 particles/mL, and the mean and mode particle size were 266.8 ± 2.1 and 186.2 ± 21.9 nm, respectively  revealed the presence of EV-associated protein CD63 A. The DU11 particles/mL starting from a pool of 500 embryos with a mean particle size of 119.2 ± 3.5 nm and a mode of 99.7 ± 6.0 nm as measured by NTA  protein and apolipoprotein A-I (ApoA-I) was detected in fractions 9 to 11 A. Additid by NTA C.9 particles/mL, starting from a pool of 500 embryos . Next, the sperm pellet was washed in IVF–Tyrode’s albumin–pyruvate–lactate (TALP) medium, containing bicarbonate-buffered Tyrode solution. Then, it was adjusted to a final concentration of 1 × 106 spermatozoa/mL using IVF–TALP medium enriched with BSA  and heparin (25 mg/mL).Frozen-thawed bull spermatozoa were separated using a 45/90% Percoll® washed bull spermatozoa. After 21 h gamete co-incubation, presumed zygotes were vortexed to remove surplus zona attached cumulus and sperm cells. The presumed zygotes were cultured in groups of 25 in 50 µL droplets of ultracentrifuged synthetic oviductal fluid enriched with non-essential and essential amino acids (SOFaa), ITS  and medium droplets were covered with mineral oil and incubated at 38 °C in 5% CO2, 5% O2, and 90% N2.After 22 h maturation, bovine oocytes were washed in 500 µL IVF–TALP and subsequently co-incubated with Percollg of ultracentrifugation for 18 h at 4 °C). On 8 dpi, 200 µL embryo-conditioned medium was collected and stored at −80 °C. To obtain 3 mL of embryo-conditioned culture medium, 15 replicates were performed with a total of 1500 presumed zygotes. One mL of pooled embryo-conditioned medium was used for each EV isolation method . The pellet was discarded, and the supernatant was centrifuged for 30 min at 30,000× g and 4 °C, again the pellet was discarded, and the supernatant was centrifuged for 3 h at 100,000× g and 4 °C. The pellet was resuspended in 100 µL of PBS and stored at −80 °C for further EV characterization.Differential ultracentrifugation was performed as described previously by Théry et al.  with a fw/v) aqueous iodixanol solution). Moreover, the gradient was prepared in a 16.8 mL open-top polyallomer tube (Beckman Coulter) by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10%, and 3.5 mL of 5% solutions on top of each other. One mL of embryo-conditioned medium was overlaid onto the top of the gradient. Subsequently, the gradient was centrifuged at 4 °C for 18 h at 100,000× g . Then, all 16 gradient fractions were divided into six samples by pooling the fractions 1–4, 5–7, 8–9, 10–12, 13–16, respectively. The pooled fractions were added to 14 mL PBS. Subsequently, the separate suspensions were centrifuged at 4 °C for 3 h at 100,000× g. The resulting pellets were resuspended in 100 µL PBS and stored at −80 °C for further EV characterization.OptiPrep™ density gradient (ODG) ultracentrifugation was conducted, as previously reported by Van Deun et al. . BrieflySepharose CL-2B  was washed three times with PBS containing 0.32% trisodium citrate dihydrate  ,23. For All samples were suspended in a reducing buffer ) and boiled for 5 min at 95 °C. Protein samples were separated by SDS polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane . Next, the membrane was blocked at room temperature with 5% skim milk/BSA TBST for 45 min. Subsequently, the membranes were exposed to CD63 rabbit , AGO-2 rabbit , and ApoA-I mouse , primary antibodies at 4 °C. After overnight incubation, the membranes were extensively washed with 0.5% Tween in PBS. Then, the membranes were incubated with the appropriate secondary antibodies , 1:3000 in 5% milk + 0.5% Tween PBS; anti-rabbit IgG , 1:4000 in 5% BSA + 0.5% Tween PBS. After a final wash step, chemiluminescence substrate  was added to the membranes. Imaging was performed using Proxima 2850 Imager .For transmission electron microscopy, each sample was deposited on precoated formvar/carbon support film copper mesh electron microscopy grids . Grids were labeled for 45 s with 1% uranyl acetate (in double-distilled water). Prepared grids were examined using electron microscopy . Moreover, images were made by Quemasa charge-coupled device camera .8–1 × 109) of the NTA Software. Graphical presentations were performed using GraphPad Prism version 7.05 .Samples were analyzed by nanoparticle tracking analysis (NTA) using the NanoSight LM10 microscope  equipped with a 455-nm laser. For each individual sample, three videos of 60 s were recorded and analyzed with detection threshold 3 and camera level 13. All videos were analyzed by NTA Software version 3.2. To achieve optimal measurements, all EV samples were diluted with PBS to obtain a particle concentration within the optimal range (3 × 10We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV190022; Consortium et al.  EV-TRACK


The AP-1 transcriptional program modulates the expression of key neuronal remodeling genes, including S100a10 (p11), linking neuronal plasticity to the antidepressant response. We find that AP-1 function is required for the antidepressant effect in vivo. Furthermore, we demonstrate how neurochemical pathways of BDNF and FGF2, through the MAPK, PI3K, and JNK cascades, regulate AP-1 function to mediate the beneficial effects of the antidepressant response. Here we put forth a sequential molecular network to track the antidepressant response and provide a new avenue that could be used to accelerate or potentiate antidepressant responses by triggering neuroplasticity.Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed drugs for mood disorders. While the mechanism of SSRI action is still unknown, SSRIs are thought to exert therapeutic effects by elevating extracellular serotonin levels in the brain, and remodel the structural and functional alterations dysregulated during depression. To determine their precise mode of action, we tested whether such neuroadaptive processes are modulated by regulation of specific gene expression programs. Here we identify a transcriptional program regulated by activator protein-1 (AP-1) complex, formed by  Major depressive disorder (MDD) is a disabling psychiatric disorder with diverse etiology, and is a leading cause of mortality and morbidity worldwide . The symc-Fos, FosB, Fra1, Fra2, c-Jun, Junb, Jund, Egr1, Nr4a1, and Npas4. These IEGs initiate and maintain gene expression profiles of crucial effector proteins, causing sustainable changes in the structure and function of mature synapses and thereby promoting behavioral plasticity. So far, among the IEGs, Creb1 and Egr1 have been previously shown to be associated with the antidepressant response [Gene expression regulation represents a general mechanism through which mature neuronal circuits control their physiology and behavior , 10. Sevresponse –13. Addiresponse –16, likeresponse , we chosresponse –21. AddiIn this study, we investigated which of the IEGs are activated in the cortex by chronic treatment with an SSRI, fluoxetine, and we elucidated the target genes regulated by these factors. Furthermore, we addressed whether the IEGs and their target genes contribute directly to the behavioral response. Our findings reveal activation of a network of molecules that are sequentially linked together to provide a robust antidepressant response.For chronic drug and JNK inhibitor treatment, BALB/cJ mice (Jackson Laboratories) were housed two per cage and fluoxetine hydrochloride (Sigma) at a dose of (0.167 mg/mL) was administered in drinking water in 1% saccharine solution to mask the taste of the drug. Saccharine alone was given to the vehicle-treated animals. Mice were treated on average for 28 days and replaced with fresh solution every 3 days. On average, the fluoxetine-receiving mice drank approximately ∼3–4 mL a day, somewhat less compared with the control mice (that received 1% saccharine), which drank approximately ∼5–7 mL a day presumably due to the taste of the drug. The drinking volume of the fluoxetine-treated mice eventually normalized to that of the control mice. The fluoxetine-treated mice thus received 16–23 mg/kg/day of fluoxetine, an effective dose that is known to produce an antidepressant response in different strains of mice [ of mice . BALB/cJFor growth factor stimulation experiments in PC12/TrkB cells and primary mixed cortical neurons, growth factors BDNF (50 ng/mL) [c-Fos or c-Jun expression, and samples were collected at 12 and 24 h for p11 expression in PC12-TrkB cells and primary mixed cortical neurons, respectively. For the BDNF- and FGF2-stimulated time course experiments, samples were collected at 2, 6, 12, 24, and 48 h after an acute application. The experiments were conducted in a 12-well plate with at least three biological replicates.0 ng/mL) , 24, FGF0 ng/mL) , EGF (100 ng/mL) , IGF (100 ng/mL) , 28, NGF0 ng/mL) , VEGF (10 ng/mL) , BMP4 (10 ng/mL) , TGFβ (10 ng/mL) , Bicucul0 ng/mL) , and KCl0 ng/mL) , all froFor inhibitor experiments in primary cortical neuronal cultures, we tested which pathways affect basal c-Fos and c-Jun transcription. We applied inhibitors to block the various molecules in the tyrosine kinase pathway. To test the BDNF- and FGF2-inducible c-Fos and c-Jun transcription, we treated cells with inhibitors 30 min before stimulation. We used inhibitor concentrations that were previously known to induce neurogenic and neurotrophic effects: TrkB  [a, 1 μM) , MAP kina, 1 μM) , PI3K  , PLCγ  , p38 MAPa, 1 μM) . All inhFor transfections of transcription factor small interfering RNA (siRNA), experiments were done in PC12-TrkB cells. To identify the transcription factor regulating p11, inhibition of transcription factors were done upon treatment with two pre-validated silencer select siRNAs (Thermo Fischer Scientific) for each transcription factor according to the manufacturer’s instructions. siRNAs were transfected using Lipofectamine RNAiMAX reagent (Thermo Fischer Scientific) according to the manufacturer’s instructions, and expression levels of the transcription factor and S100a10 was analyzed by quantitative PCR, 48 h after transfection. siRNA efficciency was calculated based on the downregulation of the transcription factor by its specific siRNA. The experiments were conducted in a 12-well plate with at least three biological replicates. The siRNAs used are Atf3, s129666, s129668, Foxo1, s136654, s136655, Lrrfip1, s173268, s173270, c-Myc, s128068, s128069, Nfkb, s135615, s135616, Stat5a s128672, s128673, Egr1, s127689, s127690, Ets1, s127719, s127721, Junb, s127982, s127983, Jund, 201434, 201435, Fosl1, s217983, s129817, Fosl2, 197390, 197391, Bhlhe40, s135199, s135201, Yy1, s128676, s128677, Sp1, s128429, s128430, Srf, s180122, s180123, Creb1, s135438, s135439, Crem, s130285, s234984, Stat3, s129048, s129047, c-Fos s66197, s66198, c-Jun, s68563, and s201552.S100a10 promoter using the Dual-Luciferase Reporter Assay system (Promega). Exon 1 of S100a10 with varying lengths of 3′UTR region were cloned upstream of a firefly luciferase reporter construct in a mammalian expression vector pGL4.11 using the SLIC method (Li M 2007). We identified the functional S100a10 promoter reflected by robust luciferase activity with the coordinates, chromosome3: 93554373–93555181, based on mouse genome assembly, mm10. Next, we identified three c-Fos and c-Jun binding motifs within this region, and mutated the three sites named mutation1, mutation2, and mutation3 and deleted mutation3. The mutated and deleted firefly luciferase constructs were transfected into confluent Neuro-2a (N2a) cells grown in 12-well culture plates using lipofectamine LTX with Plus reagent (Thermo Fischer Scientific). The renilla luciferase gene was cotransfected as a control for transfection and expression efficiency. Cells were harvested and lysed 48 h after transfection and cell lysates were assayed for firefly and renilla luciferase activity according to the manufacturer’s instructions. Firefly luciferase activity was normalized to renilla luciferase activity for each cell culture well and plotted as activity relative to control transfections. At least four biological replicates were used. For dual transfections, c-Fos and c-Jun siRNA was first transfected using RNAiMAX and then 12 h later, DNA constructs were transfected using lipofectamine LTX with Plus reagent. The cells were harvested 36 h later, when the siRNA effect is maximum. The sequences of all plasmids were verified by sequencing and restriction enzyme digestion.Assays were performed in N2A cells to identify the functional 2 and decapitated, and cortex was rapidly dissected, frozen in liquid nitrogen, and stored at −80 °C until further processing. For cell analysis, cells were scraped and collected, rinsed with PBS, flash frozen in liquid nitrogen, and stored at −80 °C. For both tissues and cell pellet, samples were sonicated at 4 °C in lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 1% SDS supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Roche), and boiled for 10 min. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein samples were diluted in equal volume of 2× LDS sample buffer (Invitrogen) and supplemented with DTT to a final concentration of 200 mM (Sigma). Twenty micrograms of protein samples were separated on 4–12% Bolt Bis-Tris precast denaturing gels (Invitrogen) and transferred onto PVDF membranes and blocked with 5% milk in TBS–0.1% Tween (TBST) solution for 1 h at room temperature. Membranes were probed with primary antibodies diluted in 5% milk–TBST solution overnight at 4 °C. Membranes were then washed and probed with horseradish-peroxidase-conjugated anti-mouse , anti-rabbit , or anti-goat antibody  for 1 h at room temperature. Membranes were developed using Pierce Western blotting substrate  and exposed on film. Exposed films were scanned, and protein bands were quantified using ImageJ Software . Protein quantities were normalized using GAPDH. All values were plotted relative to control/untreated samples. Antisera and antibodies against the following proteins were purchased from the indicated sources: p11 , GAPDH , total c-Fos , phospho c-Fos , c-Jun , phospho c-Jun .For tissue analysis, mice were anesthetized with CO2. Tissue was transferred to a dounce homogenizer and buffer containing 1% formaldehyde, 50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA was added and fixed for 10 min end-to-end rotation. Formaldehyde was then quenched by adding 0.125 mM of glycine for 5 min. Halt-protease and phosphatase inhibiter (Thermo Fischer Scientific and Roche) and chromatin immunoprecipitation (ChIP) cross-link Gold (Diagenode) were added to the buffer before homogenization. Throughout the protocol buffers were supplemented with protease and phosphatase inhibitors. Lysate was then subjected to two wash steps with the same 1× PBS and 1 mM MgCl2 at 1350g at 4 °C for 5 min. Each sample was washed with lysis buffer 1 containing 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% NP-40, 0.2% Triton X-100 after incubating for 10 min at 4 °C. The pellet was then washed with lysis buffer 2 containing 10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, after incubating at RT for 10 min. The pellet was resuspended in 1 mL shearing buffer at 1 × 107 cells, containing 10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% deoxycholate, 0.2% N-lauroylsarcosine, and 0.2% SDS. The samples were sonicated in Covaris settings duty cycle 10% and 175 cycles/burst for 3 min to achieve fragments of sizes ranging from 200 to 500 bp. To each sample, 0.2% Triton X-100 was added and spun at 20,000g @ 4 °C for 15 min. The supernatant was collected; 1% of the input sample of each ChIP reaction was collected. The sheared chromatin was immunoprecipitated with washed antibody-bound beads at 4 °C overnight. The antibody coupled beads were washed 2× with low-salt buffer containing 50 mM HEPES-KOH, pH 7.6, 100 mM LiCl, 0.5 mM EDTA, 0.2% NP-40, 0.7% deoxycholate, and then washed 2× with 50 mM HEPES-KOH, pH 7.6, 100 mM LiCl, 0.5 mM EDTA, 0.2% NP-40, 0.7% deoxycholate. Then the beads were washed 2× with 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, and eluted with 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10% SDS buffer and incubated at 65 °C for 1 h. The isolated chromatin and the input samples were immediately cleaned with the IPure kit (Diagenode). The unbound fraction of the immunoprecipitation reaction was used to validate the fragmentation on a 1% agarose gel. Purification with the IPure kit was carried out according to the manufacturer’s instructions with the addition of Proteinase K treatment (Roche) for 1 h at 55 °C after the de-crosslinking step. Immune-enriched chromatin was further purified with phenol:chloroform:isoamyl alcohol (25:24:1) and concentrated by ethanol precipitation. ChIP-sequencing libraries were prepared using the Ovation Ultralow system V2 (Nugen). The samples were further validated, and processed for single reads 75 bp sequencing on the Illumina HiSeq 2500 platform.Antibodies against c-Fos , c-Jun , and rabbit IgG antibody  were bound to Protein-G magnetic beads  for 2 h, at 4 °C  with bowtie v1.0.1 . Peak regions were called with MACS2  and reads were extended by 200 bp to account for the size of the fragment isolated by the ChIP reaction. Three-way comparisons were performed to identify locations of differential enrichment in the genome between two conditions. This was done by using the tool bdgdiff in MACS2 . IGVTools (IGV Version 2.3.52) were used to convert the pileup peak files into binary tdf files for viewing in IGV. Possible promoter and closest gene of the differential enriched peaks were annotated by R package ChIPpeakAnno; peaks overlapped 5kbp upstream or downstream of a TSS were annotated as possible promoters. We invoke MEME  to discover the motifs with the sequences from the peak regions.2 and decapitated. The PFC or the whole cortex were rapidly dissected, frozen in liquid nitrogen, and stored at −80 °C. RNA extraction from frozen samples was performed using the Trizol/chloroform technique according to the manufacturer’s instructions (Thermo Fischer Scientific). After extraction, RNA was precipitated overnight at −80 °C in isopropanol with 0.15 M sodium acetate and Glycoblue , washed twice with 80% ethanol, air-dried, and resuspended in nuclease-free water. Purified samples were analyzed using a Nanodrop 1000 spectrophotometer (Thermo Fischer Scientific) in order to assess mRNA quantity and quality.Mice were anesthetized with COGapdh. Relative expression (ΔCt) and quantification (RQ = 2 –ΔΔC) for each mRNA were calculated using the ΔΔCt method as suggested and the graphs were plotted. Calculation of standard deviation (SDΔCt = (SDtarget2 + SDref2)1/2) and error bars (RQ1 = 2(−(ΔΔCt+SDΔΔCt)) and RQ2 = 2(−(ΔΔCt-SDΔΔCt))) was performed according to ABI technical literature Part Number 4371095 Rev B. For the control samples, the RQ values are close to 1 but not exactly 1, because the ΔΔCt was obtained after substracting the ΔCt of each control sample from the average ΔCt of the biological replicates.cDNA was prepared from DNase-treated total RNA using the High Capacity RNA-to-cDNA kit (Thermo Fischer Scientific). Relative gene expression of the cDNA was assayed by qRT-PCR using pre-designed recommended Taqman gene expression assays from Applied Biosystems (ABI) following the manufacturer’s recommendations. Cycle counts for mRNA quantification were normalized to Creb1, Mm00501607_m1, Rn00578826_m1; Crem, Mm04336053_g1, Rn04338541_m1; Egr1, Mm00656724_m1, Rn00561138_m1; c-Fos, Mm00487425_m1, Rn00487426_g1; Fosl2, Mm00484442_m1, Rn00564121_m1; Fosl1, Mm04207958_m1, Rn00564121_m1; Fosb, Mm00500401_m1, Rn00500401_m1; c-Jun, Mm00495062_s1, Rn99999045_s1; Junb, Mm04243546_s1 Rn00572994_s1; Jund, Mm04208316_s1, Rn00824678_s1; S100a10, Mm00501457_m1, Rn01409218_m1; Bdnf, Mm04230607_s1, Rn02531967_s1; Fgf2, Mm00433287_m1, Rn00570809_m1; Egf, Mm00438696_m1, Rn00563336_m1; Igf, Mm00439560_m1, Rn00710306_m1; Ngf, Mm00443039_m1, Rn01533872_m1; Vegf, Mm00437306_m1, Rn01511602_m1; Tgfβ, Mm01178820_m1, Rn00572010_m1; Gapdh, Mm_99999915_g1, Rn99999916_s1; Srsf5, Mm00833629_g1; Slc1a2, Mm01275814_m1; Sirt1, Mm01168521_m1; Glul, Mm00725701_s1; Glo1, Mm00844954_s1; Crhr1, Mm00432670_m1; Adrb1, Mm00431701_s1; Abcb1, Mm00440736_m1.The following pre-designed TaqMan gene expression assays from Applied Biosystems (ABI) were used. The probe names start with Mm for mouse probes , and Rn for Rat probes (used to test in rat PC12/TrkB cells). All behavioral studies were carried out and analyzed with the experimenter blind to the treatment group. Genotypes were decoded after data were processed and analyzed. Procedures were performed as described previously: open-field test (OFT) ; noveltyt-test. For multiple group comparisons, we used one-way or two-way ANOVAs and corrections were applied using the appropriate post hoc test. In all experiments, P < 0.05 was considered significant. Bar graphs show mean values and the error bars for bar plots are standard error of the mean (±SEM). N represents the number of mice for behavior experiments and biological replicates for cell culture experiments. For behavior experiments, all studies were carried out and analyzed with the experimenter blind to the treatment group.Statistical details of each experiment are included in the figure legends. Briefly, for two group comparisons, we used two-tailed unpaired Student’s To test the hypothesis that IEGs implement specific gene expression programs to mediate an antidepressant response, we aimed to first determine the kinetics of expression of transcription factors in response to treatment with chronic fluoxetine. To do so, we used BALB/cJ mice as they are inherently anxious and produce a robust behavioral response to chronic fluoxetine treatment . We admiP < 0.01, Fig. P < 0.04, Fig. The response to treatment was assessed by using well-described behavioral paradigms for depressive behavior, including TST and NSF . The fluCreb1, Crem, Egr1, Fosl1, Fosl2, c-Fos, Fosb, c-Jun, Junb, and Jund) was performed by quantitative PCR (qPCR)  Fig. . Among aene Fig. . Surprisisorders , 12, 43.isorders –47. The lex Fig. .O-tetradecanoylphorbhol-12-acetate(TPA)-responsive element (TRE) palindromic sequence, TGAC/GTCA [The AP-1 complex (Jun–Jun and Jun–Fos dimers) functions as a transcriptional regulator by binding to a common 12-GAC/GTCA . We hypoP value) cut-off of 1.3 are shown. As expected, we found that the c-Jun target genes regulated some of the same pathways as c-Fos target genes, but in addition also bound to genes that regulated structural plasticity, such as Rho, Rac signaling, and actin cytoskeleton signaling  stand out as they are both essential for mediating the antidepressant response in the cerebral cortex and hippocampus, and play a major role in mood and depressive disorders [Next we tested whether any of the human MDD-associated genes based on GWAS studies , 53 are isorders , 55. TheSrf, Egr1, Arc, Junb, Npas4, Jund, Creb1, and other effector genes, Dusp6, Trib1, and Nptx2 in response to fluoxetine.Among the c-Fos and c-Jun targets, transcription factors were the most upregulated group across the various categories Fig. , and repTaken together, these findings indicate that the specific induction of AP-1 by antidepressant treatment initiates a transcriptional program that modulates the expression of neuronal plasticity genes which then leads to an antidepressant response. These results prompted us to characterize the pathways that regulate AP-1 transcription and activity.c-Fos and c-Jun transcription is stimulated by chronic fluoxetine only after 9 days of chronic treatment. This prompted us to investigate and identify the factors that regulate c-Fos and c-Jun transcription. The expression of c-Fos and c-Jun are known to be readily induced by growth factors, neurotransmitters, and electrical stimulation [c-Fos and c-Jun expression. To determine which factors regulate c-Fos and c-Jun transcription, we established an in vitro primary cortical culture system that allowed for efficient and simultaneous screening of multiple growth factors that could directly stimulate c-Fos and c-Jun mRNA expression. This cell culture system comprises predominantly cortical neurons and other supporting cell types, and is widely used to study physiological properties of neurons [c-Fos and c-Jun transcription, we measured their levels 2 h after stimulation (as reported previously [c-Fos mRNA expression was stimulated by BDNF (∼20-fold), FGF2 (∼4-fold), and EGF (∼1.8-fold) , as previously reported [c-Jun mRNA expression was similarly induced by KCl, BDNF, FGF2 , and EGF (∼1.2-fold). The other factors showed no effects.We show that mulation , 56, 57. neurons . To capteviously ) with a ld) Fig. . c-Fos mreported , 56. c-Jc-Fos and c-Jun transcription, we next addressed which specific signaling cascade(s) induces AP-1 transcription and activity. Growth factors act by binding to their cognate receptors and activating MAPK signaling cascades that include extracellular signal-regulated kinases (ERKs), stress-activated protein kinases (JNK/SAPK), and p38 family of kinases (P38 MAPK), all of which stimulate c-Fos and c-Jun gene expression [c-Fos and c-Jun transcription in the absence  and MAPK inhibition, and moderately by p38 MAPK inhibition. No effects were observed upon inhibition of the PLCγ, PI3K, or JNK pathway . Next, we searched for a suitable cell line that would enable robust stimulation of AP-1 by BDNF and FGF2, and, in addition, would allow for efficient and targeted transcription factor knockdown following siRNA inhibition of candidate transcription factors.To ensure that AP-1 is a specific transcription factor that is relevant for the chronic fluoxetine response, we chose to study in detail the regulation of one of the AP-1 target genes—pression . We alsoelements , we detec-Fos, c-Jun, and S100a10 mRNA by qPCR. c-Fos and c-Jun mRNA were rapidly stimulated within 30 min of BDNF or FGF2 application, with a peak induction at 2 h as observed in primary cortical cultures. In contrast, S100a10 mRNA expression was first observed at 2 h (when c-Fos and c-Jun expression were at their peak), and the highest expression was observed at 24 h after treatment.  protein and the total and phosphorylated forms of c-Fos and c-Jun were congruent to the mRNA levels, as illustrated in Fig. S100a10 mRNA (2 h) at the same time we detected the phosphorylated forms of c-Fos and c-Jun (when they are able to form the AP-1 complex)  as a suitable system to study growth factor signaling. These cells demonstrated robust stimulation of AP-1 expression in response to BDNF and FGF2 Fig. . To evalnt. Fig. . To furtex) Fig. .Fig. 4APS100a10 at 24 h, we next tested which of the transcription factors, when silenced by specific small-interfering RNAs (siRNAs), affected S100a10 transcription. We tested 22 candidate transcription factors, all of which have potential to bind to the S100a10 promoter region. The siRNA inhibition efficiency for each factor and their effect on modulating S100a10 mRNA expression are indicated in Table c-Fos, c-Jun, or both decreased S100a10 mRNA . The S100a10 luciferase promoter construct was transfected into mouse Neuro-2a (N2a) cells in the presence and absence of AP-1 siRNA  to overcome influence of variability. We observed that the JNK inhibitor had no effect on the body weight of the mice and the animals drank about 3–6 mL of fluoxetine per day  and reduced latency to bite food (P < 0.003) in the fluoxetine-treated animals compared with the vehicle controls  and NSF (P < 0.01). The injection of the JNK inhibitor itself on vehicle-treated animals did not affect their behavior  confirm a significant role for these cascades during the antidepressant response. Such neuroadaptive properties have been documented for BDNF –92; FGF2ntegrity . Consistntegrity . These rc-Fos and c-Jun in response to BDNF and FGF2-stimulation, whereas the MAPK and PI3K pathways were required for both basal and inducible expression. Also, the role of MAPK in the induction of depressive behavior has been documented before [c-Fos and c-Jun induction and Jun phosphorylation, potentially during chronic fluoxetine response. Our results show that an attenuation in the fluoxetine response occurs when the activity of JNK is inhibited, thus validating that AP-1 function is necessary for providing behavioral response to fluoxetine in mice. Although we show evidence that both c-Fos and c-Jun are necessary to regulate the transcription of genes essential for the antidepressant response, we cannot rule out the effects of the AP-1 complex formed by the Jun-Jun homodimer.We establish that AP-1 function is essential for the fluoxetine response. We chose to inhibit the JNK pathway based on key observations. First, our data showed that the JNK pathway was the only pathway that was essential for the inducible expression of d before . Second,d before . Third, S100a10, we demonstrate that the AP-1 complex comprised of c-Fos and c-Jun (and not any of the other members of the Jun-Fos protein family) is the only transcriptional activator, among 22 other transcription factors tested. Furthermore, we identified the AP-1 binding site within the S100a10 promoter, and proved with nucleotide resolution that this binding site is functionally active, and is likely important for the antidepressant response. In addition, we also identified transcriptional repressors  of S100a10 gene that may a play a role in the regulation of basal- versus inducible transcription. These findings reveal that the activators and repressors of S100a10 transcription, together, potentially regulate the cell-type-specific expression of S100a10 and its signal-dependent induction—both of which are potentially important for the antidepressant response.We show that the transcriptional regulation of the target genes by the AP-1 complex is selective. By characterizing the regulation of one of the AP-1 target genes, In summary, we have identified AP-1 target genes with known links to human MDD based on GWAS data, indicating that these molecules could be used as potential biomarkers for predicting antidepressant responses. We have linked a number of molecules including growth factors, signaling cascades, and a fully integrated gene expression program that function consecutively to activate neuronal remodeling pathways to provide the antidepressant response. Importantly the molecules, FGF2 , BDNF 1, c-Fos , and p11Figure S1Figure S2Figure S3Figure S4Figure S5Table S1Table S2Table S3Description of Table S1, S2, S3