Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and β-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado ( Deciphering molecular and cellular mechanisms using common molecular biology techniques, such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA-Seq (RNA sequencing), cDNA (complementary DNA) library synthesis, gene cloning and northern blot analysis requires the use of high-quality nucleic acids. In addition, long-read sequencing techniques require high-molecular weight, contaminant-free DNA for the generation of long contigs and full-length transcripts ,2. HowevPersea americana Mill.) is a high-value, rapidly growing horticultural crop, with production having increased by 34.8% since 2015 to 7.2 million metric tons annually [Avocado -based RNA extraction protocol has been developed to obtain a high quality and quantity of RNA from all major tissue types in avocado, including roots, callus, shoots, flowers, fruits, seeds and leaves throughout development. These protocols have been used successfully for long-read sequencing that demands high-molecular weight, intact and contaminant-free DNA and total RNA, as well as short-read sequencing, PCR and qPCR experiments. These DNA and RNA extraction protocols have also been successfully applied with additional plant species, including capsicum, cotton and finger lime (data not shown).2SO3), PVP (polyvinylpyrrolidone), incubation times and temperatures were optimised.DNA was extracted from the leaf tissue of 40 avocado cultivars see  represenCTAB lysis buffers with high salt concentrations have been developed and regularly used for recalcitrant tree species, as most commercial kits produce very low yields, if any, in avocado ,37,38. WGenerally, CTAB-based protocols suggest using a high amount of initial tissue (1 g/10 mL reaction or 0.3 mg /mL) ,43, but 2SO3 to the buffer is suggested to help eliminate these compounds [Avocado leaves contain a high concentration of phenols, saponins, flavonoids and steroids . Avocadoompounds ,51,52. Hompounds ,54. Addiompounds . Sodium ompounds ,51,52.High levels of oxidised phenolics, similar to those found in avocado, are well known to hinder DNA and RNA extraction ,20,21,22The DNA extraction method developed here is simple and yields good quality intact DNA from 40 different avocado cultivars with a 260 nm/280 nm absorbance ratio of, on average, 1.8 (1.45–2.03) see  and a 26http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 22 December 2021) for Illumina-sequenced DNA samples indicated a high Phred score of the sequenced reads and corresponding bases  of Hass avocado were collected for extracting total RNA. Variation in properties of different avocado tissues ,66,67,68The amount of tissue used was crucial. Here the use of 0.01 g (10 mg) per 1.5 mL reaction to obtain a high concentration and quality of extracted nucleic acids is recommended. For seeds, more tissue  yields more nucleic acids. However, for roots, more tissue led to degradation of the 5S ribosomal RNA band. Most other tissues did not support high quantities (>10 mg/mL) of starting material. When tested with capsicum , finger The incubation time also varied for some tissues. Unripe fruit seeds required a very long incubation time (30 min); in contrast, roots could not withstand more than 5 min of incubation at 65 °C. Leaves yielded RNA at various time and temperature ranges but had maximum efficiency between 10–25 min at 65 °C.v/v) of SDS to assist lysis but to avoid protein contamination was used. The percentage of SDS used was increased (1% v/v) in the case of capsicum fruit . Intact ribosomal RNA could not be isolated at pH 8 or 6.5. This pH range also fits with the recommended pH exposure range (6–9) by Pacific Bioscience for long-read sequencing . Isoprop® fluorometer , Bioanalyzer  and agarose gel electrophoresis . Samples  that failed to pass the cut-offs  were cleaned using additional methods. Firstly, column-based methods  are to be added. For samples with high co-precipitants, up to 5% of 2ME can be used. Adjust the pH to 7.6 using NaOH.Optimised Buffer 1-D contains 2% CTAB, 1.4M NaCL, 10 mM EDTA, 0.1M MES, 1% PEG (filter sterilized buffer). At the time of use, 0.5% Nav/v of 10% SDS and 2% β-mercaptoethanol are to be added. Adjust the pH to 7.6 using NaOH.Optimised Buffer 1-R contains 2% CTAB, 1.4M NaCL, 10 mM EDTA, 0.1M MES, 1% PEG (filter sterilized buffer). At the time of use, 0.01% Optimised Buffer 2 contains 25 mM sodium citrate, 0.5% N-lauroylsarcosine sodium salt, 1 M sodium acetate, 2% PVP and 4.2 M guanidine thiocyanate .The optional clean-up buffer contains 1 M NaCl, 0.5% SDS, 10 mM Tris (pH 8.0) and 1 mM EDTA (pH 8.0) .The other reagents used were: chloroform:isoamyl alcohol (C:IAA) 24:1; isopropanol (100%); sodium dodecyl sulfate (SDS) (10%) .*appropriate PPE including lab coat and other safety apparatus is recommended for performing these protocolsThere are two versions of this protocol, i.e., for a large (10–20 mL) volume (Protocol A) and a small (less than 2 mL) volume (Protocol B).PROTOCOL APulverise frozen tissue to powder under liquid nitrogen without the addition of buffer.Add approximately 0.2 g of ground frozen leaf tissue to 15 mL hot OB1-D (65 °C) in a 50 mL tube and incubate at 60–65 °C for 5 min.Centrifuge at 10,000 rpm  for 10–15 min at room temperature to pellet debris.Transfer the supernatant to a new 50 mL tube and add 10 µL of RNase A (10ng/µL).Incubate at 37 °C for 20–30 min. (This incubation is sufficient due to the high pH (7.6) of the buffer.)Add an equal volume of C:IAA.Mix the contents by gently inverting the tubes fifty times.g) at room temperature for 20 min.Centrifuge at 9500 rpm  and mix gently by inverting the tubes ten times. Precipitating with 95% ethanol can improve DNA quality with reduced yield for cultivars where more contaminant is precipitated.Incubate at −20 °C for 30 min (can be left overnight). Extended incubation increases yield; however, it may increase salt content in the pellet.g) for 25 min at 4 °C. Remove the supernatant.Centrifuge at 10,000 rpm  to ensure the pellet is submerged. If needed, pool tubes at this stage to increase yield.g) for 10 min. Centrifuge longer if pellet does not adhere to tube.Centrifuge at 7500 rpm  for the allotted times.Steps 8,12: Centrifuge at 14,000 rpm .Step 13: Wash pellet with 500 µL 75% ethanol.Step 16: Dissolve the pellet in 20 µL nuclease-free water.g, followed by isopropanol precipitation and an ethanol wash.Notes: If high-quality avocado DNA is not required, a crude DNA extraction method that works well with leaf tissues can be used. For this, after the incubation at step 5, an equal volume of C:IAA is added and centrifuged at 14,000 rpm/21,000× Pulverise the frozen tissue to powder without the addition of buffer.To OB1-R add 10 µL 10% SDS per 2ml buffer and 2% 2ME. Heat the buffer to 65 °C.Add approximately 10 mg of ground frozen tissue to 650 µL hot OB1-R buffer in a 2 mL tube and incubate at 60–65 °C for 5 min. g) for 10 min at room temperature and transfer supernatant to a new tube.Centrifuge at 14,000 rpm  at room temperature for 20 min.Centrifuge at 14,000 rpm  and incubate at −20 °C for 1 h or longer as needed. Longer incubation time increases yield but also increases salt concentration in precipitate. Alternatively, use a half volume of 10M lithium chloride (LiCl) instead of isopropanol and incubate overnight at 4 °C. This can improve RNA quality, however, it may result in the loss of small and complex RNAs.g) for 25 min at 4 °C.Centrifuge at 10,000 rpm  for 10 min.Centrifuge at 8500 rpm  and 0.05% v/v (24 units) of DNase 1 enzyme and incubate at 37 °C for 30 min.If DNA must be removed from the RNA sample, proceed with DNase treatment by adding 0.05% 17.Add DNase- and RNase-free water to make up 500 µL of total volume.18.Add an equal volume of TRIsure™  (use SSTE buffer if RNA degrades with the use of TRIsure™) and incubate for 3–5 min.19.g) for 20 min.Add 100 µL chloroform and centrifuge at 14,000 rpm  for 40 min.Centrifuge at 14,000 rpm . The quantity of DNA was estimated using Gel DocTM XR+ with Image LabTM Software  by comparing band fluorescence intensities to that of standard lambda DNA quantity. The total amount was calculated using the formula:  × total volume. The RNA quantity was estimated using an Agilent 2100 Bioanalyzer . The purity was measured by absorbance at 230, 260 and 280 nm using a NanoDrop® Spectrophotometer . In addition, RNA quality was estimated by the RIN value using an Agilent 2100 Bioanalyzer .Nucleic acid quality was checked on a 1% agarose gel and with capillary electrophoresis. Midori green stain  was added to agarose gels and visualized using the Gel DocPCR was performed using primers designed on contig SDSS01000523.1 at position 285719 of the avocado genome  , 400 nM primers and 50 ng of total DNA. qPCR was performed using the aforementioned primers as follows: polymerase activation at 95 °C for 3–5 min, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 65 °C for 10 s and extension at 72 °C for 10 s. Melt curves were generated by melting from 65 to 95 °C, raising the temperature by 0.5 °C and waiting for 2 s after each step for clear fluorescence capture using a Rotor-Gene Q on Rotor-Disc 72, Hilden, Germany.qPCR was performed in 20 µL reactions with 1X SensiFAST SYBRDNA was sequenced using PacBio long-read, PacBio HiFi-read and Illumina short-read sequencing techniques. For PacBio long-read sequencing, 20 µg genomic DNA was shipped to Nucleome Informatics Private Limited, India. Similarly, 20 µg genomic DNA was provided for HiFi sequencing at the Institute for Molecular Bioscience, University of Queensland, Australia. For Illumina nextra-seq sequencing and Illumina NovaSeq sequencing, respectively, using an S4 lane at an expected coverage of 20×, 2–4 µg genomic DNA was shipped to the Ramaciotti Centre for Genomics, Sydney, NSW, Australia and Macrogen, Seoul, South Korea. The RNA samples were pooled and sequenced using PacBio Iso-Seq sequencing and Illumina short-read sequencing. For Iso-Seq sequencing two libraries were prepared per sample  using Iso-Seq Express library preparation and sequenced using 2 × PacBio Sequel II SMRT Cell 8M. Illumina reads were sequenced at an expected 100× coverage using NovaSeq.Establishing an all-in-one protocol for challenging plants, like avocado, has proven difficult in the past. Here an efficient, high-quality DNA and total RNA extraction method for various avocado cultivars and tissue types has been reported. To our knowledge, this is the first report showing successful nucleic acid extraction from multiple tissues and cultivars of avocado in a comprehensive protocol. Recommendations established here could prove adaptable to extract high-quality nucleic acids from tissues from a range of difficult plant species.


Over the last decade, the knowledge in extracellular vesicles (EVs) biogenesis and modulation has increasingly grown. As their content reflects the physiological state of their donor cells, these “intercellular messengers” progressively became a potential source of biomarker reflecting the host cell state. However, little is known about EVs released from the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze their EVs proteome modulation after paraquat (PQ) stimulation, a widely used herbicide known for its neurotoxic effect. Size distribution, concentration and presence of well-known EV markers were assessed. Identification and quantification of PQ-exposed EV proteins was conducted by data-independent acquisition mass spectrometry (DIA-MS). Signature pathways of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results highlighted that EVs exposed to PQ have modulated pathways, namely the ubiquinone metabolism and the transcription HIF-1 targets. These pathways may be potential molecular signatures of the PQ-induced toxicity carried by EVs that are reflecting their cell of origin by transporting with them irreversible functional changes. Extracellular vesicles (EVs) are nanosized particles (<100 nm to <1000 nm) bounded by a lipid bilayer and exempt of nucleus that are naturally released by their cell of origin ,3,4. DueDespite the existing studies published on EVs from a wide range of cell types, including endothelial cells, there is limited information available on EVs from human brain microvascular endothelial cells (HBMECs). Indeed, HBMECs are important, as they are one of the cell types composing the blood-brain barrier (BBB) ,9. In thParaquat (PQ), a commonly used herbicide, is transported by neutral amino acid transporters in the BBB and has already been demonstrated to have significant neurotoxic effect on the brain ,19,20,21In the present study, EVs from primary human brain endothelial cells were characterized by mass spectrometry-based proteomics. The major finding of this study is that EVs exposed for 24 h to paraquat at 100 µM displayed modulation of two biological pathways, which are highly significant: the ubiquinone metabolism and the transcription of HIF-1 targets. These two biological pathways highlight that EVs carried information from their parent cells, as they are two well-described pathways impacted by the PQ.EVs from primary human brain microvascular endothelial cells were isolated using an EVtrap isolation method based on a chemical affinity capture approach, permitting to isolate small extracellular vesicles ,26.Three proteins were selected to confirm EV enrichment by western blots. PDCD6IP (Alix) and TSG101 are cytosolic proteins, whereas the calreticulin (CALR) is localized at the lumen of the endoplasmic reticulum, making it a suitable candidate as a negative enrichment control . As showTo further characterize EVs, a proteomic analysis using mass spectrometry was conducted. Overall, a total of 1452 proteins were identified . Typicalhomo sapiens” for species, “protein” for content types, “endothelial cell” for cell types and “mass spectrometry” for methods on FunRich 3.1.3 [A deep analysis of the identified proteins in EV samples highlighted that a majority of the proteins (>70%) were annotated as EV proteins in Vesiclepedia, a reference database of extracellular vesicle composition . Proteinch 3.1.3 ,30 , “extracellular vesicle”  and “extracellular organelle”   ranked terms such as “extracellular exosome” (enrichment  10−299) D with hiBefore performing EV analyses from primary brain endothelial cells after paraquat exposure, paraquat-induced toxicity was evaluated for 24 h at different paraquat concentrations on HBMECs. Cell proliferation was assessed by MTS Proliferation Assay and cell cytotoxicity by measuring LDH release. No effect was denoted on cytotoxicity or proliferation of HBMECs after PQ exposure at 1, 10 and 100 µM . Howeverp-value = 0.0222). EV concentration was also measured by NTA, resulting in 3.50 × 107 ± 4.00 × 106 particles/mL for the control group and 2.60 × 107 ± 4.20 × 106 particles/mL for the treated one . These results suggest that the size and concentration of EVs are impacted by PQ exposure. Additionally, NTA measurement demonstrated that EVs purified from primary endothelial cells (from control and treated group) were in the expected diameter range of small vesicles (<100 or <200 nm), validating the high selectivity of EV preparation [To determine whether 24 h exposure to PQ at 100 µM alters EV size distribution and concentration (particles/mL), a nanoparticle tracking analysis (NTA) was conducted. The control group presented a mean EV size of 126.70 ± 10.10 nm and the PQ-treated group at 143.90 ± 18.90 nm A, showinated one B, indicaTo evaluate proteome modifications in EVs under PQ exposure, a quantitative proteomic analysis was conducted by data-independent acquisition mass spectrometry (DIA-MS). A total of 1452 proteins were quantified of which 107 were found to be differentially expressed (DE) in PQ-treated EVs  . As illuGene Ontology (GO) enrichment analysis was performed, showing that most of the DE proteins were enriched with the highest significance in biological processes, such as GO terms related to oxidative processes  and to mitochondria  .p-value of 2.56 × 10−6) and the transcription of HIF-1 targets  pathways  ,45, actip-values. These results were in accordance with previous proteomic findings [EVs enriched by EVtrap met required EV physical characteristics according to the MISEV 2018 criteria ,26. EVs findings ,46,47, sPQ concentration was chosen based on other studies using this xenobiotic ,49,50,51The quantitative mass spectrometry analysis of EVs from PQ-exposed HBMECs demonstrated that more than a hundred proteins were significantly modulated by the xenobiotic exposition. A large number of the differentially expressed proteins (DEP) are located in the mitochondria. Similarly, Gene Ontology (GO) terms enrichment highlighted GO terms such as “ATP metabolic processes”, “mitochondrion organization” or “electron transport chain”, which are closely related to the mitochondria. In addition, a quarter of DEP were linked to the oxidative phosphorylation directly associated to the complex I of mitochondria , as well as oxidation-reduction processes. One of the most described effect induced by PQ is the generation of reactive oxygen species leading to altered biological processes related to cellular oxidation ,21,22. MFinally, enrichment pathway analysis of EVs exposed to PQ highlighted “ubiquinone metabolism” and “transcription of HIF-1 targets” as pathways enriched with the highest significance. These results suggest that paraquat modulates mechanisms such as oxidative stress and hypoxia-related pathways in EVs. As described above, PQ is an oxidative stress inducer that inhibits respiratory chain complexes in the mitochondria  . In addiIn conclusion, a qualitative proteomics study allowed us to confirm the proper isolation and enrichment of EVs from HBMECs. The quantitative portion of this study also provided the first insight into EV proteome profiles from HBMECs after a xenobiotic exposure such as PQ. Results suggested that proteins from EVs shared common modified biological pathways to their parent cell, most notably the ubiquinone metabolism and the transcription HIF-1 targets. Both pathways are mechanisms already reported in PQ-induced toxicity in brain research, suggesting their potential use as “signature pathways” to demonstrate PQ effects on the BBB.2 incubator. At 80% confluence, cells were washed three times with phosphate-buffered saline  solution containing calcium and magnesium. They were incubated at 37 °C with 20 mL of complete endothelial cell growth medium-2 containing 5% of heat-inactivated exosome-depleted fetal bovine serum  for 24 h. Three T150 were treated with paraquat  (100 µM) for 24 h. Afterwards, the medium was collected and used for the extracellular vesicle (EV) isolation. Cells were detached with Stempro Accutase  and washed three times with ice-cold Phosphate Buffered Saline , pelleted and dry-stored at −80 °C.Primary human brain microvascular endothelial cells  were cultured onto a rat tail collagen type I-coated  flask (T150) and maintained in complete endothelial cell growth medium-2  at 37 °C in a 5% COIn each T150 flask, about 10 million of human brain endothelial cells were cultured. 20 mL of cell media were recovered. Cells and apoptotic bodies were removed using centrifugation . EVs were isolated with 1 mL of cell supernatant by using EVtrap magnetic beads provided by Tymora Analytical as a suspension in water.v/v ratio of the cell supernatant. 24 μL of EVtrap magnetic beads were added. The samples were incubated by end-over-end rotation for 1 h, according to manufacturer’s instructions. Following supernatant removal using a magnetic separator rack, the beads were washed with PBS and the EVs eluted by two 10-min incubations with 100 mM of fresh elution solution. Both eluted EVs were pooled and either resuspended in 80 μL of 0.2 μm-filtered water for nanoparticle tracking analysis (NTA) or dry-stored at −80 °C.The media loading buffer were added at 1:10 ® software version 8.05.12 SP1 .NTA was carried out using a Particle Metrix ZetaView® instrument . EVs were diluted at 1/500 with 0.2 μm-filtered PBS prior to analysis. To evaluate the total particle count and the overall size, the samples were measured in scatter mode using the 488 nm and standard instrument settings . The samples were measured with ZetaViewCell and EV pellets were resuspended in 80 μL of 0.1% Rapigest  100 mM TEAB , incubated 10 min at 80 °C and then sonicated (five cycles of 20 s with breaks on ice). Samples were then spun down  and the supernatant was recovered. Protein content was measured using the Bradford assay .The equivalent of 10 μg of proteins for cell samples and for EV samples were separated using electrophoresis on a 10% T/2.6% C polyacrylamide gel and were subsequently transferred onto a PVDF membrane. Membranes were stained with Amido black to highlight the proteins and washed with water to remove the excess. Immunoblot assays were performed using an anti-mouse antibody against PDCD6IP at a dilution of 1:500 , anti-rabbit TSG101 at a dilution of 1:500  and anti-rabbit calreticulin (negative marker) at a dilution of 1:100 .w/w ratio 1:50) . The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid  . Samples were then desalted on a C18 reverse phase column , peptides were dried under vacuum and subsequently resuspended in 5% ACN 0.1% FA  (1:20)).For each sample, 5 µg proteins was reduced using TCEP  , alkylated using iodoacetamide   and digested by an overnight tryptic digestion  at 30%. Isolation windows (30) were variables with an AGC target of 2 × 106 and a maximum injection time of 54 ms. The raw DIA MS data were matched against the spectral library following the published protocol [For each sample (cells and EVs), the equivalent of 2 µg of peptides were analyzed using Liquid Chromatography–Electrospray ionization-MS/MS (LC-ESI-MS/MS) on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fischer Scientific) equipped with an EASY nLC1200 liquid chromatography system (Thermo Fisher Scientific). Peptides were trapped on a 2 cm × 75 μm i.d. PepMap C18 precolumn packed with 3 μm particles and 100 Å pore size. Separation was performed using a 50 cm × 75 μm i.d. PepMap C18 column packed with 2 μm and 100 Å particles and heated at 50 °C. Peptides were separated using a 160-min segmented gradient of 0.1% formic acid (solvent A) and 80% acteonitril 0.1% formic acid (solvent B) at a flow rate of 250 nl/min, following the published protocol . Data-Inprotocol .For the analysis of cells and EVs, protein abundances were exported from Spectronaut™, and selected proteins were tested for significance using Student’s two-tailed t-test. Proteins and peptide intensities were exported and analyzed using mapDIA. No further normalization was applied. The following parameters were used: min peptides = 2, max peptides = 10, min correl = −1. min DE = 0.01, max DE = 0.99 and experimental design = replicate design. Proteins were considered to have significantly changed in abundance with a LFDR < 0.05 and an absolute fold change (|FC|) > 1.2. Data are available via ProteomeXchange with identifier PXD024691.The list of differentially expressed proteins was then analyzed with MetaCore™ version 21.1  to highlight significantly represented biological pathways. Top 10 biological pathways were selected.® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA), whereas cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) released using a Pierce™ LDH cytotoxicity kit . Both the MTS and LDH assays were performed according to the manufacturer’s recommendations.HBMEC were seeded in a 96-well plate  and treated for 24 h with PQ at different concentrations . Cell proliferation was determined using the MTS assay .Data are reported as mean ± standard deviation (SD).


Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export.The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using  Novel drug candidates are generally designed based on the assumption that they enter cells by passive diffusion through the plasma membrane lipid bilayer. Thus, compounds that do not follow the rules predicting an efficient diffusion through the lipid bilayer are not considered drug-like and are discarded early in the drug discovery process. However, a growing body of evidence indicates that passive diffusion via the lipid bilayer is an exceptional, rather than the normal, mode of drug entry –6, with –The investigation of the carrier substrate specificity is one of the objectives of the RESOLUTE consortium, a public-private partnership that aims to study the therapeutic potential of the human solute carrier (SLC) protein superfamily . This coSaccharomyces cerevisiae plasma membrane were employed for transporter identification  based on their transporter function and plasma membrane expression  methodology. Genetic crosses were performed between a strain carrying a deletion due to replacement of a transporter gene with the pression .To allow a further characterization of our transporter double-deletion library and determine plasma membrane import routes for different xenobiotic compounds, we purchased 32 compounds that largely obey the Lipinski’s “rule of 5”  and are 10.1128/mbio.03221-21.1TABLE S1Table S1, PDF file, 0.1 MB.Xenobiotics used in this study. Compound name, catalogue numbers, and chemical properties. Download Copyright © 2021 Almeida et al.2021Almeida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 90) for each compound’s effect on yeast in liquid cultures and proceeded to select for resistance in plates with sublethal doses of each compound. We performed serial dilutions of the wild type (BY4741), the transporter double mutant, and single-mutant pools and spotted these onto YNB+Sc  agar plates with inhibitory concentrations of the commercial xenobiotics (trx2Δ::kanMX and cpr1Δ::kanMX) as controls. The xenobiotics for which we did not observe significant growth inhibition at 200 μM  in solid media were excluded from further screens  from approximately 20 strains were sequenced to identify the transporter genes that had been deleted.Xenobiotic-resistant strains from the transporter double-deletion library pool were selected by plating 10ompounds . Once 20Saccharomyces Genome Deletion Project’s barcode list (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html), we identified the genes deleted in each compound-resistant strain . For the other three 5-fluorocytosine-resistant strains selected, the deletion of FCY2 was identified by PCR with the natMX cassette (data not shown).After aligning the sequenced barcodes to the t strain . It is inha1Δ strains. Other compounds showed preferential representation of certain transporter gene deletion strains, indicating their potential involvement in compound import. Although these results suggested possible transporters, additional assays are needed to confirm these phenotypes. In fact, plate screening assays can give rise to false-positive results, requiring a larger sample size to confirm the hits. Thus, for a more comprehensive screening, with the monitoring of the fluctuation in the abundance of all strains in the pool, we performed a high-throughput assay in liquid culture.Analyzing the results in plate assays , we coulkanMX upstream barcodes from the population were sequenced for transporter gene deletion identification. Thus, we were able to identify deletions responsible for resistance (putative import route) and sensitivity (putative export route) to the test compounds  using our double mutant nonessential transporter gene deletion library. The library was cultivated in liquid cultures containing inhibitory concentrations of xenobiotics for ca. 15 generations, and the 10.1128/mbio.03221-21.10DATA SET S1Data Set S1, XLSX file, 0.3 MB.Differential abundance of barcodes between samples treated compared to untreated controls of 21 compounds. Each sheet corresponds to data from each compound, specified in the sheet name. Headlines are described on the first sheet (headline meaning). Download Copyright © 2021 Almeida et al.2021Almeida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P value adjusted for multiple tests [padj] of ≤0.1; P value ≤ 0.001; log2 fold change ≤ 0.5), we could identify deletions for genes encoding ATP-binding cassette (ABC) drug efflux pumps involved in pleiotropic drug resistance: Pdr5p, Snq2p, and Yor1p  family member involved in sterol uptake , dtr1Δ (fluconazole), qdr1Δ (5-fluorocytosine), qdr2Δ (5-fluorocytosine), and atr1Δ (5-fluorocytosine). It should be noted that some transporters of this class  have been reported to be determinants of resistance to compounds tested in this study   , 24; howonazole) ; howeverPrincipal-component analysis (PCA) allows an evaluation of the distribution of variations between replicates and conditions within an experiment. 10.1128/mbio.03221-21.2FIG S1FIG S1, PDF file, 2.8 MB.Principal component analysis of all treatments from the CGP experiment, excluding fluconazole. Exclusion of fluconazole from PCA plot revealed a different profile for 5-fluorocytosine, the positive control of the assay. Azoles still group, with a clear separation of the subgroups triazoles and imidazoles. Download Copyright © 2021 Almeida et al.2021Almeida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Some of the compounds evaluated in our assays were not cytotoxic in either liquid or solid medium at the highest concentrations tested  which prevented us from performing the downstream experiments to identify resistant strains and suggest import routes. To propose possible transport routes for all the other compounds tested, we evaluated the results obtained from both low- and high-throughput assays . In addifur4Δ deletion strain did not appear significantly abundant  signaling network . In the abundant . Howeverype F18; . The rectes I03; . It is ptes I03; , 28, whi10.1128/mbio.03221-21.3FIG S2natMX followed by ::kanMX), and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S2, TIF file, 2.5 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of tunicamycin. The upper left panel shows a test plate containing tunicamycin (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map . In all cases, we observed that nha1Δ::kanMX conferred resistance to tamoxifen, whereas the same was not always evident for nha1Δ::natMX. nha1Δ::natMX was resistant to tamoxifen in combination with approximately 50% of the transporter deletions tested: itr1Δ::kanMX, tpo5Δ::kanMX, mal11Δ::kanMX, zrc1Δ::kanMX, dnf1Δ::kanMX, or fcy2Δ::kanMX (none of which was previously identified as resistant to the drug). This suggests that there could have been a mutation in the original nha1Δ::natMX that leads to an increased sensitivity to tamoxifen and is present in half of the spores produced in the library. The same pattern was not evident for azoles. Considering the CGP assay, the most abundant strains (log2 fold change ≥ 0.5) in the tamoxifen-treated pool were the adp1Δ and tna1Δ mutants , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2 and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S3, TIF file, 2.5 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of tamoxifen. The upper left panel shows a test plate containing tamoxifen (4 days of growth). The upper right panel shows a control plate with DMSO 2% (4 days of growth). The middle panel presents a plate map  indicated that this deletion provides resistance to this compound, corroborating the involvement of Pdr5p in carbendazim uptake.Carbendazim is a benzimidazolic carbamate fungicide that acts by inhibiting the polymerization of microtubules by interaction with β-tubulin . Some calic acid , and maylic acid , 38 as pfui1Δ, arn1Δ, arn2Δ, and enb1Δ strains. Fui1p is a high-affinity uridine permease, and the transporters Arn1p, Arn2p, and Enb1p have similar cargo specificity , whereas Ftr1p is a high-affinity iron permease, which is also involved in iron homeostasis. However, ftr1Δ strains were depleted in CGP assays. Even though Arn1p, Arn2p, Enb1p, and Ftr1p are iron transporters, only the transporters with specificity for large molecules, such as Arn1p, Arn2p, Enb1p, and Fui1p appear to contribute to 1,10-phenanthroline transport. Single transporter deletions or deletions in combination with ftr1Δ do not confer resistance probably due to redundancy. Only with the deletion of at least two of the four suggested transporters can we limit the compound’s uptake to confer measurable resistance. Our results demonstrate the power of the double mutant deletion library in identifying groups of transporters that contribute to the import of the test compound.We performed validation experiments to investigate the resistance of double mutants enriched in CGP to 1,10-phenanthroline . Among tERG11 gene in S. cerevisiae (r2> 0.95) (r2 = 0.97) . Azolesrevisiae , 39, 40.2> 0.95) . These a2> 0.95) . The azo = 0.97)  and E. F = 0.97) . This ma = 0.97) –43. We hnha1Δ::kanMX deletion mutant, for example, was a top hit for five of the six azoles tested (CGP), and nha1Δ deletants were identified among the resistant colonies selected in a plate assay (low-throughput assay) for clotrimazole, ketoconazole, and difenoconazole , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same DMSO 2% plate as the experiment control. Download FIG S4, TIF file, 2.4 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of difenoconazole. The upper left panel shows a test plate containing difenoconazole (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S5, TIF file, 2.4 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of epoxiconazole. The upper left panel shows a test plate containing epoxiconazole (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S6, TIF file, 2.4 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of tebuconazole. The upper left panel shows a test plate containing tebuconazole (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S7, TIF file, 2.5 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of clotrimazole. The upper left panel shows a test plate containing clotrimazole (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map , and the lower panel shows the z-score for each quadruplicate. Green squares correspond to scores 3*SD, and orange squares correspond to between 2*SD and 3*SD. The high-density assay presents the same 2% DMSO plate as the experiment control. Download FIG S8, TIF file, 2.4 MB.High-density assay with 308 double-deletion strains (in quadruplicate) in the presence of an inhibitory concentration of ketoconazole. The upper left panel shows a test plate containing ketoconazole (4 days of growth). The upper right panel shows a control plate with 2% DMSO (4 days of growth). The middle panel presents a plate map  of itr1Δ strains to difenoconazole, epoxiconazole, ketoconazole, and tebuconazole in both solid and liquid media assays  and high-throughput assays (CGP), using a pool of the double mutant library, combined with validation assays with isolated strains. The low-throughput approach clearly yields valuable and verifiable results; however, it is laborious and demands high sampling to obtain significant data. Alternatively, we employed high-throughput assays, where pools of double mutant transporter deletion strains were grown in competition and the relative contribution of each transporter to the import or export of a given test compound was inferred from the resistance (enrichment) or sensitivity (depletion) phenotypes conferred by their deletion. CGP provided quantitative clues to the relative contribution of each transmembrane protein to the transport of different compounds across the plasma membrane.myo-inositol transporter Itr1p plays a significant role in the uptake of azoles (both triazoles and imidazoles), which is in agreement with previous work that indicated the entry of azoles into the cell is via facilitated diffusion in an ATP-independent process , and the pdr5Δ itr1Δ double mutant was also sensitive to azole treatment, indicating that an alternative import route is used in the absence of Itr1p. Furthermore, in spite of the evidence suggesting the import of azoles by Itr1p, the deletion of ITR2, a paralog of ITR1 generated by the whole-genome duplication event, did not provide a resistance profile for the strain. Both transporters are responsible for uptake of myo-inositol and have high sequence homology  , we did not observe any resistance profile for nha1Δ::natMX his3Δ::kanMX, a strain bearing the Itr1 transporter. Hence, the presence of nha1Δ as a top hit may be due to additional mutations in the strain carrying nha1Δ::kanMX.In a search for transporters that may be either secondary azole transporters or have an indirect effect on these drugs’ efficacy, we focused on and Rb+) –33. It w mutants . While tkanMX marker with barcodes) with 120 transporter gene deletions (natMX marker without barcodes). Considering all the combinations, we have double deletions of importers and exporters that can improve the identification of transport routes. We believe that this strategy for identifying which transporters are involved in the transport of specific compounds could be improved by performing pairwise crosses of all nonessential transporter gene deletions (including genes not represented in our library) barcoded in both alleles to better represent all nonessential import routes and facilitate the identification of transporter pairs working in xenobiotic import. With the development of new strategies for mapping compound import and export routes, we aim to contribute to our understanding of resistance mechanisms, which is critical for the design of drugs with continued efficacy. Furthermore, the knowledge of transporter substrate specificity may allow the design of prodrugs with enhanced targeting to the cell type of interest. Hence, we are convinced that our double-deletion library is an invaluable tool for the design of more specific and efficient therapies.Our library consists of approximately 14,000 strains constructed by crossing 122 transporter gene deletions , with or without 2% Bacto agar.dl-4-hydroxy-3-methoxymandelic acid and tamoxifen) in 100% DMSO of the compounds purchased from Sigma-Aldrich (Merck Group)  is well characterized and provides a resistant phenotype to this compound  background (MATα can1Δ::STE2pr-Sp_his5 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) background  identifying deletions in each of the following open reading frames: YAL067C, YBL042C, YBR008C, YBR021W, YBR043C, YBR068C, YBR069C, YBR180W, YBR294W, YBR295W, YBR296C, YBR298C, YCL025C, YCR010C, YCR011C, YCR028C, YCR098C, YDL199C, YDR011W, YDR046C, YDR345C, YDR384C, YDR387C, YDR406W, YDR497C, YDR508C, YDR536W, YEL063C, YEL065W, YER056C, YER145C, YER166W, YFL011W, YFL040W, YFL050C, YFL055W, YGL077C, YGL114W, YGL255W, YGR055W, YGR121C, YGR138C, YGR217W, YGR224W, YGR260W, YGR281W, YGR289C, YHL016C, YHL040C, YHL047C, YHR092C, YHR094C, YHR096C, YIL013C, YIL088C, YIL120W, YIL121W, YJL093C, YJL129C, YJL212C, YJL214W, YJR040W, YJR054W, YJR152W, YKL174C, YKL217W, YKR039W, YKR050W, YKR103W, YKR106W, YLL028W, YLL043W, YLL048C, YLL052C, YLL061W, YLR081W, YLR092W, YLR130C, YLR138W, YLR237W, YML047C, YML116W, YML123C, YMR011W, YMR177W, YMR243C, YMR279C, YMR319C, YNL065W, YNL142W, YNL268W, YNL270C, YNL275W, YNL291C, YNL318C, YNR002C, YNR055C, YNR056C, YNR072W, YOL020W, YOL103W, YOL122C, YOL158C, YOR011W, YOR071C, YOR153W, YOR192C, YOR202W, YOR273C, YOR306C, YOR328W, YOR348C, YPL036W, YPL058C, YPL092W, YPL265W, YPL274W, YPR124W, YPR138C, YPR156C, YPR192W, YPR198W, and YPR201W  glycerol and stored at −80°C. Double mutants were also pooled in the ratio of 1:1:1:1:…:1 and stored in YPD plus 15% (vol/vol) glycerol in 5-ml aliquots for competition experiments (library pools). The transporter deletion library constructed during the current study will be deposited in EUROSCARF.The construction of the transporter double mutant collection library was performed essentially as described by Tong and coworkers –51. Brie–S. cerevisiae BY4741 was inoculated into 5 ml of fresh YNB+Sc and grown overnight at 30°C with agitation. Then the culture was diluted to an optical density at 595 nm (OD595) of 0.1 in 70 μl of YNB+Sc containing different dilutions of each xenobiotic. The xenobiotics were tested at the following concentrations: 200 μM, 100 μM, 40 μM, 20 μM, 8 μM, 4 μM, 1.6 μM, 0.8 μM, 0.32 μM, and 0.16 μM. Controls containing 2% and 1% DMSO (vol/vol) were also tested. Cultures were prepared in quadruplicate in 384-well flat-bottom plates and incubated at 30°C, with linear shaking (700 rpm) in the CLARIOstar (BMG Labtech) plate reader, for 30 h with OD595 measurements every 10 min. Curves derived from the growth data were smoothed based on the moving average of the 15 closest measurements, and the growth score was calculated by multiplying the yield (maximum OD – minimum OD) by the maximum slope of the curve and dividing by the time taken to reach the maximum slope, using the data analysis software MARS (BMG Labtech). Nonlinear regression for IC90 definition was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA.Spot assays were performed in petri dishes (90 × 15 mm) containing YNB+Sc with inhibitory concentrations of a xenobiotic, as defined by growth assays in liquid cultures, with equivalent volumes of DMSO in negative-control plates. Serial dilutions (1:5 dilution) of BY4741, single mutant and double mutant library pool and selected single mutant strains were spotted onto control or xenobiotic-containing plates for selective inhibition verification and, for definition of inhibitory concentrations, onto solid media using 48-pin replicators . Plates were incubated at 30°C for 2 to 4 days, and images were registered with ChemiDoc MP (Bio-Rad).3, 104, 105, and 106 CFU of BY4741 or the double mutant library pool were plated. Plates containing YNB+Sc with DMSO were used as plating controls. After 2 days of incubation at 30°C, growth was registered with ChemiDoc MP (Bio-Rad), and resistant colonies from library pool plates were picked and transferred to fresh nonselective plates.Assays were performed on petri plates (90 × 15 mm) containing YNB+Sc agar with inhibitory concentration of xenobiotics. Onto these plates, approximately 10g. Pellets were washed with 200 μl of 70% (vol/vol) ethanol, centrifuged for 3 min at 15,000 × g, and air dried. Genomic DNA (gDNA) pellets were dissolved in 100 μl of ultrapure water, followed by centrifugation for 15 s at 15,000 × g. PCRs using Taq DNA polymerase P1011 (Sinapse Inc.) were prepared following the manufacturer’s instructions. Barcode amplification was performed using primers pairs U1 forward 5′-GATGTCCACGAGGTCTCT-3′ with kanMX reverse 5′-CATCATTGGCAACGCTAC-3′ (upstream barcode) or kanMX forward 5′-CTCCTTCATTACAGAAACGG-3′ with D1 reverse 5′-CGGTGTCGGTCTCGTAG-3′ (downstream barcode). PCR products were purified using the E.Z.N.A. gel extraction kit (Omega Bio-tek) and sequenced by Sanger Sequencing. For upstream barcode sequencing, primer pTEF seq reverse 5′- CGACAGTCACATCATGCC-3′ was used, and for downstream barcode sequencing, primer kanMX forward was used. Sequencing was performed at Myleus Biotechnology using capillary electrophoresis (ABI3730) using POP7 polymer and BigDye v3.1. Sequence analyses and barcode identification were performed based on the Saccharomyces Genome Deletion Project barcode list.Genomic DNA was prepared as described by Lõoke et al. . Cells f595 measurements every 10 min. Concentrations selected for chemical genomic profiling were those that inhibited growth of transporter deletion pools by approximately 80% while allowing the culture to reach the stationary phase in 24 h.Determination of the inhibitory concentration of xenobiotics for transporter deletion strains was accomplished by growth curve assays of library pools. These assays were performed in liquid media in 48-well flat-bottom plates with incubation at 30°C, with 500 rpm double orbital shaking in the CLARIOstar (BMG Labtech) plate reader, for 48 h with OD600 of 0.1 in 500 μl of YNB+Sc containing the xenobiotic compounds in 48-well flat-bottom plates (samples were prepared in quadruplicate). After 24 h of growth at 30°C with agitation (ca. 5 generations), OD595 was measured, and cultures were diluted 20× in 500 μl of fresh media containing the xenobiotic and allowed to grow for 12 h (ca. 10 generations) under the same conditions. The dilution procedure was repeated, and cultures were allowed to grow until stationary phase (ca. 15 generations). Cell pellets were collected, and genomic DNA was extracted using the Wizard Genomic DNA purification kit. Upstream barcodes were PCR amplified with U1 and U2 primers containing Illumina preadapters for multiplex barcode sequencing with Illumina HiSeq2500 platform by the University of São Paulo Functional Genomics Center.The transporter double-deletion library pool was grown in YNB+Sc for 12 h at 30°C with agitation. Pools were then diluted to an ODP value adjusted for multiple tests (padj) by the Benjamini-Hochberg method (≤0.1) and P value less than 0.001. For the analysis, we used thresholds of log2 fold change at ≥0.5 (for resistant strains) and ≤−0.5 (for sensitive strains). Correlation from 116 genes presented in all azole data from CGP was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA. CGP data sets with differential abundance of barcodes are presented in The quality of the generated reads was analyzed with the FastQC (version 0.11.7)  and MultP value ≤ 0.001; log2 fold change ≥ 0.5). These mutants were inoculated in 50 μl of YNB+Sc liquid medium in a 384-well plate and grown at 30°C until saturation (∼36 h). Using the Rotor HDA , cultures were stamped in quadruplicate  on plates containing solid YNB+Sc medium at inhibitory concentrations (1× and 2×) of the test compounds. Cultures were incubated for 2 days at 30°C and on subsequent days at room temperature (∼25°C). Plate photos were registered with ChemiDoc MP (Bio-Rad).Based on chemical genomic results, 308 double-transporter-deletion mutants were selected for validation  and wild-type 288 spots  were identified. For each spot, a quadrangular cut-out of a fixed area was delimited. To estimate the growth in the spot, the pixel values in this cut-out were averaged, considering the black and white scale . For each strain, the median of the four values was calculated according to the plate map. The z-score was calculated using median values, according to the median of a sample minus the mean of all median values, divided by the standard deviation of all median values. Strains that varied by more than 3 standard deviations (3*SD) from the mean were considered resistant.Saccharomyces Genome Deletion Project) and kanMX reverse 5′-CATCATTGGCAACGCTAC-3′ (for kanMX gene deletion) or natMX reverse 5′-AAGACGGTGTCGGTGGTG-3′ (for natMX gene deletion). Overnight cultures in YNB+Sc were serially diluted in a 96-well plate and stamped using a replica plater with 48 pins  in petri dishes with YNB+Sc solid medium containing inhibitory concentrations of xenobiotics. Cultures were incubated for 2 days at 30°C and on subsequent days incubated at room temperature (∼25°C). Plate photos were registered with ChemiDoc MP (Bio-Rad). Growth curves and nonlinear regression of selected deletants were performed as described for IC90 determination.Gene deletion of strains selected for validation were confirmed by PCR with “A_confirmation_primer” (PRJNA718573 (BioSample accession nos. SAMN18541664 to SAMN18541685).High-throughput sequencing data of the CGP screens have been deposited in the NCBI Short Read Archive (SRA) under BioProject accession no.


Although novaluron is an insect growth regulator with a low mammalian acute toxicity and a low risk to the environment and nontarget organisms, toxic effects of novaluron have been reported. However, no studies have yet evaluated the effect of novaluron on reproduction. Therefore, we examined the effects of novaluron on sperm functions. The spermatozoa of ICR mice were incubated with various concentrations of novaluron to induce capacitation. Then, sperm motion parameters and capacitation status were evaluated using CASA program and H33258/chlortetracycline staining. In addition, PKA activity and tyrosine phosphorylation were evaluated by Western blotting. After exposure, various sperm motion parameters were significantly decreased in a dose-dependent manner. The acrosome reaction was also significantly decreased in the high concentration groups. Sperm viability was significantly reduced at the highest concentration. In addition, PKA activity and tyrosine phosphorylation were also significantly altered. Thus, novaluron affects sperm viability, sperm motility, and motion kinematics during capacitation. Furthermore, it may promote the reduction in acrosome reactions. The physiological suppression of sperm function may depend on abnormal tyrosine phosphorylation via the alteration of PKA activity. Therefore, we suggest that it is necessary to consider reproductive toxicity when using novaluron as a pesticide. Novaluron belongs to a new class of insecticide known as benzoylphenylurea that is currently registered for the control of thrips, whiteflies, leaf miners, and other foliar-feeding insect pests of ornamental plants grown in greenhouses . NovalurAlthough novaluron is a known low-risk chemical, several researchers have reported that it has a hazardous effect on mammals ,7,8. StuFor successful fertilization, ejaculated sperm cells must undergo a special process within the female reproductive tract to acquire the full capacity to fertility, which has been termed “capacitation” ,10. In t2·6H2O, 0.36 mM NaH2PO4·H2O, 5.56 mM D-glucose, 25 mM NaHCO3, 1.78 mM CaCl2·2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, 50 µg/mL gentamicin, and 0.005 mM phenol red). To induce sperm capacitation, 0.4% bovine serum albumin was added to the BM. All chemicals were purchased from Sigma-Aldrich .Modified Tyrode’s medium was used as the basic medium (BM) . All animals were maintained at a temperature of 24 °C ± 2 °C under a 12 h light/dark cycle with appropriate humidity. The animals were provided food  and water ad libitum. Male ICR mice  were sacrificed at 8–12 weeks of age by cervical dislocation. Spermatozoa were released from the cauda epididymis into BM containing 0.4% BSA. Then, the spermatozoa were incubated with various concentrations of novarulon at 37 °C and 5% COeviously ,14,15. T1. All aneviously .The computer-assisted sperm analysis program (CASA)  equipped with a CMOS image sensor and a 2048 × 1536 (300 M pixels), 60-frame camera , and an OLYMPUS BX43 phase-contrast microscope  with a 10× objective phase-contrast mode, was used to evaluate sperm motility and motion kinematics. A 10 µL smear of sample was evaluated in a preheated (37 °C) Makler counting chamber . Total sperm motility (%), rapid sperm motility (%), medium sperm motility (%), slow sperm motility (%), progressive sperm motility (%), curvilinear velocity , straight-line velocity , average path velocity , beat cross frequency , and mean amplitude of head lateral displacement  were measured.2 for 2 h protected from light. After gently shaking the plates, the absorbance was monitored at OD 570 and 650 nm . Cell viability was measured using the ratio of OD 570 nm to OD 650 nm .To monitor cellular functions, a cell cytotoxicity assay was performed using the Cell Cytotoxicity Assay Kit . Before experiments, the assay solution was thawed and preheated to 37 °C. Sample wells were incubated at 37 °C and 5% COg for 2.5 min. Most of the supernatant was removed, and samples were kept in 135 µL, after which, 15 μL of H33258 solution (10 μg H33258/mL DPBS) was added to the remaining 135 µL of samples and incubated for 2 min at RT. Then, 250 μL of 2% (w/v) polyvinylpyrrolidone in DPBS was added, and the sample was centrifuged at 100× g for 2.5 min. The supernatant was completely removed, and the samples were resuspended in 100 μL of chlortetracycline fluorescence (CTC) solution  and 100 μL of DPBS. After incubating the samples in the refrigerator without light for 20 min, 10 μL of each sample was smeared onto slides. At least 400 spermatozoa were evaluated per slide for each sample. An OLYMPUS BX43 microscope under epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450–490/LP 515 excitation/emission filters were used for H33258 and CTC . Finally, the samples were evaluated and divided into four groups according to the capacitation status, as follows: live acrosome-reacted sperm (AR pattern), live capacitated sperm (B pattern), live non-capacitated sperm (F pattern), and dead sperm (D pattern).The dual staining method (combined Hoechst 33258/chlortetracycline fluorescence assessment) was used to evaluate capacitation status ,13,14,15g for 5 min. Sperms were stored in modified Laemmli sample buffer  at RT for 10 min and then centrifuged at 10,000× g for 5 min. The resulting supernatants were boiled at 95 °C for 3 min. The samples were then separated using a 12% SDS-PAGE gel , and then the separated proteins were transferred onto PVDF membranes . The membranes were incubated at RT for 2 h in a 3% ECL blocking agent . Next, the membranes were washed two times with PBS-T and incubated with the primary antibody overnight at 4 °C. Primary antibodies were diluted with the 3% ECL blocking agent , 1:1000; anti-phosphotyrosine antibody [PY20] (HRP) , 1:5000; anti-alpha tubulin antibody , 1:5000). After treatment, the membranes were washed four times with PBS-T. Goat anti-rabbit IgG H&L (HRP) was diluted at 1:2000 with the 3% ECL blocking agent and used as a secondary antibody for phospho-PKA substrates. Goat anti-rabbit IgG H&L (HRP), diluted with the 3% ECL blocking agent at 1:2000, was used as a secondary antibody for α-tubulin. Protein expression levels were detected by Image Quant LAS 500 , using the enhanced chemiluminescence (ECL) method. Then, the Image Studio Lite  system was used for expression analysis. The density ratios of each treatment band were normalized to α-tubulin.Phospho-PKA substrates and tyrosine phosphorylation were examined by Western blot analysis. Each sample group was washed twice with DPBS and centrifuged at 10,000× p < 0.05.One-way ANOVA using the SPSS software  was applied for the statistical analysis of all data. Tukey’s multiple comparison test was used to compare the five groups with the control. Each experiment was performed at least four times. Numerical data are expressed as mean ± SEM. The level of significance was set at p < 0.05; p < 0.05; p < 0.05; The CASA system was used to evaluate sperm motility and motion kinematics. Total motility, rapid motility, progressive motility, VCL, and ALH were significantly decreased at high concentrations of novaluron    (p < 0.05; p < 0.05; The levels of phospho-PKA substrates and tyrosine phosphorylation were measured by Western blotting. Phospho-PKA substrates were found at approximately 20, 30, 55, and 120 kDa, and phosphotyrosine substrates were detected at approximately 22, 60, and 100 kDa. The levels of approximately 20 and 30 kDa phospho-PKA substrates were significantly increased, whereas those of approximately 55 and 120 kDa substrates were significantly decreased in a dose-dependent manner (Selectively effective biorational insecticides are considered to have less risk to beneficial organisms compared with broad-spectrum pesticides . The genSperm motion patterns change to vigorous patterns during capacitation ,21. TherFor successful fertilization, the ejaculated spermatozoa must undergo capacitation during their passage through the female genital tract ,21. CapaNovaluron is well known as one of the suitable pesticides worldwide because of its low mammalian acute toxicity and reduced risks to the environment and nontarget organisms. However, our results demonstrated that novaluron induces detrimental effects on sperm functions during capacitation, and, to our knowledge, this is the first report concerning the male reproductive toxicity of novaluron. In particular, abnormal PKA activity and tyrosine phosphorylation were observed, resulting in the suppression of sperm motility, motion kinematics, cell viability, and the acrosome reaction. Therefore, we suggest that novaluron should be used considering its reproductive toxicity, and in particular, it is essential to warn people who have occupations related to novaluron use, regarding its reproductive toxicity. In addition, further studies are needed to evaluate the exact concentration for the safe use of novaluron.


Coffee husks are a major by-product of coffee production and are currently being underutilized. The aim of this work was to chemically characterize coffee husks to allow for an adequate evaluation of their potential for valorization. Blanched and non-blanched coffee husks were characterized for extractable and non-extractable phenolics, caffeine, trigonelline content, and for their polysaccharide and proximal composition. The total, soluble and insoluble fiber contents were determined, together with the husks’ technological properties. Antioxidant activity and bioaccessibility of phenolic compounds of coffee husks were evaluated. Two types of husk were studied: one comprised mostly of outer skin and pulp (CH1); and other comprised mostly of parchment (CH2). Blanching had positive effects on non-extractable phenolics, chlorogenic acid and on the bioaccessibility of phenolics, promoting small reductions in extractable phenolics, protocathecuic acid, caffeine and trigonelline contents. Blanched CH1 presented more appropriate properties than CH2 for potential applications in food. It also presented better antioxidant, hydration, and oil holding properties than those of other agri-food by-products. Tentatively identified polysaccharides included galactomannans, arabinogalactans type II, pectin and cellulose. Coffee is one of the main commodities in the world in terms of revenue, second only to petroleum, being consumed by approximately a third of the world population ,2. BraziCoffee production by-products have been studied since the beginning of the twentieth century, mostly for applications as ingredients in animal feed and composting . HoweverExtractable phenolics and other bioactive compounds were the classes of compounds more minutely studied in coffee husks, pulp and parchment, with identification and quantification of individual compounds, such as chlorogenic, ferulic and protocatechuic acids, rutin, catechin, epicatechin, anthocyanins and caffeine ,17,18,19Polyphenols have received considerable interest due to their potent antioxidant activities  and are To evaluate the potential of minimally processed coffee husks, in the form of flour, as a functional food ingredient, there is a need to determine the bioaccessibility of its bioactive compounds. Hence, this study aimed at contributing to the evaluation of coffee husk flours as a potential source of dietary fibers and bioactive compounds by tentatively characterizing their polysaccharide fractions, by determining the contents of bioactive compounds, such as caffeine, trigonelline and phenolic compounds, and by investigating the bioaccessibility of the associated bioactive phenolic compounds.Standards for chromatography analyzes  were obtained from Sigma-Aldrich , except protocatechuic acid, trigonelline  and D-(+)-xylose .Two samples of husks, CH1 and CH2, obtained by dry processing of Bourbon Arabicas were used in this study: CH1, comprised of 80% peel and pulp and 20% parchment, was provided by the Coffee Industry Syndicate of the State of Minas Gerais ; and CH2, comprised of 28% peel and pulp and 72% parchment, were provided by the Santa Inês Farm . Husks were stored in plastic containers at 20 °C. Half the samples were blanched at 90 ± 2 °C for 1 min. The blanched (CH1b and CH2b) and unblanched (CH1 and CH2) samples were dried in a convective oven for 6 h at 60 ± 2 °C and, subsequently, ground, graded in a 35-mesh sieve (Tyler), and stored at −18 °C.v/v) and 20 mL of acetone (70% v/v). After each extraction, the samples were centrifuged at 3500× g rpm for 15 min. The supernatants, comprising total extractable phenolics (TEP), were combined and the volume made up to 50 mL with distilled water [Half a gram of husk powders was placed in 50 mL Falcon tubes and extraction of phenolics was performed by adding 20 mL of methanol (50% ed water . The resm/v), and 2 mL of distilled water. Samples were stirred and let to rest for 90 min in the dark. Absorbance values were measured at 700 nm in a UV-VIS spectrophotometer  [Extractable phenolics were evaluated by the Folin–Ciocalteu method. 1 mL of extracts was combined with 1 mL of Folin-Ciocalteu reagent solution at a 1:3 ratio, 2 mL of sodium carbonate (20%  Brazil) . A calibv/v) containing 0.7 g/L of FeCl3 and kept at 100 °C for 50 min. The mixtures were centrifuged, and supernatants collected. Samples were washed twice with 5 mL of n-butanol-HCl (95:5 v/v) containing 0.7 g/L of FeCl3, centrifuged again, and supernatants collected. Volume was completed to 25 mL with butanol-HCl solution. Then, absorbance values were measured at 450 and 550 nm in a UV-Vis spectrophotometer. The sum of absorbance values was plotted against NEP concentration. The spectrophotometer was reset to zero with a white solution of n-butanol-HCl (95:5 v/v) containing FeCl3. Proanthocyanidin obtained from carob pods (Ceratonia siliqua L.) was used as standard [Dry residues from the TEP extraction were mixed with 10 mL of n-butanol-HCl . The samples were shaken at 95 rpm in an orbital incubator for 10 min at 37 °C. Subsequently, the mixtures were acidified (pH 2.5) with HCl 3 M, and 5 mL of pepsin preparation was added (13 mg of porcine pepsin in 5 mL of HCl 0.1 M). For gastric digestion simulation, samples were incubated for 1 h at 37 °C under agitation at 95 rpm and subsequently cooled in an ice bath. For the simulated small intestinal digestion, pH was adjusted to 7.5 with NaHCO3 1 M, followed by addition of 5 mL of NaHCO3 1 M containing 87 mg of pancreatin and 7 mg of bile salts. The mixture was incubated at 37 °C for 2 h under agitation, centrifuged at 3500× g rpm for 10 min, the supernatant was collected, and the volume was made up to 50 mL with distilled water. The extracts were used to quantify total extractable phenolics as previously described. The bioaccessibility percentage (% Bio) was calculated by:b is the quantity of phenolics released after in vitro digestion and a is the quantity of phenolics prior to in vitro digestion.In vitro digestion of the flour was evaluated in oral, gastric, and small intestinal phases, according to Dutra et al. . 500 mg g rpm for 15 min and supernatants combined, with volume completed to 25 mL with methanol 100%. Filtered samples were injected in a Prominence HPLC system , using a Photodiode Array (PDA) detector (203–325 nm) and a Shimadzu C18 column (4.6 μm × 150 mm)  at 50 °C. The mobile phase consisted of water and acetonitrile (92.6:7) containing 0.4% phosphoric acid (phase A) and acetonitrile containing 0.4% phosphoric acid (phase B) at a flowrate of 1.2 mL/min. Gradient elution was performed as follows: 0–8 min, 1–3% linear B; 8–12 min, 3–8% linear B; 12–15 min, 8–10% linear B; 15–20 min, 10–15% linear B; 20–25 min, 15–40% linear B; 25–30 min, 40–80% linear B; 30–35 min, 80–95% linear B; 35–35.1 min, 95–1% linear B and 35.1–42 min. –1% B isocratic. Phenolic compounds were identified by retention times and UV spectra of respective standards. Quantification was based on the standard curves.Phenolic compounds extracts were obtained according to Dutra et al. . Before Extraction of trigonelline and caffeine was conducted according to Perrone et al. . We mixeNeutral monosaccharides composition was determined by gas chromatography, according to Leão et al.  5 mg of Proximate composition of the flour was evaluated by AOAC methods . Moistur•+  and FRAP (Ferric Antioxidant Power) assays [In vitro antioxidant capacity was evaluated according to methods based on radical scavenging activity ABTS) assays ,28. For Color was analyzed by a Hunter ColorFlex colorimeter  with D65 standard illumination and 10° observer angle. CIE L*a*b* color coordinates were converted to chroma (c*) and hue angle (h).Microstructures of CH samples were observed by a JEOL JSM-5510 scanning electron microscope (SEM)  at magnifications of 1000×. Coffee husk flours were dehydrated in an oven at 40 °C, for 24 h, and placed on SEM specimen tubs with double adhesive tape and coated with a 10 nm gold layer.−1 resolution. KBr was utilized as background spectrum. Readings were carried out in dry atmosphere at 20 °C. Diffuse reflectance (DR) measurements were performed using a Shimadzu diffuse reflectance sampling accessory (DRS8000A).One milligram of flour was mixed with 10 mg of KBr and the spectra collected by IRAffinity-1 FTIR Spectrophotometer  with a DLATGS detector with a 4 cmg rpm for 30 min. The supernatant was discarded, and the sample weighed. WHC and OHC were expressed as g water or oil/g of sample. SWC and WHC were also evaluated at 37 °C in isotonic solutions (NaCl 0.9% m/v), simulating conditions inside the stomach (pH = 1.5), duodenum (pH = 8.5) and also in pH = 7 in order to verify the effect of surrounding medium on hydration properties of CH samples.Swelling capacity (SWC), water holding capacity (WHC), and oil holding capacity (OHC) were determined according to Resende et al. . For SWCp < 0.05) methods by IBM SPSS software (version 19). The normality of data was verified using the Shapiro–Wilk test.Analyses were performed in technical triplicates. Data were statistically analyzed by ANOVA and Tukey (Data for contents of extractable (TEP) and non-extractable phenolic (NEP), chlorogenic acid (CGA), protocatechuic acid (APC), caffeine. trigonelline, and for bioaccessibility of phenolics in coffee husks (CH) are shown in Blanched CH samples presented NEP values higher than those of unblanched samples, confirming that polyphenoloxidases could be inactivated by heat treatment, preserving NEP. Also, these compounds were more strongly bound to the plant matrix, being somewhat protected from the high temperature during blanching. The increase of NEP contents with blanching is attributed to the removal of soluble solids, which decreased the dry basis mass. NEP contents of all samples were significantly higher than those of other fruit by-products, such as pequi (346.84 mg/100 g), apple (1278.7 mg/100 g), kiwi (1522.0 mg/100 g), melon (316.2 mg/100 g), nectarine (1797.5 mg/100 g), pear (721.1 mg/100 g), and watermelon (305.3 mg/100 g) ,30. In aChlorogenic (CGA) and protocatechuic (PCA) acids are phenolic compounds that exhibit a variety of biological functions in the human body, such as anti-inflammatory, antioxidant, and anticarcinogenic bioactivities ,32. CGA Simulated buccal, gastric, and duodenum digestion of CH were performed to evaluate polyphenols stability under physiological conditions. Most studies have determined the bioactive properties of phenolic compounds without considering their physicochemical modifications after being submitted to digestive conditions. Besides, assessing the bioaccessibility of bioactive compounds is also important to further evaluate their actual availability in the human body. Bioaccessibility values of phenolics in coffee husks ranged from 64.10% to 70.76%, indicating most phenolic compounds in CH flours were bioaccessible. The interactions of polyphenols with lipids, proteins and cell wall polysaccharides (CPS) can significantly affect the respective bioaccessibility, bioavailability, and bioefficacy ,36. AlthCaffeine and trigonelline are important bioactive compounds in coffee beans . They arThe identification of monosaccharides is relevant for determining the structure of the polysaccharides present in coffee husks. The relative molar percentages (% mol) of monosaccharides identified in the CH samples are presented in All samples showed high percentages of xylose, indicating the presence of xylans and xyloglucans in CH. Xyloglucans also have glucose and might have galactose in their structure. Thus, samples that showed higher percentages of xylose and lower percentages of glucose and galactose (CH2 and CH2b) probably contain more xylans (or arabinoxylans) and fewer xyloglucans (xylose and glucose). Only arabinose (19.93 mol%), mannose (4.43 mol%), galactose (60.27 mol%) and glucose (15.37 mol%) were found in spent coffee grounds , and thiBlanching caused an increase in glucose percentages in CH samples. This treatment causes softening of the vegetable matrix, probably due to hydrolysis of polysaccharides. Thus, glucose in CH samples can be released after hydrolysis of polysaccharides such as hemicellulose, pectin, and cellulose at high temperatures. In general, samples showed traces of myo-inositol, and these results were similar to the contents of myo-inositol in fresh pulp of lemon, tangerine, orange, and grape (0.6–2.1%) . Myo-ino−1 (polysaccharide fingerprint region) and its second and fourth derivatives  are presented in −1 [−1 (characteristic of mannuronic acid) and rhamnose was detected in reasonable amounts in the samples, hence, it can be safely inferred that the pectic polysaccharides in coffee husks are of the rhamnogalacturonan type, since the bands at 1107 and 1016 cm−1 are characteristic of homogalacturonan content in pectin [−1 is attributed to out of plane hydroxyl vibrations characteristic of α-linkage between (1→4) galacturonic acid units in the backbone of pectin [−1 are, respectively, attributed to bending of hydroxyl in pyranose ring of pectin and to C–O and C–C stretching vibrations in pectin [−1, with the wavenumber 897 cm−1 corresponding to C1 group frequency or ring frequency, characteristic peak of β-glycosidic linkages in sugar units, 1000 cm−1 characteristic of C–O and C–C stretching in cellulose [−1 attributed to stretching vibrations of β-(1→4) glycosidic linkage of glucans [−1, with 1044 cm−1 attributed to steric interaction between side-chain arabinofuranosyl units and galactopyranosyl backbone at 1078 cm-1 in arabinogalactan type II [−1 attributed to α-(1→3) glycosidic links of side-chain arabinose [−1 attributed to stretching vibration of anomeric C–H of β-galactopyranosyl units in the arabinogalactan backbone [−1 with 1090 and 1061 cm−1 corresponding to stretching vibrations of C–O–C in β-(1→4) links of mannan polymers and 1033 cm−1 attributed to C–C bonds of mannose unit rings [−1 is characteristic of sidechain (1→6)-α-D-galactopyranosyl units in mannan backbone and the band 816 cm−1 attributed to (1→4)-β-D-mannopyranosyl units in the backbone of galactomannans [−1 correspond to the skeletal bending of galactose rings and to C–O–C bending vibration separately in galactomannan glycosidic linkages [−1 are suggestive of the presence of xyloglucans and arabinoxylans, in which the higher frequencies at 1171, 1121 and 1040 cm−1 are characteristic of xylans [−1 is attributed to ring vibration and 891 cm−1 attributed to stretching of the β-anomeric link in xyloglucans [−1, associated with linear and branched (1→4)-β-xylan, and 955 cm−1, characteristic of disubstituted xylose residues in arabinoxylans [The FTIR spectrum of CH2b in the range of 1250 to 700 cmed in −1 ,46,47. Tn pectin  and the  glucans . Arabino type II , 1138 anbackbone . Galactoomannans . The wavlinkages . The peaf xylans , the wavnoxylans .In general, samples comprising mostly the outer skin and pulp (CH1) showed values for TEP, CGA, PCA, NEP, bioaccessibility of phenolics, trigonelline, and some monosaccharides contents  higher than those of samples comprising mostly parchment (CH2), confirming its highest nutritional and bioactive potential as food ingredient. Despite the loss of some compounds  by heat treatment, others could be better preserved (NEP and CGA). However, significant amounts of TEP, PCA, caffeine, and trigonelline could be maintained even after blanching. Considering industrial production, the use of blanched husks as a food ingredient would be more favorable for product conservation due to its microbiological load reduction and enzyme inactivation. For these reasons, CH1b was selected to be chemically characterized and evaluated for its technological potential, antioxidant capacity, and microstructure.The proximal composition of CH1b  was deteThe soluble and insoluble characteristics of fibers affect their technological functionality and physiological effects . IDF con2SO4/g). The antioxidant activities of CH1b determined by different techniques were higher than or similar to those of many matrices known as a potent antioxidant, such acai (220 ± 32.9 µmol Fe2SO4/g) and spent coffee grounds (180 ± 57 µmol Trolox/g) [CH1b presented high antioxidant activity  ,55. Suchrolox/g)  and of eThe technological properties that render a by-product suitable for utilization as food ingredient were determined for CH1b and are presented in WHC represents the water quantity that remains in hydrated flours after application of an external force and SWC indicates how much the fiber matrix increases in size when water is absorbed. SWC value for CH1b (8.75 ± 0.35 mL/g) was higher than those of by-products of buriti (3.7 mL/g), mango (4.6 mL/g), passion fruit (7.2 mL/g), pineapple (6.6 mL/g), and guava (1.4 mL/g), whereas WHC value (4.08 g/g) was higher than those of buriti blanched peel (1.14 g/g) and pequi peel (3.74–3.98 g/g) ,55,57. HUnlike water hydration properties, oil retention capacity is related to the presence of hydrophobic groups in the food matrix. Proteins are characterized by presenting hydrophilic and hydrophobic functional groups. These hydrophobic groups can be associated physicochemically with non-polar chains of the oil and promote their retention. Therefore, the higher the protein content, the greater the oil holding capacity. As the protein content of CH1b was higher than those of other fruit peel by-products, OHC value for CH1b was consequently higher ,55. High2O/g respectively for pH 1.5, 7.0 and 8.5. The progressive ionization of the chains of the different polysaccharides present in the CH1b, mostly pectin  contributed to the increase of its hydration properties. Carboxylic groups and carboxylic esters are the main functional groups of pectin. Thus, the ionization of carboxylic groups in alkaline medium led to an expansion of the pectin structure due to effects of electrostatic repulsion between negative charges present in the chains [Regarding SWC and WHC results for samples evaluated under physiological conditions (isotonic solutions at 37 °C at different pH), hydration properties of CH1b increased with increasing pH from 1.5 to 8.5. SWC values were 6.87 ± 0.03, 9.02 ± 0.37 and 10.60 ± 0.52 mL/g respectively for pH 1.5, 7.0 and 8.5; and WHC values were 4.12 ± 0.02, 4.16 ± 0.01 and 4.19 ± 0.04 g HThe morphology of CH1b was observed by scanning electron microscopy . CH1b exThe chemical composition of coffee husks was investigated. Blanching of coffee husks had a positive effect on the bioactive potential of these by-products demonstrating their potential for use as a functional food ingredient. Coffee husks (CH) comprising mostly outer skin and pulp (CH1) showed higher bioactive potential and bioaccessibility of phenolics than CH comprising mostly parchment (CH2). NEP contents were higher than TEP ones, indicating most of the phenolics of CH were trapped in the plant matrix. Blanching treatment led to the loss of some compounds , but others (NEP and CGA) were better preserved, depending on the level of interaction of the compounds with the matrix and their thermal stability. The polysaccharide fraction was characterized with cellulose, pectic polysaccharides, arabinogalactans type II, galactomannans, xyloglucans and arabinoxylans being tentatively identified. The blanched CH1 sample (CH1b) showed significant potential to be used as ingredient in food products since it had similar characteristics of commercialized fiber products, predominantly yellow color (h = 67.28) and small sized particles (5–40 µm). Besides, its values for antioxidant, hydration properties, and oil holding capacity were higher than those of other fruit by-products, which were associated with the presence of phenolic compounds, insoluble fibers, and proteins, respectively. Results confirmed the high nutritional and bioactive potential of coffee husks.


The transition period is a challenging time, which combines a massive increase in nutrient requirements and leads to a negative energy balance. Therefore, disorders related to negative energy balance, such as ketosis, are more frequent. In addition, in this period, oxidative stress increases, favoring immune suppression functions and inflammation, which may lead to increased susceptibility to other diseases such as metritis and mastitis. Therefore, the combination of ionophores, such as monensin, that mitigate the accentuated negative energy balance; vitamin A, which plays an important role in supporting the immune system; and a high level of crude protein might improve immune parameters in dairy cows in the transition period. This study demonstrated that monensin and vitamin A supplementation and high crude protein levels enhanced some vitamin status and inflammatory markers when used during the late prepartum period.p ≤ 0.05. Dairy cows receiving high crude protein (CP) diets with monensin had a greater retinol-binding protein serum concentration than cows receiving high CP diets without monensin (p = 0.04). Animals supplemented with vitamin A showed lower SCC (p = 0.04) and a higher thiobarbituric acid reactive substances concentration (p = 0.06) than cows non-supplemented. Moreover, cows receiving low crude protein diets had a greater haptoglobin concentration (p = 0.01). In addition, cows fed a high crude protein diet had a greater TNF-α expression in peripheral blood mononuclear cells (p = 0.04). Animals fed diets without monensin had a greater serum haptoglobin on day 3 postpartum than those fed monensin (p = 0.01). Moreover, dietary vitamin A increased serum 13-cis retinoic acid postpartum. We conclude that vitamin A, crude protein levels, and monensin fed during the close-up period affect milk somatic cell count, some vitamin statuses, and inflammatory markers during early lactation.The objective of this study was to determine the effect of feeding different combinations of dietary vitamin A supplementation (0 or 110 IU/kg body weight), protein (10.3% or 12.2%), and an ionophore (monensin at 0 or 400 mg/day) on retinoid metabolism and immune function of dairy cows. Eighty multiparous Holstein dairy cows were studied from d −35 to +21 relative to expected parturition in a complete randomized block design with a 2 × 2 × 2 factorial arrangement of treatments. The significance of treatments was declared at  The transition period has been defined as approximately 3 weeks before to approximately 3 weeks after parturition . ManaginMonensin is an ionophore that has been used in transition dairy cows to mitigate the consequences of the negative energy balance . MonensiFurthermore, during the transition period, oxidative stress is increased, leading to immune suppression functions and inflammation, which may set the stage for other diseases such as metritis and mastitis . TherefoThe meta-analysis by Husnain and Santos  showed tAll the animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Idaho (IACUC protocol number 2014–78).n = 5), foot issues/lameness (n = 4), mastitis (n = 3), and extreme calving difficulty (n = 2). There was no relationship between the treatments and the incidence of health issues or calving difficulty because the affected dairy cows belonged randomly to different treatments . Eighty multiparous Holstein cows that completed the experiment were included in the final analyses. Cows were assigned to a treatment group in a 2 × 2 × 2 factorial arrangement of treatments in a completely randomized block design. The factors consisted of dietary protein levels (10.3% or 12.2%), vitamin A levels (0 or 110 IU/kg body weight), and monensin levels (0 or 400 mg/day). The animals were fed twice daily, one of eight dry cow ration combinations from −35 d relative to the expected calving date . Composite milk samples were obtained at the first, second, and third milking postpartum and every 3 days thereafter. Milk samples were analyzed for somatic cell count  and vitamin concentration.Liver biopsy samples were taken between ribs 12 and 13 on days −35, −3, +3, +10, and +21 relative to parturition. Tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until processing. Blood samples were collected on days −35, −21, −14, −7, +1, +2, +3, +6, +9, +12, +15, +18, and +21 by coccygeal venipuncture using 10 mL blood vacutainer tubes . Blood samples were centrifuged at 1500× cis retinoic acid and all-trans retinoic acid concentrations, 400 μL serum samples were mixed with 420 μL of acetonitrile and 20 μL of acetic acid. One aliquot of 20 μL of retinol acetate  was used as the internal standard. Samples were extracted twice with 1.5 mL of a mixture of 6.5:1.5 hexane: isopropanol and 0.2 mL of HPLC-grade water, and the hexane layer was removed via evaporation under a light flow of nitrogen gas. The residue was re-suspended in 200 μL of a 50:50 mixture of the mobile phase solutions, and 100 μL was injected into the instrument. Separation of retinoids was performed using an isocratic gradient on a Waters C18 CSH 3.5 μm column . The mobile phase consisted of two solvents. Solvent A consisted of a 10 mM ammonium acetate solution in HPLC grade water, and Solvent B consisted of acetonitrile, isopropyl alcohol, and tetrahydrofuran (45:45:10) at a flow rate of 1 mL/min. All vitamin analyses were conducted by reversed-phase HPLC  with a photodiode array detector , as described by Tsai et al. . To detecis retinoic acid and all-trans retinoic acid except retinol palmitate used as the internal standard, and samples were analyzed using a non-isocratic method. A single mobile phase consisting of acetonitrile, dichloromethane, methanol, and n-butanol (72:12:8:0.08) and a 1.25 mL/min flow rate was used.Serum retinol, α-tocopherol, and β-carotene concentrations were determined similar to serum 13-Milk retinol, α-tocopherol, and β-carotene were determined in a similar procedure used to serum, except for the extraction. Milk samples were processed by mixing 2.5 mL of milk with 5 mL of 1% pyrogallic acid solution dissolved in 200 proof HPLC grade ethanol, then mixed with 10 mL of a 50% potassium hydroxide solution. Samples were placed in a water bath for 7 min at 70 °C, cooled down, posteriorly, 10 mL of HPLC grade Petroleum Ether was added, and samples were mechanically shaken at high speed for 10 min. Specifically, 15 mL of HPLC grade water were added to each sample, and the samples were centrifuged for 10 min. Once complete, 5 mL of the upper layer was extracted and evaporated under a light nitrogen gas flow. Samples were dissolved in 100 µL of HPLC grade methanol, 400 µL of starting mobile phase , and 100 µL was injected into the instrument. Serum haptoglobin concentration was determined using an enzymatic assay . A total of 50 µL of the first reagent (stabilized hemoglobin) was added to 3.25 µL of serum, followed by 70 µL of the second reagent (chromogen). Samples were incubated for 5 min at room temperature, and the absorbance was determined in a spectrophotometer  at 600 nm. Concentrations were determined using a standard linear curve according to instructions provided by the manufacture. Serum RBP concentration was determined using a commercially available ELISA kit  according to instructions provided by the manufacturer. Briefly, 100 µL of sample was added to a pre-coated 96-well plate, and 50 µL of the conjugate (RBP4-HRP) was added to each well. The plate was incubated for 1 h at 37 °C. Upon completion of the incubation, each well was washed 5 times using a 1× wash solution. Posteriorly, 50 µL of solution A (substrate for HRP enzyme) and B (stop reagent) were added to each well, and the plate was covered and incubated for 15 min at 37 °C. After incubation, 50 µL of stop solution was added, and the absorbance was determined in a spectrophotometer  at 450 nm. The standard curve fit an exponential model using SAS, and concentration was determined for each sample. Serum concentration of malondialdehyde (MDA) was determined via colorimetric assay testing for thiobarbituric acid reactive species  according to the manufacturer’s instruction. A total of 50 µL of serum was mixed with 50 µL of sodium dodecyl sulfate (SDS) solution, and 2 mL of color reagent  were added to each sample. Samples were boiled for 1 h and immediately cooled on ice for 10 min. Samples were centrifuged, transferred on a 96 well plate, and absorbance determined in a spectrophotometer  at 532 nm. A standard linear curve was created, and MDA concentration was determined according to the standard curve. The intra- and inter-assays’ coefficients of variation were determined to serum metabolites and RBP, and when those coefficients were higher than 10%, the samples were re-analyzed.g for 1 h at room temperature, plasma was discarded, and the buffy coat and red blood cells were collected. Red blood cells were lysed using a 10% NaCl solution and water, then further washed with phosphate-buffered saline (PBS). Samples were separated, and PBMC was stored at −80 °C for further processing for gene expression.Peripheral blood mononuclear cells (PBMC) were isolated, according to Scholte et al.,  with mod® RNA , as described by the manufacturer. The RNA concentration was determined using NanoDrop ND-1000  spectrophotometer. The complementary DNA (cDNA) synthesis was carried out using Applied Biosystems High-Capacity Reverse Transcription Kit , as described by the manufacturer. Total RNA was extracted from PBMC and liver using NucleoSpin® Universal Master Mix II with Uracil-N-Glycosylase , 1 μL of Applied Biosystems 20X custom primer probe mixture , and 7 μL of RNase-free water.Real-time reverse-transcribed PCR was carried out in a 7500 Fast real-time PCR system  using custom-designed TaqMan MGB probes on targeted genes Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Intercellular Adhesion Molecule (ICAM) in PBMC. The bovine RPS9 and GAPDH were used as endogenous controls, also called housekeeping genes . Reactiop ≤ 0.05 with trends toward significance when p < 0.1. Separation of means was accomplished using the diff option within SAS to perform a pair-wise test between means. Intercept was designated as the random effect, with the subject being the cow. Several covariate structures, including compound symmetry, unstructured, autoregressive(1), variance components, and Toeplitz matrix, were tested; however, compound symmetry fit the model best, likely because sampling periods were not equidistant. Samples taken on periods previously listed in the sampling section were used for the analysis of treatment and time. Data are presented as least square means (LSM) ± standard errors of the mean (SEM). The experimental design was a 2 × 2 × 2 factorial arrangement of treatments in a completely randomized block design. Pre-planned orthogonal contrasts were used to compare individual dietary main effects , as well as two-way interaction between the main factors on the response variables measured. Secondarily, variables were compared over time. Response variables were analyzed with repeated-measures ANOVA using the MIXED model procedure . Sources of variation in the model included effects of main factors and 2-way interactions amongst the main effects. When analyzed over time, time was also included in the model. Significance was declared at Gene expression data were analyzed using delta Ct values  and are graphically presented as fold change (2-ΔΔCt) relative to the control treatment .p = 0.04, p = 0.05); cows supplemented with vitamin A and monensin had the lowest SCC. The low SCC is possibly attributed to a combination of a balanced immune response (from vitamin A supplementation) combined with improved energy metabolism because of monensin.Milk somatic cell count and linear somatic cell score were affected by dietary treatments . Milk soMonensin and other ionophores have been related to reduced incidence of intramammary infection ,26 in daS. aureus is the most common bacterium that causes mastitis [S. aureus infection. Wiedermann et al. [S. aureus. One of the main reasons neutrophil function might be diminished during vitamin A deficiency is that neutrophils and other granulocytes, developed from myeloid stem cells in the bone marrow, are mediated by retinoic-acid-binding to retinoic acid receptors to stimulate gene expression [Similar results have also been observed using the rat model. mastitis , and Wiemastitis  observedn et al.  also indpression . In genep < 0.01; data not shown) and β-carotene  concentrations over time in postpartum. These reductions corroborate Johnston et al. [p = 0.01, Milk samples were tested over five time points  during early lactation, and there was a reduction in milk vitamin A  milk retinol than cows receiving low crude protein 1 day after calving. Minimal research has been performed investigating the relationship between milk retinol and prepartum dietary crude protein. Lindberg et al. [Milk retinol was affected by dietary crude protein over time , where cg et al.  reportedp = 0.80, data no shown). However, an interaction (p = 0.04) between dietary protein and monensin on milk α-tocopherol concentration was observed (p > 0.13). Milk α-tocopherol and β-carotene followed an expected curve over time, similar to what was observed with milk retinol concentrations. Milk α-tocopherol concentration was not affected by dietary treatments when observed over time . The result indicates that cows receiving vitamin A tended to have a greater serum MDA than cows that did not. Although vitamin A and the β-carotene (precursor of vitamin A) have been shown to have antioxidant capacities [Lipid oxidation measured by TBARS assay determines the concentration of malondialdehyde (MDA) in the serum. Vitamin A supplementation affected serum MDA concentration . This difference between the results observed in the present study and the Lindberg et al. study might be attributed to the higher crude protein concentration in prepartum diets used by Lindberg et al. We [Serum retinol-binding protein showed a protein × monensin interaction . Our data follow the trend reported in the literature [No significant difference was detected among diets on serum retinol concentrations . As reported by previous studies [Similar to serum retinol concentration, no significant treatment effect was detected over time for either α-tocopherol or β-carotene , where cows receiving vitamin A and low crude protein showed a greater concentration. Furthermore, on day 3 and day 9 postpartum, cows receiving vitamin A supplementation had greater (p < 0.01) concentrations of 13-cis retinoic acid than cows that did not receive a vitamin A supplement .Dietary factors contributed to changes in serum concentrations of 13-pplement . Van Mertrans retinoic acid concentration than cows receiving the vitamin A supplement on d −35 , where cows fed with low protein diets associated with monensin inclusion had a greater 13-cis retinol acid than animals fed with low protein without monensin or high protein with monensin. Moreover, cows fed with low protein diets with monensin had a greater β-carotene than cows fed with low protein without monensin. There is no evidence of similar effects in the literature; therefore, further investigations can help to better understand the interaction of these metabolites as both have important functions in immunity.Cows receiving no vitamin A supplement had greater all-milk SCC ; there wp = 0.04, p = 0.05, cis retinoic acid, as demonstrated by Chadwick et al. [cis retinoic acid were affected synergistically to aid in the expression of ICAM, it may explain why cows receiving vitamin A had greater expression of ICAM in isolated PBMCs.An effect of high crude protein on PBMC expression of TNF-α was observed in the present study, where cows receiving high crude protein had greater  or interactions (p > 0.32). The hepatic gene expression of TNF-α and RBP were also determined; however, there was no difference between the treatment .The increase in TNF-α for cows receiving high crude protein may also provide evidence toward the increase in ICAM expression as ICAM is induced by TNF-α. According to the literature, ICAM is upregulated when retinoic acid is used as a treatment for cervical cancer in humans . The uprIn summary, dairy cows supplemented with vitamin A during the late prepartum period showed a lower milk SCC postpartum and a greater serum TBARS concentration pre- and postpartum. Moreover,, high crude protein diets led to a greater milk retinol content postpartum and a greater gene expression of TNF-α in PBMC, and low crude protein diets resulted in a greater serum haptoglobin concentration pre- and postpartum. Furthermore, the combination of high protein and monensin increased serum retinol-binding protein. Further research is required to better understand how these factors mechanistically affect the immune system and how they can be used to decrease the incidences of both mammary gland infections and metabolic disorders.