Herpesviridae and Mimiviridae, but the presence of transcripts in other DNA viruses, such as poxviruses, has not been analyzed yet. Therefore, we first established a next-generation-sequencing (NGS)-based protocol, enabling the unbiased identification of transcripts in virus particles. Subsequently, we applied our protocol to analyze RNA in an emerging zoonotic member of the Poxviridae family, namely Cowpox virus. Our results revealed the incorporation of 19 viral transcripts, while host identifications were restricted to ribosomal and mitochondrial RNA. Most viral transcripts had an unknown and immunomodulatory function, suggesting that transcript incorporation may be beneficial for poxvirus immune evasion. Notably, the most abundant transcript originated from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery.Transcripts are known to be incorporated in particles of DNA viruses belonging to the families of  The biooviruses , suggestHerpesviridae [Mimiviridae [Poxviridae comprises highly complex dsDNA viruses that encode more than 200 genes. Poxvirus replication, transcription and assembly occur in distinct cytoplasmic areas called virus factories [Except for viruses belonging to the families of sviridae ,2,3,4 aniviridae , no otheactories . Becauseactories , these vactories . The numactories , but mecactories , which hactories . Hence, D5L/I1R gene transcript, encoding an inhibitor of the cellular DNA-dependent protein kinase, which functions as a pattern recognition receptor for DNA in the host cell cytoplasm [Virion-incorporated RNAs have been analyzed in previous studies using gene array technology, PCR and nucleic-acid-sequence-based amplification (NASBA) ,2,3,4,5.ytoplasm . This sug and 4 °C for 80 min, and the virus pellet was resuspended in 1 mL of 10 mM Tris pH 9.0. The virus suspension was sonicated for 1 min, layered on a 24–40% continuous sucrose gradient and centrifuged at 26,000 × g and 4 °C for 50 min. The virus band was collected and stored at 4 °C, while the pellet was resuspended in 1 mL of 10 mM Tris pH 9.0, sonicated for 1 min and centrifuged through a fresh 24–40% continuous sucrose gradient as described before. The virus bands were pooled and concentrated at 32,900 × g and 4 °C for 60 min. The pellet was resuspended in 1 mL of 10 mM Tris pH 9.0 and purified further through a 1.23–1.29 g/mL continuous CsCl gradient at 180,000 × g and 4 °C for 4 h. IMV particles with a density of about 1.27 g/mL were aspirated and concentrated through 17 mL of a 36% sucrose cushion as described. The pellet was resuspended in 1 mL of 10 mM Tris pH 9.0 and stored in aliquots at −80 °C. The number of plaque-forming units (PFUs) was determined by a plaque assay as described previously [CPXV Brighton Red  was propagated in HEp-2 (ATCC# CCL-23) and Rat-2 cells (ATCC# CRL-1764) maintained in DMEM supplemented with 2 mM L-Gln and 5% (volume/volume) FCS. CPXV was passaged at least five times in both cell lines prior to virion purification. Purification of intracellular mature virions (IMVs) was done by rate-zonal sucrose gradient centrifugation  followedeviously . Control9 PFUs of heat-inactivated (1 h 60 °C) purified CPXV IMV particles or the according volume of heat-inactivated control sample without virus were filled up to 500 µL with 50 mM Tris pH 8.5 and pelleted at 25,000 × g and 4 °C for 30 min. The pellet was resuspended in 60 µL of buffer P1  with or without 100 µg/µL RNase A (Qiagen) and incubated at 37 °C for 30 min. Alternatively, the virus pellet was dissolved in 25 µL of 50 mM Tris pH 8.0 containing 0.25 µg of trypsin  and incubated at RT for 5 min. Trypsin was inactivated by adding Phenylmethylsulfonylfluorid  to a final concentration of 1 mM. Virus particles were pelleted at 25,000 × g and 4 °C for 10 min and resuspended in buffer P1 for RNase digestion as described. Subsequently, RNA was isolated using the NucleoSpin® RNA II Total RNA Isolation Kit  according to the manufacturer’s instructions. DNA was eluted in 40 µL of RNase-free water using the high-yield and high-concentration protocol. For digestion of viral DNA, the Ambion Turbo DNA-free™ Kit  was used. Briefly, 3 µL of Turbo DNase buffer and 1 µL of Turbo DNase were added to 27 µL of eluate and incubated at 37 °C for 30 min. To stop the digestion, 5 µL of inactivation reagent were added and incubated at RT for 5 min with occasional flipping of the tube. The inactivation reagent was pelleted at 10,000 × g for 1.5 min and 10 µL of the supernatant was used for cDNA synthesis. For first-strand synthesis, Super Script IITM reverse transcriptase  was used according to the manufacturer’s instructions, including 10 µM random hexamer primers without RNaseOut reagent. For second-strand synthesis, the NEBNext® mRNA Second Strand Synthesis Module  was used according to the manufacturer’s instructions. Double-stranded cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen) prior to library preparation. During method establishment, removal of RNA by digestion and RNA stability during DNase digestion were verified by spike-in RNA control. Briefly, 107 copies of plasmid-derived Crimean-Congo hemorrhagic fever virus (CCHF) RNA were added before RNase or DNase digestion and assayed for CCHF RNA before and after digestion by real-time PCR [Approximately 2 × 10time PCR . Moreovetime PCR .® XT DNA Library Preparation Kit (Illumina) was used according to the manufacturers’ instructions. Sequence files are available from the NCBI Sequence Read Archive under the BioProject ID PRJNA401971.Double-stranded cDNA was analyzed in the sequencing facility  using an Illumina MiSeq machine . For library preparation, the NexteraHomo sapiens reference genome assembly (GRCh38) or the Rattus norvegicus reference genome assembly (Rnor6.0), using the split read mapper TopHat2 [www.ensembl.org). Reads mapping to the host and the virus genome were identified and extracted using a custom Python script. The number of reads mapping to each gene was counted based on ENSEMBL annotations for the human and rat genomes. This included the mapping to 18S and 28S and annotation from the CPXV BR reference sequence genome, using the tool featureCounts [Trimmed reads were mapped to both the CPXV BR reference genome and either the  TopHat2  with stareCounts . Read coTo analyze the presence of transcripts in CPXV particles without prior sequence knowledge, an NGS-based protocol was established. In summary, RNA outside of virus particles was removed, RNA extracted and the viral DNA digested prior to NGS analysis . To miniSamples showed a maximum coverage of the virus genome of up to 118,673, in contrast to controls with only a maximum coverage of up to 188. This observation was conclusive, since control samples contained buffer without virus particles. Instead, controls displayed a considerable number of host reads. As some virus genes may share homology to host genes, multiple mapping of reads to the host and the virus genome was analyzed. A maximum of nine reads was identified which mapped to both the viral and the according host genome . TherefoUp to about 4500 and 2600 genes were identified in HEp-2- and Rat-2-passaged virions, respectively, including viral and host genes. However, most genes displayed very low read counts. Hence, to define a gene as a transcript hit, the cutoff for each sample was set in a way that hits included 90% of summed normalized read counts . This reD5L/I1R gene (CPXV GRI-90 nomenclature), located in one copy at each end of the genome, displayed by far the highest coverage, followed by the B8R gene (CPXV GRI-90 nomenclature). Additionally, viral genome alignments frequently contained sequences without any aligned reads verifying the absence of viral genome contaminations in preparations . Additionally, the coverage increase was not restricted to annotated CDS, but was also found before and after the respective CDS. This observation made sense in that transcripts were analyzed whose sequence may generally differ from the translated protein sequence as a result of untranslated regions at the 3′ and 5′ ends.B8R, belonged to the early class of viral genes. Early OPV promoters have been predicted to contain the consensus promoter sequence AAAA---TGAAAA---A [C8L, C10L, M1L, F3L, F4L, J5R, A34R, A36R and D1L/I5R. Terminator sequences were also identified near these transcript 3′ ends. Hence, these genes were used to verify the presence of intact transcripts. The observed coverage fitted remarkably well to the predicted transcript size limited by promoter and terminator sequence. Moreover, coverage distributions showed the expected read depletion at the ends, but mostly also a tendency to 3′ bias, meaning higher coverage towards the mRNA 3′ end . In HEp-2 samples, these mt transcripts were also identified at rather high ranks, but they were not included in the cutoff. The identification of mt transcripts may indicate that the whole mtDNA or even polycistronic mtRNA was purified during sample preparation. Although transfer RNA genes are distributed throughout the mtDNA, only few transfer RNA reads were identified. Hence, it could be excluded that whole mtDNA or even polycistronic mtRNA was purified. Virus particles propagated in HEp-2 cells contained a transcript encoding a predicted microRNA (miRNA 6087) of unknown function  12S and 16S rRNA were found in virion preparations. Further mt transcripts were identified in Rat-2 samples  primers . ControvXrn1 . DecappiXrn1 . D5/I1, the IL-18-binding protein C8, the dsRNA-binding protein F3, the inhibitor of MHC class II antigen presentation A36, the NF-κB inhibitor B13 and the chemokine-binding protein D1/I5. The I1/D5-encoding RNA was identified in extraordinarily high amounts. DNA-PK is a large cellular complex which functions as a pattern recognition receptor for cytoplasmic DNA, activating innate immune response. D5/I1 blocks binding of the DNA-PK to DNA, inhibiting recognition of poxvirus DNA [The largest number of transcript with known function was associated with viral immune evasion. Out of eight transcripts encoding immunomodulatory proteins, six were identified in both HEp-2- and Rat-2-passaged virions. These included the DNA-dependent protein kinase inhibitor (DNA-PK) irus DNA .C14L, P2L, M1L, F3L, F4L, A34R, A36R, A47R and B13R. It may be hypothesized that the incorporation of transcripts in virus particles may, at least partially, explain their immediate-early expression. Assarsson and colleagues [Viral immunomodulatory genes are generally expressed early during infection in order to rapidly prevent and counteract the host’s antiviral response ,32. Morelleagues  showed trpo30 gene [rpo30 gene (F4L) was identified as a virion-incorporated transcript in the present study. Interestingly, it was shown that reactivation of early genes seems to depend on virus assembly and, moreover, on cis- and trans-acting elements [OPV gene expression is organized in a cascade-like manner by genes with early, intermediate and late promoters. Additionally, genes with dual early/late promoters were identified . The vaco30 gene . Notablyelements ,36. Thiselements , explainSummarized, it can be stated that viral transcripts are incorporated in poxvirus particles, possibly undirected in proportion to their intracellular amount. The biological meaning of these findings, however, remains to be elucidated, e.g., whether poxvirus-incorporated transcripts are released upon infection and result in the expression of intact proteins.Herpesviridae and Mimiviridae, but the presence of transcripts in other DNA viruses, such as poxviruses, has not been thoroughly analyzed yet. By using RNA sequencing of highly purified Cowpox virus virions, this study shows that 19 viral transcripts are incorporated in poxvirus particles, possibly undirected in proportion to their intracellular amount. The function of most of these transcripts is either unknown or related to immunomodulation, including the most abundant transcript originating from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery. This suggests that transcript incorporation may enable host immune suppression even before transcription from the viral genome is initiated. However, it remains to be elucidated whether poxvirus-incorporated transcripts are released upon infection and result in the expression of intact proteins.Transcripts are known to be incorporated in infectious particles of DNA viruses belonging to the families of


Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, normally faces diverse stresses in the human host. Here, we report that a novel, basic, leucine-zipper (bZIP) protein, designated Gsb1 , is required for its normal growth and diverse stress responses. C. neoformans gsb1Δ mutants grew slowly even under non-stressed conditions and showed increased sensitivity to high or low temperatures. The hypersensitivity of gsb1Δ to oxidative and nitrosative stresses was reversed by addition of a ROS scavenger. RNA-Seq analysis during normal growth revealed increased expression of a number of genes involved in mitochondrial respiration and cell cycle, but decreased expression of several genes involved in the mating-pheromone-responsive MAPK signaling pathway. Accordingly, gsb1Δ showed defective mating and abnormal cell-cycle progression. Reflecting these pleiotropic phenotypes, gsb1Δ exhibited attenuated virulence in a murine model of cryptococcosis. Moreover, RNA-Seq analysis under oxidative stress revealed that several genes involved in ROS defense, cell-wall remodeling, and protein glycosylation were highly induced in the wild-type strain but not in gsb1Δ. Gsb1 localized exclusively in the nucleus in response to oxidative stress. In conclusion, Gsb1 is a key transcription factor modulating growth, stress responses, differentiation, and virulence in C. neoformans.The human pathogen  Cryptococcus neoformans, can lead to cryptococcal meningitis (CM), killing hundreds of thousands of people annually, especially immunocompromised patients. Nearly 1 million cases of HIV/AIDS-associated CM occur annually worldwide, causing nearly 625,000 deaths within three months of diagnosis; the majority of CM cases occur in sub-Saharan Africa1. Although the introduction of highly active antiretroviral therapy (HAART) has decreased the death rate in HIV/AIDS patients with cryptococcal infection in developed countries, CM is still a major problem in third-world countries with high HIV prevalence3. C. neoformans is found in nature, e.g., in soil, trees, and bird guano. During the progression of infection, C. neoformans encounters a number of stressors, such as temperature fluctuations, oxidative and osmotic stresses, nutrient limitation, physiological pH, and high CO2 levels in the human host4. Therefore, the ability to sense, respond, and adapt to host stressors is crucial for survival and proliferation of this opportunistic pathogen4.Infection of the central nervous system with a basidiomycete fungal pathogen, C. neoformans has evolutionarily conserved and distinct signaling pathways to counteract various environmental/host stressors. These pathways include high-osmolarity glycerol response, Ras/cAMP/protein kinase A (PKA), Rim101, Ca2+/calcineurin, target of rapamycin (TOR), protein kinase C (PKC), and the unfolded protein response (UPR)13. External stressors can activate specific signaling pathways through specific receptors or sensors that then relay their signals by engaging some other downstream signaling molecules. This leads to the activation and expression of a group of target genes via one or many specific transcription factors (TFs). Among TF families, the basic, leucine-zipper (bZIP) family is one of the largest and the most conserved family of proteins that play crucial physiological roles in various eukaryotes. The bZIP domain generally contains 60–80 amino acids, and a basic region for DNA binding and nuclear translocation, followed by a leucine-zipper motif for dimerization14. Several stress-associated C. neoformans TFs, such as Yap1, Yap4, Atf1, HapX, and Hxl1, were previously classified as bZIP proteins17. A recent systematic, functional analysis of C. neoformans TFs revealed that among 178 analyzed putative TFs, 12.7% harbor the bZIP domain18. The cryptococcal bZIP TFs characterized so far are reportedly involved in cellular responses to oxidative, osmotic, and endoplasmic reticulum (ER) stresses as well as several biological functions, including iron uptake or storage, sexual development, high-temperature growth, drug resistance, and virulence. More recently, a unique bZIP TF, named Bdr1, was shown to regulate resistance to gamma radiation in C. neoformans by controlling expression of the DNA-damage response genes19.C. neoformans null-mutant strains lacking the putative bZIP proteins, which were previously shown in silico to harbor the bZIP domain similar to that of Hac1/Xbp1 homologs13, the bZIP transcriptional factors controlling the UPR pathways in eukaryotes. We then assessed the growth phenotype of the mutant strains under several stress conditions. We report that a novel bZIP protein, designated general stress-related bZIP protein 1 (Gsb1), is involved in growth, sexual differentiation, and responses to diverse stresses, particularly against oxidative stresses, and is required for potent virulence of C. neoformans. Based on the transcriptome profiling and growth phenotypes, such as mitochondrial dysfunction and abnormal cell division, of the gsb1Δ mutant strain, we propose that Gsb1 is a major regulator required for responses to oxidative stress and during other developmental processes, including mating and cell cycle.In this study, we constructed a set of C. neoformans13, we identified four other open-reading frames (ORFs)  as putative Hac1/Xbp1 homologs, but with low similarity in the bZIP domain. These proteins have an N-terminal bZIP domain 13, the deletion mutants of CNAG_00871, CNAG_07560, and CNAG_07940 did not show any increased sensitivity to tunicamycin and DTT compared to the wild-type (WT) strain, indicating that the three bZIP proteins were not involved in the ER stress responses. Notably, however, the CNAG_07560 deletion mutant showed apparently slow growth even under a non-stressed condition (YPD medium at 30 °C) and increased sensitivity to high (39 °C) or low (16 °C) temperature  in C. neoformans. Notably, compared to the cac1Δ, cna1Δ, cpk1Δ, hog1Δ, and mpk1Δ mutant strains, which reportedly are highly sensitive to oxidative stress , SDS, and caffeine; oxidative stressors—diamide and Hgsb1Δ mutant to oxidative stress by assessing its growth in the presence of additional oxidative stressors—CdSO4, menadione, and tert-butyl-hydrogen peroxide, and nitrosative stressors—NaNO2 and hydroxyurea. The gsb1Δ mutant exhibited hypersensitivity to all the tested oxidative and nitrosative stressors  at its C-terminus, Gsb1HA  levels, leading to such hypersensitivity to oxidative stressors. Indeed, addition of the ROS scavenger N-acetyl-L-cysteine restored the normal growth of the ons Fig. . These r21. S. cerevisiae mitochondrial mutant strains, either having defective respiration or overproducing ROS, cannot grow on non-fermentative carbon sources, which mitochondria-dependent respiration essentially uses for cellular growth23. Interestingly, more severe growth defects were observed in the gsb1Δ mutant grown on non-fermentative carbon sources, such as glycerol, ethanol, and sodium acetate , FCCP (uncoupler of electron transport from oxidative phosphorylation), oligomycin (ATP synthase inhibitor), and rotenone (Complex I inhibitor). The majority of cryptococcal mutants with reduced respiration rates were reportedly hypersensitive to hypoxia-mimicking agents, such as cobalt chloride (CoCl2), or low-oxygen conditions24. No difference in growth was observed between the WT and gsb1Δ mutant in the presence of CoCl2 in culture, further suggesting that the gsb1Δ mutant does not have defective respiration ate Fig. , indicatETC Fig. . Compareion Fig. . Notablyain Fig. . S. ceregsb1Δ mutant. Interestingly, the gsb1Δ mutant accumulated the MitoTracker dye more than the WT strain, and this over-accumulation was reversed by Gsb1HA expression , we assessed whether mitochondrial integrity was compromised in the ion Fig. . BecauseGSB1 disruption resulted in retarded cell growth: two-fold slower growth rate of gsb1Δ than that of the WT strain was observed during exponential phase even under normal growth conditions on YPD  enrichment analysis revealed that the expression of some genes involved in autophagy, carbohydrate and nitrogen metabolism, cell cycle, metabolite and energy generation, and mitochondrial organization is significantly increased in the gsb1Δ mutant 30, were not induced in the gsb1Δ mutant , CNAG_02531 (CPK1), CNAG_00179 (GPA2), CNAG_00125 (CRG1), and CNAG_01855 (GPR2)31, was significantly decreased in the gsb1Δ mutant than in WT. In addition, the basal expression of a TF gene involved in mating  and a gene coding for a membrane protein  was about two-fold decreased in the gsb1Δ mutant than in WT . The gsb1Δ mutants did not form mating hyphae in unilateral [WT (α or a) × gsb1Δ (a or α)] or bilateral [gsb1Δ a × gsb1Δ α] mating processes on V8 medium, and reintroduction of the WT GSB1 gene completely restored the mating defect in the gsb1Δ mutants  and the gsb1Δ mutants (MATa and MATα) as previously described32. No Natr Neor dikaryotic cell fusion products were detected from bilateral pairing between the gsb1Δ a and gsb1Δ α mutant strains. Moreover, cell fusion products were hardly detected from unilateral mating, although a few colonies were generated  pathway and cAMP-PKA pathway, including CNAG_00125 , CNAG_07758 (meiotic cell cycle), CNAG_05771 (DNA checkpoint), and CNAG_02658 (regulation of cyclin-dependent serine/threonine kinase) in the gsb1Δ mutant and the overall reduced resistance to diverse environmental stresses, such as heat, and oxidative and nitrosative stresses, prompted us to investigate the gsb1Δ virulence in a murine model of systemic cryptococcosis established in A/J mice infected by nasal instillation. We found that the gsb1Δ mutant had attenuated virulence compared to the WT strain (H99)  Fig. . Virulen °C Fig. . Furthernts Fig. . These r33. Gsb1 contains a basic DNA-binding region in the bZIP domain as well as a nuclear export signal (NES) sequence , implying that the Gsb1–GFP fusion protein was functional in vivo. C. neoformans cells in the early exponential phase, cultivated under nonstressed conditions or under oxidative stress induced by 3.5 mM H2O2 treatment for 1 h, were analyzed by a confocal microscope. The gsb1Δ strain expressing the Gsb1–GFP fusion construct (gsb1Δ::Gsb1–GFP) exhibited green fluorescent signals throughout the cytoplasm and the nucleus , Bzp3, Bzp4, Bzp5 (CNAG_07940), Clr3 (CNAG_00871), Clr4, and Bdr1  carries a lysine residue characteristic of ATF1/CREB1 bZIP family  or CAN2 (a major CA), in C. neoformans5, but the expression of CAN2 did not change in the gsb1Δ mutant under ambient air condition (data not shown), indicating that Gsb1 is not involved in the CO2-sensing mechanisms.The ability of an organism to survive and proliferate under various environmental conditions requires many defense responses induced mostly at the transcriptional level. Through a variety of stress-activated signaling cascades, activation of different subsets of target genes in response to different environmental stimuli is regulated by specific TFs. The bZIP proteins are widely found as TFs in eukaryotes and display an array of specific functions. The 12 putative bZIP TFs, which have been analyzed for their function by deletion mutation in ion Fig. . Thus, wgsb1Δ mutant was highly sensitive to both high (39 °C) and low (16 °C) temperatures  are involved in the oxidative stress responses to maintain cellular redox homeostasis. The stress-activated signaling cascades are shown to involve the activation of a number of TFs, such as Yap1, Skn7, Rlm1, Msn2/Msn4, and Sfp1, which are required for the expression of a subset of genes counteracting the oxidative stresses37. In addition, the cellular responses to oxidative stress involve MAPK-dependent feedback regulation of the actin cytoskeleton, a known target for oxidation29. Our RNA-Seq-based transcriptome profiling of the gsb1Δ mutant compared with the WT strain in C. neoformans revealed that diverse groups of genes appeared to be regulated by Gsb1 under oxidative stress conditions, but a considerable number of genes were also subject to Gsb1 regulation under nonstressed conditions .The CRL3), CNAG_03976, CNAG_07560 (GSB1), CNAG_07940 (BZP5) on the C. neoformans serotype A H99 (MATα) background were disrupted by using double-joint PCR (DJ-PCR) combined with biolistic transformation as described previously42. The first 5′-flankng or 3′-flanking region of each ORF obtained by using the H99 genomic DNA was amplified by PCR using the primer sets detailed in the Supplementary Table NAT (nourseothricin acetyltransferase) was amplified using pNATSTM#225 and the primer sets M13Fe/B1455 and B1454/C814. The 2.03 kb fusion PCR product of 5′-flanking region of GSB1 (0.72 kb) and a part of NAT marker (1.31 kb), and the 1.42 kb fusion PCR product of 3′-flanking region of GSB1 (0.76 kb) and a part of NAT marker (0.66 kb) were generated by overlap PCR using the primer sets C824/B1455 and B1454/C827, respectively, and the combined templates of the first PCR products. Other DJ-PCR disruption cassettes were obtained by the same procedure. The 2.092-kb fusion PCR product of the 5′-flanking region of CNAG_00871 and the 1.31-kb NAT marker, and 1.452-kb fusion PCR product of the 3′-flanking region of CNAG_00871 and the 0.66-kb NAT marker were generated by overlap PCR using the primer sets C790/B1455 and B1454/C793, respectively. The 2.233-kb product of the 5′-flanking region of CNAG_03976 and the 1.31-kb NAT marker, and 1.412-kb product of 3′-flanking region of CNAG_03976 and the 0.66-kb NAT marker were amplified using the primer sets C820/B1455 and B1454/C823, respectively. The 2.105-kb product of the 5′-flanking region of CNAG_07940 and the 1.31-kb NAT marker, and 1.367-kb product of 3′-flaking region of CNAG_07940 and 0.66-kb NAT marker, were amplified using the primer sets C828/B1455 and B1454/C823, respectively. The NAT disruption cassettes were biolistically transformed into the H99 strain. Stable transformants were selected on the YPD medium containing nourseothricin (100 μg/mL), initially screened by diagnostic PCR, and confirmed by Southern blotting  strain, the 0.47-kb of the GSB1 promoter was cleaved using NheI/KpnI from pJAFS1–GSB1PT vector, which contains its promoter and terminator, and inserted between the SpeI and KpnI sites of pJAF1 vector, generating pJAF1–gsb1DP1. The pJAF1–gsb11DP2 vector harboring the gsb1Δ::NEOR disruption cassette was constructed by insertion of the 0.9-kb fragment containing the 3′ part of the ORF with its 3′-UTR region, amplified using primers C991/992, between the EcoRV and XbaI sites of pJAF1–gsb1DP1. The gsb1Δ::NEOR disruption cassette (3.3 kb), amplified by PCR using primers C904/C827 from pJAF1–gsb1DP2, was transformed biolistically into KN99a (MATa) strain. Stable transformants were selected on the YPD medium containing G418 (200 μg/mL) and confirmed by diagnostic PCR and Southern blotting (Data not shown).For the GSB1 complementation vector, the 0.49-kb promoter and 0.98-kb terminator fragments of GSB1 were amplified by PCR using the primer sets C904/C905 and C906/C907, respectively, using the genomic DNA of strain H99; the products were fused by a second PCR reaction using primers C904/C907. The 1.45-kb fusion PCR product was subcloned into pJAFS1 containing the NEO selection marker, generating the plasmid pJAFS1–GSB1PT. The untagged GSB1 ORF amplified using C908/C909 and the C-terminal HA-tagged GSB1 ORF amplified by two PCR reactions using C908/911 and C908/C912 were subcloned between NheI and NotI sites of pJAFS1–GSB1PT, resulting in pJAFS1–GSB1R1 and pJAFS1–GSB1R3CH, respectively. The vectors were excised at the single MluI site of the GSB1 terminator and reintegrated into the native GSB1 locus of the gsb1Δ strain by biolistic transformation. To construct a C-terminal Gsb1–GFP fusion vector, the HindIII/BamHI-digested GSB1 fragment, amplified using primers C1069/C1070, and the BamHI/NotI-digested GFP fragment, amplified using primers C11071/C1072, were subcloned into the HindIII/NotI sites of pJAFS1–GSB1R3CH, resulting in pJAFS1–GSB1R6CF. The GFP vectors were digested at the MluI site at the GSB1 terminator and reintegrated into the native GSB1 locus of the gsb1Δ strain.To generate a 24. Strains grown in YPD medium at initial OD600 = 0.3 for 3 h were incubated for 30 min after addition of MitoTracker Red CMXRos . Cells were washed twice in 1 × phosphate-buffered saline (PBS) buffer and collected by centrifugation at 300 × g for 3 min. Then cells were fixed in 3.7% formaldehyde in a rotator at 25 rpm for 10 min and washed three times in 1 × PBS buffer three times. For 4′,6-diamidino-2-phenylindole (DAPI) staining, an equal volume of cell suspension and PBS buffer containing DAPI  were mixed. Sections were mounted on slides in an antifade medium (Invitrogen) and examined using an inverted confocal microscope (Zeiss LSM700/Axio Scope Z1) equipped with a transmitted light detector T-PMT (Carl Zeiss).Mitochondria were stained as described previously with slight modifications13. Single-stranded cDNA was synthesized using an RnaUsScript reverse transcriptase kit (LeGene Biosciences). RNA sequencing was performed as previously reported using Illumina HiSeq250043. Quality-filtered sequence reads were mapped to the reference genome sequence of C. neoformans var. grubii H99 (RefSeq assembly accession number GCF_000149245.1) using the CLC Genomics Workbench 5.5 (CLC bio). The relative transcript abundance was calculated by counting the Reads Per Kilobase of exon model per Million mapped reads, RPKM44. The processed RNA-seq ORFs were matched with 4,296 annotated gene products from GOProfiler (https://omictools.com/goprofiler-tool) for taxonomy 235443 C. neoformans var. grubii H99 and analyzed by GOSlimViewer using the Generic GOSlim Set developed by the GO Consortium. Gene ontology (GO) analysis was performed using tools by AgBase45. Processed data were deposited in the Gene Expression Omnibus (GEO) database with accession number GSE96543.Total RNA was extracted as previously describedC. neoformans cells were stained using the SYTOX Green as described previously46. Yeast cells were fixed in 70% ethanol with slow mixing for 1 h at RT, washed in 1 mL distilled water, and incubated in 0.5 mL RNase solution  for 3 h at 37 °C. Cells were then collected, incubated in 0.2 mL protease solution  for 30 min at 37 °C. After centrifugation, yeast cells (5 × 107 cells/mL) were resuspended in 50 mM Tris-HCl, pH 7.5. The cell suspension (25 μL) was mixed with 1 mL SYTOX Green solution , sonicated briefly, analyzed on a FACSCalibur System (BD Biosciences) at 488-nm excitation. The SYTOX fluorescence data were analyzed using Flowing software 2.5.1 .For flow cytometry, 48. Animal studies were conducted in Chung-Ang University Animal Experiment Center III facility, and were approved by the Ministry of Food and Drug Safety . All experiments were performed in accordance with relevant guidelines and regulations. Six-week-old female A/JJmsSlc mice  were used in this study. For infection, strains were cultured in YPD medium overnight at 24 °C, washed twice in sterile PBS, and resuspended in sterile PBS at 2 × 106 cells per mL. Dilutions of the cells were plated onto YPD medium and incubated at 24 °C for 72 h to determine viability. Ten mice per strain were anesthetized using Zoletil (Virbac Pty. Ltd) and infected via intranasal instillation with 105 cells (in 50 µL). Survival was monitored twice daily, and moribund mice were euthanized using CO2. The Kaplan–Meier survival curves were generated using Prism 5.02 (GraphPad Software), and P values were calculated using the Mantel–Cox Log-rank test.Capsule and melanin production levels were measured as described previouslySupplementary Information


Part 1 evaluated safety. Part 2 evaluated efficacy using Simon’s optimal 2-stage design: if >2 responses were observed in Stage 1 (n = 28), 54 additional patients would be enrolled in Stage 2. If >9 responses were observed, the study was complete. Otherwise, nab-paclitaxel/gemcitabine would be compared with gemcitabine alone in Part 3. The primary endpoint was overall response rate (ORR). Secondary endpoints included duration of response (DOR), overall survival (OS), and safety.This 3-part sequential study evaluated Eighty-three patients were treated. The prespecified primary endpoint was met: the independently assessed ORR in Stages 1 + 2 was 35% ; therefore, Part 3 was not initiated. The median DOR was 8.9 months . The median OS and progression-free survival were 9.2  and 5.5  months, respectively. The 12-month OS rate was 30%. In an updated analysis, the median OS was 9.3 months and the 12-month OS rate was 32%. Longer OS was observed in patients with baseline neutrophil-to-lymphocyte ratio ≤ 5 vs > 5. The most common grade ≥ 3 adverse events were leukopenia (35%), neutropenia (34%), anemia (15%), thrombocytopenia (10%), and fatigue (13%). Grade 3 peripheral neuropathy occurred in 7% of patients (no grade 4 reported).nab-paclitaxel/gemcitabine is efficacious in Chinese patients with MPC. No new safety signals were observed.The MPACT regimen of NCT02135822, May 8, 2014. Pancreatic cancer is a growing health problem in China, where, similar to global trends, mortality nearly equals incidence , 2. Epidnab-paclitaxel in combination with gemcitabine has received approval for the first-line treatment of MPC [nab-paclitaxel/gemcitabine treatment demonstrated a significantly better OS and overall response rate (ORR) than did gemcitabine alone in 861 patients from North America, Europe, and Australia [nab-paclitaxel/gemcitabine is also recommended for first-line treatment of patients with MPC by the National Comprehensive Cancer Network guidelines, which are often followed by Chinese physicians [nab-Paclitaxel/gemcitabine may also be a suitable first-line treatment regimen for Chinese patients with MPC, despite known differences in cancer drug tolerability between Asian and white populations [nab-paclitaxel monotherapy appears largely similar between Western and Chinese populations [nab-paclitaxel/gemcitabine in Chinese patients. A phase I/II study evaluated this combination in Chinese patients with advanced pancreatic cancer, albeit at a dose and schedule different from that administered in MPACT [nab-paclitaxel 120 mg/m2 (the highest dose tested) plus gemcitabine 1000 mg/m2 on days 1 and 8 every 3 weeks was the recommended dosage/schedule for these patients. With respect to dose intensity, this regimen was comparable with the MPACT regimen and resulted in a tolerable safety profile [In the European Union and the United States, t of MPC , 6. Thisustralia , 8. The ysicians . nab-Paculations . These dulations –13. Baseulations , 15. Howin MPACT , 8, 16.  profile , 16.nab-paclitaxel/gemcitabine regimen used in the MPACT study were evaluated in Chinese patients with MPC.In this phase II study, the efficacy and safety of the Patients with histologically or cytologically confirmed metastatic pancreatic adenocarcinoma measurable by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 were enrolled in this study. Key eligibility requirements included ≥18 years of age, no prior treatment for metastatic disease, Karnofsky performance status (KPS) ≥ 70, and adequate hematologic, renal, and liver function. Patients with known brain metastases or baseline peripheral neuropathy grade ≥ 2 were excluded.ClinicalTrials.gov (NCT02135822).This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines of the International Conference on Harmonisation. Informed consent was obtained from all patients prior to study entry. The trial is registered at nab-paclitaxel/gemcitabine based on safety. In Part 1, 10 patients were to be enrolled and treated with nab-paclitaxel 125 mg/m2 intravenously (IV) plus gemcitabine 1000 mg/m2 IV once weekly for 3 weeks followed by a week of rest (qw 3/4). Safety data were evaluated after the last enrolled patient completed 2 treatment cycles or earlier if treatment was not tolerable or when ≥66% of patients tolerated ≥2 treatment cycles without dose delay or modification. If it was determined in Part 1 that nab-paclitaxel 125 mg/m2 was the recommended dose for Part 2, the 10 patients from Part 1 were counted as a portion of the Part 2 enrollment. If the initial dose level in Part 1 was not tolerated, the Part 2 starting doses were to be reduced to nab-paclitaxel 100 mg/m2 plus gemcitabine 800 mg/m2.This phase II, multicenter, 3-part sequential study was conducted at 13 sites in China. Part 1 evaluated the dose of nab-paclitaxel/gemcitabine based on a single-arm, Simon’s optimal 2-stage design [nab-paclitaxel and gemcitabine dose levels selected from Part 1. In Stage 1, the planned enrollment was 28 patients. If >2 responses were observed, an additional 54 patients would be enrolled in Stage 2 for treatment at the same dose level. In Stage 2, if >9 of 82 responses were observed, the study would be complete. If an insufficient number of responses was observed after Stage 1 or Stage 2, the study would progress to Part 3.Part 2 evaluated the efficacy of e design . Patientnab-paclitaxel/gemcitabine vs gemcitabine alone based on a randomized 2-arm design. Planned total enrollment for Part 3 was 154 patients. Patients were to be randomized 1:1 to receive the Part 1 recommended dose of nab-paclitaxel followed by gemcitabine on days 1, 8, 15, 29, 36, and 43 or gemcitabine 1000 mg/m2 IV alone weekly for 7 of 8 weeks (cycle 1). Subsequent treatments in both arms would occur on days 1, 8, and 15 of a 28-day cycle. Randomization would be stratified by liver metastasis and KPS score.Part 3 was designed to evaluate the efficacy and safety of The primary endpoint of the study was independently assessed ORR according to RECIST 1.0. Secondary endpoints included duration of response (DOR) according to RECIST 1.0, OS, safety, and tolerability. Exploratory endpoints were disease control rate (the percentage of patients achieving objective tumor response or stable disease for ≥16 weeks), serum carbohydrate antigen 19–9 levels and potential association with clinical outcomes, patient-reported quality of life using the European Organisation of Research and Treatment of Cancer Quality of Life Questionnaire-Core 30, and tumor biomarker analysis. Ad hoc analyses included progression-free survival (PFS) and potential association of baseline neutrophil-to-lymphocyte ratio (NLR) and OS. Efficacy was evaluated in the intent-to-treat population, which included all enrolled patients. Response and progression were independently assessed by a central imaging reviewer, blinded to treatment, according to radiological review by computed tomography scan or magnetic resonance imaging every 8 weeks per RECIST 1.0. Treatment continued until unacceptable toxicity or disease progression. Safety was assessed on days 1, 8, 15, and 22 of each cycle by the investigator in all patients who received ≥1 dose of study drug. Adverse events (AEs) were classified by the Medical Dictionary for Regulatory Activities version 17.0 system, and severity was evaluated according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events version 3.0. Dose reductions, delays, premature discontinuations, and clinical laboratory data were also evaluated.0: ORR ≤ 7% vs H1: ORR ≥ 19%. The hypotheses were based on the ORR results from MPACT; the observed ORR was 23%  for the nab-paclitaxel/gemcitabine arm and 7%  for gemcitabine alone. The planned sample size of 82 patients was estimated to provide 90% power at a 1-sided significance level of 0.05 [In Part 2, Simon’s optimal 2-stage design was used. The 1-sided hypothesis test on the ORR was HIn total, 83 patients were enrolled in Part 2. The baseline characteristics are described in Table nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 qw 3/4. These 15 patients from Part 1 were included in Part 2. On the basis of combined results for Stages 1 and 2, the prespecified independently assessed ORR endpoint for Part 2 was met , a separate analysis using the median NLR baseline value (3.7) was performed; baseline NLR ≤ 3.7 (n = 42) vs > 3.7 (n = 41) was also associated with a longer OS, but the difference was not significant . The median PFS was 5.5 months , neutropenia (14% and 16%), and leukopenia (11% and 13%), respectively. At least 1 nab-paclitaxel or gemcitabine dose delay occurred in 37% of patients. The median cumulative doses of nab-paclitaxel and gemcitabine were 1500 and 12,000 mg/m2, respectively. The median dose intensities of nab-paclitaxel and gemcitabine were 79 and 627 mg/m2/week, respectively. The median percentages of per-protocol dose of nab-paclitaxel and gemcitabine were 85% and 84%, respectively.For all patients, the median duration of treatment was 4.8 months, and the median number of treatment cycles was 5 . Forty-nine percent of patients had ≥1 Seventy-five percent of patients experienced ≥1 grade ≥ 3 AE (Table nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2) was efficacious and safe as first-line treatment of Chinese patients with MPC. Per protocol, the study did not progress to Part 3 because >9 responses were observed during Part 2 and the study was considered complete. Although no complete responses were observed in this study, 35% of patients had a partial response, and the median DOR was 8.9 months, indicating a durable response. The median OS was 9.2 months, and the OS rate at 1 year was 30% . The regimen appeared to be well tolerated in Chinese patients with MPC, and no new safety signals were identified compared with those observed in the MPACT population [In this phase II study, the MPACT regimen  in combination with gemcitabine 1000 mg/m2, both given weekly for 2 weeks in a 21-day cycle in Chinese patients with advanced pancreatic cancer [nab-paclitaxel 120 mg/m2, the median OS and PFS were 12.2 and 5.2 months, respectively, and the ORR was 42%. Similar to the findings in our study, common grade 3/4 toxicities that were associated with the 120 mg/m2 dose included neutropenia (17%) and thrombocytopenia (8%), and grade 3/4 sensory neuropathy occurred in only 1 patient. In a trial of Japanese patients with MPC, outcomes of treatment with the nab-paclitaxel/gemcitabine MPACT regimen were also higher/longer compared with the outcomes in the MPACT population [Although data from other studies of Chinese patients treated with c cancer . In thatpulation . These fnab-paclitaxel treatment modifications due to AEs were less frequent in the MPACT population compared with the Chinese population [nab-Paclitaxel dose reductions occurred in 41% and 49% of patients in MPACT and the Chinese study, respectively.In the current study, the most common treatment-emergent grade ≥ 3 AEs were leukopenia, neutropenia, anemia, thrombocytopenia, and fatigue. Similarly, the most common grade ≥ 3 AEs in MPACT were neutropenia, leukopenia, thrombocytopenia, anemia, fatigue, and peripheral neuropathy . The incpulation . nab-Pacnab-paclitaxel/gemcitabine in Chinese patients, one limitation was the homogeneous population. However, the impact of this limitation may have been addressed by the study’s multicenter sampling. In addition, efficacy was evaluated based on a single treatment arm rather than on a comparison of outcomes between 2 randomized groups. The results described here in Chinese patients are similar to those of the global MPACT study, though cross-trial comparisons should be interpreted with caution because of differences in factors such as patient population and usual supportive care. For example, in our study, a higher percentage of Chinese patients had a better baseline performance status (KPS of 90–100) than patients in the global MPACT population (70% vs 58%) [nab-paclitaxel/gemcitabine vs gemcitabine in this disease setting, it would only have been triggered if sufficient activity was not observed vs known historical data in Part 2. Such adaptive trial designs are generally more efficient, requiring fewer patients to answer research questions. This unique study design was particularly beneficial and relevant in this disease setting and helped to avoid enrolling Chinese patients into an inferior treatment arm, as a large global study has established the significant clinical benefit of nab-paclitaxel/gemcitabine vs gemcitabine.Results from this phase II study in Chinese patients are positive; however, several factors must be considered to put the data in perspective. Although this was a bridging study to assess the safety and efficacy of pulation 0% vs 58%nab-paclitaxel/gemcitabine regimen used in MPACT was efficacious and well tolerated in Chinese patients with MPC, supporting the use of this combination regimen in this patient population.The


For coriander, the main compounds were E-2-dodecenal, dodecanal, and octane and the highest contents were found at C2 ; while, the main two components of mint essential oil were carvone and limonene, and the highest contents were found at M1 . The present study was the first one reporting data on descriptive sensory analysis of aromatic herbs at this optimal harvest date according to the content of volatile compounds of their essential oils.Volatile composition of essential oils from dill, parsley, coriander, and mint were investigated at different harvest dates to determine the most suitable harvest time for each these herbs. Hydrodistillation (HD), using a Deryng system, was used for isolating the essential oils. Isolation and identification of the volatile compounds were performed using gas chromatography-mass spectrometry (GC-MS) instrument. The results of gas chromatography-flame ionization detector (GC-FID) analysis (quantification) showed that the main components in the essential oil of dill shoots were α-phellandrene, dill ether, and β-phellandrene, and the optimal harvest date was D2 . For parsley shoots, the main compounds were 1,3,8- The production of aromatic herbs, such as oregano, marjoram, rosemary, thyme, lavender, or peppermint, is a rising sector in the Mediterranean countries. These plants have many applications, such as ornamental cropping, perfumery, or food and pharmaceutical industries; these later applications are linked to the beneficial health effects of their essential oils, which have antimicrobial, antifungal, insecticidal, and antioxidant properties ,2.Anethum graveolens L.) is an important aromatic herb, which is used as flavoring and seasoning of various foods, such as salads, sauces, soups, sea foods, and especially pickled vegetables [Petroselinum crispum) and coriander (Coriandrum sativum) are two culinary herbs commonly used to enhance the flavor of many dishes of the cuisines of China, Mexico, South America, India, and South East Asia [Mentha piperita L.) is a famous aromatic and medicinal herb used in traditional and folk medicines in the world for its antimicrobial and antioxidant properties [Dill (getables . Parsleyast Asia . Peppermoperties .M. piperita L.) essential oils have been obtained by steam distillation in a Clevenger-type apparatus [et al. [Anethum graveolens L.) by using solvent extraction technique.In addition, culinary herbal extracts and essential oils have become increasingly popular as alternative sources of natural preservative agents, largely because herbs are widely cultivated, effective, and safe for consumption . Essentipparatus , while m [et al. . Besides [et al.  isolatedmonoterpenes, monoterpenoids, and phenylpropanoids. In lower amount alcohols, sesquiterpenes, sesquiterpenoids, aldehydes, and esters were also found [etc. [It is well-known that the presence of essential oils and their composition determine the specific aroma of plants and the flavor of the resulting condiments ,8. The mso found ,10,11,12nd [etc. ,14,15.Therefore, the aim of this study was to analyze the volatile composition of the essential oils and the sensory quality of different aromatic herbs  grown in different Mediterranean regions of Spain in order to stablish the best harvest time according to the highest content of essential oils and the optimum sensory quality.Four aromatic herbs  were grown under conventional agricultural practices and conditions according to the recommendations of farmers located at Mediterranean regions of Spain according to their experience. These conditions are described as follows:Anethum graveolens L. Cv. ELLA) were sown on the 7 September 2014 in expanded polystyrene (EPS) trays and placed in a greenhouse located at Santomera  until 4 October 2014. Then, plantlets were transplanted into a commercial orchard placed at Sucina . Dill samples at the commercial stage were harvested at two different dates within the same plant on the 26 November 2014 (D1: 80 days after sowing) and 28 February 2015 (D2: 174 days after sowing).Dill seeds  were sown on the 2 September 2014 in expanded polystyrene (EPS) trays and placed in a greenhouse located at Santomera  until 24 September 2014. Then, plantlets were transplanted into a commercial orchard placed at Sucina . Parsley samples at commercial stage were harvested at three different dates within the same plant on the 19 November 2014 (P1: 78 days after sowing), 5 January 2015 (P2: 144 days after sowing), and 25 February 2015 (P3: 175 days after sowing).Parsley seeds (Coriander sativum Cv. MARINO) were directly sown on the commercial orchard placed at Sucina . Coriander has only one harvest for each plant and in these regions three crops per year are sown, grown, and harvested. The first crop (C1) was planted on 30 September and harvested on 19 November 2014; the second crop (C2) was planted on 22 October 2014 and harvested on 5 February 2015; and, the third crop (C3) was planted on 29 December and harvested on 25 February 2015.Coriander seeds (Mentha piperita L.) of 5–7 cm were sown on 7 August 2014 in polyethylene trays and placed in a greenhouse located at Santomera  until 6 October 2014. Then, plantlets were transplanted to a commercial orchard placed at Sucina . Mint samples at commercial stages were harvested at two different dates from the same plant on the 11 December 2014 (M1: 133 days after sowing) and 5 February 2015 (M2: 189 days after sowing).Mint cuttings , which is suitable for aromatic herbs crops. Soil was uniformly silty-loam in texture, with a low content in organic matter (1.22%), medium salinity conditions (3.35 mS·cm−1), and good levels of sulfates (37.83 meq·L−1) for aromatic herb development.The irrigation water was of good quality, highlighting its slightly basic pH (7.91), and its proper electrical conductivity  of 60 kg·ha−1, and K (K2O) of 160 kg·ha−1 for dill development. For parsley, the total amount of fertilizers was 300 kg·N·ha−1, 190 kg·P·ha−1 (P2O5), and 350 kg·K·ha−1 (K2O). Regarding coriander, the fertilization was carried out with a total amount N of 275 kg·ha−1, P (P2O5) of 170 kg·ha−1, and K (K2O) of 310 kg·ha−1. Finally, the total amount of fertilizers was 200 kg·N·ha−1, 130 kg·P·ha−1 (P2O5), and 230 kg·K·ha−1 (K2O).Along the development of crops, fertilization was carried out with a total amount of N of 130 kg·ha−1). After the mixture started boiling, heating was maintained for 1 h. A cold refrigerant was used to condense the vapors, and 1 mL of cyclohexane was added to the Deryng apparatus at the beginning of the hydrodistillation process to retain the essential oil distilled from the samples of herbs shoots. After 60 min of extraction, the solvent, enriched with the volatile compounds, was transferred into a 2.5 mL vial, after drying it over anhydrous sodium sulfate (Na2SO4), and kept at −15 °C until the gas chromatography-mass spectrometry (GC-MS) analyses were conducted. The extractions were conducted in triplicate.Hydrodistillation (HD), using a Deryng system (the Polish version of the Clevenger apparatus), was used for isolating the essential oil in fresh herbs . About 15.0 g of freshly chopped herbs shoots  were put in a 500 mL round bottom flask, together with 1.0 g sodium chloride (NaCl), 150 mL of distilled water, and 50 µL of benzyl acetate as an internal standard  80 °C for 0 min; (b) increase of 3 °C·min−1 from 80 °C to 210 °C and hold for 1 min; (c) increase of 25 °C·min−1 from 210 °C to 300 °C and hold for 3 min. The temperatures of the injector and detector were 230 °C and 300 °C, respectively.Analysis and identification of the volatile compounds were performed using a Shimadzu GC-17A gas chromatograph coupled with a Shimadzu QP-5050A mass spectrometer detector . The GC-MS system was equipped with a TRACSIL Meta.X5 (95% dimethylpolysiloxane and 5% diphenylpolysiloxane) column . Analyses were carried out using helium as carrier gas at a column flow rate of 0.3 mL·minAll compounds were identified using three different analytical methods: (1) comparison of experimental retention indexes (RI) with those of the literature; (2) GC-MS retention times . The quantification was obtained from electronic integration measurements using flame ionization detection (FID). Benzyl acetate (1000 mg·L−1) was added as internal standard at the beginning of the distillation procedure to simulate the behavior of volatile compounds; this chemical was used as an internal standard after checking that it was absent in herbs, it separates well from other volatiles, it possesses similar FID and MS response factors to most of the volatiles in the aromatic herb essential oil, it is stable at high temperatures, and does not react with water. Calibration curves were performed with the following compounds  as representative of each chemical family: α-phellandrene (monoterpenes), α-terpineol (terpenoids), trans-β-caryophyllene (sesquiterpenes), dill ether (terpene ethers), nonanal , myristicin (phenylpropanoids), bornyl acetate (esters), 1-decanol , undecane ; the correlation coefficients (R2) for all compounds were >0.995, and results were expressed as mg·kg−1 fresh weight, fw.The semi-quantification of the volatile compounds was performed on a gas chromatograph, Shimadzu 2010, with a flame ionization detector (FID). The column and chromatographic conditions were those previously reported for the GC-MS analysis. The injector temperature was 200 °C and nitrogen was used as carrier gas , with ages between 23 and 56 years old. Panelists belonged to the Food Quality and Safety research group of the Universidad Miguel Hernández de Elche and had over 1000 h of evaluation experience; they had been trained in descriptive evaluation of aromatic herbs ,9.−1 = 6), citrus , pine , spicy , earthy , and woody . The key sensory descriptors  are: herb-ID, green grass, citrus, spicy, and pine; earthy and woody are not positively correlated with the herb quality. Reference products of these attributes with different intensity, were prepared and provided to the panel.An odor profile method was used to describe the dill samples. During two preliminary orientation sessions of 90 min, panelists discussed about the main odor characteristics of the herbs and agreed on their use of odor attributes. During these orientation experiments, panelists evaluated different coded samples of Spanish fresh aromatic herbs together with samples from field. Panelists agreed that the odor of the samples could be described using seven attributes: aromatic herb(dill/coriander/mint/parsley)-ID , green grass  was the program used for the statistical analyses.To compare the experimental data two consecutive tests were performed: (i) one-way analysis of variance (ANOVA), and (ii) Tukey’s multiple range. Homogenous groups and the least significant difference (LSD) were determined at significance level of p-cymene, α-pinene, trans-β-ocimene, and myristicin. In several studies, α-phellandrene was predominating in the “leaf” essential oil of dill from Romania, Egypt, and Finland [Dill was planted on 24 September 2014 and the first harvest date for commercial purpose was on 26 November 2014 (D1) while the second harvest date was on 11 February 2015 (D2). After isolation of the essential oil of dill shoots, 18 compounds were identified by GC-MS . The ide Finland ,16,17,18 Finland  found 22et al. and Radulescu et al. [u et al. ,19 who r−1 in D1 to 474 mg·kg−1 in D2 (an increase of 38.6 %), where the concentration in dill ether changed from 46.2 mg·kg−1 in D1 to 62.9 mg·kg−1 in D2 (an increase of 36.1 %) and from 46 mg·kg−1 (D1) to 60 mg·kg−1 (D2) for β-phellandrene (an increase of 30.4 %). Zlatev [Volatile composition of dill essential oil at two commercial stages were investigated in the current study and the total concentration of volatile compounds was higher in commercial stage of D2 as compared to D1 stage; these trend was also true for the three main components . For α-phellandrene the concentration changed from 342 mg·kg. Zlatev  and El-G. Zlatev  found thet al. [et al. [et al. [Parsley was planted on 24 September 2014, the first harvest date for commercial purposes was on 19 November 2014 (P1), the second harvest date was on 5 January 2015 (P2), and the third harvest date was on 25 February 2015 (P3). Eighteen compounds were identified in the essential oil of parsley and the chemical classification of these 18 compounds was as follows: monoterpenes (nine compounds), sesquiterpenes (three compounds), terpenes (two compounds), monoterpenoids (one compound), monoterpene alcohol (one compound), esters (one compound), and phenylpropanoids (one compound). The main eight compounds of the essential oil of parsley shoots were: 1,3,8-p-menthatriene (38.4%–48.8%), β-phellandrene (22.2%–29.5%), myristcin (6.2%–11.1%), myrcene (5.8%–6.5%), terpinolene (4.2%–5.0%), limonene (2.7%–3.3%), α-pinene (1.6%–2.3%), and α-phellandrene (1.4%–2.7%). Vokk −1, as compared to 414 and 418 mg·kg−1 at P2 and P3 stages, respectively. The main two components were 1,3,8-p-menthatriene and β-phellandrene, which represented 68%–72% of the total concentration of essential oil of parsley. 1,3,8-p-Menthatriene had the highest concentration, 222 mg·kg−1, at the P1 stage, while the highest concentration of β-phellandrene was found at 122 mg·kg−1 P2 harvest. For myristicin and myrcene, the highest concentration was in found at P2 stage, while for limonene was P3 and for terpinolene P1  was planted on 30 September 2014 and the harvest date was 19 November 2014, the second crop (C2) was planted on 22 October 2014 and harvested on 5 January 2015 and the third crop (C3) was planted on 29 December 2014 and harvested on 25 February 2015.−1, mean of all treatments), E-2-dodecenal (mean of 26.9 mg·kg−1), dodecanal (22.0 mg·kg−1), octane (18.6 mg·kg−1), 1-decanol (5.13 mg·kg−1), undecanal (4.30 mg·kg−1), and E-2-tridecenal (3.54 mg·kg−1). Given the results in E-2-dodecenal, dodecanal, and octane represented a big percentage of the total concentration of volatile compounds in the essential oil of coriander shoots (70.9%–82.5%). The highest concentrations of E-2-dodecenal (39.9 mg·kg−1), dodecanal (24.4 mg·kg−1), and octane (20.7 mg·kg−1) were found at C2 harvest, while the maximum value of decanal was at C3 with 36.4 mg·kg−1  the most abundant compounds are E-2-decenal, E-2-dodecenal, decanal, dodecanal, E-2-tridecenal and tetradecenal; these compounds have characteristic green, soapy, and cilantro-like aromas and are particularly important in the overall aroma of the C. sativum herb [GC-MS was used to identify the chemical composition of essential oil of coriander plant. Twenty-four compounds were identified  and the  mg·kg−1 . Nurzyńsvum herb ,24. ThesMentha piperita L.) was planted on 6 October 2014 and the first harvest date was on 11 December 2014 (M1) while the second harvest date was on 5 February 2015 (M2). After isolation of the essential oil of mint shoots, 27 compounds were identified by GC-MS  (a decrease of 24.7 %), while the concentration of limonene increased from 590 mg·kg−1 in M1 to 735 mg·kg−1 in M2 (an increase of 24.6 %) , while the concentrations of limonene and  24.6 %) .Mentha arvensis L.) the content of menthol was not affected by the planting date or harvesting schedule but menthone signficantly decreased with the delay in harvesting [According the previous studies, the harvest date and harvest time can affect the water content, the concentrations of menthol and menthofuran and the yield of limonene, menthol, and menthofuran in Mentha canadensis ,26. Besirvesting .Mentha piperita (peppermint) showed in the composition of its essential oil a higher content of monoterpenes D-carvone (58.79 %) and limonene (28.29 %) [Mentha piperita grown in Norway [The major components of peppermint essential oil were menthol 30.35 %), menthone (21.12 %), and trans-carane (10.99 %) according to previous studies [ %, menthlimonene .29 % [28-1) and can be affected by a number of agronomic  [Volatiles directly affect the sensory quality of fresh fruits, vegetables and aromatic herbs. Within the sensory quality, the odor (perception of volatile compounds with the food outside the mouth)  plays ang stage) ,32 and tg stage) . The qua−1 of its essential oil, were characterized by high intensity of dill-ID (herb-ID in Dill samples (D2), with a total number of 18 volatile compounds accounting a total concentration of 649 mg·kg−1 of its essential oil, were characterized by high intensity of parsley-ID (herb-ID in Parsley samples (P1), with total number of 18 volatile compounds accounting a total concentration of 455 mg·kg−1 of its essential oil, were characterized by high intensity of coriander-ID (herb-ID in Coriander samples (C2), with a total number of 24 volatile compounds accounting a total concentration of 134 mg·kg−1), were characterized by high intensity of mint-ID , with the highest number of volatile compounds (27) and the higher concentration of these compounds in its essential oils  however, for parsley (19 November 2014) while, for coriander (5 January 2015), and for mint (11 December 2014), there are relevant aspects which must subjected to further studies such as, different irrigation treatments, different plant densities, or fertilization conditions. In addition, the effect of pre-harvest treatments with organic compounds may be employed.


Danio rerio) embryos were incubated at 24, 28 or 32 °C until first feeding. Larvae originating from each of these three temperature regimes were further distributed into three challenge temperatures and exposed to lipopolysaccharide (LPS) in a full factorial design . At 24 h post LPS challenge, mortality of larvae incubated at 24 °C was 1.2 to 2.6-fold higher than those kept at 28 or 32 °C, regardless of the challenge temperature. LPS challenge at 24 °C stimulated similar immune-related processes but at different levels in larvae incubated at 24 or 32 °C, concomitantly with the down-regulation of some chemokine and lysozyme transcripts in the former group. Larvae incubated at 24 °C and LPS-challenged at 32 °C exhibited a limited immune response with up-regulation of hypoxia and oxidative stress processes. Annexin A2a, S100 calcium binding protein A10b and lymphocyte antigen-6, epidermis were identified as promising candidates for LPS recognition and signal transduction.In many fish species, the immune system is significantly constrained by water temperature. In spite of its critical importance in protecting the host against pathogens, little is known about the influence of embryonic incubation temperature on the innate immunity of fish larvae. Zebrafish ( Moreover, the mucus contains various antimicrobial substances, such as mucins, lysozymes, proteases, apolipoproteins, natural antibodies, and matrix metallopeptidase. Also, a variety of antimicrobial peptides are present, including cathelicidins, piscidins, defensins, hepcidins, and pardaxins, which not only function in pathogen cell lysis but also have roles in phagocytic chemotaxis, mast cell degranulation, and phagocytosis3. In most cases, the above surface barriers and associated factors are sufficient to defend the host against pathogens. If this first line of defence is breached, pathogens will encounter additional humoral immune mediators. Some mucosal antimicrobial components, such as lysozymes, proteases, complement components, also have functions in fish blood. For instance, lectins not only opsonise pathogens and prevent them from adhering to mucosal surfaces, but also activate the complement system in blood4. The complement system is crucial in targeting and lysing pathogens. For instance, complement component 3b (C3b) opsonises pathogens and presents them to phagocytic leukocytes, while C5b, C6, C7, C8, and C9 are able to form the membrane attack complex and lyse pathogens5. In addition to humoral regulators, cellular components also have key roles in the host defence. Once triggered by pathogens, leukocytes proliferate rapidly within a short time and are led by chemoattractants to infected sites. Cytokines, including interleukins (ILs), tumour necrosis factors (TNFs), chemokines, and interferons (IFNs), are also released by phagocytes; these are essential for regulation of pro- and anti-inflammatory responses6.In teleosts, the innate immune system is extremely important for host defence. The integumental physical barrier, which consists of skin, gill, gut and associated mucus are effective in preventing pathogens from adhering to the surface of fish7. To date, 17 TLRs have been identified in teleosts, and some paralogues have been subjected to extensive duplications8. Following signal transduction, the nuclear factor-κB (NF-κB) is activated and released from its inhibitor protein IκB through phosphorylation and then transferred into the nucleus where it binds to DNA to activate the transcription of pro-inflammatory cytokine genes, such as tnfα, il1β, il6, and ifnγ9.Pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) on the surface of leukocytes and other cells involved in the innate immune response. Toll-like receptors (TLRs) are one of the most important PRR families conserved among vertebrates. In vertebrates, they are phylogenetically grouped into six major families and each TLR family recognizes distinct PAMP ligand types10. Also, teleosts lack lymph nodes and germinal centres, which results in poor antibody affinity maturation and a limited immunoglobulin (Ig) repertoire10. Compared to the innate immune system, the adaptive immune system in fish takes longer to become fully functional11. For instance, in zebrafish (Danio rerio) the thymus is morphologically mature at 3 weeks post-fertilization (wpf), T cells are detectable at 4–6 wpf, and secreted Igs are measurable at 4 wpf12. The adaptive immune system also requires additional time to respond. For example, in Japanese eel (Anguilla japonica) at least two weeks are needed for the antibody production13. Therefore, teleosts rely heavily on the innate immune system, particularly during their early ontogeny.Teleosts lack some immune organs that are important in mammalian host defence. For instance, the bone marrow, which is the main immune organ of mammals for production of haematopoietic stem cells is absent in teleosts; its function is replaced by the pronephros and thymus14. It has been demonstrated that the early thermal environment has a profound influence on various phenotypes in adults, such as muscle growth15, swimming performance16, reproduction17, thermal tolerance18, and sex determination19. However, little is known about the influence of environmental temperature on the developmental plasticity of the innate immune system. A few studies have focused on how temperature affects the post-larval stages of fish, but have not examined embryogenesis21. The only exception is a recent study in sea bream  reporting the persistent thermal effect of embryonic development on the plasticity of the hypothalamus-pituitary-interrenal axis and immune function in adult fish22.As most fish are ectotherms, their body temperature changes following ambient thermal fluctuations. This is extremely challenging for the innate immune system during early stages of ontogeny in spite of their high developmental plasticity23. We used LPS to mimic a bacterial challenge and the mRNA transcriptome was analysed to evaluate the global innate immune response at early larval stages.In this study, we investigated the thermal plasticity of innate immunity in zebrafish during early development. Lipopolysaccharide (LPS) is an endotoxin from Gram-negative bacteria with well characterized immunostimulatory and inflammatory properties in fishMortality rates in control groups ranged from 0 to 3%  were found in LPS-challenged larvae compared to their respective controls   indicated that the first principal component (PC1) explained 57% of the total variance, while PC2 explained 15% of the total variance Fig. . MoreoveIn larvae incubated and challenged with LPS at 24 °C, a number of immune processes were enriched by up-regulated DEGs, including “response to bacterium”, “myeloid leukocyte activation”, “leukocyte chemotaxis”, “defence response”, and “response to wounding” Fig. , Table 3Salmonella infection”, “adipocytokine signalling”, “TLR signalling”, “cytokine-cytokine receptor interaction”, and “apoptosis” were enriched by up-regulated DEGs , interleukin 13 receptor, alpha 1 (il13rα1), and interleukin 6 signal transducer (il6st), pro-inflammatory mediator genes nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha a (nfκbiαa), suppressor of cytokine signaling 3b (socs3b), suppression of tumorigenicity 14 (colon carcinoma) a (st14a), fosl1a, and jun B proto-oncogene a (junba), and chemokines chemokine (C-C motif) ligand 34a, duplicate 4 (ccl34a.4) and cxcl18b (Table lysozyme (lyz), macrophage expressed 1, tandem duplicate 2 (mpeg1.2), cathepsin H (ctsh), cathepsin S, ortholog 2, tandem duplicate 2 (ctss2.2), and apolipoprotein A-IV b, tandem duplicate 1 (apoa4b.1), pro-inflammatory transcripts E74-like factor 3 (elf3), leukotriene A4 hydrolase (lta4h), and caspase b, like (caspbl), chemokine transcripts ccl20a.3 and ccl20b (Table il1β (2.1-fold), cxcl8b.1 (2.3-fold), and CCAAT/enhancer binding protein (C/EBP), beta (cebpβ) (1.8-fold) , TIMP metallopeptidase inhibitor 2b (timp2b), leukocyte cell-derived chemotaxin 2 like (lect2l), tnfrsf11b, interferon regulatory factor 6 (irf6), and junba were up-regulated between 2.1- and 6.3-fold. The expression of some heat shock protein (HSP) genes such as heat shock 60 protein 1 (hspd1), hspa5, hsp90b1, and antioxidant genes such as glutathione peroxidase 1b (gpx1b), glutathione S-transferase omega 2 (gsto2), microsomal glutathione S-transferase 3b (mgst3b) was enhanced 1.6–2.5 fold , mmp13a, ptgs2b, fosl1a, heparin-binding EGF-like growth factor a (hbegfa), insulin-like growth factor binding protein 1a (igfbp1a), lye, and anxa2a were up-regulated, whereas mucin 5.1, oligomeric mucus/gel-forming (muc5.1), and muc5.2 were down-regulated in all three temperature groups . In contrast, incubation at a high temperature (32 °C) had a negligible effect on the subsequent ability of first-feeding larvae to cope with LPS challenge. Low temperatures have been demonstrated to negatively influence the innate immune parameters, such as lysozyme activity20, respiratory burst activity21, opsonisation capacity21, blood leucocyte profiles24 and complement activity21 in adult fish. However, this cannot be generalised to all teleosts, since enhanced innate immune parameters, including blood leucocyte percentages20, phagocytic kidney macrophage proportion24, and complement activity24 have been observed in fish kept in low temperatures. This could be due to different properties of innate immune parameters or distinct sensitivities of different fish species to their environmental temperature, as described in Atlantic halibut strains20. It should be stressed that the above studies of thermal acclimation in fish were carried out at months or years post fertilization, when both the innate and adaptive immune systems were fully developed and functional. Therefore, they could not fully reflect the developmental plasticity of innate immunity during early ontogeny. Our study, focusing on the early life of zebrafish, found a negative effect of a low incubation temperature (24 °C) on the innate immune response of larvae to LPS challenge compared to 28 °C or 32 °C.Animals display thermal plasticity during their embryonic development, which tends to improve their performance at that particular temperature compared to that of animals exposed to other thermal conditionsil1β, cxcl8a, ptgs2b, cebpβ, fosl1a) and processes . The up-regulation of the inflammatory negative mediator transcripts nfkbiαa25 and socs3b26, and the down-regulation of the pro-IL-1β processing transcript caspbl27, implies that the anti-inflammatory response could also be elicited. The anti-inflammatory response is a protective mechanism to quench excessive inflammatory signals, and to avoid pathophysiological consequences, such as sepsis28. Moreover, the down-regulation of antimicrobial transcripts  and immune-related processes  indicates a decreased effectiveness of the innate immune response to LPS. Cationic lysozymes bind to negatively charged LPS at a stoichiometry lysozyme:LPS molar ratio of 1:3, resulting in the LPS structure transition from non-lamellar cube to the multilamella with reduced endotoxicity29. A significant drop of 2.3-fold in lyz expression can thereby weaken this neutralization effect. Apolipoproteins, a main group of high-density lipoproteins, neutralize LPS activity either by opsonizing its endotoxic lipid A domain or via blocking LPS-binding protein30. A drop (1.7-fold) in transcript levels of apoa4b.1 suggests a decrease in the host capacity to neutralise endotoxic LPS. In addition, CXCL8a, CXCL8b.2, CXCL18b, and CCL34a.1 have been reported to have higher expression levels in susceptible channel catfish  than in resistant fish when challenged with Edwardsiella ictaluri32. The up-regulation of cxcl8a, cxcl8b.1, cxcl18b, ccl34a.4 with a fold-change between 1.7 and 2.2 may have contributed to an increased sensitivity of the larvae to LPS challenge. Both up- and down-regulated immune transcripts and processes in larvae incubated and exposed to LPS at 24 °C resulted in an intermediate mortality rate of 53.5% compared to other temperature groups  and processes  were up-regulated in comparison to the respective controls. The regulation trends of these immune transcripts and processes were similar to those in larvae incubated and challenged with LPS at 24 °C  that experienced cold exposure from 30 °C to either 23 °C, 17 °C or 10 °C over 1, 2, or 3 days, respectively, the expression profile of approximately 3,400 unique genes was affected35. To evaluate the potential effect of temperature decrease, a comparison was performed between control larvae (without LPS treatment) that experienced a temperature decrease from 32 °C to 24 °C and those kept at a constant 24 °C (Supplementary Table cold inducible RNA binding protein b (cirbpb) (1.6-fold) and one temperature responsive process (“response to temperature stimulus”). In particular, the nuclear receptor nuclear receptor subfamily 1, group d, member 1 (nr1d1) transcripts, which code for proteins involved in both circadian and thermogenic pathways through mediation of brown adipose tissue in response to cold exposure36, were up-regulated 3.5-fold. It has been demonstrated that the modulation of physiological metabolism occurs to mitigate the effect of temperature decrease37. As expected, some HSP transcripts (hspb associated protein 1 (hspbap1) and hsp70l) were down-regulated, as well as the antioxidant gene transcripts cytochrome P450, family 24, subfamily A, polypeptide 1 (cyp24a1) and gpx1a. Nonetheless, none of these processes or transcripts were significantly regulated when the LPS treatment was taken into account, indicating that the potential effect from temperature decrease on LPS-stimulated immune response was minimal. Moreover, 10 out of 33 DEGs and all (15 out of 15) GO processes were directly or indirectly related to immunity in larvae incubated at 32 °C and challenged with LPS at 24 °C, suggesting that a more effective immune response may be elicited in larvae from the 32 °C incubation temperature group compared to their counterparts incubated during embryonic development at 24 °C. These results explain the lowest mortality rate (20.5 ± 4.7%) of larvae incubated at 32 °C and challenged with LPS at 24 °C among all the temperature groups  was expressed at higher levels, suggesting a limited activation of the innate immune response by LPS. Nevertheless, the abundance of transcripts of some genes with important roles in inflammatory response was changed. For instance, irg1l transcript levels were up-regulated 6.3-fold. Its homolog gene, Irg1, is inducible by LPS in mouse macrophages, and encodes cis-aconitate decarboxylase to catalyse the production of the antimicrobial itaconate38. IRG1 is also involved in suppressing LPS-mediated sepsis and pro-inflammatory cytokine production in mouse39. The up-regulation of timp2b (4.4-fold) could either activate the pro-inflammatory NF-κB pathway in human melanoma cells, protecting cells from apoptosis40, or exert the anti-inflammatory function by inhibiting NF-κB activity in murine microglial cells, to suppress the production of nitric oxide, TNFα, IL1β, and reactive oxygen species (ROS)41. Transcript levels of the cytokine gene, high mobility group box 1b (hmgb1b), which is involved in pro-inflammatory response42, necrotic cell death43, and sepsis44, were down-regulated 1.5-fold. lect2l was up-regulated 2.7-fold; LECT2 has a neutrophil chemotactic activity specifically in the liver45. The protein encoded by hspd1 (1.7-fold up-regulation) plays a critical role in regeneration and wound healing of both hair cells and caudal fins of zebrafish larvae46. The oxidative stress and antioxidant response of larvae were affected as well, with the induction of hypoxia processes , hypoxia inducible (myoglobin (mb), igfbp1a) and antioxidant genes . In fact, the regulation of ROS and antioxidant activities by LPS has been demonstrated in zebrafish embryos48. Taken together, the limited inflammatory response and the induced hypoxia and oxidative stress could contribute jointly to the high mortality rate (85.9 ± 2.3%) of larvae incubated at 24 °C and challenged with LPS at 32 °C  were enriched, compared to their respective controls Fig. , and non °C Fig. .oxidative stress responsive serine-rich 1 (oser1) and reactive oxygen species modulator 1 (romo1) were up-regulated 1.5-fold. On the other hand, transcripts of several antioxidant genes, including gpx1a, glutathione S-transferase, alpha tandem duplicate 1 (gsta.1), gsto2, glutathione S-transferase pi 1 (gstp1), gstp2, peroxiredoxin 6 (prdx6), and NADPH oxidase organizer 1a (noxo1a) were down-regulated 1.5–2.4 fold, as compared to larvae kept at constant 24 °C. We also noticed the up-regulation of HSP transcripts, such as serpin peptidase inhibitor, clade H, member 1b (serpinh1b), hsp90aa1.1, hsp90aa1.2, crystallin, alpha A (cryaa), DnaJ heat shock protein family member A4 (dnaja4), heat shock cognate 70 (hsc70), and the down-regulation of antifreeze protein type IV (afp4), and cold inducible RNA binding protein a (cirbpa) (Supplementary Table 49. Another report in two-banded seabream (Diplodus vulgaris), white seabream (Diplodus sargus), European seabass (Dicentrarchus labrax) and thinlip grey mullet (Liza ramada) showed that protective mechanisms, including the production of HSPs, and the antioxidant activity of glutathione S-transferase, catalase, and lipid peroxidation, can be enhanced to alleviate the effects from temperature increase and associated oxidative stress50. In our study, there were no significant differences in mortality rates of control larvae when the temperature changed from 24 °C to 32 °C, suggesting a limited effect of the temperature increase per se. Nevertheless, we cannot exclude a possible interaction between challenge temperature and LPS treatment.An increase in water temperature could lead to hypoxic conditions, which further promote the production of ROS, causing oxidative stress and affecting physiological activities. In control zebrafish larvae experiencing a temperature increase from 24 °C to 32 °C, transcripts of 51 but alternative receptors other than TLR4 for LPS signal transduction can exist in teleosts. Some other fish-specific TLRs, such as TLR21 and TLR22, have been proposed as LPS receptor candidates52. However, no TLR genes showed significantly different expression in the present study. Some non-TLR receptors are also known to be involved in LPS signal transduction, such as beta-2 integrins53, scavenger receptor54, and C-type lectin55. GO analyses and InterPro annotation identified some up-regulated transcripts with potentially similar functions in zebrafish larvae exposed to LPS. CD44 molecule a (cd44a) codes for a protein with a C-type lectin-like domain and the genes transmembrane protease, serine 4a (tmprss4a) and tmprss13b encode scavenger receptors. The products of proteoglycan 4b (prg4b) and integrin, alpha V (itgav) genes display scavenger receptor and integrin activities, respectively. Further experimental evidence is needed to support their potential roles in sensing LPS.It has been demonstrated that the regulation of immune signalling pathways in response to LPS is well conserved between teleosts and mammals56, while murine HSP60 was able to bind to its specific receptor on the macrophage surface independent of TLR4, but its subsequent cytokine response was dependent on TLR457. Another study in Chinese hamster (Cricetulus griseus) ovary cells revealed that HSP70 and HSP90 were involved in sensing LPS signal from CD14 and transferring to the downstream receptors58. Our data showed the up-regulation of hspd1 and hsp90b1 in larvae incubated at 24 °C and challenged with LPS at 32 °C, suggesting their possible roles in LPS signalling. Moreover, the expression of anxa2a and its receptor gene s100a10b was up-regulated in all three temperature groups. Annexin has multiple functions, including modulation of reactive oxygen species59 and regulation of the inflammatory response triggered by TLR460. Its new function as a TLR2 ligand was recently reported in mouse61. It is also noteworthy that the transcripts of lye were up-regulated between 2.4- and 3.6-fold in all three temperature groups. Lye is constitutively expressed in immune and epithelial cells62, with pleiotropic functions in extracellular signal transduction, phagocyte activation, and inflammatory response63. The direct interaction between LPS and these genes should be investigated to ascertain their involvement in LPS recognition and signalling cascade in fish.Heat shock proteins have been implicated in LPS signal transduction. Human HSP60 contains a specific region for LPS bindinganxa2a, s100a10b, and lye), whose transcripts were up-regulated in larvae from all the temperature groups are promising receptor candidates in LPS signal transduction. These results substantially increase our understanding of the thermal plasticity of the innate immunity in zebrafish during their early development and have broader implications for fisheries and aquaculture in the context of global climate change.In summary, we demonstrated that both embryonic incubation and challenge temperatures affected the innate immune response to LPS in zebrafish larvae Fig. . The lowAll animal procedures were conducted in compliance with the guidelines provided by the Norwegian Animal Research Authority (FOTS ID 8387) and approved by the Nord University (Norway) ethics committee.Zebrafish (AB strain) were maintained in a recirculating system  under standard husbandry conditions, including a stable temperature of 28 ± 0.5 °C and photoperiod of 12 h light: 12 h dark. Adult fish were fed SDS Small Granular diet  for maintenance, and SDS 400 for conditioning prior to spawning.64. One-third of the medium was changed daily and larvae were not fed throughout the experiment. When 75% reached the first-feeding stage . LPS was prepared as a stock solution at 10 mg/L in standard phosphate-buffered saline . The remaining individuals (controls) were immersed in distilled water containing the same dose of phosphate-buffered saline (200 µL). Larvae were kept in open 500 mL beakers immersed in fish tanks at 24 °C, 28 °C or 32 °C  at a density of 149 ± 32 larva per 200 mL . The ANOVA assumptions of normality and equal variance of the data were verified by Kolmogorov-Smirnov test and by Levene’s test, respectively. Statistical significance was determined at p-value < 0.05. Mortality rates were presented as mean ± standard deviation (s.d.). The experiment was repeated three times using randomly selected broodstock fish from the same laboratory population.The experimental design is illustrated in Fig. At 24 h post LPS challenge, three LPS challenge replicates and three control replicates from each of the three incubation × challenge temperature groups  were chosen for further transcriptomic analyses. All three incubation × challenge temperature groups showed significantly different mortality rates following LPS challenge Fig. . Larvae Samples were homogenized at 6,500 rpm for 2 × 20 s in a Precellys 24 homogenizer . Total RNA was extracted from whole larvae following the QIAzol protocol . RNA concentration, purity and quality were determined using the NanoDrop 1000  and the TapeStation 2200 .TruSeq libraries were prepared from total RNA according to the manufacturer’s protocol . After purification with oligo-dT beads, mRNAs were washed and fragmented into an average length of 508–541 base pairs. The first strand of complementary DNA was synthesized with random hexamer primers , while the second strand was synthesized by Second Strand Master Mix . All 18 libraries were barcoded and normalized with the KAPA library quantification kit . The pooled libraries were then denatured according to the NextSeq System Denature and Dilute Libraries Guide  and loaded at 11 pM on a NextSeq 500 reagent cartridge  for 150 cycle, paired-end sequencing at the Nord University genomics platform (Norway).https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adapter removal using cutadapt (http://cutadapt.readthedocs.io/en/stable/guide.html) with the parameters: -q 30,25 --quality-base = 33 --trim-n -m 20. Clean reads were mapped to the zebrafish transcriptome (GRCz10.86.chr.gtf) and genome (GRCz10.dna.toplevel.fa) from Ensembl (http://www.ensembl.org) using TopHat265 with parameters: -r 100 --mate-std-dev 100. Mapped reads were counted against the reference transcriptome (GRCz10.86.chr.gtf) by HTSeq-count (http://htseq.readthedocs.io/en/release_0.9.1/), and further used for differential expression analyses by DESeq266 to compare LPS-treated versus control groups. DEGs were determined by DESeq2 with an adjusted p-value < 0.05 (Benjamin-Hochberg method). DEGs with a |fold change| ≥ 1.5 were subjected to Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses by clusterProfiler67. Graphical representation was achieved using ggplot2 and pheatmap R packages.Raw RNA-seq data were converted to FASTQ format with bcl2fastq2 , followed by quality control using FastQC (Supplementary Tables S1-S7 and Figures S1-S2


Alkaliphilus metalliredigens strain QYMF is an anaerobic, alkaliphilic, and metal-reducing bacterium associated with phylum Firmicutes. QYMF was isolated from alkaline borax leachate ponds. The genome sequence will help elucidate the role of metal-reducing microorganisms under alkaline environments, a capability that is not commonly observed in metal respiring-microorganisms. Alkaliphilus metalliredigens QYMF is a strict anaerobic bacterium isolated from alkaline borax leachate ponds with concentrations of sodium and boron that ranged from 0.04 to 0.53 M and 0.19 and 0.28 M, respectively, at U. S. Borax Company . QYMF is capable of utilizing Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, and Cr(VI) as electron acceptors during growth with yeast extract or lactate as electron donors. Optimum growth conditions for QYMF with the metals listed above were observed at pH 9.6, 35°C, and a NaCl concentration of 20 g/L in the presence of 2 g/L of borate , A. transvaalensis , and A. peptidifermentans . Comparison of SSU rRNA gene shows that QYMF has 93 to 96% nucleotide identity to these sequenced Alkaliphilus species. The genome sequence of QYMF contains 5,016 identified putative genes, of which 4,801 are protein coding genes with functions predicted for 3,130 genes, a total of 31 rRNA genes (11 are 5S rRNA gene and 10 are 16S and 23S rRNA gene each), 106 tRNA genes, and 78 other RNA genes.The genome sequence was determined with the Sanger sequencing method by the U.S. DOE Joint Genome Institute (JGI). The completed genome was curated and annotated as previously described –9. Putat–ars operons that encodes arsenite efflux permeases (Acr3), whose mechanism of transport was characterized by Fu et al. (aroA gene of QYMF, which encodes for the enzyme 5-enopyruvylshikimate-3-phosphate synthase, was shown to have the potential in developing glyphosate-resistant crops (Saccharomyces cerevisiae to rice grains (Although growth of QYMF with arsenic has not been characterized, the leachate ponds from where QYMF was isolated contained As concentrations of approximately 1.7 mM. Genome analysis of QYMF indicates the presence of genes encoding arsenical resistance proteins and two novel u et al. , 11. Recnt crops . This ime grains . Future A. metalliredigens was deposited at GenBank, National Center for Biotechnology Information, under accession number CP000724.


Proper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.Cga), αT3–1 , and LβT2 , to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.We evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.We found the common mRNA α-subunit of LH and FSH, called Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show the gonadotrope-specific genes  Cga, glycoprotein hormones also known as αGSU) is detectable at E11.5 in the mouse, followed by the nuclear receptor Steroidogenic Factor 1 (Sf1 or Nr5a1), and the gonadotropin-releasing hormone receptor (Gnrhr). Finally, the gonadotropin hormone beta subunit mRNAs emerge at E16.5 for Lhb and E17.5 for Fshb . In. InCga. Cga, Gnrhr, Lhb, and Fshb in these model cell lines and demonstrate that these promoters are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show exciting new evidence of gonadotrope-specific chromatin changes taking place during development using immortalized model gonadotrope cell lines. Although the cell lines utilized remain an in vitro system, representative of developing gonadotrope cell lines, we show a progressive opening of the gonadotrope-specific gene promoters,