Here we show a unique hybrid chemical doping method that results in high transmittance uniformity in a layered graphene-polymer nanocomposite with suppressed defect-induced carrier scattering. This layer-by-layer hybrid chemical doping results in low Rs (15 Ω/sq at >90% transmittance) and 3.6% transmittance uniformity (300–1000 nm) compared with graphene (17%), polymer (8%) and ITO (46%) films. The weak localization effect in our nanocomposite was reduced to 0.5%, compared with pristine (4.25%) and doped graphene films (1.2%). Furthermore, negligible Rs change (1.2 times compared to 12.6 × 103 times in ITO) and nearly unaltered transmittance spectra were observed up to 24 GPa of applied stress highlighting mechanical flexibility of the nanocomposite film.A traditional transparent conducting film (TCF) such as indium tin oxide (ITO) exhibits poor mechanical flexibility and inconsistent transmittance throughout the UV-VIS-NIR spectrum. Recent TCFs like graphene films exhibit high sheet resistance (R Numerous exotic behaviors such as the anomalous quantum Hall effect, the Klein paradox, coherent transport, extraordinarily high mobility, optical modulation, and persistent photoconductance are observed in graphene10. High quality, flexible transparent conducting films (FTCF) are among the crucial building blocks for optoelectronic technologies ranging from flexible touchscreens to rollable displays, flexible light-emitting devices, and flexible energy conversion applications. Traditional transparent conducting films (TCF) such as indium tin oxide (ITO) are not suitable for flexible electronics technology upgrades due to their poor mechanical flexibility13 and inconsistent transmittance near the UV-VIS-NIR region4. Several nanomaterials such as nanowires17, conducting polymers19, metal-polymer layered hybrids20, and carbon nanotubes21 have shown some potential to replace ITO. Nevertheless, there are numerous challenges in these materials, for example, CNTs and metallic nanowires suffer from long-term instability, uneven surface morphology, and high contact resistance. Conducting polymers and metal hybrids are inadequate due to their large sheet resistance (Rs).Monolayers of carbon atoms packed into a honeycomb lattice in two-dimensional (2D) space (graphene) demonstrate extraordinary electronic and optoelectronic properties22. Mechanically exfoliated suspended graphene flake exhibits remarkable mobility (2.3 × 105 cm2/V s)10. However carrier mobility plummets (2 × 103−2 × 104 cm2/Vs) significantly due to the substrate-induced scattering upon transferring graphene layers onto external substrates23. Carrier scattering can be reduced in graphene (mobility of 8 × 104 cm2/Vs) by transferring it on top of a 2D hexagonal boron nitride (h-BN) or epitaxial ferroelectric gate oxides materials (1.4 × 105 cm2/Vs) as demonstrated previously24. However, most of these results were obtained in nanometer scale devices at low temperatures. Unfortunately, large-scale graphene synthesized by different methods25 results in crystalline graphene domains separated by grain boundaries, carbon vacancies, hexagonal lattice defects, and a randomly distributed structural ripple over the surface. These defects, ripples, and grain boundaries, along with substrate induced interfacial trap charges, react as carrier scattering centers and drastically reduce carrier mobility. Numerous adaptations have been made to overcome these challenges for flexible transparent conducting films, such as the use of large area graphene synthesized by chemical vapor deposition (CVD)26, interconnected graphene networks27, reduced graphene oxide films29, graphene Langmuir–Blodgett hybrid films30 and graphene-polymer nanocomposites33. However, a comparable TCF performance to that of ITO (Rs 10 to 20 Ω/sq at >90% transmittance) has not been achieved to date due to intrinsic defects, ripples, and grain boundary effects in graphene-based nano-carbon materials.Graphene is ideally suited as a transparent conduction layer due to extraordinarily high carrier mobility in atomically thin 2D space, transmittance up to 97.6%, and exceptional mechanical flexiblity2 substrates exhibits sheet resistance in the order of few hundred ohms with very high transmittance (around 97%)26. Sheet resistance can be reduced to ~30 Ω/sq (at 90% transmittance) by using chemical doping methods34. However, this value is nearly double that of conventional ITO performance as described above. A novel, comprehensive approach is required that incorporates high quality defect-free large-area graphene, improved graphene stacking, more efficient chemical doping, and non-graphene materials integration to overcome the TCF performance bottleneck.CVD-grown single layer graphene transferred onto SiOs in graphene-polymer nanocomposite that exhibits superior flexibility and transmittance properties. A unique chemical doping method was employed to achieve resistance quenching in the graphene-polymer nanocomposite film while maintaining high optical transparency (15 Ω/sq Rs at >90% transmittance). Furthermore, nominal changes in the nanocomposite film transmittance and Rs (1.2 times compared to 12.6 × 103 times change in the ITO) were obtained under severe applied mechanical stress up to 24 GPa. These outcomes highlight the strong application potential of novel hybrid nanocomposite materials for highly conductive, transparent and flexible electronics technologies.In this investigation, we demonstrate ITO comparable Rs at >90% transmittance. A 200 nm thick ITO thin film typically shows a transmittance spectrum as demonstrated in Fig. s of graphene and PEDOT:PSS thin films (with different thicknesses) transferred on top of polyethylene terephthalate (PET) substrate were compared with ITO performance in Fig. s however, decreases with increasing thickness, from 353 Ω/sq to 172 Ω/sq respectively  of pristine graphene sulfonimide (TFSA) have been previously investigated for graphene films34. Here we have compared these doping methods for graphene-polymer nanocomposites and optimized an effective strategy to decrease film Rs while maintaining the 90% transmittance for practical applications. A 30 mM dopant concentration was sufficient to achieve Rs saturation in previous investigations38. Therefore, 30 mM dopant (AuCl3 and TFSA) concentrations in nitromethane solution were spin coated (4000 rpm for 2 min) on top of the film, followed by hot plate annealing at 80 °C for 10 minutes. Figure 3, AuCl3, and TFSA doped single layered graphene films show similar values near 550 nm  which could be due to the formation of a gold nanocluster from Au0 and Au3+ ions38. These nanoclusters significantly decreased transmittance in AuCl3 doped single layered graphene coated with 46 nm polymer film throughout UV-VIS-NIR region. Figure 34. Two and three layers of stacked graphene films resulted in a reduction in transmittance of around 94.8% and 92.4% respectively as expected. In comparison, 46 nm, 241 nm, and 1260 nm thick PEDOT:PSS films show transmittance in the range of 96.7%, 91.7%, and 81.3% respectively at 550 nm  both exhibit irregular transmittance spectra over the spectral window between 300 nm to 1000 nm see Fig. . Graphen3, AuCl3, and TFSA doping methods respectively. It should be noted that the single layered graphene doped by HNO3 results in marginally higher transmittance of 98.1% compared to pristine graphene; this could be due to the etching and defect generated by the strong HNO3 acid solution34. In comparison, AuCl3 doping does not increase defects in graphene however, resulted in aggregation of gold Au0 and Au3+ ions on the film surface38. These aggregated nanoparticles scatter with incident light and reduce film transmittance. Different polymer film thicknesses were used on top of the chemically doped graphene films to further decrease Rs. Different thicknesses of the graphene-polymer nanocomposite in the graphene/AuCl3/graphene/AuCl3/polymer structures . TFSA doped nanocomposite with 90.6% transmittance results in Rs around 69 Ω/sq. Rs can be further reduced up to 37 Ω/sq by increasing nanocomposite thickness; however, transmittance reduces to less than 90% (88%). It is evident from the Fig. s without compromising much of the optical transmittance in nanocomposite films compared to the AuCl3 doping method. Therefore, a novel hybrid chemical doping method was developed, incorporating TFSA doping  and AuCl3 doping  respectively. This unique doping strategy enabled us to achieve Rs around 15 Ω/sq with 90.7% transmittance in the graphene/TFSA/graphene/AuCl3/TFSA/polymer/TFSA nanocomposite structure (“mixed doping strategy” legend). Increasing the nanocomposite film thickness from this point could decrease Rs further  due to less nanocluster formation from Au0 and Au3+ ions, however Rs was not considerably lowered (35 Ω/sq). Moreover, the transmittance variation in this structure was obtained around 8.4%. TFSA doping was introduced between graphene layers while keeping other layers identical (in the graphene/TFSA/graphene/AuCl3/TFSA/polymer/TFSA nanocomposite structure) in order to decrease the sheet resistance further without compromising transmittance below 90% , high transmittance (>90%), low transmittance variations (<4%), and transmittance maximum/minimum in the visible region demonstrate the advantages of this nanocomposite structure. Two time use of AuCl3 doping in the graphene/AuCl3/graphene/AuCl3/TFSA/polymer/TFSA nanocomposite structure resulted in comparable Rs (17.8 Ω/sq) however, transmittance reduced below 90% (89.1%). Three-layered graphene in the nanocomposite (graphene/TFSA/graphene/AuCl3/TFSA/graphene/AuCl3/polymer/TFSA) resulted in significant reduction in film transmittance up to 86% and resulted in similar Rs (14 Ω/sq). An optical micrograph of the chemically doped graphene-polymer nanocomposite on PET substrate is shown in Fig. The transmittance spectra of hybrid doping methods are compared in Fig. see Fig. . EncouraITO Fig. . This reITO Fig. . Low Rs 3 doping as described earlier. TFSA dopant solution consists of fluorine, nitrogen, and sulfur atoms38, whereas gold ions were observed previously in AuCl3 dopants34. Therefore, F1s, N1s, C1s, S2p, and Au4f XPS spectra were compared from higher to lower binding energies respectively  peaks were observed in DGPN and DG samples as shown in Fig. 2) peaks verified TFSA doping in DGPN and DG samples both for N1s and C1s spectra near 399.4 eV and 285.3 eV respectively. Moreover, nitrogen related bonds such as N-O (402 eV), -NH- (400.8 eV), and C = NH+ (401.6 eV) were observed in the N1s deconvoluted spectrum and confirmed TFSA doping in DGPN & DG samples respectively ; S2P3/2 (163.8 eV) peaks form PEDOT; and S2P1/2 (168.6 eV), S2P3/2 (167.8 eV) peaks form PSS, similar to previous investigations40. Sulfur related peaks from TFSA doping were also found in the DG sample, as shown in row 2 of Fig. 1/2 at 164.9 eV, and S2P3/2 at 163.8 eV). All of this evidence suggests strong TFSA doping in DGPN and DG samples compared to pristine graphene, where no fluorine, nitrogen, and sulfur-related peaks were observed  and Au3+ (near 82.9 eV) related peaks in the Au 4f XPS spectrum, similar to the previous investigations34. Although AuCl3 doping was used in DGPN sample however, the doping layer was covered with two-fold TFSA dopant layers and the 57 nm thick polymer layer; this could be the reason for the absence of gold-related peaks in Au 4f XPS spectrum. It should be noted that all of the samples showed strong C-C peaks (near 284 eV) from graphene however, PG samples showed conventional Sp3 carbon (nearly 284.4 eV), C-O-C (285.6 eV), and C-O=C (288.6 eV) related peaks in pristine graphene similar to the previous investigations39  and optoelectronic nanocomposite properties as demonstrated in this study.Charge transfer during the chemical doping process can be monitored using X-ray photoelectron spectroscopy (XPS). Figure see Fig. . Furthersee Fig.  of used 2/Si substrate  in the temperature range from 300 K to 2 K in the absence of a magnetic field. At 300 K, pristine graphene (PG) devices showed Rxx close to 570 Ω, whereas Rxx reduced to 200 Ω in DG due to chemical doping. Further resistance quenching was observed (102 Ω) in nanocomposite (DGPN), similar to the observed Rs trends. At low temperature (2 K), the Rxx value increased rapidly in PG (from 570 Ω to 647 Ω) and increased exponentially in DGPN (from 102 Ω to 270 Ω). In contrast, the value of Rxx dropped in DG (from 200 Ω to 168 Ω). These low temperature rapid Rxx increases in DGPN and PG could be attributed to mesoscopic resistance fluctuation effects near the neutrality point in graphene41. Furthermore, the mesoscopic resistance fluctuation effects are related to the increasing phase coherence length known as the weak localization effect in graphene42. This weak localization effect can be further confirmed by the sharp Rxx increase near zero magnetic field (B = 0). Figure xx with an applied magnetic field in the DG sample in the temperature range from 2 K to 90 K. The weak localization peak observed in Rxx near B = 0 resulted from electronic coherent backscattering effects similar to previous reports42. All of the samples  show weak localization peaks in Rxx near B = 0 at 2 K temperature  was similar to the film resistance as discussed in Fig. xx was observed in PG (~637 Ω at B = 0), followed by DGPN (~402 Ω at B = 0), and DG (168 Ω at B = 0). One of the intriguing facts to be noted here is that the carrier coherent backscattering caused by the weak localization effect was significantly reduced in the doped graphene-polymer nanocomposite (DGPN) sample. This was manifested by the reduction of weak localization peak height (at B = 0) in DGPN: up to 0.5% compared to 4.25% in PG and 1.2% in DG samples. These results strongly suggest that the reduction in carrier scattering and consequent resistance quenching in DGPN could be due to the reduction of grain boundaries, carbon vacancies, lattice defects and structural ripple-related carrier scattering processes.It is evident from the above investigations that graphene-polymer layered nanocomposites exhibit improved optoelectronic properties compared to its close counterparts such as graphene, polymer, and ITO films for FTCF applications. In addition, low-temperature transport measurements were conducted by pattering DGPN, DG, and PG samples in Hall-bar geometry on SiOsee Fig.  however,−5 S . In comparison, the chemically doped graphene sample exhibits surface conductance one order of magnitude higher in the range of 1.79 × 10−4 S  under applied compressive stress up to 23 GPa in a 100 nm thick ITO film and DGPN on a flexible PET substrate. Initially, ITO and DGPN films demonstrated approximately 10 and 15 Ω/sq sheet resistances respectively. The change in ITO sheet resistance remained small (1.07 times) up to very low applied compressive stress (6 GPa) whereas it increased sharply up to 12.6 × 103 times at 23 GPa. In this stage the ITO film demonstrated a very high Rs value of 4.2 GΩ/sq, suggestive of significant damage to the film. Previous investigations observed similar resistance increments in ITO film under applied stress  while maintaining high optical transparency (>90% transmittance at 550 nm) in nanocomposite films (comparable to ITO and lowest so far among carbon-based FTCF). Moreover, the nanocomposite shows tremendous transmittance uniformity (3.6%) in the VIS-NIR range (300 nm to 1000 nm) compared to graphene (17% in three-layered graphene film), polymer (8% in 100 nm thick) and ITO (46% in 200 nm thick) films. Furthermore, the carrier coherent backscattering and consequent resistance quenching in the nanocomposite were significantly reduced : up to 0.5% compared to 4.25% in pristine graphene and 1.2% in doped graphene films. Moreover, the nanocomposite film shows a nearly identical transmittance spectrum and negligible resistance change (1.2 times) under 24 GPa applied stress compared to 12.6 × 103 dopant was dispersed in nitromethane (Sigma Aldrich) and the supernatant was used as a spin coating solution  to implement chemical doping. High conductive PEDOT:PSS polymer solution (HIL-1005 from Sigma Aldrich) was spin coated on top of the doped graphene layers for graphene-polymer nanocomposite synthesis. Spin coating speed (from 1000 rpm to 6000 rpm for 60 sec) was controlled to achieve desired PEDOT:PSS thickness (from 46 nm to 1260 nm thick films respectively). Each nanocomposite layer was annealed at 80 °C for 10 min under atmospheric conditions during the synthesis process. A 30 watt oxygen plasma was used for 20 min for Hall-bar device patterning of the nanocomposite layers. 5 nm Cr and 100 nm Au thin films were deposited using e-beam metal evaporation process for device contacts. The sheet resistance of the fabricated samples was measured by four probe Van-Der-Pauw technique using a semiconductor characterization system (Keithley 4200). Commercially available ITO coated glass was used for transmittance and sheet resistance comparisons. A 100 nm thin ITO film was deposited on PET substrate for compressive stress comparison using RF sputtering system. AFM measurements were conducted using an SPA 300 HV, Seiko Instrument Inc. system. Field emission scanning electron micrographs were obtained from a JEOL JSM-6700F FE-SEM system. Transmittance spectra were obtained by using a UV–Vis spectroscopy system (Ocean Optics).Single layered graphene was synthesized using a low-pressure chemical vapor deposition (LP-CVD) system on 100 µm thick Cu foil. First, Cu foil was annealed for 5 hrs at 1020 °C under a 145 sccm argon and 29 sccm hydrogen gas mixture followed by chemical polishing using Cu etchant solution . Graphene growth was conducted at 1000 °C for 30 min under 500 mTorr with a 113 sccm methane and 12 sccm hydrogen gas mixture. The graphene layer was transferred onto the target substrate using the polymethyl methacrylate (PMMA) thin film and deionized water method. Commercially available PMMA (Sigma Aldrich) solution was spin coated on top of the graphene/copper foil with 700 rpm for 7 seconds followed by 1500 rpm for 60 seconds. This was floated on top of the commercially available Cu etchant solution (CE100) for 30 min and deionized water was used to wash etchant residues. Subsequently, the Gr/PMMA films were transferred to the target substrates and acetone was used to remove PMMA layers. 30 mM TFSA and AuClSupplementary Information


PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma , aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma  and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3′-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated  Epstein-Barr virus (EBV) is one of the most prevalent human viruses. The interplay between EBV replication, latency, and immune control can be disrupted, evoking prolonged proliferation of EBV-infected lymphocytes and their malignant transformation , 2. SincPD-L1 and/or PD-L2, suggesting a close link between PD-L1/PD-L2 genetic alterations and the therapeutic response to these agents , were enrolled in this study according to the protocols approved by the Institutional Review Boards  and the CNACS algorithm, respectively, as previously described [P-value >0.1; (ii) with <4 supporting reads in tumor; or (iii) present in any of control samples. SV breakpoints were visually inspected using Integrative Genomics Viewer (IGV). Candidate focal CNAs (< 20 Mb in length) were also manually reviewed and further filtered by removing those with <3 probes.SVs and focal copy number alterations (CNAs) affecting PD-1 ligands were explored using targeted-capture sequencing with a custom SureSelect library  designed for capturing the entire sequences of the escribed , 17, 18.For the detection of EBV sequence, after sequencing reads were mapped to the EBV genome (NC_007605), the number of the EBV-aligned reads in proper pairs were enumerated and divided by the number of total reads uniquely mapped to the human reference genome (GRCh37). Then, the obtained ratio was evaluated for the cut-off value of 0.00015%, which was determined so that all EBV-positive cases with DLBCL and extranodal NK/T-cell lymphoma (ENKTL) confirmed by in situ hybridization (ISH) for EBV-encoded small RNA (EBER), LMP1 immunohistochemistry (IHC), or Southern blot were included, while minimizing EBV-negative cases. RNA sequencing (RNA-seq) data from the Cancer Genome Atlas (TCGA) cohort were analyzed in a similar manner, where the detection of EBV reads was considered as positive. Southern blot for EBV genome was performed according to a standard procedure.P-value ≤0.01; (ii) ≥4 variant reads in tumor; and (iii) allele frequency in tumor ≥0.025 were adopted and filtered by excluding (i) synonymous single nucleotide variants (SNVs); (ii) known variants listed in the 1000 Genomes Project (October 2014 release), NCBI dbSNP build 131, National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) 6500, the Human Genome Variation Database (version 2.0), or our in-house single nucleotide polymorphism (SNP) database, unless they were listed in the COSMIC database (v70). SVs and focal CNAs were analyzed in the same manner as described above.Whole-exome sequencing data for ENKTL (accession number SRP057085) were obtained from the National Center for Biotechnology Information Sequence Read Archive and applied for the detection of somatic alterations using the Genomon pipeline, as previously described . CandidaTargeted-capture sequencing was performed using a custom SureSelect library (Agilent Technologies) for 140 lymphoma-associated genes  were downloaded from the TCGA data portal (https://tcga-data.nci.nih.gov) for DLBCL and stomach adenocarcinoma samples.As previously reported, we analyzed 10,210 TCGA samples from 33 cancer types, for which RNA-seq data were available from the Cancer Genomic Hub (https://cghub.ucsc.edu), to search for 3′-UTR-disrupted nscripts . To evalIHC for PD-L1 was performed on formalin-fixed paraffin-embedded tissue sections using antibodies directed against the N-terminal  and C-terminal  domains of PD-L1. IHC for Ki-67 was performed using a mouse monoclonal antibody . The antigen–antibody complexes were visualized with Histofine Simple Stain MAX PO . Some sections were double-stained with PD-L1 (E1J2J) and CD3, using a mouse monoclonal antibody  with PolyView IHC reagent   and observed with a fluorescence microscope BX51 .FISH analyses were performed on unstained slides with bacterial artificial chromosome (BAC) clone-derived DNA probes for the 5′- and 3′-parts of PD-L1 and PD-L2 3′-UTR disruptions, as previously described [PD-L1 and PD-L2 3′-UTR single guide RNA (sgRNA) targeted sites were designed manually and checked in silico. The pSpCas9(BB)-2A-GFP (pX458) vector expressing Cas9 (Addgene plasmid 48138) was digested with BbsI and ligated to the annealed sgRNA oligonucleotides. HEK293T (obtained from the RIKEN Cell Bank) and T2  cells were transfected with indicated vectors using X-tremeGENE 9 DNA Transfection Reagent  and the Neon transfection system , respectively. To validate CRISPR/Cas9-mediated disruption of PD-L2 3′-UTR, genomic PCR flanking the breakpoint region was performed. The sgRNA and primer sequences are listed in Supplemental Tables CRISPR-mediated gene editing was used to generate escribed . BrieflyTo detect surface PD-L1 or PD-L2 expression, cells were stained with allophycocyanin (APC)-conjugated anti-CD274 (29E.2A3) and anti-CD273 (24F.10C12) antibodies , and analyzed on FACS LSR Fortessa . The data analyses were performed with FlowJo software .t-test with a Welch’s correction. The molecular features between EBV-negative and -positive tumors were compared using the Fisher’s exact test, Cochran–Armitage trend test, or Brunner–Munzel test. P-values were considered statistically significant at <0.05. In box plots, the center line and lower and upper hinges correspond to the median, and the first and third quartiles (25 and 75 percentiles), respectively. The upper and lower whiskers extend from the upper and lower hinges to the largest or smallest values no further than 1.5× inter-quartile range from the hinges.Statistical analyses were performed with R3.4.2 software . For functional assays, statistical significance was assessed by Student’s two-tailed https://github.com/Genomon-Project/.The code for the Genomon pipeline is available at PD-L1/PD-L2-involving genetic abnormalities were interrogated using targeted-capture sequencing in a total of 384 samples from different lymphoma subtypes, including DLBCL (n = 75), follicular lymphoma , mucosa-associated lymphoid tissue lymphoma , mantle cell lymphoma , PMBCL (n = 10), PTCL-not otherwise specified , and EBV-positive T-cell and NK-cell proliferations (n = 94)  and 6 (2%) samples, respectively, while focal gains or amplifications involving these genes were found in 30 (8%) samples , underscoring the importance of the deregulated expression of these PD-1 ligands in clonal selection and expansion of these tumors.FL, n = 4, mucosa-lymphoma  (1 of 35lymphoma  (1 of 23lymphoma  (1 of 13lymphoma  (3 of 6)PD-L1-involving SVs, which were detected in both B- and T/NK-cell lymphomas, those involving PD-L2 were found exclusively in B-cell lymphomas , including ENKTL (19 of 81 samples), aggressive NK-cell leukemia , and systemic EBV-positive T-cell lymphoproliferative disorder   of ENKTL cases  Fig. . Among t lesions . Additiomalities . In accomalities , the mosP53 Fig. . Moreoveses Fig. , even thses Fig. , suggestPD-L1 SVs was previously reported in a unique form of HTLV-1-mediated PTCL, ATL [PD-L1/PD-L2-involving abnormalities among EBV-associated lymphomas and stomach adenocarcinoma, another EBV-related cancer. Among 75 DLBCL cases in our cohort, 27 cases (DLBCL49-75) were positive for EBV by Southern blot and/or targeted-capture sequencing, and thus considered as EBV-positive DLBCL  than in EBV-negative DLBCL (1 of 48) (P < 0.05)   Fig. . The obs07) Fig. . Similar07) Fig. , as well07) Fig. . These rTET2 and DNMT3A mutations, while showing very low frequencies of CD79B and MYD88 mutations as well as CDKN2A loss-of-function alterations  -Rb pathway, which can explain the absence of CD79B, MYD88, and CDKN2A alterations in EBV-positive DLBCL, although EBV-positive DLBCL is characterized by an activated B-cell (ABC) immunophenotype and prominent NF-κB activation [TET2 and DNMT3A mutations in EBV-positive DLBCL indicate the possible involvement of deregulated DNA methylation and demethylation processes in this disease [PD-L1 3′-UTR truncation, which is associated with Ki-67 positivity in ENKTL, TET2 disruption is also reported to cause aberrant proliferation in an antigen receptor-dependent manner [PD-L1/PD-L2 and FAS alterations suggests different roles of these molecules in escape from T-cell mediated immune surveillance against virally infected cells.Another finding of significant importance is a distinct pattern of somatic alterations in EBV-positive DLBCL, providing new insight into the genetic basis of virus-induced lymphomas. EBV gene products, such as LMP1, have been reported to strongly activate NF-κB pathway in a ligand-independent manner, and hinder p16 (encoded by tivation , 35. Fre disease . In addit manner . These aPD-L1/PD-L2 genetic alterations in NHLs.Taken together, our findings contribute to understand the pathogenesis of EBV-associated lymphomas and exploit a new diagnostic strategy to identify patients who are likely to respond to PD-1/PD-L1 blockade therapy by detection of EBV and/or Supplemental dataSupplemental tables


The tree-like graphene dendrites with well-controlled morphology can be directly grown on both the Si and the SiO2 surfaces of the substrates by using methane and hydrogen as precursors. The graphene dendrites on SiO2/Si substrates can be directly used in the fabrication of the electronic device. The conductivity and the Hall mobility of graphene dendrites are ~ 286 Scm−1 and ~ 574 cm2(Vs)−1, respectively. Young’s modulus of graphene dendrites is up to 2.26 GPa. The developed method avoids the need for a metal substrate and is scalable and compatible with the existing semiconductor technology, making graphene dendrites be very promising in nanoelectronic applications.The long-standing interest in graphene has recently brought graphene-derived materials including graphene hydrogel, graphene fiber and graphene paper into sharp focus. These graphene-derived materials show outstanding properties in mechanics and physics. In this paper, for the first time, we demonstrate the novel synthesis of graphene dendrites on SiO Because of the excellent physical and chemical properties, graphene has attracted tremendous attention since it was found by mechanical cleavage of highly ordered pyrolytic graphite (HOPG) in 2004 [Graphene is a kind of two-dimensional 2D) crystal material with sp in 2004 . To dateD crystal in 2004 . At the  in 2004 –14.Graphene dendrite is a new type of graphene-derived material, which has a tree-like crystal structure. Generally, the dendrite structure can be formed in a non-equilibrium state during the crystal growth process. To date, a variety of materials such as metal, alloy and metal oxide have been demonstrated to form the dendrite structures –18. Thes−1 and was used as the electrode in supercapacitors [2/Si or quartz) for fabricating electronic devices [Though the dendrites are the ubiquitous crystal form in freezing alloys and super-cooled melts, the graphene oxide dendrite and graphene dendrite are not synthesized until 2015 , 23. The devices –26.2/Si substrates by chemical vapor deposition (CVD) using methane and hydrogen as precursors. Without using any catalyst, the tree-like graphene dendrites with high density were directly grown on dielectric substrates. This method does not require the metal substrate, and thus is compatible with the fabrication process of electronic device. The fabricated graphene dendrites show a good conductivity of ~ 286 Scm−1, which is about 6.5 times higher than that synthesized by the electrochemical method. The Hall mobility of the graphene dendrites is up to ~ 574 cm2(Vs)−1 by the Hall effect measurement. Moreover, the graphene dendrites show excellent mechanical properties with Young’s modulus up to 2.26 GPa. The developed technique is compatible with the existing semiconductor technology, thus will be very useful in nanoelectronic applications such as biochemical sensors, nano-electromechanical systems, as well as molecular electronics.In this work, we develop a facile strategy to directly fabricate graphene dendrites on SiO2 layer were used as the substrates. These substrates were sequentially cleaned by acetone, anhydrous ethanol, and deionized water before they were loaded into the CVD reaction system. The detailed experimental apparatus and experimental process are schematically illustrated in Fig. 4 and H2 were introduced into the CVD reaction system, following the vacuum reached as low as 1 × 10−4 mbar. The flow rate of CH4 and H2 was 25 sccm and 15 sccm, respectively. As the temperature was increased to more than 950 °C, CH4 began to decompose and deposit on the substrates thus acting as the carbon source for the graphene dendrites. When the growth process was completed, the samples were rapidly cooled down to room temperature at a rate of ~ 100 °C/min by exposing the cube in air. The gas emissions from the CVD reaction system were burned in a pyrolysis furnace and then discharged into the air.N-type Si wafers with a 300 nm thickness SiO2/Si substrates was characterized utilizing the scanning electron microscopy . The energy dispersive spectroscopy (EDS) was applied for element analysis of graphene dendrites. X-ray photoelectron spectroscopy (XPS) spectra of the samples were measured using a Thermofisher ESCALAB 250 with monochromatized Al Kα X-ray radiation. The as-grown graphene dendrites were evaluated using a confocal Raman spectroscopy (LabRAM HR800) in a backscattering configuration with a 532 nm laser for excitation. The selected area electron diffraction pattern (SAED) and transmission electron microscopy (TEM) images of graphene dendrites were acquired using transmission electron microscopy  with an acceleration voltage at 200 kV. The electrical properties (ISD-VSD characteristics) were measured with a semiconductor parameter analyzer (PDA FS360) coupled with a probe station (PEH-4) at room temperature. The mechanical properties of the graphene dendrites were characterized by Atomic force microscopy  in PeakForce Quantitative Nanomechanical Mapping (PFQNM) mode in air.The morphology of the graphene dendrites on SiO2/Si substrates using a low-pressure CVD system. In this CVD system, the growth parameters of the graphene dendrites can be precisely controlled. Figure 2/Si substrates at a different temperature from 980 °C to 1050 °C. The growth temperature can greatly affect the configuration and the length of the graphene dendrites. As can be seen in Fig. 2 surface of the SiO2/Si substrates at 1050 °C as shown in Fig. 2/Si substrate [The graphene dendrites were synthesized on the SiOubstrate .Fig. 2S2 surface increases from ~ 1 to ~ 8 μm  was also performed to further illustrate the detail structural characterization of the graphene dendrites prepared on the Si and SiOtes Fig. . The pea network . The peaubstrate . Figure l carbon . The weal carbon .Fig. 5X−1), G peak (~ 1580 cm−1), and 2D peak (~ 2680 cm−1) of graphene are observed on both Si and SiO2 surface of the SiO2/Si substrates. The G peak is the characteristic of the carbon sp2 structure and the 2D peak is the second order of zone boundary phonons. The D peak is a defect-related peak reflecting the disorder of graphene [2 surface, the intensity of the G peak is much higher than that of the 2D peak, indicating that graphene dendrites grown on both Si and SiO2 surfaces have a multilayered structure. In addition, as the growth temperature increases from 980 to 1050 °C, the peak intensity ratio of ID/IG decreases from 1.92 to1.81, indicating that the quality of the graphene dendrites was improved with the increase of growth temperature . For the electrical measurement, the samples were placed into a probe station. Two tungsten microprobes (10 μm in diameter) were used as the source and the drain electrodes and placed directly on the SiOS and L are the cross-section and length of the graphene dendrites. The conductivity σ is calculated by the formula:Where the −1.Based on the above analysis, the electrical conductivity of the dendrites is ~ 286 Scm2 surface with van der Pauw structure were carried out at room temperature. Hall resistance (Rxy) as a function of the magnetic field strength has been shown in Fig. RH is calculated by the formula:Electronic transport measurements on the SiOt is the thickness of the sample, and Rxy is the longitudinal resistance. Hall coefficient is − 1.2 cm3/C.Where The resistivity of the graphene dendrites is extracted by the equations:ρ is the resistivity of the sample, Rxx is the longitudinal resistance, f is the van der Pauw factor, and its value is close to 1 thus neglected. The obtained conductivity is ~ 474 S/cm, which is comparable to the value of ~ 286 Scm−1 measured by FET.Where Furthermore, we further calculate the Hall mobility with the following formula:2/Vs, which is much higher than that of the nanocrystalline graphene thin film reported previously [The Hall mobility of graphene dendrites is calculated to be ~ 574 cmeviously .To evaluate the mechanical properties of the graphene dendrites, AFM imaging in PFQNM mode was used to investigate Young’s modulus of the graphene dendrites. The measurements were carried out under ambient conditions at room temperature. Figure In order to obtain Young’s modulus, a fit of the retraction curve was implemented using the Derjaguin-Muller-Toporov (DMT) model .6\documF-Fadh represents the force on the cantilever relative to the adhesion force, R is the tip end radius, and d is the deformation of the sample. The result of the fit is the reduced modulus E*. Young’s modulus can be calculated by the following equationwhere the vs and vtip are Poisson’s ratio of the samples and tip, respectively, the Es and Etip are Young’s modulus the samples and tip, respectively. Sample with a scanning size of 2.0 μm × 2.0 μm was tested. As shown in Fig. where the −1 [4 Sm−1 (250 Scm-1) [−1 [−4 GPa), and is also comparable to that of graphene fibers of 420 MPa (0.42 GPa) [We compare the mechanical and electrical properties of different types of graphene-derived materials as shown in Table −1  and 2.5 0 Scm-1)  and grapm-1) [−1 . For the.42 GPa)  and ~ 77.42 GPa) . In comp2 surfaces on SiO2/Si substrates using a CVD method. The morphology of the graphene dendrites can be regulated by the growth temperature and growth time. Raman spectra and TEM analysis indicated that the graphene dendrites have a multilayer structure. The graphene dendrites show excellent electrical properties with the conductivity of ~ 286 Scm−1 and Hall mobility of ~ 574 cm2(Vs) −1. The graphene dendrites also show a good mechanical performance with Young’s modulus up to 2.26 GPa. The method avoids the need for a complicated and skilled post-growth transfer process and is compatible with current existing semiconductor technology, and thus is very promising in nanoelectronic applications.In this work, we have successfully achieved direct growth of graphene dendrites on both Si and SiO


Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections.The online version of this article (10.1186/s13567-019-0661-z) contains supplementary material, which is available to authorized users. Parasitic nematodes have evolved complex mechanisms to participate in host immunomodulatory and to evade host immune surveillance. During infection, these organisms have not simply warded off the host immune attack; but rather, nematodes interfere with, direct and modulate immune responses in favor of their own survival. Actually, nematodes have evolved immunosuppressive or immunomodulatory molecules to influence the function of cytokines, which can play an important role in protecting the host from infection of pathogens including parasites .Hp-TGM), released by the parasite H. polygyrus, could mimic TGF-β function and induce Foxp3 expression [BmMIF-1 and Bm-MIF-2, derived from Brugia malayi, have parallel functions of human MIF and regulate the host anti-inflammatory response [Recent studies have revealed potential relationships between host cytokines and parasite ESPs , 3. A nopression . Studiesresponse .Haemonchus contortus infection [H. contortus could elicit an unequivocal Th2 response, exemplified by the upregulation of IL4 mRNA transcription that could be observed in the abomasum [Cytokine IL4 is a regulator of the adaptive immune response, which plays a critical role in the type 2 immune response against nfection . Early wabomasum . Furtherabomasum .H. contortus excretion and secretion products (HcESPs) inhibited the functions of goat PBMCs in vitro, particularly these molecules significantly inhibited PBMC proliferation. Strikingly, HcESPs at different stages of the life-cycle of H. contortus could bind to goat peripheral blood mononuclear cells (PBMCs) in vivo [H. contortus 14-3-3 isoform 2 (rHcftt-2) protein from HcESPs could decrease the production of IL4 [In the infection, parasitic nematodes could regulate the host immune system by excreting ESPs. In previous studies,  in vivo , 10. Furn of IL4 .HcESPs have an immunomodulatory effect on host immune responses, the functions of several individual ESP components are not yet clear. In particular, whether certain proteins or proteins from HcESPs could antagonize the function of the goat IL4 is unclear. In this study, we reported that a novel 136-aa protein, transthyretin domain containing protein (HcTTR), could bind to goat recombinant IL4 (rIL4) and suppress IL4-induced PBMC proliferation and downregulate the transcription of genes in the IL4-activated JAK/STAT pathway, representing a potential antagonist against goat IL4.Although studies have shown that Local crossbred goats without helminth infection are used for experimental animals, aged 9–12 months, fed daily with sterile feed and water, maintained in individually ventilated cages, and housed at Nanjing Agricultural University Experimental Animal Center.Sprague Dawley (SD) rats (body weight ~ 220 g) were purchased from the Experimental Animal Center of Jiangsu, PR China .Peripheral blood mononuclear cells were acquired from the goat jugular vein using a standard Ficoll-Hypaque  gradient centrifugation method and cultured as previously described , 13. ThrHcESPs were prepared as previously described using a standard procedure [H. contortus were collected from the abomasum of sacrificed goats and maintained in RPMI 1640 medium overnight. Subsequently, the supernatants were harvested, filtered with a 0.22 μm filter and concentrated with a 3-kDa filter . The recombinant protein and prokaryotic expression vector for goat IL4 (GenBank Acc. No. U34273) was constructed and kept in our laboratory . rIL4 promoted the proliferation of PBMCs at 25 μg/mL as described previously [HcESPs and rIL4 were checked by 12% SDS-PAGE, followed by Coomassie blue staining  and rIL4 (IgG-rIL4) were obtained from SD rats as previously described [HcESPs or rIL4 mixed with Freund’s complete adjuvant. After 2 weeks, SD rats were immunized with Freund’s incomplete adjuvant mixed with the corresponding proteins at the same doses for four times at 1-week intervals. The sera containing specific antibodies were collected at 10 days after the last injection.Polyclonal antibodies against escribed . First, HcESPs, 200 μg rIL4 and 600 μg HcESPs were incubated together at 4 °C overnight, then 2 μg rat normal IgG  and 40 μL Protein A/G PLUS-Agarose beads were added and incubated at 4 °C for 30 min. The beads were pelleted by centrifugation at 1000 × g for 5 min at 4 °C, then the precipitate was discarded, and the supernatant was divided into two equal portions: Group A and Group B. Group A was incubated with IgG-rIL4, and Group B was incubated with rat normal IgG overnight at 4 °C. Immune complexes in each group were isolated using 20 μL of protein A/G plus agarose. Immunoprecipitates were collected by centrifugation at 2500 rpm for 5 min at 4 °C. The supernatants were carefully aspirated and discarded, and the pellet was washed 4 times with phosphate-buffered saline (PBS). After the final wash, the pellets were resuspended in 1× SDS loading buffer. The immunoprecipitates obtained from Group A and Group B were used to confirm the interaction between HcESPs and rIL4 in vitro by Western blotting using IgG-HcESPs as the primary antibody.For the Co-IP assay, Protein A/G PLUS-Agarose Immunoprecipitation Reagent was utilized in this study . To obtain the binding proteins of goat IL4 from The immunoprecipitates obtained from Co-IP were sent to Shanghai Applied Protein Technology, Co. Ltd. for in-solution trypsin digestion and LC/MS–MS analysis using Q Exactive . The raw files of the MS test were searched for the corresponding database using Mascot 2.2 software , and finally, the results of the identified proteins were obtained. The bound proteins with unique peptides greater than or equal to 2 were selected from the identified proteins to increase the confidence of the results.LEU2, KanR) (Table H. contortus) were inserted into the prey vector pPR3N  with the N-terminal half of ubiquitin (Nub-G) domain. The primer sequences are given in Table The interactions between binding proteins and IL4 were further validated using a DUAL membrane pairwise interaction kit . Goat IL4 cloned from full-length cDNA (goat PBMCs) were inserted into bait vector pDHB1 . Transformed colonies were selected in SD-LW medium and incubated for the growth of positive transformants. Several independent positive transformants were selected and recultured in SD-LW broth at 30 °C until the OD546 of the culture reached 1.0. Fifty microliters of each diluted culture  was applied to SD-LW and SD-LHAW selection plates and incubated at 30 °C for 2–3 days for protein–protein interaction assays. The plasmid pDHB1-IL4 co-transformed with the control plasmids pOstI-NubI and pPR3-N into yeast were set as controls.S-transferase domain containing protein (HcGST); transthyretin domain containing protein (HcTTR); calponin actin-binding domain containing protein (HcCab), the genes were amplified and cloned into plasmid vector pET-32a for transfection of BL-21 (DE3) competent cells. The primer sequences for PCR amplification are shown in Table ® Resin Chromatography kit  as previously described, and the concentration of proteins was tested by the Bradford method [E. coli was purified using the same method. Endotoxins were removed from the recombinant proteins using Detoxi-Gel Affinity Pak Prepacked columns . The purified proteins were stored at −70 °C until further usage.For each candidate, including glutathione d method . The cel6 cells/mL) with no treatment served as a blank group. Cells exposed to empty pET-32a protein were set as a negative control group, and cells treated with rIL4 (25 μg/mL) were set as a positive control group. PBMCs treated with a serial concentration of candidate proteins  were used as a control. In addition to the above control group, PBMCs exposed to rIL4 (25 μg/mL) together with candidate proteins  were set as the experimental group, followed by incubation at 37 °C and 5% CO2 for 72 h under dark conditions. Then, 10 μL of CCK-8 solution was added to each well of a 96-well plate with cells. After incubation for 2 h, the absorbance values at 450 nm (OD450) were measured using a microplate reader . The cell proliferation index was calculated by the following formula: OD450 treatment groups/OD450 control. OD450 in negative controls was set as 100%. Three independent experiments were performed in this test with three technical replicates of each group.IL4 has a variety of biological activities that can stimulate B cell proliferation and differentiation, as well as T cell proliferation , 20. CCK2) of all targets and endogenous reference genes were verified to be similar by real-time PCR (Table Ct) were used for the comparative Ct (2Ct−ΔΔ) method to calculate the level of gene expression. Three independent experiments were performed in this experiment with three technical replicates of each group.To confirm the effects of binding proteins on goat IL4, further investigation was carried out at the molecular level. Real-time PCR was performed to detect the transcription of IL4R, JAK2 and STAT6. Group settings were consistent with the description of cell proliferation assays. The primers IL4R, JAK2 and STAT6 for qPCR are listed in Table CR Table . All datP values of ≤ 0.05 were defined as statistically significant. The following symbols were used to indicate the degree of significance: *P < 0.05, **P < 0.01, ****P < 0.0001; ns: non-significant. All experiments were repeated a minimum of three independent times.Statistical analysis was performed using the GraphPad Premier 6.0 software package . One-way ANOVA was used for comparisons of three or more groups. Data are represented as the mean ± the standard deviation (SD). HcESPs were collected by Co-IP. Western blot analysis of the immunoprecipitates showed that HcESPs could bind with rIL4 in vitro. In addition to the heavy and light chains of rat normal IgG, there were many clear bands in the nitrocellulose filter (NC) membrane, whereas there was no band in the negative control  score ≥ 20. Seven binding proteins of goat IL4 were recognized as follows: HcENO, HcGST, HcGB, HcTTR, HcCab, HcGAPDH, and HcPEPCK .To further confirm the results of Co-IP, YTH screening assays were performed independently between different pairs of bait and prey vectors. The gene of goat IL4 was cloned into the C-terminal half of ubiquitin (pDHB1), and the HcGST, HcTTR, and HcCab could actually bind with IL4, and the results of the reverse tests further illustrated the interaction of IL4 with these three proteins  would allow the yeast strain to grow on SD-AHLW selective medium. When pPR3N-GST, pPR3N-TTR and pPR3N-Cab were co-transformed with pDHB1-IL4, the yeast strain NMY51 grew on SD-AHLW , HcTTR (rHcTTR) and HcCab (rHcCab) were expressed and purified by nickel chelating chromatography through affinity for the hexa-histidine tag. They were approximately 41.2 kDa, 35.4 kDa and 34.1 kDa, respectively  on the biological functions of rIL4. The results demonstrated that rHcTTR (co-incubation with rIL4) treatments significantly suppressed the proliferation of PBMCs in a dose-dependent manner compared with the effects observed for the positive control group  = 17.8, P < 0.0001)  = 29.2, P < 0.0001)  = 13.61, P < 0.0001)  = 7.057 P = 0.0003)  = 2.070, P = 0.0954) by rHcTTR co-incubated with rIL4  = 2.479, P = 0.0523) by rHcTTR co-incubated with rIL4  could adjust multiple functions of goat monocytes [HcESP proteins could bind to cytokine IL4 by co-IP assays. This result indicated that the antagonists of goat IL4 probably existed in HcESPs.Previous studies on unctions , 23, 24,unctions –28. Moreonocytes . In the HcGST and HcTTR were binding proteins to IL4. Previous studies have shown that GSTs from Trichinella spiralis, Schistosoma mansoni, and bovine filarial parasite (Setaria cervi) were immunogenic and could elicit protective immunity [HcTTR, with partial similarity to transthyretin, contained a TTR-52 domain belonging to the transthyretin-like (TTL) family. TTLs have been confirmed to be widespread in nematode genes  [H. contortus could be recognized by the serum from naturally immunized sheep [HcGST and HcTTR played important roles in the immunity and immune regulation of nematode infections. HcCab belongs to the Calponin superfamily but its role in immunity is still unclear and needs to be further researched.The yeast two-hybrid assays in this research further confirmed that immunity –32. HcTTcaninum) , 33–36. ed sheep  and had HcTTR alone could not increase or decrease the proliferation of the cells compared with the control. However, co-incubation of rHcTTR with rIL4 significantly inhibited the proliferation of PBMCs in a dose-dependent manner. Conversely, rHcGST and rHcCab did not decrease the promotion of IL4 to cell proliferation. This result indicated that the binding of rHcTTR to IL4 could block the function of IL4 to induce cell proliferation.Cell proliferation assays showed that rIL4 could promote the proliferation of goat PBMCs. rHcTTR and rIL4 compared with the other groups. These results indicated that rHcTTR could impede the activation of this pathway by IL4. This result, together with the yeast two-hybrid assay and the cell proliferation assay, suggested that rHcTTR could significantly decrease the functions of IL4 to induce cell proliferation in vitro and was an antagonist of goat IL4.The JAK–STAT signaling pathway is a signal transduction pathway stimulated by cytokines, which is involved in many important biological processes, such as cell proliferation, differentiation, apoptosis and immune regulation –39. IL4 HcTTR of HcESPs was an antagonist of goat IL4. The recombinant protein could bind to goat rIL4 and significantly inhibit the biological activity of rIL4 on goat PBMC proliferation and block the activation of the JAK–STAT signaling pathway by IL4 in vitro. This study might reveal a new mechanism for parasite immune evasion. However, the functions of the other binding proteins to IL4 identified by Co-IP and yeast two-hybrid assays should be further investigated.In our study, we confirmed that the Additional file 1.Reverse test for the interaction between goat IL4 andHcENO,HcGST,HcGB,HcTTR,HcCab,HcGAPDH,HcPEPCK. A Interaction between HcENO and IL4. B Interaction between HcGST and IL4. C Interaction between HcGB and IL4. D Interaction between HcTTR and IL4. E Interaction between HcCab and IL4. F Interaction between HcGAPDH and IL4. G Interaction between HcPEPCK and IL4. The vectors pOstI and pPR3N were set as positive controls and negative controls, respectively. The construct pairs of HcENO with IL4, HcGST with IL4, HcGB with IL4, HcTTR with IL4, HcCab with IL4, HcGAPDH with IL4, and HcPEPCK with IL4 were carried in NMY51. Each experiment was run in triplicate.


B) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ET Stroke is ranked as the second largest cause of death worldwide after coronary heart disease, and it accounts for almost 1.1 million deaths each year in the European Union , 5-hydroxytryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1), and thromboxane A2 (TXA2) in the cerebral arteries , has been proven a particularly useful tool in regards to investigating the ETB receptors , which is part of the mitogen-activated protein kinase (MAPK) pathway. U0126 is a specific inhibitor of phosphorylation by MEK1 and MEK2 at ICAccording to the scientific literature, U0126 is found to be selective for MEK1/2  and investigated functional ETB receptor upregulation as the primary endpoint. The secondary endpoint was depolarizing induced contractility, assessed by a depolarizing 60 mM K+ stimuli. We therefore, aimed to determine any potential effects of cremophor EL  as a drug delivery system of U0126 in the same ex vivo model. Finally, we present a toxicology study in rats, which include the dose chosen as the optimal treatment dose, when based on the ex vivo data.The current study aimed to determine the optimal dose of U0126 ad libitum. Rats were generally housed in Eurostandard cages (Type VI with 123-Lid) 2–6 together. All procedures were performed at the Department of Clinical Experimental Research and were approved by the Danish animal ethics committee.For the organ culture data, 40 male Sprague-Dawley rats (NTac:SD) purchased from Taconic (Denmark) was used. The animals were maintained at a 12/12-h light-dark cycle (with dark beginning at 7 am) and housed at a constant temperature (22 ± 2 °C) and humidity (55 ± 10%), with food and water ex vivo and to determine the lowest effective concentration of U0126. U0126 was dissolved in DMSO to a final concentration of 10 mM. This formulation of U0126 was further diluted with DMEM (Dulbecco’s modified Eagle’s media) to obtain a concentration of 100 µM from which 1, 3, 10 and 30 µM solutions were prepared with paired DMSO controls. In the experiments with cremophor, a stock solution of 1% was prepared in DMEM and further diluted to the desired concentrations. For the U0126 in cremophor, a 200 µM stock in 1% cremophor (sonicated for 45 min to create micelles) was created and further diluted to create the desired concentrations.The rat basilar artery was used to determine the concentration-response relationship ex vivo pharmacology studies.BAs were carefully dissected from the brains and cut into 4 equal sized segments (∼1.2 mm long). The segments were incubated for 48 h in DMEM with U0126, DMSO or cremophor, and the media was changed after 24 h. After 48 h of incubation, the segments were used for 3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. The buffer was continuously aerated with 5% CO2 to maintain a pH of 7.4 and the wires were separated for isometric pretension at 2 Nm−1 and were then allowed to equilibrate at this tension for 30–45 min. The segments were exposed to a 60 mM potassium buffer obtained by a partial substitution of NaCl with KCl in the above-described isotonic buffer.For measurements of contractile responses, a wire myograph (Danish Myograph Technology A/S) was used to record the isometric tension in segments of isolated arteries , in the concentration range of 10−14 to 10−7 M  or one-way ANOVA with Holm-Sidak post-test (to control) when comparing concentration effects (U0126/DMSO cremophor). All the analysis was done using GraphPad 8.02 software . The relative log EC50/log IC50 is by four-parameter non-linear regression and is the concentration corresponding to a response midway between the estimates of the lower and upper plateaus. The Emax is the maximal contraction in the concentration-response curve. Contractile responses of each segment were adjusted according to the length of the artery and are expressed as mN/mm (Nm−1).Data are expressed as mean ± standard error of the mean (S.E.M.), and Rattus norvegicus) were purchased from Hilltop Lab Animals, Inc. . Animals were purchased with approximate weight ranges of 230–250 g and 180–200 g for the males and females, respectively on arrival. The animals were obtained with an intracerebroventricular (i.c.v.) cannulae preimplanted by the vendor. Animals were individually-housed due to the i.c.v. cannulae in plastic static micro-isolator cages. General procedures for animal housing and husbandry met all regulations concerning use of animals in research including the U.S. Department of Agriculture regulations (9 CFR Ch. 1) implementing the Animal Welfare Act (7 USC 2131 et seq.) and the recommendations of the National Research Council’s Guide for Care and Use of Laboratory Animals . This study was conducted according to a research proposal approved by the Institutional Animal Care and Use Committee of Comparative Biosciences, lnc.Seventy-two SD-HLA® (SD)CVF®  outbred rats . Each group was dosed i.c.v. with either 0.5% cremophor alone or test article (U0126 in 0.5% cremophor) at 0.057 µg, 0.57 µg, and 1.14 µg in a dose volume of 15 µL per dose containing 0.5% cremophor. The doses (µg) correspond to 1 µM, 10 µM, and 20 µM in the injected volume, respectively. Animals were dosed twice daily (approximately every 12 h) on day 0–3, and once daily on day 4–7. In-life assessments included a daily clinical observation, body weight measurements , a weekly food consumption, and a functional observation battery test (FOB). Approximately 90 min after the last single dose on day 7, the animals were subjected to terminal blood collection and subsequently euthanized and necropsied. Histological evaluation of hematoxylin and eosin-stained tissue sections was performed on the brain from all the animals and included evaluation of the injection site , the contralateral (uninjected) left lateral ventricle)) and of four additional brain levels. A full list of organs was also evaluated histologically for all dose groups. Formulations were made fresh every four days and kept refrigerated until use.B receptor upregulation in an organ culture model . In addition, 3 × 10−5 M U0126 was significantly lower than 10−6 M U0126 (p < .001), but not compared to its paired DMSO control. For the log EC50 values for S6c, we did not observe any inhibitory effect of 10−6 M U0126, while at 3 × 10−6 M of U0126 there was a significant shift in the log EC50 value and further significant shifts at every half-log increase in the U0126 concentration . Concentrations of cremophor 0.25% (0.019 M) or higher had significant effects on the Emax . The log IC50 value for U0126 shifting the maximal potassium contraction (K+ Emax) was relatively high (−4.97 to −3.66 log [U0126] M). For cremophor, the calculated log IC50 value for the effect on the K+ Emax was −2.15 to −1.77 log M . To further investigate any synergistic effects of cremophor combined with U0126, we compared four selected groups: (I) 0.1% (0.008 M) cremophor, (II) 3 × 10−6 M U0126 in 0.1% (0.008 M) cremophor, (III) 3 × 10−6 M U0126 in DMSO, and (IV) 10−5 M U0126 in DMSO (−6 M U0126 in 0.1% (0.008 M) cremophor  was significantly more potent for S6c inhibition than both the matched cremophor control , as well as the matched U0126 concentration in DMSO . Even though it has previously been shown that U0126 prevents upregulation of receptor expression levels using both qPCR, immunohistochemistry  and cremophor  also affected the cell metabolism, we analyzed BAs at baseline (fresh) and after 48 h in organ culture. There were no significant differences in the metabolism in the fresh BAs from all three groups. Following 48 h there was no change in the metabolism (12.2 ± 12.4%) in the DMSO (0.14 M) control. BAs incubated with 100 µM U0126, had the metabolism reduced by 93.4 ± 0.7% of its internal control. In relation to the cremophor, the metabolism was also reduced, but only with 42.3 ± 4.7% compared to its control. The absolute absorbance values and intra sample trend can be seen in −6 M U0126 and 0.1% (0.008 M) cremophor. Since the CSF volume in rats is estimated to be around 90 µl  should not be higher than 3 × 10in vivo toxicology data on neurological markers, observations (once daily), body weights (prior to the first dose and at necropsy), food consumption (once weekly), and functional observational battery testing (FOB). There were four groups of animals : (I) 0.5% (0.039 M) cremophor alone, (II) low (10−6 M), (III) middle (10−5 M), and (IV) high (2 × 10−5 M) dose of U0126, all in 0.5% (0.039 M) cremophor. Although the value of systemic toxicology study on i.c.v. administration is limited, we still include the table showing that there were no observed differences across treatment groups regarding body weight, food consumption, or organ weights (Table S4).We obtained In the neurological observations the 0.5% cremophor, low and middle dose of U0126 were well tolerated and we observed no changes to any parameters in these groups. At the highest dose of U0126, there were three  out of 16 animals demonstrating clear neurologic signs. Two high-dose females showed signs of neurological impairment during day 6 with FOB testing: one high-dose female was first noted to have a right head tilt and was circling in a clockwise direction before the second daily dose on day 2. This animal had a head tilt for the duration of the study, also noted during day 6 with FOB testing, but the circling movements were only observed in conjunction with the second dose. Another high dose female was noted to have hindquarters rotating clockwise (hind feet circling with forepaws stepping in one spot) during day 6 with FOB testing. Neither animal showed any other abnormal signs during day 6 with FOB testing. One high-dose male showed slight palpebral closure (∼ ¼) during dosing on day 7, but the animal had not shown any abnormality during day 6 with FOB testing. None of the other animals had any abnormal FOB testing results, and the behavioral test results were virtually identical in these animals.Histological changes, consisting primarily of inflammation of varying severity, were observed in the right lateral ventricle (injection site) and to a lesser extent, the contralateral ventricle of brains from animals in all groups. Small multifocal aseptic abscesses were observed in association with the injection site. Variable multifocal subacute inflammation was also noted at the other brain section levels in the neuropil and in the meninges. The incidence, distribution, and severity of inflammation were similar in all groups  and most−5 M U0126 are both well tolerated. Compound-related CNS toxicity was observed initially before the second dose on day 2 (i.e. prior to the 4th dose) at the high dose (2 × 10−5 M). Hence the toxicology data support our ex vivo organ culture study, suggesting that the maximal i.c.v. administration injected in vivo dose should be 0.5% cremophor (in aCSF) combined with 10−5 M U0126, resulting in an effective concentration of around 2 × 10−6 M U0126 in 0.1% cremophor.The toxicology study on the i.c.v. administration indicate that 0.5% (0.0039 M) cremophor alone (in aCSF) and 0.5% (0.0039 M) cremophor combined with 10in vivo vehicle and drug delivery system (cremophor) for inhibiting the functional upregulation of ETB receptors. The optimal dose of U0126 was further tested in a toxicology study. Below we will discuss the possible interaction between U0126 and cremophor, and its relation to possible dose selection for in vivo studies.In this study, we have investigated the optimal concentration of the MEK1/2 inhibitor U0126 and a suitable ex vivo experiments with arterial organ culture and in vivo stroke models  did not only reduce absolute contractility, but also negatively affected cellular metabolism  with an effective concentration lower than 0.1% (see below). In epithelial cells, concentrations of 5 mg/mL and above were toxic at incubation times relevant to the present study, while in endothelial cell culture cremophor had negative effects at concentrations higher than 0.1 mg/mL  and thereby prevents PKC activation , while the inhibitory effect on PKC in a cell assay, was observed at around 1 µM , that did not show any neurological or other clinical signs of toxicity  were all comparable, excluding it from being a cremophor specific effect and supporting it being a procedural effect.The rationale for the i.c.v treatment is to give the compound as close as possible to the arteries/arterioles in the brain. Generally, the i.c.v. approach is not desirable due to the risks associated with inserting a catheter. Nevertheless, when a patient is diagnosed with SAH, such a catheter is already inserted in the current operation protocols to release intracranial pressure. Hence the injection of the MEK1/2 inhibitor U0126 can be given in the early phases of the SAH.B receptor upregulation, and we have seen that the concentration with an optimal effect  at doses showing effects in the ex vivo experiments. These data will be important in determining the optimal dose for humans in treating SAH.In conclusion, concentrations up to 10


Phytoseiulus persimilis, used worldwide as a biological control agent of the spider mite Tetranychus urticae. The biocontrol efficacy of P. persimilis decreases under dry conditions, partly because P. persimilis eggs are sensitive to drought. We exposed P. persimilis adult females from two different strains to constant and variable humidity regimes and evaluated the hatching rate of their eggs in dry conditions, as well as the survival and oviposition rates of these females. Whereas the eggs laid by P. persimilis females exposed to constant high humidity did not survive in dry conditions, females exposed to constant low humidity started laying drought-resistant eggs after 24 h of exposure. Survival and oviposition rates of the females were affected by humidity: females laid fewer eggs under constant low humidity and had a shorter lifespan under constant high and constant low humidity. The humidity regimes tested had similar effects across the two P. persimilis strains. Our results demonstrate that transgenerational phenotypic plasticity, called maternal effect, allows P. persimilis females to prepare their offspring for dry conditions.The ability of an organism to adapt to short-term environmental changes within its lifetime is of fundamental importance. This adaptation may occur through phenotypic plasticity. Insects and mites, in particular, are sensitive to changes in temperature and humidity, especially during the juvenile stages. We studied the role of phenotypic plasticity in the adaptation of eggs to different relative humidity conditions, in the predatory mite The online version of this article (10.1007/s00442-019-04556-0) contains supplementary material, which is available to authorized users. Long-term adaptations, through natural selection, may lead to a match between organisms and their environment Darwin . Short-tNasonia vitripennis  are exposed to short day length and low temperature, the majority of their larval offspring will enter diapause . P. persimilis is the most frequently applied predator for biological control of two-spotted spider mites , and its efficacy is dependent on temperature and humidity conditions  on the phytoseiid predatory mite  Sabelis . HoweverP. persimilis eggs under stressful humidity conditions and estimate the plasticity of these strategies, (2) evaluate the effects of different humidity levels on P. persimilis adult females and (3) evaluate the degree of genetic variation for potential maternal strategies in P. persimilis.The three main objectives of this study were to: (1) investigate whether maternal strategies enhance the survival of P. persimilis eggs have a different sensitivity to constant and variable humidity conditions . To evaluate the effects of different humidity levels on P. persimilis females, we focused on their oviposition and survival rates, two traits determining population growth and biocontrol efficacy. As there may be trade-offs between fecundity and survivorship , and reared in two Petri dishes, containing around 100 individuals each  infested with T. urticae. The pot was kept in a climate chamber  at 55 ± 2% RH during the day (16 h) and 65 ± 2% RH during the night (8 h), at 25 °C (photoperiod L16:D8). The bean plants were replaced once a week and spider mites were provided as food twice a week. The pot with bean plants was placed in a tray filled with sunflower oil to prevent mite dispersal. The mixed strain was reared in these conditions during 17 months before the experiments started. To control for the influence of P. persimilis female age, we used even-aged cohorts of young adult females (7 days since the egg stage) for all experiments. These females were collected as eggs from the two strains, and kept for 7 days in two separate Petri dishes, in a climate cell at 70 ± 2% RH and 25 ± 1 °C. Inside the Petri dishes, an agar layer  and a cucumber leaf disk infested with spider mites provided optimal conditions for the development of these eggs to adults.Two P. persimilis eggs survive at 65% RH and 25 °C , “constant high” (95% RH), “variable 1” (successive cycles of 8 h at 65% RH and 16 h at 95% RH), and “variable 2” (78 h at 65% RH followed by 24 h at 95% RH)  at the bottom of each cup. For humidity treatment “constant low”, the cups were placed upside down on top of a wire platform (2.5 × 2.5 cm spacing) inside a closed plastic box (40 L × 25.5 W × 16.5 H cm). The relative humidity inside the box was regulated with a humidifier (Cigar Oasis Excel), to achieve constant 65% RH (average 64.7 ± 1.6% RH). For humidity treatment “constant high”, the cups were placed upside down on an agar layer at the bottom of a closed plastic box, in which the relative humidity was maintained at 95% RH (average 96.2 ± 1.7% RH). For humidity treatment “variable 1”, two additional plastic boxes were set up using the same methods: one box at 65% RH and one box at 95% RH. The cups were kept 8 h per day in the box at 65% RH (photoperiod L8:D0), and 16 h per day in the box at 95% RH (photoperiod L8:D8). For humidity treatment “variable 2”, the cups were kept during 78 h in the same box as humidity treatment “constant low”, followed by 24 h in the same box as humidity treatment “constant high”. All boxes containing the cups were placed in a climate cell at 70 ± 2% RH and 25 ± 1 °C (L16:D8 photoperiod). The females were provided with fresh T. urticae  ad libitum every day.One replicate consisted of 15–20 adult females per treatment and per strain. The females were placed in individual plastic cups , covered by a lid with a fine gauze (gauze-width 90 µm). To provide females with an oviposition substrate, a thin layer of cardboard with hairy surface facing up was fixed with a double-sided tape (TesaP. persimilis eggs (between 0 and 6 h old) after 6, 21, 30, 54, 78, and 102 h of female exposure to each humidity treatment . To achieve this, all eggs present in each cup were removed 6 h before collecting the eggs. 6 h later, the freshly laid eggs were collected from each cup and placed with a brush in a hole  in a platform made of polymethyl methacrylate (PMMA) (17.5 L × 15 W × 4.5 H cm) containing 30 holes (one egg per hole). Each hole had a fine gauze at the bottom (gauze-width 90 µm), to ensure contact with ambient air, and was then covered with a coverslip to prevent the larvae from escaping after hatching. The PMMA platforms  were placed in a climate chamber at 60 ± 1.6% RH, 25 ± 1.7 °C and L16:D8 photoperiod. Egg hatching rate  was recorded 72 h after placing the platforms in the climate chamber. A data logger (LogTag TRIX 8) was placed in the climate chamber and in each box containing the cups, to measure relative humidity and temperature. For each humidity treatment and each strain, this protocol was repeated five to seven times.We collected freshly laid We assessed the oviposition rate of the females exposed to the four humidity treatments over 4 days. We counted the number of eggs laid by each female after 24, 48, 72, and 96 h of exposure. For each humidity treatment and each strain, the oviposition experiment over 4 days was repeated five to ten times, with 15–20 females per replicate.We also assessed the oviposition rate of females exposed to humidity treatments “constant low”, “constant high”, and “variable 1” during 10 days. Females from the commercial strain were exposed to the three humidity treatments, while females from the mixed strain were exposed to treatment “variable 1” only. One replicate consisted of 20 females and we carried out three replicates per treatment and per strain. On days 6 and 7, females exposed to humidity treatment “variable 1” spent 5 h at low humidity (photoperiod L5:D0) and 19 h at high humidity (photoperiod L11:D8), instead of 8 h at low humidity and 16 h at high humidity, for logistic reasons. The cups from humidity treatment “constant high” were changed after 5 days, to avoid development of fungi inside the cups.T. urticae  as food. On days 6, 7, 13, 14, and 20, females exposed to humidity treatment “variable 1” spent 5 h at low humidity (photoperiod L5:D0) and 19 h at high humidity (photoperiod L11:D8), instead of 8 h at low humidity and 16 h at high humidity, for logistic reasons. The cups from humidity treatment “constant high” were changed every 5 days, and the cups from humidity treatment “variable 1” were changed after 10 days, to avoid development of fungi inside the cups. To make sure that the potential stress caused by the transfer of the females to new cups did not affect their survival or oviposition rate, we also changed the cups of humidity treatments “constant low” and “variable 1” every 5 days during 20 days, for one replicate. We did not observe an influence of changing cups on the survival or oviposition of the females.The same females used in the oviposition experiment over 10 days were used for the survival experiment: after an exposure of 10 days to humidity treatments “constant low”, “constant high”, and “variable 1”, females were kept under the same conditions during ten additional days. Their survival rate was assessed over these 20 days. Females were supplied every day with ample fresh P. persimilis eggs during 102 h. We also looked at the following interactions, “humidity treatment × exposure time of females” and “humidity treatment × strain”. In a second model, we compared the effects of the humidity treatments “constant low” and “variable 2”, as well as the factors strain and exposure time of females, on the egg hatching rate during the first 78 h . We also looked at the interaction “humidity treatment × strain”. We then compared, in a third model, the effects of the two humidity treatments “constant low” and “variable 2”, as well as the factor strain, on the egg hatching rate after 102 h of female exposure . We also looked at the interaction “humidity treatment × strain”. In all three analyses, the response variable was expressed as a proportion (number of eggs hatched/number of eggs tested) for each replicate. For all analyses, we used a generalized linear mixed model (GLMM) with a binomial error distribution and a logit link function. The variables humidity treatment, exposure time of females, and strain were expressed as fixed effects in the models. The replicates which had been performed at the same date were assigned to the same replicate number. The variable replicate was expressed as a random effect in the models (by-replicate random intercept), to take into account the fact that individuals within the same replicate were potentially correlated. Since there was overdispersion in the data for the first two models, we introduced a “per-observation” random effect. For all analyses, we used the model-fitting method of the maximum likelihood (Laplace approximation) and used likelihood-ratio tests to select the most parsimonious models.For the egg hatching experiment, we carried out three analyses. In a first model, we studied the effect of the factors humidity treatment , strain, and exposure time of females on the hatching rate of For the oviposition experiment, we first analysed the total number of eggs laid per female over 4 days, comparing humidity treatments “constant low”, “constant high”, and “variable 1” for both strains. We also looked at the interaction “humidity treatment × strain”. Data from females which died before the fourth day of the experiment were not used. In total, data from 586 females were analysed. Thereafter, we analysed the total number of eggs laid per female over 10 days, comparing the three humidity treatments  for the commercial strain, and comparing both strains for humidity treatment “variable 1”. Data from females which died before the tenth day of the experiment were not used. In total, data from 135 females were analysed for the commercial strain, and from 53 females for the mixed strain. For both analyses, we used a generalized linear model (GLM) with a Poisson error distribution and a log link function. We considered a multiplicative dispersion parameter in the variance (quasi-Poisson error distribution), and we found an estimated parameter smaller than 1; therefore, we decided upon a Poisson model with dispersion parameter equal to 1, leading to more conservative conclusions. The variables humidity treatment, strain and replicate were included as fixed effects in the two models. In the analysis of oviposition rate over 10 days, we had only three replicates for each combination of humidity treatment and strain, making a reliable estimate of variance between replicates tenuous Crawley . TherefoP. persimilis females, during 20 days. The data were right censored because the females that were still alive at day 20 or died from “handling accidents” during the experiment were censored. For each humidity treatment and each strain, all females that were observed during the same 20 days were grouped under the same replicate number. To account for a possible correlation between observations grouped in the same replicate, we used a shared gamma frailty model, with gamma-distributed shared frailties at replicate level  of The statistical analysis was performed in R (R version 3. 5. 1).P. persimilis females significantly affected the hatching rate of their eggs at 60% RH . While the hatching rate of eggs laid by females exposed to constant high humidity remained between 0.03 (− 0.02 to + 0.04) and 0.06 (− 0.03 to + 0.08)  during the whole experiment, hatching rate of eggs laid by females exposed to constant low humidity increased from 0.11 (− 0.06 to + 0.12) after 1 h of exposure, to 0.98 (− 0.04 to + 0.01) after 102 h of exposure. The hatching rate of eggs laid by females exposed to treatment “variable 1” increased to a lesser extent: from 0.04 (− 0.03 to + 0.05) after 1 h of exposure to 0.43 (− 0.18 to + 0.20) after 102 h of exposure. Strain did not significantly affect egg hatching rate  and there was no significant interaction between effects of humidity treatment and strain . For humidity treatment “variable 2”, during the first 78 h of exposure, egg hatching rate was the same as for humidity treatment “constant low” . However, after 102 h of female exposure to treatment “variable 2” , egg hatching rate suddenly decreased. After 102 h of female exposure, hatching rate of eggs laid in treatment “variable 2”  was significantly lower  than hatching rate of eggs laid in treatment “constant low”  , and there was no significant interaction between effects of humidity treatment and strain . It is important to notice that, in the egg hatching experiment, we collected around one egg per female after 6, 21, 30, 30, 54, 78, and 102 h of exposure to each humidity treatment. Thus, the proportion of drought-resistant eggs reported here (egg hatching rate at 60% RH) strongly correlates with the percentage of females that laid drought-resistant eggs.Humidity treatment of χ2 = 430.28; df = 2; P < 2 × 10−16, Figs. χ2 = 136.02; df = 2; P < 2 × 10−16, Fig. χ2 = 8.27; df = 1; P = 0.004), and there was no significant interaction between effects of humidity treatment and strain . On average, over 4 days of exposure to humidity treatment “variable 1”, females from the mixed strain laid significantly more eggs than females from the commercial strain (P = 0.046). To investigate further this strain effect on oviposition rate, we compared the oviposition rate of the two strains over 10 days for humidity treatment “variable 1”. The factor strain also had a significant effect on the oviposition rate over 10 days . On average, over 10 days under humidity treatment “variable 1”, a female from the mixed strain laid 3.4 eggs more than a female from the commercial strain.Humidity treatment significantly affected the oviposition rate over 4 days . More specifically, this ratio was significantly different between humidity treatment “variable 2” and the three other treatments . Females exposed to humidity treatment “variable 2” laid a significantly higher number of eggs during the last 24 h compared to the first 78 h, whereas for the three other treatments the number of eggs produced stayed relatively constant over 4 days. On average, 34% (− 4% to + 5%) of the eggs laid by a female exposed to humidity treatment “variable 2” were laid during the last 24 h, while between 25% and 29% of the eggs laid by a female exposed to humidity treatments “constant low” , “constant high” , or “variable 1”  were laid during the last 24 h . Strain had no effect on this ratio , and there was no significant interaction between effects of humidity treatment and strain . Overall, the oviposition rate experiment showed that P. persimilis females lay fewer eggs when exposed to constant low humidity than when exposed to constant high or variable humidity.Finally, humidity treatment had a statistically significant effect on the ratio “number of eggs laid on day 4/number of eggs laid over 4 days” . Around 70% of P. persimilis females were still alive after 20 days under humidity treatment “variable 1”, whereas only less than 35% and 25% of the females survived after 20 days under constant low and constant high humidity, respectively. Females exposed to constant low humidity survived a bit longer than the ones exposed to constant high humidity; however, this difference was not statistically significant (P = 0.07). Strain did not affect survival of the females under humidity treatment “variable 1” (P = 0.32). Overall, the survival experiment showed that the lifespan of P. persimilis females is shorter under constant low and constant high humidity, compared to variable humidity conditions.The humidity treatment had a statistically significant effect on the survival of P. persimilis females has a strong effect on drought resistance of their eggs. The size of this effect depends on the duration of female exposure. When females are exposed to constant low relative humidity (65% RH at 25 °C) during 102 h, almost all females lay drought-resistant eggs. However, when females are exposed to constant high relative humidity (95% RH at 25 °C) during 102 h, only 3–6% lay drought-resistant eggs. These results demonstrate a strong phenotypic plasticity in P. persimilis females with regard to relative humidity: they are capable of sensing unfavourable humidity conditions, and prenatally prepare their offspring to cope with drought. This mechanism for transgenerational phenotypic plasticity, or maternal effect, is defined as the ability of a female to alter its offspring’s phenotype, allowing it to survive in a specific environment (Bernardo Dermacentor variabilis (Say) (Acari: Ixodidae), the ability to absorb water vapor from the air is under maternal control , they had a similar response to the relative humidity treatments tested in this study. These results indicate that there was probably little variation for the production of drought-resistant eggs between these strains and that there might be little room for improvement of the adaptive maternal strategies described previously.P. persimilis female lifespan depends on relative humidity conditions in their environment. We recorded a shorter lifespan for P. persimilis females exposed to constant low humidity. More surprisingly, females exposed to constant high humidity had the shortest lifespan. A previous study showed that when both T. urticae and P. persimilis were exposed to 100% RH, their activity diminished gradually with time, and more than 90% of the mites ceased activity after 4 h  if these eggs are exposed to high humidity (75% RH at 25 °C) for at least 7 h during their development  to start laying drought-resistant eggs and that an interruption of this exposure will delay the response of the females.Although these maternal strategies seem to be beneficial for P. persimilis female has her own life-experience and personality, influencing her behaviour Below is the link to the electronic supplementary material.