Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of Kv4.2, restores nociception in Nav1.7 knockout (Nav1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Nav1.7 and activates Nav1.9 but does not affect Nav1.8. This toxin produces pain in wild-type (WT) and Nav1.7-KO mice, and attenuates nociception in Nav1.9-KO mice, but has no effect in Nav1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Nav1.9 activation and offers pharmacological insight into the relationship of the three Nav channels in pain signalling.The sodium channels Na v1.7 leads to congenital insensitivity to pain in humans. Here the authors found that activation of Nav1.9 can restore nociception in Nav1.7 knockout mice, revealed by a venom-derived peptide as a probe.Loss of function of Na However, pain is also a part of the body’s warning mechanism, cautioning humans to take action to prevent further tissue damage4. The voltage-gated sodium channel (Nav) Nav1.7 is known to play critical roles in the regulation of peripheral pain. Homozygous or compound heterozygous loss-of-function mutations in Nav1.7 lead to congenital insensitivity to pain (CIP)6, whereas gain-of-function mutations cause episodic pain  in humans9. This evidence indicates that selectively blocking Nav1.7 may be useful to relieve pain. Sustained efforts have been made to develop selective inhibitors of this channel, some of which have shown efficacy in clinical studies, although larger clinical trials are needed to definitively assess efficacy13. On the other hand, compared with painful, painless may be more serious. CIP individuals cannot distinguish between sharp and dull stimuli, leading to self-mutilation and painless fractures, and some individuals are even unable to detect temperature differences6. Therefore, therapeutics that restore the pain responses in Nav1.7-related CIP would be useful. Notably, Nav1.7 might not be a suitable direct target to achieve such an effect because of its loss of function in affected individuals; therefore, an alternative strategy should be developed. Nav1.7 and two other Nav subtypes, Nav1.8 and Nav1.9, are preferentially expressed in the peripheral terminals of sensory neurons14. More recently, genetic and functional studies have illustrated that mutations in Nav1.8 and Nav1.9 cause human pain disorders, providing direct clinical evidence linking these two channels to human pain17. Considering that the three channels play distinct roles in the generation and propagation of action potentials (APs)20, they might regulate pain signaling cooperatively, and the elucidation of their relationship in pain regulation may be helpful for the treatment of Nav1.7-related CIP.Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, and is a serious public health issuev modulators and potential therapeutic compounds26. Specific modulators are not only novel drug candidates for therapeutics but also powerful pharmacological tools to probe the physiological roles of Navs28. In this study, we screened pain-inducing compounds from animal venoms and discovered that a spider peptide toxin, HpTx1, was able to rescue the pain response in Nav1.7 knockout (Nav1.7-KO) mice by activating Nav1.9. HpTx1 was previously identified as an inhibitor of the voltage-gated potassium channel (Kv) Kv4.229, but our study showed that it is also a Nav modulator. Here, we provide pharmacological evidence that cross talk among Nav1.7, Nav1.8, and Nav1.9 may affect AP firing and pain signaling, thereby establishing an important role for Nav1.9 in pain perception.Animal venom is a rich source of Nav1.7-related CIP, we first fractionated 15 crude venoms (from ten spiders and five snakes) using semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) and collected a total of 110 fractions (5–10 fractions per venom). Six of these fractions were identified to have pain-inducing activity, with a fraction from the venom of the spider H. venatoria exhibiting the strongest efficacy  with Nav1.8-Cre mice, which led to the specific deletion of Nav1.7 in Nav1.8-positive sensory neurons . These mice showed deficits in mechanical pain responses but no alteration in thermal pain behavior  littermate mice triggered robust nocifensive behaviors, such as licking and biting of the injected paws. Furthermore, the pain-inducing effect was further validated in evoked pain models     dorsal root ganglion (DRG) neurons from WT mice by using current-clamp recordings. Four parameters related to AP firing, including resting membrane potential (RMP), current threshold (rheobase), amplitude, and firing frequency, were determined in the experiments. As shown in Fig. .1) Fig. . The inpv1.7-KO mice. As shown in Fig. n = 28, P = 0.006) and decreased the rheobase by 16.6 pA , but did not alter AP amplitude   value measured at 1.2 ± 0.3 μM  currents expressed in human embryonic kidney (HEK) 293T cells with an IC50 value of 0.51 ± 0.12 μM  or inactivation  of this channel  channels, Nav1.8 and Nav1.9, were examined. As shown in Fig. v channels in mouse small DRG neurons. Specifically, steady-state inactivation (SSI) was not significantly altered  currents in ND7/23 cells  of HpTx1 was determined to be 0.47 ± 0.08 μM  currents expressed in ND7/23 cells . However, the voltage dependence of the SSI was significantly shifted by approximately 13.4 mV in the presence of 0.75 μM HpTx1   and hNav1.9 sequences. Nav1.9 channels in ND7/23 cells and DRG neurons have distinct posttranslational modifications and auxiliary subunits that are known to affect the pharmacological properties and physiological properties of Nav channels36. However, for both TTX-R Nav currents in DRG neurons and Nav1.9 currents in ND7/23 cells, their window currents were obviously improved in the presence of HpTx1  and acid-sensing ion channels (ASICs) , but significantly increased rheobase by 10.4 pA . Furthermore, HpTx1 had no effect on AP amplitude  or input resistance . These effects differed from those observed in WT and Nav1.7-KO mice, and these differences could be interpreted to be due to the inhibition of Nav1.7 currents by HpTx1.We next tested the effects of HpTx1 on the membrane excitability of small DRG neurons from Naent Fig. , n = 25.v1.9-KO mice  mice (simultaneous knockout of Nav1.7 and Nav1.8). As shown in Fig. v1.8-KO mice by 2.6 mV  without affecting rheobase , AP amplitude  or firing frequency  and decreased rheobase by 11.6 pA  but had no effect on AP amplitude  or firing frequency  s3b-s4 segment in the formation of receptor site 3 is crucial for fast inactivation40. In this study, our data showed that HpTx1 slowed the development of the fast inactivation of Nav1.9  of Nav1.9 was replaced with the corresponding domain of Nav1.8 (named Nav1.9/1.8 DIV s1–s4)  conferred the efficacy of HpTx1  significantly reduced the toxin activity . Accordingly, chimaera channels of Nav1.7 were constructed. A substitution of the DII s3b-s4 region of Nav1.7 with the corresponding region of Nav1.8 (Nav1.7/1.8 DII s3b-s4) caused this channel to be completely insensitive to HpTx1  increased the efficacy of HpTx1  conferred toxin sensitivity with an IC50 value of 3.0 ± 0.6 μM  are manifested in these two mouse mutants. However, WT mice, which express Nav1.7 and Nav1.9, displayed pain responses with HpTx1 treatment, indicating that enhanced Nav1.9 activity might conceal the analgesic activity derived from the inhibition of Nav1.7. Our data suggest that the inhibition of Nav1.7 by HpTx1 treatment may be considered analogous to KO of Nav1.7 to some extent. One possible explanation for the ability of HpTx1-induced Nav1.9 activation to overshadow the analgesic activity derived from HpTx1-induced Nav1.7 inhibition based on the role of Nav1.7 and Nav1.9 as AP threshold channels44 is that HpTx1-bound Nav1.9 largely depolarized the RMP of DRG neurons, potentially compensating for the absence of Nav1.7 and leading to AP generation. Studies have proven that a moderately depolarized RMP contributes to the decrease in the current threshold for AP generation by promoting the opening of other ion channels 46. Our data demonstrated that HpTx1 depolarized RMP by 2.0 mV and 3.2 mV in WT and Nav1.7-KO mice, respectively, leading to a reduced current threshold and increased AP and finally enhancing the membrane excitability of some small DRG neurons 52. On the other hand, Nav1.9 does not contribute much to the amplitude of APs44, suggesting that enhancing Nav1.9 activity is unable to compensate for the loss of Nav1.8, which contributes to the majority (58–90%) of the inward current during the rising phase of an all-or-none AP in nociceptive sensory neurons53. Indeed, our data showing that HpTx1 failed to affect AP firing in Nav1.8-KO DRG neurons and pain sensitivity in Nav1.8-KO mice are consistent with this explanation  significantly increases Nav1.9 mRNA and current density60, the endogenous molecule contactin promotes the surface expression of Nav1.962, and some inflammatory mediators, such as prostaglandin E2 (PGE2), GTP, and histamine, can markedly increase the current density of Nav1.9 currents and AP firing64. Therefore, enhancing the activation of Nav1.9 by regulating these endogenous molecules may be a feasible strategy for treating CIP. However, the possible risks caused by activating Nav1.9 should be noted. Similar to some gain-of-function mutations of Nav1.9, activated Nav1.9 might result in spontaneous pain in humans52, while hyperactive Nav1.9, similar to the L811P and L1302F mutants, may lead to pain insensitivity46.Potentiated pain sensitivity should be beneficial for human NaH. venatoria, and the freeze-dried crude venom was stored at −20 °C prior to analysis. Lyophilized venom was dissolved in double-distilled water. Every time, 10 mg dried venom was purified by semipreparative reverse-phase HPLC using an Ultimate® XB-C18 column  on the Hanbon HPLC system . The following linear gradient of solvent A (0.1% formic acid in acetonitrile) in solvent B (0.1% formic acid in water) was used at a flow rate of 3 ml min−1: 15% A for 5 min, then 15–60% A over 45 min. Absorbance was measured at 215 nm, and fractions were collected and lyophilized before storage at −20 °C. The target fraction was subjected to the second round of RP-HPLC  using a XB-C18 column  with a slower increasing acetonitrile gradient  to obtain the purified HpTx1.The venom was obtained by electrical stimulation of female spiders of TM 5800 system, Applied Biosystems, USA). The entire amino acid sequence of a peptide was obtained by automated Edman degradation using an Applied Biosystems 491 pulsed-liquid-phase sequencer from Applied Biosystem Inc.The molecular weight of a peptide was analyzed by MALDI–TOF-TOF MS spectrometry (AB SCIEX TOF/TOFv1.7 (hNav1.7) and rat Nav1.8 (rNav1.8) clones and beta subunit (β1 and β2) clones were kindly gift from Dr. Theodore R.Cummins . hNav1.7 and rNav1.8 were subcloned into the vectors pcDNA3.1 and pCMV-blank vectors, respectively. hNav1.9 was subcloned into the pEGFP-N1 vector. The C-terminal of hNav1.9 was linked a GFP to construct a fusion protein channel (hNav1.9-GFP)48. The detail methods of mutagenesis are provided in the Human Na−1 penicillin and 100 µg ml−1 streptomycin in a 5% CO2 incubator at 37 °C. Cells were trypsinized, diluted with culture medium, and grown in 35-mm dishes. When grown to 90% confluence, ND7/23 cells were transfected with hNav1.9-GFP or hNav1.9-GFP mutants using the transfection kit X-tremeGENE HP DNA Transfection Reagent  according to the manufacturer’s instructions. Transfected cells were first maintained at 37 °C with 5% CO2 for 24 h, and then incubated at 29 °C with 5% CO2 for 20 h before use in electrophysiology experiments. Transfections of hNav1.7 and hNav1.7 mutants together with β1 and β2-eGFP and other ion channels  into HEK293T cells, and rNav1.8 and rNav1.8 mutants together with eGFP into ND7/23 cells were performed by using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instruction. Six hours after transfection, the cells were seeded onto poly-D-lysine-coated coverslips (Thermo Fisher Scientific) and maintained at 37 °C in 95% CO2 for 24 h before whole-cell patch-clamp recording. The green fluorescent was used for visual identification of individual transfected cells.ND7/23 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U mlv1.7, Nav1.7-KO, Nav1.8-KO Nav1.7/Nav1.8-DKO, or Nav1.9-KO mice were euthanized via cervical dislocation under anesthesia. Dorsal root ganglion (DRG) neurons were collected from the lumbar spinal cord L4–L5. DRG neurons were dissociated by enzymatic treatment with collagenase (1 mg ml−1) and trypsin (0.3 mg ml−1) at 37 °C for 30 min. cells were seeded onto poly-L-lysine-coated coverslips and cultured in DMEM (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco) and at 37 °C in a humidified incubator with 5% CO2 for 3 h before whole-cell patch-clamp recording. Note that male and female mice in half were used in each experiment.Six to eight-week-old C57BL6 WT, fNaWhole-cell patch-clamp recordings were performed at room temperature (25 ± 2 °C) using an EPC-10 USB patch-clamp amplifier operated by PatchMaster software  or Axopatch 200B amplifier (Molecular Devices). Fire-polished electrodes (2.0–2.5 MΩ) were fabricated from 1.5-mm capillary glass using a P-97 puller . Capacity transients were canceled; voltage errors were minimized with 80% series resistance compensation. The liquid junction potential was corrected using Axopatch 200B series amplifier, the Henderson equation was used to calculate the junction potential based on the ionic strength of bath and pipette solution. During the whole-cell recording performed in the EPC-10 USB amplifier, the liquid junction potential was not corrected. Voltage-dependent currents were acquired with Patchmaster at 5 min after establishing a whole-cell configuration, sampled at 30 kHz, and filtered at 2.9 kHz. The configuration of electrophysiology recording buffer and stimulation pulse are provided in the + currents.For electrophysiology experiments, the stock solution of HpTx1 was diluted with fresh bath solution to a concentration of tenfold of the interested concentration, 30 µl of the concentrated peptide was diluted into the recording chamber (containing 270 µl bath solution) far from the recording pipet (the recording cell), and was mixed by repeatedly pipetting to achieve the specified final concentration. TTX and capsaicin were dissolved in DMSO to make 1 mM stock solutions. The final concentration of DMSO did not exceed 0.1%, which was found to have no significant effect on Nav1.9-KO mice have been described earlier56. The floxed Nav1.7 mice and Nav1.8-Cre mice are described by Nassar et al.30. These mice were housed at the constant temperature of 24 °C and 50–60% humidity under controlled conditions of 12 h light/dark cycles and provided with free access to laboratory-standard food and water.Nan = 3) and female (n = 3) mice were used on behavioral testing. Before testing, mice were habituated on an elevated platform of the mesh floor and plastic testing chambers for 30 min. HpTx1 was dissolved in saline and administered at doses of 1 μM and 10 μM, respectively. HpTx1 was injected into the hind paws of mice , and the seconds of licking/biting behavior were immediately recorded during a 20 min period following the injection. Nocifensive responses were assessed by the total seconds of licking/biting behavior during 20 min.Seven to eight-week-old male . It was recorded three times by using radiant light heat onto the plantar side of the paw with an interval of at least 5 min. In order to avoid injury to the mice, a cutoff of 20 s was set.The mechanical withdrawal threshold was assessed by recording the max force that was continuously applied until paw withdrawal , and recorded three times with an interval of at least 5 min. Note that all of the behavioral tests were double blind.v1.9 . Then the sections were incubated with biotinylated griffonia simplicifolia Lectin I isolectin B4  for 30 min at room temperature. After incubation, sections were washed three times for 5 min in PBST (0.05% Tween20). Finally, incubated with Alexa-Fluor 488-nm  and Alexa-Fluor 594 Streptavidin . After being washed three times for 5 min in PBST, sections were mounted with coverslips. Fluorescence images were acquired with the FV1000 confocal microscope .DRG neurons were collected from the lumbar spinal cord (L4–L5). The DRG were fixed in 4% PFA in PBS for 2 h 30 min at 4 °C then cryoprotected in PBS containing 30% sucrose overnight at 4 °C. The DRG were frozen in OCT, and cryosectioned at 12 μm. The sections were permeabilized in PBS containing 0.5% TritonX-100) for 10 min, and were blocked with 10% goat serum for 1 h. The sections were incubated for 24 h at 4 °C with polyclonal rabbit anti-NaAll of the animal experiments were performed in accordance with the Guidelines for Laboratory Animal Research set by Hunan Normal University and Huazhong University of Science and Technology. The experiments were approved by the Institutional Animal Care and Use Committee of the College of Medicine, Hunan Normal University and Ethics Committee of Huazhong University of Science and Technology.n represents the number of animals or cells examined. One-way ANOVA and two-way ANOVA were used to assess the difference between multiple groups. Two grouped data were analyzed by the Kolmogorov–Smirnov normality test before t test analysis. If the data were normally distributed, a parametric t test was used. Otherwise, a nonparametric t test was used. In figure legends, statistical method to a specific experiment is mentioned, and the F, t, df (degree of freedom) and p-values are also shown. Significant levels were set at p < 0.05 and the exact p-values are presented in Supplementary Data Data were analyzed with PatchMaster v2x73 (HEKA Elektronik), Clampfit 9 , IgorPro6 , Prism 7 , FV10-ASW  and Office Excel 2010 . All values are shown as mean ± S.E.M., and Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Reporting Summary


The Organization for Economic Cooperation and Development included CeO2 NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO2 NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO2 NM-based diesel additive , burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L−1) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L−1 aged CeO2 NMs evaluated by the H2DCF-DA probe. Electron microscopy showed no internalization of aged CeO2 NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO2 NMs could exert in vitro biological effects on human spermatozoa and generate ROS.Cerium dioxide nanomaterials (CeO Nanoparticles (NPs) and nanomaterials (NMs), particularly metal oxide NMs, are increasingly used in many fields of everyday life, e.g., food packaging, cosmetics, textiles, electronics, and even biomedicine. Extensive usage of NMs in various areas has raised human health concerns, mostly in terms of occupational exposure ,2. Consi2 NMs have been increasingly used in Europe and elsewhere as fuel-borne catalysts in diesel engines [TM from Energenics Europe Ltd. The addition of CeO2 NMs in diesel has been reported to increase the fuel combustion efficiency [2, CO, the total particulate mass, formaldehyde, acetaldehyde, acrolein, and several polycyclic aromatic hydrocarbons [TM is known to be used, an increase in ambient Ce-based NMs was observed with a concentration of ∼0.3 ng·m−3 and aerodynamic diameters peaking at 150 nm [2 NMs that are not representative of the emission in diesel exhaust. Indeed, once released in the atmosphere after combustion in a diesel engine, Ce is in the form of CeO2 NMs with different physical-chemical properties compared to pristine CeO2 NMs  [2 NMs on human health represents an important step in the safety assessment required from Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and from the Organization for Economic Cooperation and Development (OECD’s guidelines 2011) [Among all, CeO engines ,7 as theficiency  and decrocarbons  during cocarbons ,11. In tt 150 nm . To dateubility) , which cubility) ,14 and pes 2011) ,17.2 NMs on reproductive organs and germ cells. Pristine CeO2 NMs (5–40 nm) have been shown to cross the blood–testis barrier and accumulate in the testis  of rats following 28 days of inhalation in vivo  [2 NMs (27.62 ± 3.01 nm) at the highest doses of 20 or 40 mg/kg body weight [2 NMs (7 nm) on human sperm cells and mouse gametes after exposure to 10 µg·L−1 [Atmospheric pollution is known to affect numerous physiological functions, including human reproduction and fertility, and it is reported to be related to lower semen quality ,20,21,22y weight . This acy weight . Our tea0 µg·L−1 ,27. Sign0 µg·L−1 , and a s2 NMs , which does not reflect a realistic exposure route of men and women to Ce-based NMs that are likely released in diesel exhaust. However, the WHO’s International Agency for Research on Cancer classified diesel engine exhaust as carcinogenic to humans [2 NMs as part of a priority list of NMs whose potential toxicity require urgent evaluation [2 NMs must be evaluated.Most of the previous studies were conducted with pristine CeOo humans , and thealuation . Hence, aluation  of combu2 NM-based diesel additives (EnviroxTM). Combining biological assays with physico-chemical characterisation, we addressed two questions: (i) Do combusted CeO2 NMs from a diesel additive induce genotoxic effects towards human and rat sperm cells following in vitro exposure to low concentrations? (ii) Were the mechanisms of genotoxicity and the interactions with sperm cells different from pristine CeO2 NMs?This study was designed to evaluate the potential genotoxicity induced by in vitro exposure of human and rat sperm cells to low concentrations of combusted commercialised CeO2 NMs were recovered from EnviroxTM, Energenics Europe Ltd., Oxfordshire, UK, which is a fuel-borne catalyst scientifically and commercially proven CeO2 NM-based diesel additive supplied by Energenics Europe Ltd. EnviroxTM was combusted following the protocol published in [TM suspensions at 396,750× g and 20 °C for 1 h and removed the supernatant to recover the pellet containing CeO2 NMs. The pellets were freeze-dried  for 5 days. The dried samples were crushed, and a total amount of 1.2 g was introduced in a furnace at 850 °C  [TM  was prepared in Milli-Q water at 10.15 g·L−1 of CeO2. The combusted EnviroxTM contained CeO2 NMs with an average TEM size of 19 ± 10 nm, a larger polydispersity than the uncombusted ones, and a polyhedral shape [CeOished in . Brieflyal shape . We used2 NMs in FertiCult® medium  for the in vitro culture of mammalian gametes by inductively coupled plasma mass spectrometry (ICP-MS) . We incubated aged CeO2 NMs at room temperature (RT) in the culture medium for 2 and 5 h at three concentrations . After incubation, the suspensions were ultra-filtered at 3 KDa  at 3000× g for 1 h and by ICP-MS; triplicates were performed for each concentration.We assessed the dissolution of aged CeORat sperm cell collection. After sacrifice, we collected and cut the epididymis to enable the exit of sperm in HTF-BSA culture medium  for 1 h at 37 °C and CO2 5%.Human sperm collection. We used frozen human sperm from healthy fertile donors. After thawing, we aliquoted the preparation and centrifuged it for 10 min at 420 g. The supernatants were discarded, and the pellets were exposed to various exposure conditions.Ethical authorization for animal sampling of gametes was obtained from the National Ethics Committee on Animal Experimentation (2018061110211950-V2 #15447). We used Sprague Dawley rats, Oncins France Strain A (623OFA), which were purchased from Charles River Laboratories, France. Sexually mature male rats of 60 days old were housed with free access to food and water until sacrifice. Human sperm cells were purchased from GERMETHEQUE biobank, which obtained informed consent from each donor for inclusion of samples in the biobank and for their use in research experiments regarding human fertility in accordance with the 1975 Helsinki Declaration on human experimentation.For oxidative stress analysis, the principle of Replacement of the 3R rule  was applied and human sperm only was used.2 NMs  for 1 h at 37 °C and 5% CO2. FertiCult® medium was used as a negative control and 110 µmol·L−1 H2O2 as a positive control. The H2O2 concentration was chosen based on previous studies [We exposed human and rat sperm to four different concentrations of aged CeO studies ,27,31. A studies , and befWe performed the alkaline comet assay according to the procedure described by Singh  and adapStatistical Analysis. The data presented the medians of % Tail DNA values, with 1st and 3rd quartiles. We performed a linear mixed model analysis with “condition” (exposure condition) as a fixed effect and “cells” (sperm cells) within the replicate slide as a random effect using linear mixed effects regression (lmer) function of R software, version 3.6.0  to compare DNA damage among the various conditions. Pairwise differences of least-squares mean for all conditions were post-hoc assessed. Statistical significance was set at p < 0.05.2 NMs in vitro exposure on the generation of reactive oxygen species (ROS), at the exposure concentration that had induced the higher DNA damage, i.e., 1 µg·L−1. At least three different experiments were performed. We used a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) probe [® culture medium, we centrifuged human sperm for 10 min at 420× g, discarded the supernatant and added 10 µmol·L−1 H2DCF-DA [2 for 45 min to enable internalization of the permeable probe. Then, the sperm cells were washed and exposed to 1 µg·L−1 CeO2 NMs for 1 h at 37 °C and 5% CO2. We used H2O2 110 µmol·L−1 as a positive control and FertiCult® medium as a negative control. After 1 h, we recovered the motile sperm by swim-up selection. For vitality staining, we exposed the sperm to 4′,6-diamidino-2-phenylindole (DAPI) at 0.5 µg·mL−1 immediately prior to analysis by flow cytometry . The results are only based on a live sperm population and expressed as the percentage of DCF-positive cells (expressing the fluorescence) in 100,000 events per condition in each replicate experiment .We investigated the effect on human sperm of aged CeOA) probe . The expA) probe ,37. AfteH2DCF-DA  at 37 °C−1 of aged CeO2 NMs and selected by swim-up after 1 h. Non-exposed sperm cells were used as the control. Recovered motile sperm cells were washed with 0.1 M phosphate buffer, then fixed with glutaraldehyde 2.5% in 0.1 M phosphate buffer during 30 min at RT, and finally rinsed 3 times with 0.1 phosphate buffer. Samples were post-fixed with 2% osmium tetroxyde in 0.1M phosphate buffer during 30 min and washed 3 times with 0.1 M phosphate buffer. Progressive dehydration with 50% to 100% Ethanol bath was performed before embedding in Epon 812 (epoxy resin) from 33% to 100% EPON. Ultrathin sections (60 nm) were obtained using Ultracut-E ultramicrotome  and contrast was performed using Uranyl acetate 5% for 12 min and dried at room temperature. Pictures were obtained using a JEM 1400 transmission electron microscope  at 80 kV with a Megaview III camera under iTEM Five software . This procedure was described in [Human sperm were exposed in vitro to 1 and 100 µg·Lribed in . 2 NMs in abiotic FertiCult® medium (pH 7.2–7.5) was studied after 2 and 5 h of incubation at 10, 1000, and 100,000 µg·L−1. Regardless of the concentrations tested, no dissolution of the CeO2 NMs could be measured by ICP-MS in abiotic conditions. Below 3 kDa, the concentrations of dissolved Ce species were under the limits of detection . The dissolution (<3 kDa) of CeO−1 of aged CeO2 NMs. After exposure, all viability rates exceeded the normality threshold as stated by the WHO criteria [p < 0.05, for the differences compared versus *: negative control (NEG), a: vs. 10 µg·L−1 CeO2 NMs, b: vs. 100 µg·L−1 CeO2 NMs, c: vs. 1000 µg·L−1 CeO2 NMs  (−1)  compared to the exposure to 10, 100, and 1000 µg·L−1 aged CeO2 NMs; (p < 0.001). We also observed a significant difference between 10 and 1000 µg·L−1 aged CeO2 NMs in rat sperm (p < 0.05) and a significant difference among the three highest tested concentrations in human sperm (p < 0.05). The variability of biological data in the 3 independent experiments is presented in In rat- and human-exposed sperm cells, all exposure concentrations induced significantly higher DNA damage than the negative control ( < 0.05) a,b. Furt−1 aged CeO2 NMs, the percentage of DCF-positive live sperm (mean ± SEM= 13.1 ± 3.9) significantly increased compared to the negative control (5.21 ± 2.9) (p = 0.047) (After 1 h of in vitro exposure to 1 µg·L= 0.047) .2 NM aggregates near the sperm plasma membrane after the exposure to 1 µg·L−1 CeO2 NMs  exposed mice to a single dose of 162 μg of CeO2 or TiO2 NMs in 50 μL of 2% serum in nano-pure water [3 for TiO2 assuming 9% alveolar deposition [2 NMs in a nose-only exposure system for a single 4-h exposure (27–39.8 μg/m3). They used a scanning mobility particle sizer to estimate the average mass concentration and compared it with the physical sampling measurements based on the filter packs [One major drawback of the currently used studies and models to evaluate the toxicity of NMs for humans is the lack of relevance of the exposure scenario  . Indeed,re water . This siposition ,42. Li  and Préaubert et al. (2015) [2 NMs (1 µg·L−1) was found to induce the highest DNA damage in both human and rat sperm cells . In that case, smaller aggregates would be more prone to interact with the cells and enhance their biotransformation, biological, and toxicological effects [Here, we highlighted the importance of the dose and more particularly of the low dose. We expected a moderate release of CeO. (2015) ,26. The rm cells a,b. Thess  and oxidative damage . Oxidati2 NMs is a major challenge since they are known to exhibit pro- and antioxidant properties. For example, Das et al. (2007) [2 NMs  could reduce oxidative stress as free radical scavengers, whereas Auffan et al. (2009) [2 NMs (7 nm) to damage fibroblast DNA at very low doses (6–1.2 × 106 µg·L−1). The determining factors are the stoichiometry, oxido-reduction state of Ce, pH of the medium, presence of H2O2, etc. [Understanding the mechanism of action of CeO. (2007)  and Niu . (2007)  showed t. (2009)  highlighO2, etc. , but it O2, etc. , size anO2, etc. .−1 aged CeO2 NMs induced a significant increase in ROS generation. The H2DCF-DA probe becomes fluorescent on oxidation and is purported to directly monitor reactive oxygen and/or nitrogen species (ROS/RNS) [−1 of pristine CeO2 NMs was significantly decreased by the addition of an antioxidant (L-ergothioneine) in the exposure medium [2DCF-DA probes [Herein, we demonstrated in human sperm that 1 h of in vitro exposure to 1 µg·LROS/RNS) . ONOO- aROS/RNS) ,61. Our e medium . L-ergote medium , which cA probes ,61. We h−1, aged CeO2 NMs were found in close contact with the plasma membrane of the head of the spermatozoa. This interaction with the sperm head associated with the highest sensitivity of the H2DCF-DA probe when oxidants are generated near the plasma membrane or in the cytoplasm [2 NMs. Indeed, the head of the spermatozoa expresses various ion channels  [2+ and Pb2+ into a mature spermatozoa [3+ and Ce3+ act as T-type calcium channel antagonists and competitively bind and block the Ca2+ binding sites [At 1 µg·Lytoplasm  can help and K+) ,64, whicrmatozoa ,65,66. Mng sites ,68, whic® medium. However, reductive dissolution of nanocrystalline Ce(IV)O2 into Ce(III) is pH-dependent at pH < 7 [2 NMs in the vicinity of the cells. More information on the biodistribution and biotransformation of CeO2 at the scale of the cell membrane is required to validate the hypothesis that the observed oxidative stress can be attributed to the possible biotransformation of CeO2 at the surface of the plasma membrane.Here, we did not observe any abiotic dissolution of Ce in the Ferticultt pH < 7 ,70,71 ant pH < 7 ,70. Indet pH < 7 . Such an2 NMs (mostly monodisperse and homogeneously coated), which are not representative of CeO2 NMs that are likely released during use. One strength of this study is the use of combusted EnviroxTM to mimic real exposure to CeO2 NMs, which are potentially released from diesel vehicles equipped with an active nano-catalyst for soot abatement. With an over-combustion in a single-cylinder engine with mechanical fuel injection [2 NMs evolve compared to those initially incorporated in the diesel additive: the size of the CeO2 crystallites significantly increased  without detectable Ce(III) in the structure, and no organic compounds remained at the surface. This transformation upon combustion highlights that at each stage of the nano-enabled product life cycle , the mechanisms and kinetics of interactions between the released CeO2 NMs and the aqueous media  and exposed organisms will differ [To date, few studies have explored the DNA damage and oxidative stress induced by pristine CeOnjection  or in annjection , the phyl differ . Hence, l differ ,74. Thisl differ  towards l differ ,76.2 NMs (production stage of the life cycle) and combusted diesel additives (usage stage of the lifecycle). At both lifecycle stages, a higher genotoxicity attributed to oxidative stress was observed at the lowest doses towards human sperm cells [2 NMs are likely more dispersed, which increases the contact surface between NMs and cells [Based on previously published data and the current study, we compared the impacts towards gametes of pristine CeOrm cells ,27. For nd cells .2 NMs, which are similar to the ones released by combustion in a diesel engine. In vitro exposure to the lowest concentration of combusted CeO2 NM induced oxidative stress in human sperm cells after the interaction with the plasma membrane. While important from a mechanistic standpoint, this study remains limited by its in vitro nature. However, in vitro exposure is a relevant design since spermatozoa can encounter nanoparticles in the male gonads or female genital tract. In vivo studies after a low-dose exposure to aged NMs will help us more closely approach realistic exposure conditions. Then, we will be able to decipher the transfer of CeO2 NMs at different stages of the lifecycle to various organs in animals, their permeation of biological membranes, accumulation in reproductive organs, and their impact on embryonic development, as conducted with pristine NMs [aP) [2 NMs associated with other organic compounds generated during diesel combustion.We demonstrated for the first time the DNA damage induced in human and rat sperm cells after exposure to low concentrations of CeOtine NMs ,78,79,80tine NMs  should btine NMs . Some ofNMs [aP) . An even


Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V  and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis. The fundamental regulation of DNA within eukaryotes is coordinated through highly conserved histone proteins that package DNA into the nucleus. Proteins that regulate post-translational modifications of histone proteins or of DNA, or that control nucleosome assembly, disassembly, or movement, are frequently targeted in cancer . More reWe set out to address the role of mutations in histone H3, one of the most highly conserved proteins in eukaryotes, using the highly tractable fission yeast. This system excels for evaluation of the biological effects of mutations divorced from confounding effects of additional mutations in tumors and can be stripped of the complexity of additional wild-type histone H3 variants within cells. We previously reported that fission yeast manipulated to express only G34R mutant histone H3 (H3-G34R) showed defective H3K36 acetylation (H3K36ac) and reduced levels of H3K36me3 . These cHere, we investigate other mutants of H3G34. H3-G34V mutation is less frequent than G34R in pediatric high-grade glioma, but the tumors occur in the same cortical location in patients of similar age . Based o+hht2 and +hhf2)  representing strains with three copies of H3/H4 (Fission yeast have three genes that code for a single histone H3 protein . Strainsd hhf2+) , which md hhf2+) . Previou) (hht2+  to gener)  are carried out by a single enzyme, Set2 , whereas H3-G34V cells were not. G34V cells were also not sensitive to DNA alkylation , whereas both G34R and set2Δ cells were sensitive. Finally, H3-G34V cells were unaffected by exposure to the DNA topoisomerase I ligase inhibitor CPT, whereas H3-G34R and set2Δ showed slight or pronounced sensitivity, respectively. At the concentrations used, these genotoxins predominantly affect DNA replication or require DNA replication to inflict damage. Thus while H3-G34R and set2Δ are sensitive to several forms of replicative stress, H3-G34V cells are not. Since both H3-G34V and H3-G34R have reduced K36me3, the sensitivity of H3-G34R cells to replicative stress cannot be attributed to a reduction in H3K36me3 but may be due to loss of H3K36ac.Altered H3K36 post-translational modification is associated with defective repair of DNA damage . Consist+set2 are sensitive to γ-IR and IR mimetics  break repair efficiency in H3-G34V cells, since H3-G34R and A repair . leu1-32ored see . In compct in HR . Howevern rad51Δ . Togethe insults .HP1 displayed high frequencies of Ch16 loss , similar to H3-WT cells (1.2%).H3-G34R cells exhibit genomic instability . Since Hss 8.5%, . H3-G34Rss 8.5%, , whereasset2Δ cells showed no or little chromosome mis-segregation, respectively , compared to wild-type cells (0.5%) and clr4Δ cells that lack heterochromatin (17.5%). We conclude from these assays that the genome of H3-G34V cells is relatively stable when compared with H3-G34R cells.As an alternate assay to measure chromosome segregation defects, we quantified chromosomal DNA segregation in late anaphase cells (with a spindle length >10 microns) . Since fThe surprising differences in the behavior of H3-G34V and H3-G34R mutants prompted us to ask whether H3-G34R phenotypes are caused by the large size or the basic charge of the arginine substitution, since valine is, like glycine, small and uncharged. To address this question, we generated a panel of sole copy H3-G34 mutant strains, with G34 replaced by the bulky uncharged residue tryptophan (W), the negatively charged glutamine (Q), the positively charged lysine (K), and methionine (M) . Using tset2Δ or H3-G34V mutant cells  were sensitive to the alkylating agent MMS and to topoisomerase inhibition CPT. We found that MMS sensitivity was not altered in strains bearing the bulky H3-G34W, G34M, or the negatively charged G34Q substitutions . This wont cells . These eSince H3-G34K mutants were sensitive to DNA damage, we tested whether they showed genomic instability in the absence of exogenous DNA damage. Monitoring chromosome missegregation during anaphase, we found that H3-G34K cells had elevated levels of chromosome missegregation (7.4%), which were reproducibly higher than seen in H3-G34R cells (5.9%) . In contrad51 mutant allele and monitoring epistasis between H3-G34K and rad51Δ, we found that double mutant H3-G34K rad51Δ cells were more sensitive to both MMS treatment and bleomycin treatment than either single mutant alone , similar to the results we obtained previously  called ST domains  were rep2Δ cells . In this2Δ cells . In cont2Δ cells . We furtcontrols .+fah1 and +grt1 expression, we confirmed downregulation of ST gene expression in gcn5Δmst2Δ cells  wild-type cells. Notably, the proportion of cells with visible knobs and their number per cell was significantly elevated in H3-G34R but not in H3-G34V cells -stained ‘knobs’ . Knob fo4V cells , and as 4V cells . A previ4V cells . Since G4R cells .mst2Δgcn5Δ relative to WT controls  and two wild-type genes  from their endogenous loci , which like the sole copy H3-G34R mutant, grew less well in the presence of HU . We alsoOur finding of the dominance of HU sensitivity of H3-G34R mixed copy strains led us to ask whether the H3-G34R mutation dominantly affected chromosome segregation or HR-mediated DNA repair. In contrast to the chromosome missegregation seen in H3-G34R and H3-G34K sole copy strains, there was no evidence of increased chromosome loss in the mixed H3 backgrounds for either H3-G34K or H3-G34R . There wgrt1 and fah1 transcripts, we validated the repression of subtelomeric genes in both H3-G34R and H3-G34K mixed background strains . We note that the overall numbers of genes that are differentially expressed in the (3xH3) background are reduced compared with the number of differentially regulated genes in the sole copy strains  vs 160 genes in H3-G34R, and 142 genes differentially expressed in H3-G34K (3xH3) vs 223 in H3-G34K when using FDR 5% and FC of 1.5), suggestive that the presence of wild-type H3 partially overrides some transcriptional effects of the mutants. To determine whether the mutations exert dominant effects on expression of particular genes, we looked for overlap between genes that are differentially expressed in the single copy and mixed copy strains. Using the more relaxed criteria of p<0.05, we found that the majority of genes influenced by mutation in the (3xH3) background were also differentially regulated in the sole copy backgrounds . Manhatt strains . ExtendiIn this work, we probe the consequence of mutation of Glycine 34 in histone H3 when the mutant provides the only source of H3, and identify phenotypes that dominate when mutants are co-expressed along with wild-type H3. We found a surprising diversity of phenotypes with H3-G34 mutants differentially affecting post-translational modification of nearby K36, DNA damage sensitivity, and transcriptional control.Given that a single enzyme, Set2, mediates all H3K36 methylation in fission yeast, it is intriguing that we only saw a reduction in H3K36me3, and only in some H3-G34 mutant backgrounds. Set2 may be unable to catalyze the di-methyl to tri-methyl transition on H3K36 when H3G34 is substituted by V, R, or K, but in the absence of a direct assay of in vitro H3K36 methylation activity of purified or recombinant Set2 on the mutant histones, this remains speculative. However, consistent with a defect in tri-methylation, a very recent report shows that activity of the H3K36 tri-methylase SETD2 is blocked on nucleosomes bearing H3.3 G34 mutations, whereas NSD2-mediated di-methylation is unaffected , althougleu1 reduces local levels of K36ac and disrupts HR at that locus. Similar explanations have been suggested for local dominant roles of other histone mutants identified in cancer  have reduced H3K36me3, reduction in K36me3 is not causal of the HR defect. However, like H3-G34R mutants, Gcn5 mutants are reduced for H3K36ac, are HU sensitive, and are also defective for HR ; so thesn cancer . UnfortuAnother possibility would be that the unique phenotypes of H3-G34R mutants are due to the generation of a novel site of arginine methylation within the H3 tail. We have used mass spectroscopy to explore this possibility, but found no evidence for methylation of H3-G34R in fission yeast .In contrast to H3-G34R cells, which are not sensitive to γ-IR, both H3-G34V and H3-G34K are sensitive to γ-IR and to IR-mimetics but there was no dominant effect since mixed copy strains were not sensitive to IR mimetics. This may mean that local perturbation of chromatin structure from deposition of the mutant histones is insufficient to yield IR sensitivity in the presence of wild-type H3.gcn5Δ and mst2Δ similarly show repression of ST domains  domains correlating with enhanced silencing of these regions. These mutants also caused chromosome missegregation, but whether these phenotypes are linked is not clear. Cells defective for  domains , and we  domains . In mito domains . One posWhat is clear from many organisms is that recruitment of Shugoshin proteins relies on Bub1 kinase-mediated phosphorylation of histone H2A on S121  . In fissWe note that an interesting functional link between H3 and Shugoshin has also been made in budding yeast, where the homolog of Sgo2, SGO1 has been shown to bind histone H3. Although not reliant on G34 or K36 residues of H3, SGO1 binds a ‘tension-sensing motif’ at residues 42–45 on H3, which is necessary for Sgo1 recruitment to centromeres and for efficient chromosome segregation during mitosis . Intrigucis on the H3.3 tail  media with appropriate supplements (Fission yeast were maintained on rich (YES), or plements . PMG is S. pombe) with 1x FLAG was cloned into pET28a in frame with N-terminal 6x HIS tag by GenScript to generate JP-2587.Codon optimized Gcn5 (E. coli and the cDNAs were synthesized by IDT and were cloned into pCDF duet (Novagen) in frame with an N-terminal 6x HIS tag for bacterial expression: vectors JP-2395 (WT), JP-2489 (G34R), JP-2490 (G34V), and JP-2902 (G34K).Fission yeast histone H3  were codon optimized by use of codon utilization analyzer 2.0 for expression in E. coli by ‘The Histone Source’ at Colorado State University following standard procedures for histone purification (Recombinant pombe histones were purified from fication .S. pombe Gcn5 was purified by the St. Jude protein production core using BL21 (DE3) transformed with JP2587. Four 1 L cultures were grown in LB medium with Kanamycin to an OD600 of about 0.6, then expression was induced with 0.1 mM IPTG and incubation continued for 16 hr at 16°C. Cell pellets were collected by centrifugation. Cells were suspended in 50 mM Tris.Cl pH 7.9; 500 mM NaCl, 10% glycerol with complete EDTA-free protease inhibitors (Roche) and lysed by two passages through a microfluidizer (Microfluidics Inc). After clarification by centrifugation, the protein was applied to a 1 mL HisTrap column  and Gcn5 eluted with a gradient of imidazole. Proteins were visualized by SDS-PAGE and fractions containing Gcn5 were pooled and dialyzed against 20 mM Tris.Cl, pH 8.0; 200 mM NaCl; 10% glycerol, and 1 mM TCEP.Recombinant 6 cells/mL in 4× YES media and grown at 25°C to a density of 3.6 × 107 cells/mL and harvested by spinning at 3000 rpm for 5 min. Cells were washed with H2O containing 10 mM sodium butyrate, followed by NIB buffer . The pellet was frozen on dry ice and stored at −80°C. For lysis, the pellet was resuspended in 3 mL of NIB and transferred to two 7-mL bead beater tubes along with chilled acid-washed glass beads. Samples were cooled on ice and bead beaten twice for 2 ½ min at max power with 5 min on ice in between. The sample was collected by ‘piggy backing’ into a 50-mL Oak Ridge tube at 3000 rpm at 4°C in a benchtop centrifuge. The sample was then pelleted by centrifuging at 13,000 rpm for 10 min at 4°C in a Beckman Avanti centrifuge J-30I centrifuge using a JA25.50 rotor. The pellet was washed in 15 mL of NIB before resuspension in 10 mL of 0.4N H2SO4, sonication for 45 s at max power and incubation for 2 hr on a rotating wheel at 4°C. The sample was pelleted by centrifuging at 13,000 rpm at 4°C for 10 min and the supernatant was transferred to a new tube along with 5 mL of 5% buffer G (5% guanidine HCl and 100 mM potassium phosphate buffer pH 6.8) where the pH was adjusted to 6.8 using 5N KOH. 0.5 mL of Bio-Rex pre-equilibrated in 5% buffer G was added to the sample and incubated at room temperature with rotation overnight. The resin was then washed twice with 20 mL of 5% buffer G and incubated with 3 mL of 40% buffer G (40% guanidine HCl and 100 mM potassium phosphate buffer pH 6.8) for 1 hr at room temperature to elute the bound protein. Buffer exchange and concentration was performed against 5% acetonitrile with 0.1% TFA to a final volume of 150 µL and the sample was stored at −80°C. Protein concentration was measured and 10 µg of sample was electrophoresed and stained with coomassie blue for quality control.Histones were purified from fission yeast following a previously described purification protocol with some modification . A 150 mXenopus are reported in Xenopus peptides and pombe H3G34R have been previously reported  is calculated as:sI is the intensity of a specific peptide state and pI is the intensity of any state of that peptide.Each acetylated and/or propionylated peak was identified by retention time and specific transitions. The resulting peak integration was conducted using Xcalibur software . The fraction of a specific peptide , recombinant S. pombe H3 and mutants were mixed with recombinant Gcn5 in a reaction buffer containing 100 mM ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37°C. Experiments were performed utilizing excess S. pombe histone H3 , excess acetyl-CoA (200 µM), in the presence of S. pombe Gcn5 (1 µM). Reactions were quenched at time intervals  with 3x volume TCA and boiled at 95°C for 5 min. Samples were analyzed via UPLC-MS/MS analysis, as described in the section 'Acetylation mass spectrometric analyses: UPLC-MS/MS analysis'. The complete experiment was performed three times.To determine the activity of ade6-216 allele, which can complement the ade6-210 allele present within the strain background. Loss of Ch16 causes loss of complementation of function of +ade6 and accumulation of red pigment when cells are grown on limiting adenine media. Strains containing Ch16 were grown in PMG–Leu (to maintain Ch16) at 32°C to a density of 5 × 106 cells/mL. Cells were diluted in PMG (no additives) to a final concentration of 5−10 × 103 cells/mL. Cells were plated onto PMG agar supplemented with amino acids and nucleobases with limiting  adenine, incubated at 25°C for 5 days and then transferred to 4°C to let red color develop. Ch16 loss frequencies were calculated by counting half-sectored colonies (and those with >50% red but <100% red) and dividing by the total number of white, white sectored, and less than half red colonies, excluding red colonies from the analysis as they have lost the minichromosome before plating. Results represent data from multiple (2-4) independent cultures of cells, and 5−10 × 103 colonies were scored for each strain. Samples were assigned a code so identity was masked for this experiment.Minichromosome loss. The minichromosome (Ch16)  bears anLagging chromosome analysis. Chromosome mis-segregation frequencies were obtained as previously described . Over 40Denatured extracts in 2× SB. Whole cell extracts (WCE) were made as published previously .6 cells/mL at 30°C in YES. Cells were collected by spinning at 3000 rpm for 3 min and washed 3× with 1 mL of ice-cold NIB (described above). After washing, the supernatant was discarded, and the final chromatin pellet was resuspended in 1 mL of NIB. Five hundred microliters of this material was mixed with 500 µL of 4× SDS sample buffer and heated at 98°C for 10 min; 10–20 µL was loaded onto a 12% SDS-PAGE gel for analysis.Fifty milliliters of cells were grown to a density of 6 × 10Anti-H3 . Anti-H4 (millipore 05–858 lot no. 2020541). Anti-FLAG M2 monoclonal (Sigma F1804), anti-tubulin (TAT1 kind gift from Keith Gull) , Abnova (PAB31320), Rockland (600–401-I89), and Thermo Fischer (MA5-24672).th Gull) . Anti-H3Peptide sequences used for assessment of K36 methyl Abs are listed in Slot blots used a twofold serial dilution series with 150 µL of 50, 25, 12.5, 6.25, 3.125, and 1.6 µM peptides spotted on activated PVDF 0.2-micron membrane using a 48-well BioRad Bio-Dot SF apparatus. Spots were air dried for 5 min before staining with Ponceau S stain to verify equal peptide loading. The membrane was blocked in 5% BSA in TBST at RT for 1 hr, incubated with primary Ab for 1 hr at room temperature, washed with TBST, incubated with HRP-conjugated anti-rabbit secondary Ab for 30 min, washed with TBST and then developed with enhanced chemiluminescence and images captured by LI-COR imaging. Anti-H3 K36 methylation Abs used are listed above in "Antibodies used".All western blot quantification was done using LI-COR Image Studio software.+act1 or +clr4 loci as a ratio of signal from input DNA; or Sgo2-FLAG association with subtelomeric regions, or centromeric regions relative to +.act1 Primer sequences used for q-PCR are listed in nda3-KM311 background used asynchronous cells grown at 32°C or cells blocked in prometaphase by 8 hr incubation at 18°C.ChIP assays were performed similarly to 6 cells/mL. The cells were pelleted by centrifugation and washed in DEPC H2O. The pellet was resuspended in 750 µL TES Buffer  along with an equal volume of 5:1 phenol:chloroform pH 4.7 and incubated at 65°C for 1 hr with vortexing for 10 s every 10 min. The samples were then cooled on ice and centrifuged for 5 min at 13,000 rpm. The aqueous phase was transferred to a 2-mL phase lock tube and an additional phenol:chloroform extraction was performed. After centrifugation, the aqueous phase was transferred to a new tube and re-extracted using an equal volume of chloroform. To precipitate the RNA, the aqueous phase was transferred to a 2-mL microcentrifuge tube and three volumes of ice cold ethanol and 1/10th volume 3M NaOAc pH 5.2 were added and the samples were kept at −20°C overnight. The next day, samples were centrifuged at 14,000 rpm and 4°C for 15 min to pellet the RNA. The pellet was washed with ice cold 70% ethanol and air dried for 30 min. A Turbo DNAse (Ambion) reaction was set up with 100 µg of RNA in a 150 uL reaction containing 5 uL of Turbo DNAse. The reaction was incubated at 37°C for 30 min and another 5 µL of Turbo DNAse was added and incubated for an additional 30 min prior to the removal of the DNAse using 50 uL of inactivation beads. An RNeasy Mini kit (Qiagen) was used to further clean up and concentrate the RNA, which was eluted in a final volume of 30 µL of DEPC H2O. Five micrograms of total RNA was diluted to 10 µL, and 2 µL of a 1:20 dilution of ERCC RNA Spike-in mix (Invitrogen) was added to each sample.Hot phenol extraction was used to prepare the RNA ; 25 mL cS. pombe (v2.29) genome using STAR (v2.5.3a)  following manufacturer instructions (Illumina). RNA was quantified using a Quant-iT assay (Life Technology). The quality was checked by 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or LabChip RNA Pico Sensitivity assay (Perkin Elmer) before library generation. Libraries were prepared from 2 µg of RNA. Libraries were prepared using the TruSeq Strand Total RNA Library Prep Kit, beginning at Elution 2 – Fragment – Prime step immediately preceding cDNA synthesis according to the manufacturer's instructions (Illumina) with the following modifications the 94°C Elution 2 – Fragment – Prime incubation was reduced to 5 min and the PCR was reduced to 11 cycles. Libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) or Kapa Library Quantification kit (Kapa Biosystems). One hundred cycle paired end sequencing was performed on an Illumina HiSeq 4000 (single copy strains) or on an Illumina Novaseq 6000 (triple copy strains). Three biological replicates were used for each strain analyzed. The total RNA was sequenced using stranded protocol with 2 × 100 bp setting. The paired end reads were mapped to v2.5.3a) . Reads cv2.5.3a) . Raw couv2.5.3a) . Externa2 machine. Transcript levels were normalized to +adh1 or +act1 transcripts.Real-time q-PCR was performed on random primed cDNA generated from two independent RNA preps for each strain as previously described  using prS. pombe cells were cultured in liquid minimum medium with supplements (EMM2 5S) at 26°C with shaking to the early logarithmic phase. Cells were pelleted by gentle centrifugation, and chemically fixed by re-suspending in a buffer containing 4% formaldehyde , 80 mM HEPES-K, 35 mM HEPES-Na, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, and 15 mM 2-mercaptoethanol, pH 7.0. After fixation for 10 min at room temperature, cells were washed with PEMS  three times, then digested with 0.6 mg/mL zymolyase 100T  in PEMS at 36°C for 5 min. Next, cells were treated with 0.1% Triton X-100 in PEMS for 5 min and washed three times thereafter with PEMS. Cells were incubated with 0.2 µg/mL DAPI in PEMS for 10 min, and then cells were resuspended with nPG-Glycerol  diluted with 1:1 with PEM (PEMS without sorbitol) and mounted on a clean 18 × 18 mm coverslip . The slides were observed with 3D-SIM using a DeltaVision|OMX microscope version two or SR  equipped with a 100x UPlanSApo NA1.35 silicone immersion objective or 60X UPlanApo NA1.42 oil immersion objective lens . Reconstruction of 3D-SIM was performed by softWoRx  with Wiener filter constants of 0.003 using a homemade optical transfer function. Conspicuously condensed, DAPI-stained bodies were counted as knobs by visually inspecting each optical section of 3D-SIM images. Experiments were performed with three biological replicates for each strain, and a cumulative total of 189–293 nuclei were examined for each strain. Error bars represent the SEM.Knobs were observed as described previously . The S. 4 cells in spot 1. Plates were incubated at indicated temperatures. For chronic exposure assay, a fivefold serial dilution was spotted onto PMG complete agar plates ± HU, on YES agar plates with DMSO or DMSO and CPT, and on YES agar plates ± MMS. Plates were photographed after 4–5 days incubation at 30°C (CPT and MMS), or 6–7 days at 25°C (HU). All experiments were repeated at least twice.Fivefold serial dilution assays were performed using exponentially growing cells and were spotted on agar plates using a plate replicator with 2 × 106 cells/mL were irradiated using a Cobalt source, and 100 µL samples were taken following increments of 200 Gy exposure (Cells at a density of 5 × 10exposure . Cells wleu1-32 mutant cells (that bear a single nucleotide mutation in leu1 that renders cells auxotrophic for leucine) with a fragment of wild type +leu1 (amplified using JPO 183/JPO 3480) and scored +leu1 transformants that can only arise by HR of the wild-type leu1 fragment into the mutant allele (+leu1 plasmid DNA (JP1050). The experiment was performed 3–4 times, with error bars representing SEM. Strain identities were masked.We transformed t allele . The ratStatistical analyses were performed in GraphPad Prism version 7.03, using ordinary one-way ANOVA or repeated measures ANOVA and Dunnett’s multiple comparisons test. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This article uses yeast to examine the phenotypes of Gly 34 mutations in the histone H3 amino terminal tail. These mutations are found in human cancers. Surprisingly mutation of Gly 34 to different amino acids gives rise to different phenotypes. This suggest that these different mutations are likely to have different effects in cancer cells as well.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Jerry L Workman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor.Thank you for submitting your article "Surprising Phenotypic Diversity of Cancer-associated mutations of Gly 34 in the Histone H3 tail" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Summary:This is an exhaustive study of different phenotypes associated with Histone H3-G34 mutations in a fission yeast model. Because mutations at this site occur in certain human cancers, teasing apart their different phenotypes in a model system helps to understand their potential effects in pathology. The phenotypes vary widely, suggesting a key role for this residue in a variety of genome maintenance functions.Essential revisions:This is overall a technically very well done paper with a variety of methods to examine different mutations in H3-G34. The strength is the consistent approach applied to numerous mutations. However, as there is no single response, it's rather descriptive overall. We have no major concerns about the data, but feel that the conclusions need to be tempered in two areas where the assays were not direct.1) In the absence of NHEJ repair assays it needs to be noted that conclusions about NHEJ proficiency based on drug sensitivity are indirect.2) The authors imply that the H3G34 mutants affect the activity of the Set2 H3K36 methyltransferase. In the absence of an in vitro H3K36 methylation assay on the mutant histones with recombinant or affinity purified Set2 the authors need to note that this conclusion is speculative as they have not measured it directly. Essential revisions:This is overall a technically very well done paper with a variety of methods to examine different mutations in H3-G34. The strength is the consistent approach applied to numerous mutations. However, as there is no single response, it's rather descriptive overall. We have no major concerns about the data, but feel that the conclusions need to be tempered in two areas where the assays were not direct.1) In the absence of NHEJ repair assays it needs to be noted that conclusions about NHEJ proficiency based on drug sensitivity are indirect.2) The authors imply that the H3G34 mutants affect the activity of the Set2 H3K36 methyltransferase. In the absence of an in vitro H3K36 methylation assay on the mutant histones with recombinant or affinity purified Set2 the authors need to note that this conclusion is speculative as they have not measured it directly.As requested, we have tempered the language in respect to the conclusions drawn about NHEJ proficiency, and about Set2 activity (Results and Discussion respectively). These modifications have been tracked along with inclusion of a new grant funding Atsushi Matsuda, an addition to the Materials and methods section and inclusion of the key resource table. I hope it is acceptable to keep separate tables for the fission yeast strain list (Supplementary file 1) and list of peptides used (Supplementary file 2).


Crassostrea gigas, are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively “µvars”), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of C. gigas juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower  than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 108). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.Pacific oysters,  Aquaculture provides a substantial contribution to food production throughout the world. Farming of aquatic species contributed over 80 million tons of food in 2017, resulting in a revenue of ~USD 230 billion globally . DistincCrassostrea gigas) are among the top 30 principal aquaculture species with a total revenue of ~USD 1 billion in 2017 [C. gigas sales accumulated USD 89 million and contributed to 31% of total oyster sales in 2018 [C. gigas in France [C. gigas (60–100%) in New Zealand hatcheries [Globally, Pacific oysters  and MSX disease (caused by Haplosporidium nelsoni) have both historically devastated Eastern oyster (C. virginica) aquaculture in the US [Breeding oyster lines for resistance to pathogens has become a useful tool to mitigate losses . Dermo dn the US . Selectin the US ,38. Thisn the US ,41,42,43n the US ,17. HoweObservational data indicate that the OsHV-1 µvar may have increased virulence compared to the reference OsHV-1  and thatC. gigas families for yield and survival in estuaries along the West Coast since 1996, with seven generations of oyster families produced so far [C. gigas juveniles to three variants of OsHV-1: a California reference OsHV-1 (OsHV-1 TB), an Australian µvar (AUS µvar), and a French µvar (FRA µvar). The AUS µvar and FRA µvar isolates used in this study are the same as those used in previous comparative studies [The Molluscan Broodstock Program (MBP) through Oregon State University has been selecting d so far . More red so far . Of thes studies ,45.8, 1.09 × 107, and 7.67 × 106 total copies of OsHV-1 DNA for the FRA µvar, the AUS µvar, and OsHV-1 TB, respectively. Consequently, oysters exposed to the French µvar were exposed to 23 and 33 times more OsHV-1 viral DNA copies than the AUS µvar and OsHV-1 TB, respectively, on day zero.Groups of sub-adult oysters were injected with one of the three OsHV-1 variants and incubated to allow viral shedding for 24 h, which resulted in varied concentrations of OsHV-1 for each inoculum. For all three treatments, each petri dish was inoculated with 60 mL of corresponding exposed seawater (from the sub-adult oysters), resulting in exposure to 2.51 × 102 = 1295, df = 3, p < 0.0001). Family 60 had higher mortality in the controls than when exposed to OsHV-1 TB (7.4% higher), and therefore was removed from further analysis (see p > 0.05). The survival probability for oysters exposed to the FRA µvar (42.6 ± 1.5%) or the AUS µvar (71.3 ± 1.4%) was lower than those exposed to OsHV-1 TB and the controls (p < 0.0001). Oysters exposed to the FRA µvar had lower survival probability than those exposed to the AUS µvar (p < 0.01). Hazard ratios indicated that exposure to the FRA µvar or AUS µvar increased risk of mortality compared to the controls, while exposure to OsHV-1 TB did not increase risk of mortality (p < 0.05) and similar to each other . The survival probability of family 11 (88.8 ± 2.5%) was greater than all other families (p < 0.05), followed by family 67 . The survival probability of family 53 (63.0 ± 3.8%) was greater than family 5 , and the survival probability of family 74 (54.6 ± 3.9%) did not significantly differ from family 53 or family 5 (p > 0.05).Subsets of high-performing  and low-performing  families based on a previous study  were cho2 = 77.6, 21.4, 63.3, 52.7, 103, respectively, df = 3, p < 0.0001). For each individual family, survival probability when exposed to OsHV-1 TB did not significantly differ from the controls (p > 0.05), except for family 53, which had a higher survival probability when exposed to OsHV-1 TB than the controls (p < 0.001). However, each oyster family experienced lower survival probability when exposed to either the FRA or AUS µvar (9.3–100% lower) relative to the controls and OsHV-1 TB (p < 0.01), except Family 11, where survival probability when exposed to the AUS µvar or OsHV-1 TB was equivalent (p > 0.05). For families 5 and 67, exposure to the FRA µvar caused 40% and 21% lower survival probabilities than exposure to the AUS µvar, respectively (p < 0.05). Family 11, 74, and 53 all had equal survival probability when exposed to either µvar (p > 0.05).Individual survival probability of each of these five families varied depending on the virus to which they were exposed (p = 0.03) and the FRA µvar . Oyster survival also correlated between the AUS µvar and the FRA µvar . The survival of oysters exposed to OsHV-1 TB in the present study also correlated with survival to OsHV-1 TB in the field  and survival to the same FRA µvar in another laboratory study  by Divilov et al. [p > 0.05) by Divilov et al. [Survival of all 20 oyster families was positively correlated among all viruses. The survival of oysters correlated between those exposed to OsHV-1 TB and both the AUS µvar , the AUS µvar (n = 20), and the FRA µvar (n = 22) were (mean ± SE) 3.83 × 107 ± 3.04 × 107, 2.11 × 107 ± 3.51 × 106, and 1.22 × 107 ± 2.32 × 106 copies per oyster, respectively. These loads are equivalent to a 5.00 and 1.94 times increase in OsHV-1 loads per dead oyster relative to original inoculum added for OsHV-1 TB and the AUS µvar, respectively, and a 20.57 times decrease from the original FRA µvar inoculum.Subsets of high-performing  and low-performing  families based on a previous study  were chop < 0.0001; all comparisons) compared to those exposed to OsHV-1 TB and controls, which were similar . Higher copy numbers were detected in oysters exposed to the FRA µvar compared to the AUS µvar . OsHV-1 infection in live oysters did not differ among families  or between days . Of these five families, live oysters exposed to either the FRA or AUS µvar accumulated higher OsHV-1 viral copy numbers (p < 0.0001) and OsHV-1 TB (p < 0.0001); the AUS µvar and OsHV-1 TB were similar on Day 2 (p < 0.05). On day four, the OsHV-1 DNA quantified in the water of oysters exposed to FRA and AUS µvar did not differ (p < 0.05), but water for families exposed to OsHV-1 TB had 52,500 and 11,100 times fewer viral copies than FRA and AUS µvar (p < 0.0001). When averaged across all viruses, increased copy numbers were detected in the water of Family 5 as compared to Family 11 (0.05), Family 53 (p < 0.01), Family 74 (p < 0.05); no other significant differences were detected between families.The same subset of five families was used to investigate the concentration of OsHV-1 DNA in petri dish water. The presence of OsHV-1 in water of oysters exposed to the FRA and AUS µvar on days four and six indicated continued viral shedding, as opposed to water of oysters exposed to OsHV-1 TB, which greatly decreased in OsHV-1 DNA concentration on day 6. The best-fit linear model included a fixed effect for family, virus, day, and the interaction for virus by day . On day This is the first study to directly compare the survival and infection of Pacific oysters to OsHV-1 µvars with a non-µvar, OsHV-1 TB, using experimental infections in the laboratory. Exposure of oysters to either the FRA µvar or the AUS µvar caused significantly lower survival probabilities, higher cumulative mortality, and increased the risk of death compared to exposure to OsHV-1 TB. The survival probability of oysters exposed to OsHV-1 TB did not differ from the controls. Low levels of OsHV-1 were detected in some control plates. A routine health exam using a random sample of the sourced juvenile oysters found no detectable OsHV-1 DNA via PCR see , and the7 copies/µl of OsHV-1 in dead animals) and cause mortality  of susceptible sub-adult oysters over four days [7 ± 3.04 × 107). Taken together, this suggests that the viral inoculum used was virulent, but did not cause infection in all oysters. It is possible that the initial viral dosage was not high enough to cause infection. Alternatively, the families tested may be less susceptible to OsHV-1 TB, supported by the observed concordance in survival between oysters in the present study and those exposed to OsHV-1 TB in the field (rho = 0.50) by Divilov et al. [Prior to the emergence of OsHV-1 µvar, laboratory transmission of OsHV-1 to juvenile oysters was never achieved. Transmission of OsHV-1 between larval oysters was well established ,49, and our days ; no othev et al. . AlthougSurvival to a pathogen can occur through three mechanisms: inability to become infected (decreased susceptibility), the ability to control a pathogen once infected (increased resistance), or a lack of disease despite infection (increased tolerance) ,53. The 8 ± 1.63 × 108 copies per oyster (mean ± se), or ~35–70 million copies per 5–10 mg wet weight, which is similar to susceptible lines in other studies [7 ± 2.13 × 106 copies per oyster; ~1.5–3 million copies per 5–10 mg we weight). The threshold of increased risk of death from OsHV-1 µvar infection is considered 8.8 × 103 copies/mg of oyster tissue [5 copies/mg of tissue, the OsHV-1 µvar is considered the principal cause of death [5 copies per mg threshold. Thus, it is possible that a longer experimental time in this study would have resulted in increased mortality in these surviving individuals with high copy numbers [Oyster families in the present study that appeared to be “resistant” based on potential high survival to the FRA or AUS µvar still accumulated high amounts of OsHV-1 DNA in live tissue. Due to the small size of oysters in this study, we are able to present our data in OsHV-1 copies per oyster with additional data on copies/ng of DNA see . Based o studies ,41,44,47r tissue , and wheof death . Concent numbers . These rMany oyster families varied in survival depending on which variant they were exposed to, and high survival in relation to one variant did not always result in high survival in relation to the other two. These results demonstrate the importance of testing oyster families to multiple variants of OsHV-1 prior to determining resistance. Oyster families 11, 40, 46, and 72 did show high survival (>65%) when exposed to all three variants . In previous studies, oyster lines resilient to a French OsHV-1 reference variant have been shown to also display resistance to OsHV-1 µvars ,41. Thes8, 1.09 × 107, and 7.67 × 106 total copies of the FRA µvar, the AUS µvar, and OsHV-1 TB, respectively). Each virus was allowed to incubate in donor oysters for as long as possible; however, due to time constraints, the AUS µvar and OsHV-1 TB did not incubate long enough to shed the same amount of virus as the FRA µvar. The inoculum for the FRA µvar contained the highest viral dose, suggesting that donor juvenile oysters shed this variant at a faster rate than the AUS µvar, which in turn was shed faster than OsHV-1 TB. Additionally, the concentration of OsHV-1 in petri dish water on day four of the experiment was similar for oysters exposed to the FRA and AUS µvar, despite differences in initial viral doses and greater viral loads in the tissue of live juvenile oysters exposed to the FRA µvar. By day four, less oysters were in the petri dishes exposed to the OsHV-1 FRA µvar  indicating that fewer oysters contributed similar copy numbers to the water. Viral shedding is an important mechanism for viral spread and key in understanding the pathogenesis of each variant: in other aquatic herpesviruses such as channel catfish virus (CCV), koi herpesvirus (KHV), and salmonid herpesvirus-3 (EEDV), viral shedding is key to dispersal [Our results also highlight key questions regarding the pathogenesis of different OsHV-1 variants that will help improve future research and better inform breeding programs. Variations in viral shedding rate likely led to different initial viral doses for each treatment  until 24 h before trials began.Juvenile oysters (Sub-adult oysters to be used as donor oysters for OsHV-1 inoculum (~20 mm) were shipped overnight (25 June 2018) on ice from Hog Island Oyster Company, Humboldt Bay, California: an area known to be free of OsHV-1 . The oysExperiments were conducted with permission from the Arizona state veterinarian and the United States Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS). The inocula were created following methods of Kirkland et al.  and heldC. gigas via injection into the adductor muscle of 1 × 106 copies of either FRA µvar, AUS µvar, or OsHV-1 TB (These inocula were then used to infect 20 sub-adult diploid sHV-1 TB . InjectisHV-1 TB . n = 20) were placed into petri dishes for each of 20 C. gigas families  was extracted using the Zymo Quick DNA Miniprep Plus Kit following the solid tissue protocol. ® 7500 Fast Real-Time PCR System. The 20 µL reaction consisted of 10 µL of SYBR Green Master Mix, 15 µg of BSA, 5.9 µL of PCR water, 400 nM of forward and reverse primer, and 2 µL of sample DNA. The thermocycler protocol consisted of an initial denaturation step at 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s, and a final melt curve analysis consisting of continuous fluorescence monitoring during a 20 min temperature ramp to 95 °C that was held for 15 s. All qPCRs included a standard curve generated from dilution of a plasmid constructed in our laboratory from 3 × 107 down to 3 copies per reaction, the limit of detection for this assay [™ PicoGreen dsDNA Assay Kit (Molecular Probes) with fluorescent intensity  measured using a Molecular Devices FilterMax F5 microplate reader with SoftMax Pro 64 software . An OsHV-1-specific qPCR test was run on all extracted homogenates, inocula, oyster tissues, and water samples to quantify total OsHV-1 viral gene copy numbers as a proxy for viral infection. The qPCR protocol followed methods outlined in Burge and Friedman , targetiC. gigas using the primers CG 18S F (5′-CAG CGA AAG CAT TTG CCA AG-3′) and CG 18S R (5′CAC CCA CCG AAT CAA GAA AGA G 3-’) [Oyster tissue samples that were negative for OsHV-1 DNA were run in a second qPCR to ensure the presence of amplifiable DNA by amplifying the 18S gene of A G 3-’) . The masAll statistical analyses were performed using R version 4.0.2  . An alph10 (x + 1) transformed to normalize them prior to analysis. The package MuMIn [p < 0.05) followed by a multiple comparison using the Wilcoxon Rank Sum Test with a “holm” probability adjustment [To investigate the effects of treatment on concentrations of OsHV-1 in tissues and water, we used linear models. All combinations of linear models were run to determine which factors contributed to accumulation of qPCR copy number in live oysters or water, with fixed effects for virus, oyster family, day, and their interaction. All qPCR data were logge MuMIn  was usedjustment .Correlations were run between survival data in the present study and the corresponding oyster families exposed to the FRA µvar via bath exposure and OsHV-1 TB in the field by Divilov et al. . CorrelaWe examined the resistance of 20 oyster families to three variants of OsHV-1: a French µvar, an Australian µvar, and a non-µvar from Tomales Bay, California. Importantly, we have defined the desirable breeding trait for disease resistance as increased survival and decreased susceptibility or a lack of viral replication within the host. Our findings highlight the importance of the inclusion of qPCR viral loads as part of breeding programs focused on OsHV-1 resistance to identify tolerant oyster families, as increased survival does not always indicate resistance. OsHV-1 has historically spread through unclear sources. Minimizing the spread within and between species and geographic regions is key to mitigating the impact of this disease, and asymptomatic reservoir populations of oysters threaten the transmission of this virus. Our data demonstrate the importance of testing oyster families against multiple OsHV-1 variants, as high survival in relation to one variant does not always determine high survival in relation to another. Although no family in the present study showed resistance to all three variants, further breeding of oyster families with correlations in survival among multiple variants is promising for the development of multi-variant resistant oysters. Finally, we pose multiple areas of improvement for OsHV-1 research. These include investigations into variant shedding rates and the minimum number of virions needed to induce infection, as well as testing the effects of multiple variants under different environmental conditions and oyster life stages to ensure that laboratory results translates to resistance in the field. As new variants of OsHV-1 continue to emerge and devastate Pacific oyster populations, persistent research in these areas will be crucial to maintaining this economically and ecologically important species.


The material is fabricated using epitaxial growth of an ultrathin layer of gallium nitride on zinc oxide microtetrapods followed by decomposition of sacrificial ZnO and oxidation of GaN which according to the results of X-ray diffraction (XRD) and X-ray photoemission spectroscopy (XPS) characterizations, is transformed gradually in β-Ga2O3 with almost stoichiometric composition. The investigations show that the developed ultra-porous Aerogallox exhibits extremely low reflectivity and high transmissivity in an ultrabroadband electromagnetic spectrum ranging from X-band (8–12 GHz) to several terahertz which opens possibilities for quite new applications of gallium oxide, previously not anticipated.In this paper, fabrication of a new material is reported, the so-called Aero-Ga The materials transparent for a certain electromagnetic bandwidth are key components for many industries such as aeronautic, space, telecommunications, etc. ,2. They β-Ga2O3 semiconductor is studied intensively for power electronics [β-Ga2O3, there are other Ga2O3 polymorphs, all with a small index of refraction (less than 2) [2O3 since only a few results on Ga2O3 transistors are reported  , indicatted , hydrogen chloride  and hydrogen  acquired from Geschaftsbereich Linde Gas, Germany, were used as source materials and carrier gases during the growth process. In the source zone, at high temperature (T = 850 °C) the GaCl is formed as a result of chemical reactions between gaseous HCl and liquid Ga. The gaseous GaCl and NH3 reacted with each other in the react zone, where initially the temperature was kept at 600 °C for 10 min to initiate nucleation of GaN on the surface of ZnO microtetrapods, and then increased up to Tg = 850 °C for 10 min to produce GaN layer. In the process of GaN growth, the HCl, NH3 and H2 flow rates were equal to 15, 600 and 3600 smL/min, respectively. In the process of HVPE growth of GaN, the ZnO sacrificial template is being decomposed due to corrosive atmosphere at high temperature leading to the formation of microtubular structures representing aero-GaN [2O3 or Aerogallox.The technological route for the fabrication of aero-Gaemplates . The ZnOemplates . GaN wasaero-GaN . At the 2O3 thin films crystal structure and phases were investigated using a Bruker AXS D8 Advance X-ray diffractometer  in a standard θ–2θ Bragg–Brentano configuration with a monochromatic Cu Kα1 (λ = 0.15406 Å) radiation. A 40 kV beam voltage and 40 mA beam current were used. For acquisition a linear position-sensitive semiconductor detector  in 0D-mode was used, beam optics were Göbel mirror, 6 mm slit, Soller 2.5°; detector optics were 6 mm slit, Soller 2.5°. Diffraction pattern data were collected between 20° and 50° with step lengths of 0.025°, the sample was measured in powder form. The aero-GaN and aero-Gaν = 1486.6 eV) operating at 150 W . Spectra were recorded from a 100 × 100 µm2 analysis area and at 160 eV (survey) pass energy, whereas core level spectra were recorded using pass energy of 20 eV. Operating pressure was 6 × 10−7 Pa. Prior to the measurement, the surfaces were sputtered using an Ar+ beam operated at 2 keV and 0.84 µA for 2 min. Surface charging was compensated using low energy (~5 eV) electrons and adjusted using the charge balance plate on the instrument. Three different spots were analyzed for each sample. All the spectra were processed with CasaXPS (ver. 2.3.20). Spectra were calibrated setting hydrocarbon C1s at 285.0 eV. The surface composition was evaluated from the survey spectra, after a Tougaard U3-type background subtraction, using relative sensitivity factors provided by the manufacturer. Peak fitting was performed with no preliminary smoothing. Symmetric Gaussian–Lorentzian (70% Gaussian and 30% Lorentzian) product functions were used to approximate the line shapes of the fitting components.The chemical composition at surfaces was studied by means of X-ray Photoemission Spectroscopy, XPS  equipped with a monochromatic Al Kα source  connected to a WR90 waveguide-based set-up suitable for measurements in the X-band  [a = 22.86 mm and b = 10.16 mm. Since the dimensions of the cavity are a bit larger than the aero-Ga2O3 pellet, we made use of a supporting flange to fix the sample in the cavity, as to avoid electromagnetic radiation due to imperfect coupling between the two waveguides, hence affecting the reliability of the performed measurements.For the microwaves and terahertz characterizations of the material, bulk samples were prepared in the form of rectangular pellets (20 mm × 10 mm × 2 mm). The freestanding samples were exposed to electromagnetic radiation. The microwave characterization of the aero-Ga2.4 GHz) . A schem3 of aero-Ga2O3 were investigated. Room-temperature terahertz spectra of complex dielectric permittivity ε*(ν) = ε’(ν) + iε”(ν) were measured in the range ν = 10–100 cm–1 in a quasi-optical arrangement (in an open space with no waveguides used) with the help of commercial time-domain spectrometer TeraView TPS 3000 . The spectra of the real and imaginary parts of dielectric permittivity are determined in the transmission geometry via measurements of the complex transmission coefficient (amplitude and phase) of the plane-parallel samples; standard expressions for electrodynamics of a plane-parallel layer are used [−1 using a standard Fourier-transform spectrometer Bruker Vertex 80v.For terahertz measurements the samples were fixed on metallic holders covering 6 mm aperture, the radiation passing through the sample in free space. Samples with different densities from 70 to 110 mg/cmare used . In addi2O3 (c). The inset pictures are the respective photos of the pellets of 20 mm × 10 mm × 2 mm (L × W × H), where one can easily distinguish the change in color of the material after each technological step .2O3, the mixture phase of GaN and Ga2O3 , (002), (−1−11), (1−11), (202), (600), (1−12) and (−510), respectively. Comparing the different patterns, the qualitative trend of GaN oxidative phase change occurring at different stages of the thermal synthesis processes can be appreciated. While both nitride and oxide phases co-exist after the thermal step at T = 800°C in air for 1 h (both reflection peaks sets for wurtzite GaN and monoclinic Ga2O3 are found in the diffraction pattern), only reflections from the oxide remain after annealing at T = 950°C. Formation of monoclinic Ga2O3 in thin structures was already observed for annealing temperatures as low as 750 °C [nd Ga2O3 b that waro-Ga2O3 c obtaines 750 °C .β-Ga2O3 surface chemical analysis by means of XPS. Survey spectra . The presence of Zn 2p doublet  and transmission (S12/S21) coefficients at/between the two ports, respectively. The measured S-parameters of the Aerogallox sample . One can notice that the reflection coefficient is better than −10 dB all over the band of interest, whereas the insertion loss has a maximum value better than −0.12 dB in both cases without and with aero-Ga2O3. In other words, the presence of the aero-Ga2O3 inside the cavity between the two X-band waveguides does not affect at all the transmission of the microwave signal, which means the aero-Ga2O3 is completely (within our accuracy) electromagnetically transparent in the X-band. In 3 and the high density was 110 mg/cm3. Comparative analyses with aero-GaN samples with the density of 15 mg/cm3 were performed. Broad-band terahertz-infrared transmissivity spectra for two samples with low and high densities are shown in −1 is caused by intensive absorption between 200 and 800 cm−1  do not depend noticeably on the sample density. A few more resonances are observed above 800 cm−1, most intensive at ~3500 cm−1. Origin of observed narrow absorptions seems to be related to the presence of impurities such as hydrogen [Two samples of Aerogallox with different densities were studied—the low density equals to 70 mg/cmhydrogen .2O3 material drastically differ from those of parent bulk Ga2O3 crystal as well as from any other bulk semiconductor. For example, typical semiconductors Ge, GaAs and Si have refractive indexes at 300 GHz n = 3.99, 3.59 and 3.43, respectively [2O3, n ≈ 1.07 . From 2O3 and previously studied aero-GaN [2O3 real permittivity ε′ is dispersionless and imaginary permittivity ε” is small and approaches zero with frequency decrease, both indicating absence of any absorption process, aero-GaN demonstrates strong increase of ε′ and ε” toward low frequencies. The origin of corresponding pronounced absorption is associated with the polarizability of the 3D architecture of mutually interpenetrated GaN aerotetrapods, with the ZnO-GaN interfaces, and finally with dynamics of relatively big complexes of tetrapods. No such contribution exists in the present Aerogallox nanomaterial. Aero-Ga2O3 is characterized by very small imaginary permittivity ε” and dielectric losses tanδ = ε”/ε′ , GaAs (n = 3.59) or silicon (n = 3.43); corresponding reflection coefficients fall in the range 30–36%. Along with high value of transmissivity, this low reflectivity of the material may be beneficial for future applications of Ga2O3.In ectively , while taero-GaN . While i = ε”/ε′ a that ar= n + ik b close t= n + ik b R=[(n−12O3 with nanometer thick walls, and demonstrated that the gallium oxide skeleton is of crystalline β-phase with almost stoichiometric composition. The new nanomaterial is shown to exhibit ultra-low reflectivity and high transparency in an extremely wide range of the electromagnetic spectrum, covering the X-band and THz region, up to 3 THz. The disclosed novel properties of aero-Ga2O3 open possibilities, in premiere, for the use of gallium oxide in IoT applications.We developed a highly porous ultra-lightweight three-dimensional nanoarchitecture consisting of interconnected microtubes of Ga