P. aeruginosa is an opportunistic pathogen of humans. With roughly 10% of its genes encoding transcriptional regulators, and hundreds of small noncoding RNAs (sRNAs) interspersed throughout the genome, P. aeruginosa is able to fine-tune its response to adapt and survive in the host and resist antimicrobial agents. Understanding mechanisms of genetic regulation is therefore crucial to combat pathogenesis. The previously uncharacterized sRNA PA0805.1 was overexpressed in P. aeruginosa strain PAO1, resulting in decreased motility, increased adherence, cytotoxicity, and tobramycin resistance. In contrast, a ΔPA0805.1 deletion mutant had increased susceptibility to tobramycin under swarming conditions. Omic approaches uncovered 1,121 transcriptomic and 258 proteomic changes in the overexpression strain compared with the empty-vector strain, which included 106 regulatory factors. Downstream of these regulators were upregulated adherence factors, multidrug efflux systems, and virulence factors in both transcriptomics and proteomics. This study provides insights into the role of the sRNA PA0805.1 in modulating bacterial adaptations. Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date posttranscriptional regulation has not been extensively investigated. Small noncoding RNAs (sRNAs) are hypothesized to play posttranscriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. One of these, sRNA PA0805.1 (which was 5-fold upregulated under swarming conditions), when cloned, transformed into wild-type (WT) PAO1, and overexpressed, led to broad phenotypic changes, including reduced swarming, swimming, and twitching motilities, as well as increased adherence, cytotoxicity, and tobramycin resistance. A ΔPA0805.1 deletion mutant was more susceptible to tobramycin than the WT under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty-vector strain by transcriptome sequencing (RNA-Seq) and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles showed 1,121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, and sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD. Therefore, the sRNA PA0805.1 appears to be a global regulator that influences diverse bacterial lifestyles, most likely through a regulatory cascade.IMPORTANCEP. aeruginosa is an opportunistic pathogen of humans. With roughly 10% of its genes encoding transcriptional regulators, and hundreds of small noncoding RNAs (sRNAs) interspersed throughout the genome, P. aeruginosa is able to fine-tune its response to adapt and survive in the host and resist antimicrobial agents. Understanding mechanisms of genetic regulation is therefore crucial to combat pathogenesis. The previously uncharacterized sRNA PA0805.1 was overexpressed in P. aeruginosa strain PAO1, resulting in decreased motility, increased adherence, cytotoxicity, and tobramycin resistance. In contrast, a ΔPA0805.1 deletion mutant had increased susceptibility to tobramycin under swarming conditions. Omic approaches uncovered 1,121 transcriptomic and 258 proteomic changes in the overexpression strain compared with the empty-vector strain, which included 106 regulatory factors. Downstream of these regulators were upregulated adherence factors, multidrug efflux systems, and virulence factors in both transcriptomics and proteomics. This study provides insights into the role of the sRNA PA0805.1 in modulating bacterial adaptations. Pseudomonas aeruginosa, a Gram-negative opportunistic and nosocomial pathogen, is highly motile and undergoes swarming motility in response to specific environmental conditions, namely, a semisolid environment with amino acids as a nitrogen source. These conditions are relevant to human lung diseases such as pneumonia and cystic fibrosis (CF). P. aeruginosa is a common CF pathogen, and the incidence of multidrug resistance has increased in recent years  are noncoding RNA species, usually around 40 to 500 bp in length in evolving . Typicalevolving ; howeverevolving . Translaevolving . In addievolving , 10. sRN effects . In manyruginosa , but subruginosa , 12. NevPrior research in our lab identified 20 sRNA species that were dysregulated under swarming conditions . One of araC promoter in the arabinose-inducible pHERD20T vector and transformed into wild-type (WT) PAO1 H103. PA0805.1 was overexpressed after induction with arabinose 28.1-fold ± 1.9-fold under swarming conditions .Specific quantitative reverse transcriptase PCR (qRT-PCR) analysis demonstrated that the transcript for PA0805.1 was upregulated under swarming conditions (versus swimming) 5.0-fold ± 1.7-fold. In contrast, it was downregulated in biofilm cells by 4.8-fold ± 3.8-fold . Since bAt the time of the assay, arabinose was added to induce expression. Motility assays showed that PA0805.1 overexpression had antimotility effects resulting in partial reductions in each of swarming (reduced to 36% ± 3% of EV), swimming (51% ± 10%), and twitching (61% ± 3%) motility .l-glutamine and no d-glucose [DMEM] and LB) showed little difference between strains  cells . Overexp10.1128/mSystems.00204-20.1FIG S1n = 3. Download FIG S1, TIF file, 0.2 MB.Growth curves of PA0805.1 in three different media showed little difference from EV. Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rpoD). MIC assays performed with the equivalent medium in microtiter trays showed little difference . Download FIG S2, TIF file, 0.5 MB.Tobramycin kill curve showed that swarm cells overexpressing PA0805.1 survived better than swarm cells with EV in the presence of tobramycin with no arabinose. Averages ± standard errors are depicted. Paired Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00204-20.4TABLE S14)2SO4. (n = 3). Download Table S1, DOCX file, 0.02 MB.Tobramycin MIC (micrograms per milliliter) in BM2 glucose with 0.1% Casamino Acids and no  in the PA0805.1 overexpression strain compared to EV, including 140 with increased abundance and 118 with decreased abundance (P ≤ 0.05 and absolute FC ≥ 1.5) revealed by RNA-Seq, with 401 downregulated and 720 upregulated  P ≤ 0.05 and absolute FC ≥ 1.25. Download Table S2, XLSX file, 0.6 MB.Transcripts and proteins with differential abundance in PA0805.1 versus EV by RNA-Seq and proteomics. Two analyses are shown for reference using proteomic cutoffs of (i) Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mexXY, a known efflux pump mediating resistance to aminoglycosides  were downregulated, and in other studies we showed that these can also lead to tobramycin resistance  were upregulated . qR. qRmexXYegulated . Further RNA-Seq . Moreovesistance  4, 16)., 16.mexXegulated . To confegulated .10.1128/mSystems.00204-20.6TABLE S3rpoD (n = 5). Download Table S3, DOCX file, 0.02 MB.Additional genes verified by qRT-PCR. The strains overexpressing PA0805.1 and EV were harvested from BM2 glycerol swarm plates with 1% arabinose, and expression was normalized to Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the fimX transcript and response regulators pilGH could cause the downregulation of twitching motility proteins PilIJK and the type 4 fimbrial biogenesis proteins PilAD, PilMNOPQ, PilEVWX, and PilY1-2 . Asi. AsifimXA operon . Regulat RNA-Seq , which c RNA-Seq . The upr and flp . Lastly,dherence . CollectlasAB and piv, which are cytotoxic proteases. Other upregulated virulence factors were type VI secretion system (T6SS) genes, rahU, alginate and phenazine biosynthetic genes, pslABCDEFGHIMN, and rhlABC , with 128 genes similarly downregulated , or upregulated , while 3 were regulated in opposite fashions (quadrants II and IV). This might relate in part to the differing abilities of the two methods since RNA-Seq was more sensitive and detected in total transcription from 5,194 genes, while proteomics identified in total 2,366 proteins . It is worth noting that transcripts for extracellular proteins were more likely detected in the RNA-Seq data since wash steps were employed prior to proteomics. Conversely, since sRNAs act by posttranscriptional regulation, it was expected that there would be changes in protein abundance with no corresponding difference in RNA transcript levels, while a single translationally dysregulated regulatory protein might control the expression of hundreds of genes. Lastly, due to the fact that transcripts and proteins have different half-lives, a good correlation between RNA-Seq and proteomic data is not necessarily expected  Venn diagram showing unique and common genes and proteins between the two methods; (B) scatterplot showing log2 FC values for the common genes and proteins. Download FIG S3, TIF file, 0.4 MB.Comparison of RNA-Seq and proteomics data using a cutoff Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the As mentioned above, PA0805.1 was upregulated 5.0-fold ± 1.7-fold under swarming versus swimming conditions . A deletion mutant of PA0805.1 was constructed and showed no dramatic motility phenotype, but it was more susceptible to tobramycin than the WT under swarming conditions and when complemented substantially restored tobramycin resistance . These dBAD promoter of pHERD20T)  . The tobbdlA was also upregulated 1.9-fold  than exsD, and downregulation of exsD could potentially be a mechanism to compensate for downregulation of T3SS.ExsD, a negative regulator of T3SS, was downregulated 3.8-fold in the RNA-Seq and 1.9-fold in the proteomics data set . ExsD bicapB  and predictive methods are indicated. Download Table S4, DOCX file, 0.02 MB.sRNA targets predicted Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lasAB and piv, were upregulated, likely resulting in the observed increased cytotoxicity. Lastly, the multidrug efflux systems mexXY and mexGHI-opmD were upregulated, which might contribute to tobramycin resistance. The sRNA PA0805.1 thus modulates important adaptations in P. aeruginosa, including motility, virulence, and antibiotic resistance.In this study, it was shown that overexpressing the sRNA PA0805.1 resulted in broad transcriptional and proteomic changes, most likely through a hierarchical regulatory cascade . A totalP. aeruginosa strain PAO1 H103 was grown in Luria-Bertani broth and BM2 minimal medium . LB overnight cultures were diluted 1/50 and grown to mid-log phase .Escherichia coli TOP10 and selected with 50 μg/ml of kanamycin (TOPO). Plasmid was subsequently isolated according to instructions with the Thermo Fisher kit and digested with the restriction endonucleases EcoRI and KpnI. After the fragment was gel extracted, it was ligated into the similarly digested vector pHERD20T with T4 DNA ligase (Thermo Scientific), transformed into TOP10 E. coli, and selected with 100 μg/ml of ampicillin. Plasmid sequences were confirmed by Sanger sequencing at the UBC Sequencing and Bioinformatics Consortium.PAO1 WT genomic DNA was isolated as specified in the Qiagen DNeasy blood and tissue kit protocol. Three hundred nanograms of DNA was PCR amplified using the cloning primers PA0805.1 F and PA0805.1 R described in 10.1128/mSystems.00204-20.8TABLE S5Table S5, DOCX file, 0.02 MB.Primers used in this study. Download Copyright © 2020 Coleman et al.2020Coleman et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa PAO1 was transformed with both EV pHERD20T and vector overexpressing PA0805.1 according to the method of Choi et al.  and then conjugating this construct along with the helper plasmid pTNS3, using the BAD promoter of the plasmid pHERD20T (4)2SO4 as the nitrogen source and 20 mM potassium succinate (pH 7.0) as the carbon source, unless otherwise indicated. For swarming assays, plates were solidified with 0.5% (wt/vol) agar, 0.1% Casamino Acids was used as the nitrogen source, and 0.4% (wt/vol) glycerol was used as the carbon source, unless otherwise indicated. Swimming and swarming BM2 plates were composed of 25 ml of medium per plate and dried for 1 h. In contrast, LB medium was used for twitching motility, with 1% agar and 10 ml of medium per plate, and dried overnight. Arabinose was included where indicated for plasmid induction. All plates were stab (swim and twitch) or spot (swarm) inoculated with 1.5 μl of mid-log-phase bacteria. After inoculation, plates were incubated 16 to 20 h at 37°C (unless otherwise indicated) and imaged on the ChemiDoc touch imaging system (Bio-Rad).The concentration of agar and nitrogen source in BM2 were varied to allow for different kinds of motility. Glucose (0.4% [wt/vol]) was often replaced with an alternative carbon source (as indicated) since glucose represses expression from the PpHERD20T . SwimminSwarming BM2 plates containing 0.4% (wt/vol) glycerol, 1% (wt/vol) arabinose, and 0.1% (wt/vol) Casamino Acids were grown for 20 h at 37°C. For RNA isolation, the edge of the swarm front was harvested with a plastic loop and transferred to RNAprotect bacteria reagent (Qiagen), pelleted, and stored at −80°C. For protein isolation, the edge of the swarm front was harvested with a plastic loop and transferred to phosphate-buffered saline , washed three times with PBS, and stored as a pellet at −80°C.Pellets were lysed by resuspension in 3 mg/ml of lysozyme dissolved in Tris-EDTA (TE) buffer . RNA isolation then proceeded according to the manufacturer’s instructions using the RNeasy mini kit (Qiagen). Eluted RNA was further purified with the TURBO DNA-free kit (Thermo Fisher). Two independent runs of RNA-Seq were performed with a total of 5 biological replicates for each strain.P. aeruginosa PAO1 were mapped to its genomic sequence using STAR v2.6.1a. Read counts for individual genes were obtained using HTSeq-count v0.9.1. Significantly differentially expressed genes  were identified using DESEQ2 1.20.0 and were then used for further analysis.RNA samples were depleted of rRNA using the RiboZero bacterial kit (Illumina). Libraries of cDNA were prepared using the KAPA stranded total RNA kit (Kapa Biosystems) and sequenced on an Illumina HiSeq 2500. Fastq reads, determined using FastQC v0.11.7 and MultiQC v1.6.dev0, for swarming of g for 10 min. Protein concentration was determined using the Pierce detergent-compatible Bradford assay kit (Thermo Fisher Scientific). All protein samples were processed and handled using the single-pot solid-phase-enhanced sample preparation (SP3) protocol described below. Prior to SP3 treatment, two types of carboxylate-modified SeraMag Speed beads (GE Life Sciences) were combined in a ratio of 1:1 (vol/vol), rinsed, and reconstituted in water at a concentration of 20 μg of solids per μl. Initially, 200 μg of lysate was reduced with 10 mM  dithiothreitol for 30 min at 60°C followed by alkylation using 50 mM  iodoacetamide for 45 min in the dark at room temperature. After that, 20 μl of the prepared bead mix was added to the lysate and samples were adjusted to pH 7 using HEPES buffer. To promote protein binding to the beads, acetonitrile was added to a final concentration of 70% (vol/vol) and samples were incubated at room temperature on a tube rotator for 18 min. Subsequently, beads were immobilized on a magnetic rack for 1 min. The supernatant was discarded and the pellet was rinsed twice with 200 μl of 70% ethanol and once with 200 μl of 100% acetonitrile while on the magnetic rack. Rinsed beads were resuspended in 65 μl of 50 mM HEPES buffer (pH 8) supplemented with trypsin–Lys-C mix (Promega) at an enzyme-to-protein ratio of 1:25 (wt/wt) and incubated for 16 h at 37°C. After overnight digestion, supernatant containing peptides was transferred into a fresh tube and subsequently measured for peptide yield using the Pierce quantitative fluorometric peptide assay (Thermo Fisher Scientific).Bacterial cell pellets were resuspended in lysis buffer . The cells were sonicated three times for 15 s per cycle with 1 min of cooling on ice between each cycle. The insoluble cellular debris was removed by centrifugation at 17,000 × Representative samples containing 85 μg of peptides were adjusted to the same concentration using 50 mM HEPES (pH 8) and labeled with 10-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific). The TMT10 reporter channels were sequentially assigned in increasing reporter mass as TMT0 to TMT9. Four TMT10 channels (TMT0 to TMT3) were assigned to samples from the EV strain and three TMT10 channels (TMT7 to TMT9) to samples from the PA0805.1 strain. This represented four biological replicates for the EV strain and three replicates for the PA0805.1 strain. In short, 0.8 mg of each TMT channel was first dissolved in 41 μl of dimethyl sulfoxide (DMSO) before addition to the corresponding peptide digests. The labeling reaction mixture was incubated at room temperature for 60 min. Following incubation, samples were quenched for 15 min with the addition of 8 μl of 5% hydroxylamine. Finally, labeled samples were mixed at equal volumes and desalted using SOLA HRP SPE cartridge (Thermo Fisher Scientific) prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS).18  column packed in-house in Pico-Frit (100 μm by 30 cm) capillaries . Peptide separation was done using the following gradient: 3 to 5% increase of phase B over 4 min, 5 to 7% over 3 min, 7 to 25% over 197 min, 25 to 60% over 25 min, and 60 to 90% over 1 min, with final elution of 90% phase B for 10 min at a flow rate of 300 nl/min.Analysis of TMT-labeled peptide digests was carried out on an Orbitrap Q Exactive HF-X instrument . The peptide mixture was resuspended in 0.1% formic acid prior to injection. The sample was introduced using an Easy-nLC 1000 system (Thermo Fisher Scientific) at 2 μg per injection. Mobile phase A was 0.1% (vol/vol) formic acid, and mobile phase B was 0.1% (vol/vol) formic acid in 80% acetonitrile (LC-MS grade). Gradient separation of peptides was performed on a Cm/z were acquired at a resolution of 120,000 (at m/z 200), with a maximum ion injection time of 60 ms and an automatic gain control (AGC) target value of 3E6. For MS2 scan triggering, up to 20 of the most abundant ions were selected for fragmentation at 32% normalized collision energy, with the intensity threshold kept at 5.7E4. Automatic gain control (AGC) target value for fragment spectra was set at 1E5, which were acquired at a resolution of 45,000, with a maximum ion injection time of 88 ms and an isolation width set at 0.7 m/z. Dynamic exclusion of previously selected masses was enabled for 30 s, charge state filtering was limited to 2 to 6, peptide match was set to preferred, and isotope exclusion was on.Data acquisition on the Orbitrap Q Exactive HF-X instrument was configured for the data-dependent method using the full MS/DD−MS/MS setup in a positive mode. Spray voltage was set to 1.85 kV, funnel radio frequency (RF) level at 40, and heated capillary at 275°C. Survey scans covering the mass range of 350 to 1,500 www.uniprot.org). The one-dimensional (1D) LC-MS run was converted into an MGF file using the Proteome Discoverer bundled tool and was searched against the PAO1 database using X!tandem (cyclone 2012.10.01.1). Peptide identification settings were standard for the instrument: single missed cleavage tryptic peptides were permitted, with a parent and fragment mass tolerance of 10 ppm. A fixed posttranslational modification of C + 57.021 was applied, and variable posttranslational modifications, including N-terminal acetylation, deamidation, phosphorylation, and oxidation, were permitted. Peptide assignment into source proteins was managed by X!tandem.A January 2019 reference database of PAE PAO1 (taxon 208964) was downloaded from UniProt .Peptide level TMT10 reporter tag intensities were integrated across a window of ±3 mDa each and corrected for isotopic overlap between channels using the supplied batch-specific correction matrix. Protein level quantitation required at least two unique peptides with expectation values of log(e) ≤ −1.5 each, yielding highly confident protein assignments of at least log(e) ≤ −3. The sum of peptide level TMT10 reporter tag intensities for each protein was converted into a logt test function in Excel between population averages. The P scores were not subjected to multiple-testing corrections, and any differences with P values of <0.05 were considered candidates for biological exploration. Differences between normalized population means were scaled back into a log2 scale by multiplying them by an average system-wide SD of 2.26.Differential analysis between normalized sample populations (PA0805.1 versus EV) was conducted using the Welch qC) values were normalized to the housekeeping gene rpoD or 16S, as indicated, using the threshold cycle (ΔΔTC) method. qPCR primers used are described in Swarming or swimming BM2 plates containing 0.1% (wt/vol) Casamino Acids and the desired carbon source were grown overnight at 37°C. RNA was isolated and DNase digested as described above and quantified on a NanoDrop ND-1000 spectrophotometer. RNA was then diluted to 1 ng/μl and 5 μl was used in a total reaction volume of 25 μl. The qScript one-step SYBR green quantitative reverse transcriptase PCR  was used, and samples were run on a LightCycler 96 (Roche). Quantification cycle  glucose and 0.1% (wt/vol) Casamino Acids and no (NH4)2SO4 at 100 μl/well in 96-well polystyrene round-bottom plates. After 24 h of incubation at 37°C, the minimal concentration to inhibit visible bacterial growth was reported as the MIC.Bacteria were seeded at 5 × 10600 of 0.03 in 90% LB supplemented with 5% (wt/vol) arabinose and seeded at 100 μl/well in 96-well flat-bottom polystyrene plates. After 4 h of incubation at 37°C, unattached cells were removed by discarding the media and rinsing three times with distilled water (dH2O). A total of 105 μl of 0.1% crystal violet was added and incubated with shaking for 20 min at room temperature, then the plates were rinsed three times with dH2O, and the crystal violet was solubilized by adding 110 μl of 70% (vol/vol) ethanol and shaking for 20 min at room temperature. Then the absorbance at 595 nm was read in an Epoch plate reader (BioTek).Overnight cultures were diluted to a final ODl-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco). After cells reached 80 to 100% confluency, they were washed once with PBS , trypsinized with 0.25% trypsin-EDTA (Gibco), and diluted in medium before counting. HBE cells were seeded at 2 × 104/well in a 96-well plate and grown again to confluency (2 to 3 days). A total volume of 200 μl per well was used. Then the medium was changed to Dulbecco’s modified Eagle medium with l-glutamine and no d-glucose  supplemented with 1% FBS and 1% sodium pyruvate 1 to 2 h prior to infection. Next, bacterial cultures were prepared by pelleting overnight cultures, washing once with PBS, and resuspending in DMEM (no glucose)-1% FBS-1% sodium pyruvate with or without 1% (wt/vol) arabinose. Bacteria were diluted in the same medium. Next, the medium of the HBE cells was removed and replaced with a suspension containing 3 × 105 CFU/ml of bacteria. The coculture was incubated at 37°C with 5% CO2 for 16 h, followed by monitoring the release of lactate dehydrogenase (LDH) as an indicator of cytotoxicity as described below. Cells treated with 2% (vol/vol) Triton X-100 (Fisher Scientific) in DMEM (no glucose)-1% FBS-1% sodium pyruvate were used as a positive control for the LDH assay.Human bronchial epithelial 16HBE14o- (HBE) cells between passages 14 and 40 were grown in minimum essential medium with Earle’s salts (1×)  supplemented with 10% fetal bovine serum , 2 mM Plates were centrifuged for 5 min at 1,000 rpm in an Eppendorf 5810 R centrifuge (15 A version), and 50 μl of supernatant was removed and mixed with 50 μl of solution as indicated in the cytotoxicity detection kit (Roche) assessing release of LDH  and incubated for 10 min at room temperature in the dark. Then the absorbances at 492 and 900 nm were read in the Epoch plate reader (BioTek). Next, the absorbance at 900 nm was subtracted from the absorbance at 492 nm. Percent cytotoxicity was calculated by subtracting controls  from coculture values and then normalizing to the Triton X-100 control.600 of 0.05 in each of the three media listed below and seeded in 96-well round-bottom plates at 100 μl/well. They were incubated at 37°C with shaking at a frequency 567 cpm (3 mm) in a Synergy H1 microplate reader, and the OD600 was read every 30 min. Media used were (i) liquid BM2 swarming medium , (ii) 90% LB with 5% arabinose, and (iii) DMEM with no glucose but with 1% FBS, 1% sodium pyruvate, and 1% arabinose.Overnight cultures were diluted to a final ODP ≤ 0.05 for IntaRNA2, P ≤ 0.05 for TargetRNA2, and top 100 for RNAPredator. Only targets predicted by more than one tool were considered.sRNA targets were predicted using three tools: IntaRNA2 , RNAPredGSE137738. Proteomics data were deposited in MassIVE under index number MSV000084373.RNA-Seq data were deposited in GEO under the accession number


Catalão is a Portuguese dry-cured traditional sausage, highly appreciated for its distinctive sensory properties. The aim of this study was to evaluate the impact of a 25% salt reduction on Catalão manufactured with either purebred Alentejano (Al) or crossbred Iberian × Duroc (IDr) pork meat, on its physicochemical and microbiological stability, texture parameters, and sensory attributes. No significant effect of salt reduction or genotype was observed for pH, aW, and microbiological parameters. PUFA content was significantly higher for Al Catalão, particularly due to the content in linoleic and linolenic fatty acids. IDr 3% NaCl samples had the highest mean n6/n3 PUFA ratio, and the highest mean values for the atherogenicity and thrombogenicity indices, showing that both genotype and salt content influence these nutritional indices. Texture profile of Catalão was significantly influenced by salt content and genotype. Al samples were less adhesive, cohesive, and easier to chew. Low-salt Catalão was harder, more adhesive, and less cohesive, with lower resilience and higher chewiness values. Regarding sensory attributes, salt content influenced the product aroma, with reduced-salt sausages being evaluated as significantly less aromatic. Overall, a 25% salt reduction did not have a significant impact on the quality, stability, and sensory evaluation of Catalão. Traditional cured meat products are appreciated worldwide because of their sensory characteristics. These sensory characteristics are related to meat selection, recipe, and processing of sausages among other factors . In the Traditional dry-cured meat sausages have been considered delicatessen products, but have lately been regarded as potentially unhealthy foods for several consumers, due to nutritional characters, associated to their high fat and salt contents ,5,6. HigCatalão sausages [The production of low-salt, dry-cured meat sausages must assure not only safe and stable products, but also that consumers’ appreciation will be not affected. Previous studies on sausages  had alresausages .Catalão, a dry-cured meat traditional sausage produced with either purebred or crossbred Iberian pork meat, on its stability  and texture, as well as on its sensory attributes.The aim of the current work was to evaluate the impact of a 25% salt reduction on Catalão, a cured traditional meat sausage was manufactured in a local factory of the Alentejo region  with two different meat raw materials, either Alentejano purebred (Al) or crossbred Iberian × Duroc (IDr) pork meat.Piper nigrum L.) (0.15%), white pepper (0.15%), cumin (Cuminum cyminum L.) (0.10%), disodium diphosphate (0.04%), pentasodium triphosphate (0.04%), NaNO3 (0.003%), and KNO2 (0.003%). Nitrates and nitrites were added in the form of the commercial additive NITROS 5/5 . Regarding salt content, Catalão sausages were manufactured with the commercial formulation usually  and with a 25% salt reduction . Therefore, four different groups of sausages were produced: Al 3% NaCl, Al 4% NaCl, IDr 3% NaCl, and IDr 4% NaCl.Meat was manually cut into large pieces, mechanically minced (5.0 × 5.0 mm), and mixed with 5% minced backfat (5.0 × 5.0 mm), white wine (3.50%), sodium chloride (NaCl), black pepper  with a horseshoe shape.After two days under refrigeration at 5 °C and 90% relative humidity, for ripening purposes, the meat batters were stuffed into cleaned and desalted natural pork casings, with 36–38 mm. Quercus ilex L.) wood, for two days, approximately 7 h/day. The drying process took place in a controlled environment chamber (7 °C and 80–85% relative humidity) until a 35% weight loss was reached, which took about 18 days. Three independent production batches of Catalão were manufactured for each group, with duplicate samples  being collected after 35% weight loss . Sausages were immediately processed for physicochemical, microbiological, and sensory analyses.The general curing procedure of meat sausages occurred in two steps: smoking and drying. The sausages were indirectly exposed to smoke generated by burning oak  [Listeria monocytogenes (ISO 11290-2) [Escherichia coli (ISO 16649-2) [Sausages were prepared for microbial analysis according to ISO 6887-2 . MicrobiSO 6579) ; Listeri11290-2) ; mesophi11290-2) ; lactic 11290-2) ; staphyl11290-2) ; enteroc11290-2) ; Escheri16649-2) ; and mol16649-2) . All micW) was determined using a Rotronic Hygrometer station (Rotronic Hygroskop DT) previously calibrated at 20 ± 1 °C with EA00-SCS, EA50-SCS and EA80-SCS Humidity Standards .pH values were measured in triplicate in each homogenized sample with a pH meter  equipped with a pH electrode  according to the procedures described in ISO 2917 . Water aTotal chloride content was quantified according to the Volhard method (ISO 1841-1)  and exprCatalão sausages was performed using a Dionex 100 accelerated solvent extractor (ASE)  using the following extraction procedure: aliquots of approximately 300 mg of sausages was combined with 6 g diatomaceous earth , and the mixture was transferred to a 34 mL stainless steel extraction cell fitted with two cellulose filters. Further, the extraction procedure carried out with a mixture of chloroform/methanol (60:40 (v/v)  containing 100 mg·L−1 BHT   as an antioxidant at 100 °C and 12.4 MPa. Two static extraction cycles were carried out for 5 min each. The solvent was removed under vacuum using a Vacobox B-177  equipped with a vacuum controller B-720, a rotavapor R-114  attached to a water bath B-480 afforded a crude residue. Next, crude residue was dissolved with 1 mL of chloroform and an aliquot of 100 µL was taken into a glass tube and the solvent was removed under a stream of nitrogen. The residue obtained was saponified in methanolic NaOH solution (0.5 N) at 70 °C for 15 min. Fatty acids were derivatized with boron-trifluoride-methanol solution (10 g BF3 L1CH3OH)  in order to give fatty acids methyl esters (FAMEs) as described by Morrison and Smith [Each sausage was cut into pieces and grounded in a mechanical mill, lyophilized, placed in a glass flask, and stored at 4 °C until use. The extraction of fatty acids from nd Smith . FAMEs wnd Smith . FAMEs wTexture Profile Analysis (TPA) was performed at room temperature (20 ± 1 °C) on five replicates per sample using a Stable Micro System TA-Hdi  as described previously ,24,25. USensory analysis was performed at the University of Évora, Portugal, in a Sensory Test Room prepared according to the environment requirements described in ISO 8589 . SelecteCatalão sausages (4 groups × 3 batches × 2 samples) were analyzed. Data were presented as means ± standard error deviation. Analyses of variance (ANOVA) were performed for the factors genotype and salt content.Data analysis was performed using Statistica version 12 software . A total of 24 No significant differences were observed between batches, proving that the authors effectively analyzed three independent production batches. Consequently, the factor batch was not further considered for statistical analysis, and all replicates were analyzed together. Moreover, no significant interaction was observed between the two factors, genotype and salt content, and thus, one-way ANOVAs were performed for each factor separately.Catalão cured sausages had a pH range from 5.46 to 5.60  implicated in the protection of the meat product [ to 5.60 , indepen product . Further product ,32,33.Catalão is summarized in The composition of fatty acids methyl esters of Catalão sausages were oleic (C18:1), palmitic (C16:0), stearic (C18:0), and linoleic (C18:2) acid  and linolenic (C18:3) acids (p < 0.05), essential fatty acids for human health, that must be provided by the diet, because they cannot be synthesized by humans [As observed in :2) acid , in decr:2) acid . Howevery humans .Catalão sausages  for Catalão sausages made with Alentejano (Al) raw meat (8.66–8.97%), particularly due to the content of linoleic (C18:2) and linolenic (C18:3) fatty acids  and the thrombogenicity (TI) indices, were also calculated according to Larqué and co-workers [Based on the results obtained from the FAMEs profile of -workers . AccordiConcerning n6/n3 PUFA ratios, high values were obtained (14.94–19.78), since the recommended n6/n3 PUFA ratio should not exceed a value of 4 . ScientiCatalão samples studied in this work, these values being very similar to other AI reported for fermented sausages made of pork meat from various breeds [The Atherogenicity Index (AI) expresses the relationship between the sum of the main SFA (pro-atherogenic) and that of the main MUFA and PUFA (anti-atherogenic) . The AI s breeds .p < 0.001). On the other hand, AI and TI show higher values for lower salt concentrations (p < 0.05).The Thrombogenicity Index (TI) can be defined as the ratio between the pro-thrombogenic SFA and the anti-thrombogenic MUFA and PUFA . RegardiThe values obtained for both AI and TI are generally low, when compared to the literature ,41,42. Ip > 0.05). Therefore, no major differences were found in the fatty acids profile regarding salt content, that may justify the differences depicted in these indices.Despite of the significant differences observed for Atherogenicity and Thrombogenicity Indices associated to an increased salt content, the relative abundances of SFA and MUFA do not show significant differences with salt content , cohesiveness (p < 0.01), springiness and chewiness (p < 0.05)  (Texture profile of  < 0.05) . Cured m < 0.05) . When saRegarding sensory analysis, the results of different attributes evaluated by the panelists are shown in Catalão sausages (The main differences detected by the sensory panel in sausages  were relp < 0.05), already reported in previous studies [Salt content influenced the product aroma. Products with lower salt content (3%) presented significantly lower aroma intensity ( studies ,44,45,46Catalão sausages. This may be considered an advantage, because it means that the product was not unsalted or tasteless. All in all, reduced-salt Catalão sausages are stable without significant changes in the analyzed microbial groups, without a significant damage in texture or fatty acids profile. Therefore, a 25% salt reduction, with reference to the salt content of the original Catalão sausages available in the market, can be beneficial, provided that manufacturing procedures, leading to a weight loss of 35% in the final product, are observed.Besides a lower aroma intensity, no other significant differences were perceived by assessors regarding reduced-salt Catalão sausages was studied. Generally, a 25% salt reduction did not have a significant impact on the stability, safety, and sensory appreciation of Catalão sausages, which thereby retained their quality.The effect of salt reduction and genotype on the quality and safety of dry-cured W, and microbiological parameters. Regarding genotype, the content in PUFA was significantly higher for Alentejano Catalão sausages, particularly due to the content in linoleic and linolenic fatty acids. Both genotype and salt content had influence in the n6/n3 PUFA ratio, and the atherogenicity and thrombogenicity indices, with IDr 3% NaCl samples showing the highest mean values for all three indices. Texture profile of Catalão was significantly influenced by salt content, with Alentejano samples being less adhesive, cohesive, and easier to chew. Low-salt Catalão sausages were harder, more adhesive, and less cohesive, with lower resilience and higher chewiness values. The texture profile of Catalão was also influenced by genotype, with significantly lower adhesiveness, cohesiveness, springiness and chewiness values for the Alentejano sausages. Regarding sensory attributes, salt content influenced aroma intensity, with reduced-salt sausages being evaluated as significantly less aromatic, which had been reported before for similar products.Furthermore, no significant effect of salt reduction or genotype was observed for pH, aBased on the presented results, and following the WHO recommendations, sausage manufacturers may be advised to safely reduce salt content, without compromising their safety and sensory attributes.


Following the drinking period, mice were evaluated for neuroinflammatory cytokine response or tested for cognitive and behavioral deficits. Results indicate no difference in alcohol consumption or preference following rmTBI as compared to sham; however, increases in the neuroinflammatory cytokine response due to alcohol consumption and some mild cognitive behavioral deficits after rmTBI and alcohol consumption were observed. These data suggest that the cytokine response to alcohol drinking and rmTBI + alcohol drinking is not necessarily aggregate, but the combination does result in an exacerbation of cognitive behavioral outcomes.The relationship between alcohol consumption and traumatic brain injury (TBI) often focuses on alcohol consumption increasing the likelihood of incurring a TBI, rather than alcohol use outcomes after TBI. However, patients without a history of an alcohol use disorder can also show increased problem drinking after single or multiple TBIs. Alcohol and mild TBI share diffuse deleterious neurological impacts and cognitive impairments; therefore, the purpose of these studies was to determine if an interaction on brain and behavior outcomes occurs when alcohol is consumed longitudinally after TBI. To examine the impact of mild repetitive TBI (rmTBI) on voluntary alcohol consumption, mice were subjected to four mild TBI or sham procedures over a 2 week period, then offered alcohol (20%  Alcohol use disorder (AUD) and traumatic brain injury (TBI) independently represent major health problems in the United States. Each year, over 14.5 million people are diagnosed with AUD ,3. HowevThe majority of TBI cases, up to 90%, are concussions, also called closed head injuries, which are classified as “mild” ,13. MildThe intertwined presentation of mTBI and AUD, exacerbated recovery times for TBI after alcohol drinking, similar cognitive and behavioral deficits, and common neuroinflammatory processes suggest that these disorders may have shared neural mechanisms that can be targeted to reduce damage or improve cognitive and behavioral outcomes. These longitudinal experiments investigated the effects of mTBI on alcohol consumption using a well-characterized mouse model of repetitive mTBI (rmTBI)  and a weSixty-four adult (8–12 weeks old) male C57BL/6J mice  were used, with 12 mice per behavioral condition and four mice for the nonmanipulated RT-qPCR control. Food and water were freely available throughout the duration of the experiment, and all mice were maintained on a 12 h reverse light/dark cycle (6:00 p.m./6:00 a.m.) in a temperature- and humidity-controlled animal facility. Mice were transported to a separate room in the same building followed by a short habituation period (20 min) prior to rmTBI induction and behavioral assays, while all drinking measures occurred in the home cage. Following the final drinking session (24 h) or behavioral task (1 h), the mice were given a lethal dose of isoflurane, so that the brains could be cleared of blood via transcardial perfusion with phosphate-buffered solution and extracted for assessment of neuroinflammatory response.These experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of the University of South Florida, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. IS00002500.To model the rmTBI observed in humans, a conservative but validated model of closed head injury protocol was used . Mice wev/v) ethyl alcohol (ethanol) with tap water to a 20% (v/v) concentration. Solutions were presented using sipper tubes featuring a ball bearing nozzle [Voluntary alcohol consumption was determined using the well-established drinking in the dark (DID) protocol, two-bottle choice variant ,29,30. Ag) following collection and the alcohol concentration measured using an Analox Instrument analyzer . Plasma samples were analyzed immediately or stored at −20 °C until analyzed.Blood samples were taken once per week immediately following the Day 4 drinking session via a standard submandibular bleed procedure. This procedure was very quickly conducted by an experienced researcher and did not require anesthesia. Samples were centrifuged . An aliquot (5 µL) of RNA was diluted in 495 µL of DEPC-treated water in order to measure RNA concentration and purity. Each sample was assessed with ultraviolet (UV) spectroscopy utilizing the absorbance measured at 260 and 280 nm . The remainder of the RNA sample was stored at −80 °C until further use.Frozen (whole) brains were brought to room temperature; TRIzol  was added and tissue was homogenized until no solid pieces were visible. Homogenized samples were incubated at room temperature for 5 min to allow for complete dissociation of the nucleoprotein complex. Chloroform was added to each sample, thoroughly mixed, and allowed to incubate another 2–3 min. Samples were centrifuged at 12,000× The cDNA synthesis reaction was prepared according to manufacturer provided protocol included in the iScript Advanced cDNA Synthesis Kit for RT-qPCR . Briefly, a working solution was made using the provided 5× iScript Advanced Reaction mix, iScript Reverse Transcriptase, and water. The sample RNA template was added to each well with the working solution. Samples went through reverse transcription by being held at 46 °C for 20 min and then 1 min at 95 °C to inactivate the reverse transcriptase. Samples were stored undiluted at −20 °C if not used for rt-PCR immediately. 2O. The samples were added to the plate in duplicate. Samples were then placed in iCycler MyIQ Real-Time PCR Detection System  and heated to the protocol-specified temperatures for 35 cycles. Quantitative PCR was conducted via manufacturer provided protocol for SsoAdvanced Universal SYBR Green Supermix . Briefly, a working solution made using the provided SsoAdvanced Universal SYBR Green Supermix (2×), forward and reverse primers specific to the house gene (18S) or target gene , and nuclease-free HPrimer sequences used included TNF-α forward (F): 5’–GACCCTCACACTCAGATCATCTTCT, TNF-α reverse ®: 5’–CCTCCACTTGGTGGTTTGCT; IL-1β F: 5’–CTGGTGTGTGACGTTCCCATTA, IL-1β R: 5’–CCGACAGCACGAGGCTTT; IL-6 F: 5’–GGCCTTCCCTACTTCACAAG, IL-6 R: 5’–ATTTCCACGATTTCCCAGAG. Cognitive and behavioral tasks included the Serial Spatial Discrimination Reversal Learning (SSDRL) task measuring simple discrimination and behavioral flexibility and an integrated open-field (OF) task measuring spontaneous locomotor activity and novelty preference; see The SSDRL task uses a partially submerged T-maze , in which mice were required to learn to escape from the water  using a platform at one of the arms ,32,33,34All mice started testing on their nonpreferred arm. This was determined by allowing the animal to choose freely between the two arms on the first test trial and then the escape platform was placed in the opposite arm. Errors included entering the incorrect arm, re-entry to the starting stem, and backtracking through the correct arm without escaping. Because the criterion to reverse the contingencies was six correct trials, if an animal completed two consecutive trials during the final trials of the day, an additional four trials were added in an attempt to allow the animal to reach the criterion. If the mouse made an error during one of the extra trials, testing ended for the day. These tasks assessed locomotor activity , preference for novelty, and working memory (via recognition of familiar object and time spent with novel object). On Day 5, mice freely explored the arena for 3 min to habituate. Approximately 30 min later, an additional 3 min session took place to assess locomotor activity. On Day 6, a 3 min exploration included a novel object. After a 30 min delay, a second object was added to the arena and the mouse was allowed another 3 min to explore. The objects were solid-colored, hard plastic geometric toys  approximately 1.25 in tall. The objects were epoxied to a thin ceramic tile, ensuring the mouse could interact with but not move the toy. The arena and all objects were cleaned with 40% ethanol wipes to sanitize and deodorize between mice. p-value less than 0.05 was set to evaluate significance unless otherwise indicated. Outliers were identified and removed following the use of Grubbs’ test for a single outlier. Where appropriate, an unpaired t-test or a one-way ANOVA with corrected multiple comparisons (Holm–Šídák) was used to compare between groups. Blood alcohol content (BAC) was analyzed with a simple correlation. Means are reported ± the standard error of the mean (SEM). All analyses were conducted with Prism . A −ΔΔCT method comparing changes in house gene (18S) to target gene in controls and experimental groups [Relative quantification of gene expression of cytokines  was calculated as fold change using the 2l groups . IL-1Β aM = 3.67 g/kg ± 0.38) and rmTBI (M = 2.84 g/kg ± 0.30) groups, t(22) = 1.70, p = 0.052 or alcohol preference Sham (M = 84.17% ± 3.717%) and rmTBI (M = 2.92% ± 5.89%), t(22) = 1.62, p = 0.060. However, these results suggest that there could be a small decrease in alcohol drinking after rmTBI, but this difference was not replicated in the intake measures of the second experiment, and the effect size (R = 0.116) was minimal; therefore, it was not considered a meaningful trend. Results of the BAC analysis showed a positive Pearson’s correlation for both sham  and rmTBI  groups.Comparison of the sham (M = 41.15 mg/dL) and rmTBI (M = 22.22 mg/dL) average BAC did reveal a difference between groups, t(46) = 1.99, p = 0.026, indicating differing levels of intoxication following the 4 h drinking period. The results of the voluntary alcohol intake (g/kg), alcohol preference, and BACs fell within expected range for the DID, two-bottle choice protocol [As shown in protocol ,37,38,39F = 4.50, p = 0.025, for control , sham + alcohol drinking (M = 2.67 ± 0.39), and rmTBI + alcohol drinking (M = 1.98 ± 0.24). Holm–Šídák’s multiple comparisons test confirmed a statistical difference between control and sham + alcohol drinking (adjusted p = 0.024); however, the increase observed after rmTBI + alcohol drinking did not meet the statistical threshold after multiple correction comparisons to control or sham + alcohol drinking groups (adjusted p = 0.206 for both comparisons). Alcohol drinking increased cytokine expression for TNF-α, IL-1β, and IL-6. TNF-α expression more than doubled following 2 weeks of alcohol drinking, F = 7.17, p = 0.010). As shown in M = 1.01 ± 0.09), sham (M = 37.97 ± 7.54), which was statistically significant after Holm–Šídák’s multiple comparisons test (adjusted p = 0.009). Surprisingly, the rmTBI + alcohol drinking group did not reach the statistical threshold despite a more than 20-fold increase compared to control  or to the sham + alcohol drinking group (adjusted p = 0.111). There was an even greater increase in IL-6 expression, F = 7.39, p = 0.009, after sham + alcohol drinking (M = 95.62 ± 18.98) compared to control (M = 1.12 ± 0.32) (adjusted p = 0.008). Again, the rmTBI + alcohol drinking group (M = 60.28 ± 13.23) did not meet the statistical threshold in adjusted comparisons to control (adjusted p = 0.059) or sham + alcohol drinking (adjusted p = 0.111). The large differences but lack of statistical significance for the IL-1β and IL-6 multiple comparisons are likely attributable to the reduction in sample size and, therefore power, in an effort to conserve the sample and compare both targets on a single 96-well plate. However, the effect size  was large enough to lend confidence that the results are meaningful and worthy of further exploration in future studies. An additional evaluation of IL-1β and IL-6 expression showed similar patterns to TNF-α, but with notably higher fold changes following sham + alcohol drinking  and rmTBI (M = 3.74 g/kg ± 0.41) groups, t(22) = 1.059, p = 0.129, or in alcohol preference between sham (M = 74.42% ± 4.522%) and rmTBI (M = 66.08% ± 5.01%), t(22) = 1.226, p = 0.115. As shown in F = 0.87, p = 0.431, control (M = 48.50 ± 6.51), sham + alcohol drinking (M = 38.50 ± 5.53), and rmTBI + alcohol drinking (M = 48.73 ± 6.93). Similarly, no differences were observed in the novelty preference task for percent time spent with the novel object, F = 1.16, p = 0.077, control (M = 46.75% ± 6.45%), sham + alcohol drinking (M = 45.64% ± 8.80%), and rmTBI + alcohol drinking (M = 59.50% ± 3.64%). During the battery of cognitive and behavioral tasks, locomotor activity  was similar for all three groups, F = 2.81, p = 0.076, control (M = 17.91 ± 1.87), sham + alcohol drinking (M = 20.36 ± 2.48), and rmTBI (M = 27.09 ± 3.81). However, the cognitive deficits became readily apparent in the number of errors made through the first reversal, F = 4.231, p = 0.024. The number of errors for the control (M = 17.27 ± 1.73) and sham + alcohol drinking (M = 17.55 ± 2.26) groups was similar, but the rmTBI + alcohol drinking group (M = 27.27 ± 3.86) showed an increase compared to the other two groups (adjusted p = 0.047 in both corrected comparisons). Finally, to compare the percentage of mice able to achieve at least one reversal on the first training day, an exact binomial test was conducted for each group with the expected outcome of 100% success. All control mice were able to achieve at least one reversal on the first training day, in contrast to 83% of sham + alcohol drinking mice (p < 0.001), and 75% of rmTBI +alcohol drinking mice (p < 0.001). The SSDRL task revealed decreased cognitive behavioral performance for the alcohol drinking and rmTBI + alcohol drinking groups. For the rmTBI + alcohol group, the number of trials needed to reach the first reversal was slightly increased but did not statistically differ, In both experiments, the DID two-bottle choice protocol produced consistent alcohol intake. However, there was no evidence for elevated voluntary alcohol consumption or an escalated drinking pattern following this model of rmTBI as compared to sham. In fact, the data suggested that there might be differing intake patterns over the 4 h drinking period, resulting in different end-point BAC levels. In our study, the sham mice had higher BAC levels at the end of the 4 h drinking period, and only sham mice reached intoxicating BAC levels at the collection point. While these data are very enticing and suggest differing intake patterns after rmTBI, perhaps due to differing subjective effects of alcohol after injury, this would need to be investigated via time course intake and BAC measures in future studies. Results from the current study are in contrast to other studies that showed bimodal changes in alcohol drinking or elevated drinking after TBI ,41. TherAlternatively, rodent strains have different voluntary alcohol consumption patterns that could account for the different outcomes. Sprague-Dawley rats are generally considered a low-alcohol preferring model, whereas the C57BL/6J mice used in the current study normally drink high volumes of alcohol without training or forced consumption ,43,44,45It was expected that alcohol drinking would elevate neuroinflammatory markers in the current study. This hypothesis was supported by the observed elevated expression of TNF-α, IL-1Β, and IL-6 cytokines in the sham + alcohol drinking group. It was also predicted that the combination of rmTBI and alcohol consumption would together compound the independent increase in cytokine expression observed after alcohol exposure and in TBI models. However, despite the rmTBI group consuming similar amounts of alcohol, there was not a significant increase in expression for any of cytokines probed, suggesting a more complicated cytokine response from the combined neuronal insult of alcohol drinking and rmTBI. The recruitment of cytokines can be incredibly complex and occurs via the Toll-like receptor (TLR) or the receptor for advanced glycation end products (RAGE) pathways, each with unique intracellular mechanisms that lead to altered gene expression of cytokines . TNF-α aIn acute doses, alcohol has been reported to suppress some of the TLRs, thereby attenuating proinflammatory cytokines ,57,58. HTo further complicate assessing the response to neural insult via alcohol and/or injury, the recruitment of cytokines can independently occur at neurons, microglia, and astrocytes, each of which may be mediated by different factors ,62. It sThe results of our cognitive behavioral battery supported the hypothesis that rmTBI + alcohol drinking together would lead to worse cognitive and behavioral outcomes compared to alcohol drinking alone. This was most evident in the decreased percentage of subjects in the sham + alcohol drinking group that were able to achieve a reversal of the simple discrimination rule in the first training day, with a further decrease for the rmTBI + alcohol subjects. Looking more closely at the data, the rmTBI + alcohol drinking group was able to achieve reversal with only slightly more trials, but these mice committed more errors compared to the sham + alcohol drinking and control groups. This suggests that the rmTBI + alcohol drinking mice had mild cognitive deficits that resulted in loss of behavioral flexibility and increased perseverative errors. Our data did not show any changes in locomotor activity or novelty preference, which suggests the observed impairments are not due to motor difficulties or basic short-term memory deficits. While animal models of repetitive mTBI do often demonstrate poor cognitive and behavioral outcomes after multiple mTBIs ,65,66, pAs previously discussed, any alteration in alcohol administration, timing or severity of injury, or rodent strain could explain the incongruent outcomes reported here and throughout the literature. For instance, it is possible that the doses of alcohol consumed in this study were not sufficient to elicit more dramatic alcohol-induced deficits in behavioral flexibility that have been previously observed ,33,34,70Future studies should consider the administration of alcohol at low, moderate, and high doses following rmTBI to better control alcohol-mediated effects. Researchers using animal models could also use highly innovative measures such as diffusion tensor imaging (DTI) imaging to assess and correlate the integrity of white-matter tracts after rmTBI to executive functions such as reversal learning and working memory. Lastly, this study only addressed cognition and behavior post injury in alcohol-naïve mice, whereas, in humans, it is more likely that alcohol exposure starts prior to injury. Future studies should consider pretreatment of alcohol and possibly even the use of alcohol-dependent mice with the rmTBI model to assess changes in cognition and behavior. Our results show a deleterious impact of alcohol drinking on neural cytokine response, but suggest a more complex interaction when rmTBI and alcohol drinking were combined. Further, our data demonstrate mild behavioral flexibility deficits and increased perseverative errors in mice after rmTBI + alcohol drinking compared to alcohol drinking alone. Together, the current studies highlight the importance and complexity of investigating comorbid presentation of rmTBI and alcohol drinking.


Schizophyllum commune strains that originated from Serbia (SRB) and Italy (IT). Submerged cultivation of grown mycelia (M) and fermentation broth (F) of ethanol (EtOH) and polysaccharide (PSH) extracts lasted for 7, 14, 21 and 28 days. For AChE activity Ellman method was performed, while for antioxidative activity, sevendifferent assays were conducted: DPPH, ABTS, FRAP, SOA, OH, NO together with total phenolic content. Antimicrobial screen, LC–MS/MS technique and FTIR measurements were performed. Different isolates exhibited different AChE activity, with PSH being the strongest , while in EtOH extracts, IT stood out . PSH extracts (7 days) exhibit significant antioxidative activity (AO), opposite to EtOH extracts where 14 and 21days periods stood out. Only tw extracts showed antibacterial activity. Following LC–MS/MS analysis p-hydroxybenzoic and gallic acids were the most abundant phenolics. PSH extracts demonstrated remarkable results, making this study debut and introducing S. commune as a valuable resource of AChE inhibitors.In recent years, fungi have been recognized as producers of acetylcholinesterase (AChE) inhibitors, agents important for the prevention of Alzheimer’s disease (AD). This study aimed to examine the AChE inhibitory, the antioxidative and antibacterial activity of two different  Reactive oxygen species (ROS) represent the most potent free radicals since they can have a destructive effect on various cells and cause oxidative stress ,2. The iIn recent years, mushrooms have been recognized as producers of non-alkaloid agents that inhibit acetylcholinesterase enzyme (AChE) ,11 as weSchizophyllum commune  is a widely distributed wood—decaying basidiomycete—that is present on all continents except Antarctica, but it is more common to be found in warmer regions with high humidity [S. commune originated from Serbia (SRB) and Italy (IT). Both isolates were cultivated in submerged conditions in order to examine and compare the activity of mycelium biomass (M) and fermentation broth (F).humidity . Thanks humidity ,19,20, ohumidity . ConsideS. commune Fries 1815.  were collected near Bologna, Italy, in 2016 and in Zmajevac (Fruška Gora mountain) in Serbia in 2012. Mushroom identification was carried out by studying the fungal morphology macroscopically  and microscopically [https://www.pmf.uns.ac.rs/en/research/groups/profungi/), of the ProFungi laboratory at Department of Biology and Ecology-DBE, Faculty of Natural Sciences-PMF, University of Novi Sad-UNS. The isolates were referenced under the following numbers: 0043 and 0047, respectively, for S. commune SRB and IT.Two dikaryon strains of the wild-growing opically . Mycelia2 in size, were transferred into 100 mL of fermentation broth that contained 0.5 g peptone, 3.7 g glucose, 0.17 g maltose, 0.17 g fructose, 0.17 g xylose, 0.5 g yeast extract, 0.1 g K2HPO4, 0.05 g MgSO4x7H2O and 0.005 g vitamin B1. Incubation on a rotary shakerat 120 rpm, 26 °C and in the light  lasted for 7, 14, 21 and 28 days. Every 7 days, three replicates of SRB and IT isolates were filtered  and lyophilized at −80 °C under vacuum , F for 72h and M for 48h. Afterward, samples were grounded to a fine powder  and kept in dark bottles at room temperature until further use. Based on the measured masses of mycelium and lyophilized filtrate, appropriate growth curves were made (w/v). Polysaccharide (PSH) extracts were prepared according to Ren et al. with some modifications [Mycelia of SRB and IT strain from culture collection were cultivated on malt agar  at 26 °C for 12 days in a thermal incubator in the dark, each isolate in triplicate. A sterilized fermentation medium was prepared in Erlenmeyer flasks (500 mL narrow throat flasks), in which 5 plaques of pure culture mycelia, approx. 1 cmere made . The masications . The modsample and Acontrol stand for the absorbance of the tested and control samples, respectively. All measurements were performed in triplicate, and the results were presented as IC50 and IC90 values, with lower values corresponding to higher AChE activity of the sample. The reference inhibition time is a period of 10 min or 600 s, respectively.In vitrodetermination of AChE inhibitory activity of PSH and EtOH extracts was done according to Ellman et al. adapted for the use in 96-well microplates . As an a2−), hydroxyl (OH·) and nitric oxide radical (NO·) was assessed for EtOH. Synthetic antioxidant propyl gallate (PG) was used as a positive control for RSC assays, and all assays were completed in triplicates. The results were presented as the mean values ± standard deviations (SD).2.4.1. DPPH AssayFor the in vitro evaluation of the AO of PSH and EtOH extracts, the scavenging effect on 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzthiazolin)6-sulfonic acid (ABTS) radicals, in addition to ferric reducing antioxidant power assay (FRAP assay) of extracts was determined. Additionally, the capability to neutralize superoxide anion . The mixture was kept in the dark at room temperature for 30 min, and the absorbance was measured at λ = 515 nm . The following Equation (2) was used for the calculation of radical scavenging capacity (RSC):sample and Acontrol stand for the absorbance of the tested extracts and control. After regression analysis, results were presented as IC50 values (µg/mL) .The DPPH free radical scavenging assay was carried out according to the previously described procedure . Briefly+ into its neutral, colorless form, was carried out according to Arnao et al. [+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM K2S2O8, and after incubation (12–16 h in the dark at the room temperature) on the day of the assay, ABTS solution was diluted with EtOH to achieve absorbance between 0.800and 0.900 at λ =734 nm. 10µL of fungal extract (EtOH and PSH) was added to 290 µL of ABTS solution and mixed. The sample absorbance was read at λ = 734 nm after a 5-min incubation at room temperature, while Trolox was used to calculate the standard curve. The ABTS radical scavenging activity was expressed as mg of Trolox equivalents per g dry weight (mg TE/g d.w.).The ABTS test, based on spectrophotometric monitoring of the transformation of the blue-green colored cation radical ABTSo et al. . ABTS+ w3x6H2O and acetate buffer (pH 3.6) in ratio 10:1:1. For the assay, 10 µL of each extract was mixed with 225 µL of FRAP reagent and 22.5 µL of dH2O. The absorbance was measured after 6 min at 593, while ascorbic acid (AA) was used to calculate the standard curve. Reducing power of analyzed fungal extracts (EtOH and PSH) was expressed mg AA equivalents per g dry weight (mg AAE/g d.w.).FRAP assay was done by the method of Benzie and Strain . The fre2− the activity of tested EtOH extracts was evaluated by a previously described method [O2-was calculated according to the formula in 50 values obtained ± SD (μg/mL).The antiradical Od method , based o· was determined by the modified method of Halliwell and Gutteridge [2O2, 100 µL FeSO4 and 100 µL 2-deoxyribose-D-ribose were mixed with different concentration of EtOH fungal extract (10 µL) and 2.7 mL of phosphate buffer (pH 7.4). Samples were incubated for 60 min at 37 °C, and 200 µL of EDTA (ethylenediaminetetraacetic acid) and 2 mL of TBA reagent  were added in a mixture. The absorbance of the reaction mixture (pink complex) was noted at λ = 532 nm, and results were expressed as IC25 values ± SD (µg/mL).RSC activity of OHtteridge . 100 µL 25 ± SD (µg/mL).The concentration of nitrite ions was determined according to Green et al. . The reaStaphylococcusaureus and Bacillus cereus) and Gram-negative (Escherichia coli and Pseudomonasaeruginosa) bacteria strains from American Type Culture Collection (ATCC). Antimicrobial screen included agar-well diffusion and disc-diffusion assays in the evaluation of the antibacterial activity of EtOH fungal extracts of stock concentration, 10% (w/v). Evaluation of activity was performed according to the CLSI procedure [In vitroantibacterial susceptibility, assays were performed using 2 Gram-positive  was performed by the method of Singleton et al. . Extract−1 wave number range.FTIR spectra of the EtOH and PSH spectra were recorded on a Thermo-Nicolet Model 6700 spectrophotometer  equipped with Smart Orbit (Diamond) ATR accessory and OMNIC 7.3 software. Spectra were recorded in the 400–4000 cmv/v/v). Components were visualized with alkaline silver nitrate [For analysis of the monosaccharide composition of PSH extracts, paper chromatography was applied. Each lyophilized PSH extract (2 mg) was hydrolyzed with 2 M TFA (2 mL) in a sealed tube at 100 °C overnight. Samples were concentrated by evaporation under reduced pressure (below 50 °C) to dryness. Residual TFA was removed by two evaporation cycles with 0.5 mL of isopropanol, and the final residue was dissolved in 0.02 mL ofdistilled water and analyzed by PC on Whatman No.1 chromatography paper (descending method) in the solvent system ethyl-acetate:pyridine:water  pressure 40 psi, drying gas (N2) flow 9 L/min and temperature 350 °C, capillary voltage 4 kV, negative polarity. Data were acquired in dynamic MRM mode, using the optimized compound-specific parameters . For all the compounds, peak areas were determined using Agilent MassHunter Workstation software Qualitative Analysis (ver. B.03.01). Calibration curves were plotted, and concentrations of samples calculated using the OriginLabs Origin Pro (ver. 9.0) software.The quantification of the selected phenolic compounds in PSH and EtOH extracts of ć et al. . To obta50 and IC25 values were determined by the linear regression analysis of RSC . The data that have a normal distribution were subjected to two-way analysis of variance (ANOVA)and multivariate analysis of variance (MANOVA). Tukey’s test was used to determine significant differences (p < 0.05) between the extracts. Nonparametric Friedman tests and posthoc LSD tests were used for data that do not have a normal distribution. The correlation between antioxidant capacity and total phenols content was established using the Pearson’s product-moment correlation  and Spearman correlation . The statistical analysis was performed using the IBM SPSS Statistics software version 22.0 for Windows.All assays were performed in triplicate. The results were expressed as mean ± SD. ICS. commune, where the highest concentration range (1%) overtakes IC90  was 87.92%.The strongest potency was observed for PSH extracts of kes IC90 A. Result0 values B. The ICS. commune  showed different AChEI activity. The PSH extracts of the SRB strain showed higher activity compared to the IT strain, while in EtOH extracts, the opposite was obtained (90 79.73 ± 26.34 µg/mL), while among EtOH, the strongest extract was F of IT strain after 14 days of incubation (IC50 0.8 ± 0.6 µg/mL).Different strains of obtained . Among Pp < 0.05) in inhibition of AChE when it comes to the incubation period, as opposed to EtOH extracts.By comparing the inhibitory activity of M and F, higher AChEI activity was observed in the F within EtOH extract, while in PSH extracts M was stronger. Among EtOH extracts, differences in activity were related to the incubation period, and the strongest activity was shown after 14 days of incubation, apart from F extract originating from SRB, which showed the strongest activity after 28 days of incubation. PSH extracts did not show a statistically significant difference  and 7 days of cultivation (IC50 18.92 ± 6.12 for SRB), while within F extracts those incubated 7 days distinguished .All tested samples exhibited antiradical activity, with PSH being more active . Among P50 55.43 ± 1.89 µg/mL for IT isolate and IC50 55.96 ± 1.31 µg/mL for SRB isolate, respectively), and then on the 28th day, it began to decline for both isolates. Among M extracts, the analyzed antiradical activity had variations, but nevertheless, on day 14, they proved to havethe best ability to neutralize free radicals .For EtOH extracts, the AO of F increased linearly from the 7th to the 21st day when it reached its highest activity  showed the ability to “capture” ABTS radicals, with the PSH extracts being more active . When wep < 0.05) incubation period did not increase the reduction potential of the extracts.The ferric reducing ability of PSH and EtOH extract for both isolates was evaluated and compared . By comp2−. SRB strain was more potent compared to IT, while F extracts of both strains showed much higher antiradical activity  showed the best OH• neutralizing activity after 28 days of incubation , apart from M extract of IT strain which displayed the highest activity after 7 days (IC25 20.20 ± 1.36 µg/mL) (EtOH extracts of 6 µg/mL) . Summari25 . F extract of SRB strain after 14 days of incubation demonstrated antibacterial activity (MIC 0.62% and MBC 0.31%) only against E. coli.Studies on the MIC and MBC for EtOH extracts demonstrated that only two extracts among all from SRB strain exhibited antibacterial activity, while IT strain was not active. The F extract of SRB strain after 21 days of cultivation demonstrated the highest antimicrobial activity (MIC and MBC < 0.31%) against 2 –0.79), while a close correlation (p < 0.05) in OH assay in both isolates (r2 0.81 for IT and r2 0.71 for SRB) was obtained. In addition, a positive correlation was detected between FRAP assay and TP content (r2 0.69 for IT).TP content is presented in −1 was characteristic of the presence of β-glucan due to O-substituted glucose residues [−1 in each sample indicated that these polysaccharides had pyranose rings [−1 corresponded to the characteristic frequency of protein as well as to the bending vibration O-H of associated water. This strong vibration was overlapped by specific absorption of aromatics (C=C and C=O stretch vibrations) and indicated the presence of phenolic compounds [−1 corresponded to O-H groups of phenolics, too. In the anomeric region (950–700 cm−1), the characteristic weak absorption at 890 cm−1 in all samples corresponded to the presence of β-glycosidic linkages (C1-H deformation mode) [−1 in B and C samples was also related to β-glycosidic bonds [The spectra of all PSH samples investigated (residues . The strse rings . An inteompounds . Frequenon mode) . Furtheric bonds .–1 correlate with the valence vibrations of the OH groups of phenolic compounds. Among F samples of SRB extracts, the intensity of individual absorption bands was different. Namely, the intensity of bands in the region 1000–1200 cm−1 related to valence vibrations of CO and CC polysaccharide structures is reduced compared to the intensities of other absorption peaks. This indicates a decrease in the proportion of carbohydrate polymers relative to other compounds present in the mixture . The absence of the characteristic ester band in the region 1730–1740 cm−1 in all analyzed FTIR spectra indicates that the examined extracts do not contain uronic acids as part of their structural characteristics.The FTIR spectrum of the EtOH extract  showed aThe presence of a large amount of D-glucose with smaller amounts of D-galactose and traces of D-mannose was confirmed in investigated hydrolysates by PC.p-Hydroxybenzoic acid was detected in all investigated samples, while richer content was found in IT isolates . The most abundant compound was gallic acid found in SRB F extract (75.77 µg/g). In comparison, EtOH extracts contained most of the analyzed phenolic acids, whereas F extracts showed higher phenolic content than M .In all examined PSH and EtOH, among 45 investigated phenolics, 4 compounds were quantified using LC–MS/MS procedure . Other pS. commune . Though some subtle distinction exists, the FTIR spectrum of PSH extracts (Inhibition of AChE enzyme was determined and compared for PSH and EtOH extracts of both investigated strains of  commune . All PSHextracts  indicatep < 0.05), while among EtOH incubation period had a greater impact (p = 0.04)on the AChEI activity, with 14 and 28 days being the highest .Among EtOH extracts, stronger AChEI was observed for IT F extracts after 14 days of cultivation, while among PSH extracts, SRB M expressedgreater inhibition potential after 28 days of incubation. Furthermore, the incubation period did not show a statistically significant difference in PSH extracts  among PSH and EtOH samples from SRB in DPPH and FRAP assays. EtOH samples from OH assay stands out since in NO and SOA tests, except F extracts, much lower antiradical activity was recorded (25 10.80 ± 0.54 µg/mL) while among SRB samples, the activity of F and M  for the same incubation period was almost eight times lower.AO of PSH and EtOH extracts was evaluated in vitro using standard antioxidant assays . Based orecorded . The hig2 0.81 for IT and r2 0.71 for SRB). Moreover, these results suggest the potential use of this mushroom for the prevention of oxidative stress since OH is physiologically active and toxic [Coprinus species demonstrated a positive correlation, which may be due to the application of different solvents [S. commune showed three times lower anti-DPPH activity (IC50 153.1 ± 0.02µg/mL) [50 18.56 µg/mL [50 49.34 ± 0.65 µg/mL). EtOH extracts from submerged fermented mycelium (10 days) demonstrated stronger scavenging capacity as reported by Tripathi and Tiwary with IC50 31.40 ± 0.05 µg/mL [S. commune from Thailand showed higher DPPH activity (inhibition of DPPH ranged from 34.29 to 81.00%) with fermentation broth being twice as active [T. versicolor exhibited RSC activity 50 times lower [Results of OH assays for EtOH extracts are comparable with TP content since a high positive correlation was obtained (rnd toxic . DPPH assolvents .EtOH and02µg/mL) , while E05 µg/mL . In othe05 µg/mL ,53,54. Ns active , which imycelium  days demLC–MS/MS procedure of extracts from the fruiting body of this mushroom species detected only protocatechuic and hydroxybenzoic acids in higher amounts , while lS. aureus and B. subtilis) [E. coli [S. commune was active against E. coli as well [Antibacterial activity was very low as only two extracts showed activity. In a previous study, methanolic extract of culture filtrate incubated in submerged conditions for 10 days was active against two Gram-positive bacteria (ubtilis) , which i[E. coli ,60,61,62 as well , suggestFindings from this study suggest that different compounds, both primary and secondary, could be responsible for inhibition of AChE enzyme as well as for detected AO and antibacterial activities that strongly point out to a likelihood of synergistic activity of compounds in the extracts . Based oS. commune. Most of the analyzed submerged samples exhibited AO, which is in direct relation to the characteristics of strain’s growth as it can point to the moment of entry in secondary metabolism, while in AChEIactivity, PSH extracts demonstrated remarkable results and represent first data for this species. Taken all together, PSH extracts obtained significant AO within samples incubated for 7 days, opposite to EtOH extracts where a 14- and 21-day cultivation period stood out, which was in accordance with LC–MS/MS phenolic analysis. Differences in activities between two isolates and different types of extracts, together with variations of phenolics content, indicate the important role of both genetic and environmental factors in metabolite production, as well as possible synergistic effect of primary and secondary metabolites. This study introduced S. commune as a potential natural antioxidant producer and indicated that the submerged fermentation might serve as an effective alternative method for producing bioactive agents, e.g., pharmaceuticals and nutraceuticals. In summary, this ubiquitous mushroom species should be considered as a valuable alternative source for future palliative therapy of AD.To the best of our knowledge, this is the first report describing the AChE inhibitory activity of PSH extracts of submerged extracts of