The second harmonic generation response at the heterointerface is either substantially enhanced or almost entirely quenched by an underlying ferroelectric domain wall depending on its chirality, and can be further tailored by the polar domains. Unlike the extensively studied coupling mechanisms driven by charge, spin, and lattice, the interfacial tailoring effect is solely mediated by the polar symmetry, as well explained via our density functional theory calculations, pointing to a new material strategy for the functional design of nanoscale reconfigurable optical applications.Complex oxide heterointerfaces and van der Waals heterostructures present two versatile but intrinsically different platforms for exploring emergent quantum phenomena and designing new functionalities. The rich opportunity offered by the synergy between these two classes of materials, however, is yet to be charted. Here, we report an unconventional nonlinear optical filtering effect resulting from the interfacial polar alignment between monolayer MoS 2 and a ferroelectric oxide thin film.The heterointerface between complex oxides and van der Waals materials presents a versatile platform for exploring new functionalities. Here, the authors report an unconventional nonlinear optical filtering effect resulting from the interfacial polar alignment between monolayer MoS An even broader spectrum of functional properties can emerge at the heterointerface between these two distinct materials, a territory yet to be fully explored. Like the ferroelectric oxides, monolayer (1L) transition metal dichalcogenides (TMDCs) such as MoS2 are noncentrosymmetric and possess polar axes. The associated functional phenomena, including piezoelectricity16 and polar metal switching17, have drawn substantial research interests recently. When 2D TMDC is interfaced with a ferroelectric gate, the spontaneous ferroelectric polarization offers the unique opportunity to induce nonvolatile charge modulation in the channel20. Combining the polarization doping with nanoscale ferroelectric domain patterning further allows local tuning of the electronic22 and optical properties25 of the vdW channel layer. Beyond the charge-mediated interfacial coupling, synergy between the polar nature of TMDCs and ferroelectric has never been explored to date.The heterointerface between two functional materials presents a powerhouse of various emergent quantum phenomena and novel functionalities. Two notable examples are the complex oxide epitaxial interfaces2 and a neighboring ferroelectric PbZr0.2Ti0.8O3 (PZT) thin film. The heterostructure exhibits either strong enhancement or substantial quenching of the reflected second-harmonic generation (SHG) response at the ferroelectric domain walls (DWs), which reveals the intricate coupling of the polar axis of MoS2 with the chiral rotation of the surface dipole at the DWs, as modeled via our density functional theory (DFT) calculations. Unlike the extensively studied interfacial coupling mechanisms driven by charge, spin, and lattice18, this tailored SHG signal is solely mediated by symmetry, pointing to a widely applicable strategy for achieving designate optical functionalities in noncentrosymmetric materials.In this work, we report an unconventional nonlinear optical filtering effect enabled by the polar symmetry of 1L MoS2–ferroelectric heterostructure. 1L MoS2 flakes were mechanically exfoliated from bulk crystals on Gel-Films and identified via the frequency difference Δ between the 2 was identified on the Gel-Film by polarized SHG measurements  (10 nm) buffer layers serving as the bottom electrode (“Methods”). The PZT films are (001)-oriented with out-of-plane polar axis  and down  DWs are along the [100] ([010]) orientation of PZT. During transfer, the a-axis of MoS2 (zigzag orientation) was aligned with the horizontal DWs ([100] orientation of PZT) (see “Methods” for transfer details). As shown in Fig. 2 top layer does not alter the underneath domain structure. This is not surprising as the PZT film is exposed to the ambient condition prior to the transfer, where the polarized surface-bound charge can be well screened by charged adsorbates27. The MoS2 flake is deposited on top of the domain structure via a dry-transfer approach, which should not affect this surface screening layer on PZT.Figure rum Fig. . The cry2 obtained from the regions on the Pup and Pdown domains, with the corresponding PL mapping shown in Fig. Pup domain exhibits higher PL intensity, narrower peak width, and a reduced ratio between the trion and neutral exciton populations , we observed strong SHG response (~400 nm) from the 1L MoS2 flakes on Gel-Films, which conforms to the rotational symmetry of the lattice  electromagnetic wave propagating along the polar axis  facilitated by the DW. The width of the detected SHG signal is about 300–400 nm, which approaches the diffraction limit at this wavelength and the resolution of the SHG microscope (“Methods”). To determine the orientation of p||, we performed SHG imaging with an analyzer applied at various orientations, i.e., making angle φ = 90°, 45°, and 0° with respect to the incident light polarization (x-axis). As shown in Fig. p|| is residing in a plane normal to the DW, similar to the Néel-type chiral DW31. In bulk PZT, the 180° DWs are known to be at the unit cell scale34, and such chiral DW is not energetically favorable. Continuous rotation of local dipoles, however, can be stabilized at the surface of PZT thin films by depolarization field35, resulting in a net lateral polarization. For both MoS2 on Gel-Films and bare PZT, the SHG signals detected in the transmission mode exhibit qualitatively similar behavior as in the reflection mode  and no analyzer applied. As expected, we observed strong SHG intensity from MoS2 on the uniformly polarized Pup and Pdown domains. Unlike the PL data  DWs , the SHG response is substantially quenched. In Fig. 2 flake shows several cracked regions resulting from the transfer, exposing the bare PZT underneath. The fact that the SHG intensity at the even-numbered DWs is comparable to these regions indicates that the emission from MoS2 is close to be entirely canceled by the presence of these DWs. The tailoring of the reflected SHG signal at the DW is a robust effect and has been observed in multiple 1L MoS2 samples. Similar tuning pattern is also observed on three- and five-layer MoS2 flakes on PZT and is absent in bilayer and four-layer MoS2  studies have revealed a flux-closure polar structure at the surface of the DW in PZT thin films35 and even emergence of polar vortices in PbTiO3/SrTiO3 superlattices7, where theoretical modeling has pointed to the dominant role of the interface contribution to the polar anomaly7.Next, we compared the SHG response of 1L MoSces Fig.  due to t35, considering a triangle-shaped flux-closure domain structure that hosts continuous electric dipole rotations at the surface of a 180° DW  tip  and in-plane (Pin) polarization were calculated via DFT within the local density approximation  inside this triangular area can be decomposed to the in-plane P||  (x-component) and out-of-plane z-component). The net in-plane dipole moment can then be estimated by integrating P|| over the volume of the flux-closure domain VPZT. We thus deduced the in-plane dipole per unit length as:w and depth h of the triangle domain to be 2.5 and 3 nm, respectively, based on the TEM result35, and l is the lateral extension of the DW.To examine the feasibility of the interfacial polar coupling scenario, we exploited a phenomenological model to estimate the net in-plane polarization at the DW based on the TEM result DW Fig. . Compare2, which belongs to the D3h point group. The polar displacement in the unit cell can generate three equal polarizations along those three polar directions, leading to zero net polarization. However, when one of the polar axis is coupled to a neighboring dipole, the rotational symmetry is lifted. Using DFT, we estimated the polarization of MoS2 along one polar direction to be 2 of h1 = 3.11 Å, we obtained the dipole moment per unit length for the area above the flux-closure DW in PZT  DWs, on the other hand, p|| is not coupled to any of the polar axes of MoS2. The SHG responses of PZT and MoS2 remain to be independent, and we only observe the weak SHG from PZT that is filtered by the MoS2 top layer.We then considered the polar property of 1L H-MoSPZT Fig. :2\docume2 and the DW, we created square domains on PZT with different stacking angles (θ) with respect to the same MoS2 top layer. Figure θ = 0°,15°,30°, and 45° relative to x-axis in the clockwise direction. For bare PZT, the SHG response is uniform at all DW regions, independent of their orientations  aligned along x-direction. As shown in Fig. 2 and DW has a clear impact on the reflected SHG intensity, suggesting that the heterointerface acts as an unconventional light polarizer.To further test the proposed scenario based on the interfacial polar coupling between MoSons Fig. . We then2/PZT heterointerface. As the thickness of MoS2 and the depth of the flux-closure region at PZT DW (h) are well below the optical wavelength, the susceptibility tensor (or the contracted d-tensor) of the composite system equals to the sum of the adjacent layers: d-tensors for MoS2 and DW, respectively. For 1L MoS2 with D3h point group symmetry, the second-order d-tensor can be expressed as29:d-tensor can be written as39:39. In our work, the crystallographic axes of PZT coincide with the experimental reference frame , where the [001] orientation of PZT is along the z-axis . The interfacial composite tensors at the Pup and Pdown domains are given by:The net SHG response for each of these DWs can be well modeled using the nonlinear electromagnetic theory, considering the second-order nonlinear optical susceptibility tensors for the MoSxis Fig. . We firsPZT Fig. , with thd-tensors for the four DWs  via a rotation matrix transformation  DWs become appreciable  and odd (Iodd) horizontal DWs, defined as (I − Ieven)/(Iodd − Ieven). It clearly illustrates that the signal contrast is either tailored by the uniformly polarized domains or the DWs in these two detection modes.While bare PZT domains . This is challenging as the PZT films thicker than 50 nm tend to relax the epitaxial strain through forming a-domains32, making the local polarization orientation not well defined. The SHG contrast between the regions on the Pup and Pdown domains, on the other hand, depends sensitively on the light polarization and can reverse the relative strength  via off-axis radio frequency magnetron sputtering. The LSMO layer was deposited at 650 °C in 120 mTorr process gas composed of Ar and O2 (ratio 2:1). We then deposited the PZT layer in situ at 490 °C in 150 mTorr process gas (Ar:O2 = 2:1). The PZT films are c-axis oriented with out-of-plane polar axis  SrTiO2 flakes were mechanically exfoliated on elastomeric films (Gel-Film® WF × 4 1.5 mil from Gel-Pak) from bulk single crystals. Selected flakes were transferred on top of the patterned domain structures using an all-dry transfer technique40. The Gel-Film with exfoliated MoS2 was flipped upside down and anchored with a high-precision XYZ manipulator. The PZT sample was placed on a rotatable hot plate. We then aligned the MoS2 sample with the patterned domains under an optical microscope with submicron precision. The uncertainty of stacking angle θ is 2°–6°. The details of the sample alignment during transfer can be found in Supplementary Note 1L and few-layer MoSk of 1–5 Nm−1, resonant frequency fo of 60–100 kHz). The coercive voltage of the PZT films is about +2 V (−3 V) for the Pup (Pdown) state  at room temperature. An Arx–y plane, which is placed above the 1” diameter stage opening. The incident light was transversely polarized and directed to the sample surface along −z direction, and the excited SHG signals were collected in both reflection (+z) and transmission (−z) geometries by photomultiplier detectors (photomultiplier tubes (PMTs)). Before the SHG signals enter the PMTs, the excitation laser beam was filtered out by an IR cut filter (OD >4 @ 692–1100 nm). A long working distance (WD) condenser (NA 0.8/WD 5.7 mm) was used for the transmission signal collection. In the LSM, the transmission and reflection modes share the same focus plane using the same type of objective lens , so both measurements can be performed simultaneously. The focusing to the MoS2/PZT interface was performed through the reflected mode. The signal was first collected with no analyzer inserted and then with an analyzer inserted in different angles with respect to the polarizer orientation. The analyzer is a Thorlab LPVISE100-A with operating wavelength range of 400–700 nm. The band-pass filter used for SHG imaging is a Semrock FF01-390/40 . The diffraction limit of the excitation laser beam (spot size) was estimated to be λ/2NA = 380 nm. Owing to the second-order nonlinearity of the SHG light, the spatial resolution was estimated to be ~300 nm.The experimental set-up for SHG imaging is shown in Fig. 2. The SHG mapping results were expressed in terms of arbitrary units and were proportional to the actual SHG intensity detected by the PMTs. The intensity level can be directly compared if they were taken at the same laser power.The SHG mapping plots, unless otherwise specified, are the raw data collected by the PMTs without modification. During the SHG measurements, the PMTs in the LSM were set on the photon count mode, so the responses of the PMTs are proportional to the number of actual SHG photons detected by the PMTs but are not calibrated to the light intensity in units of W/mSupplementary InformationPeer Review File


Aspergillus awamori on the growth, intestinal histomorphology, immune parameters and antioxidant enzyme activity of growing rabbits. The rabbits of 5 weeks of age  were allotted into four groups (12 rabbits per group) and fed four experimental diets enriched with A. awamori at 0, 50, 100 and 150 mg per kg diet for 8 weeks. After the feeding trial, an equal number of male APRI rabbits  were housed in metabolism cages and fed the test diets for 14 days for the digestibility trial. All A. awamori‐enriched diets induced a significant increase in the average body weight and weight gain of rabbits and significantly decreased the feed conversion ratio. In rabbits fed with A. awamori at 100 or 150 mg per kg diet, protein, lipid and fibber digestibility coefficients significantly increased compared with the control group. Intestinal villi measurements (length and thickness) were also enhanced in all rabbits fed with A. awamori. Adding A. awamori as a food supplement generally did not affect rabbit haematology and blood biochemistry values; however, at 150 mg per kg diet, it significantly increased the levels of haemoglobin and total protein, as well as red blood cell count. Furthermore, all enriched diets significantly increased rabbits’ phagocytic activity and their phagocytic index. Rabbits fed with A. awamori also showed decreased malondialdehyde but increased catalase activity. In conclusion, A. awamori administered as feed supplement at 100–150 mg per kg of growing rabbits’ diet enhances their growth, intestinal health and nutrient digestibility, and it raises the levels of their immune and antioxidative responses.In this study, we explored the effects of dietary administration of  In this study, we have explored the effect of dietary administration of Aspergillus awamori on growth, intestinal histomorphology, immune parameters and antioxidant enzyme activity of growing rabbits.  Intensified cultures, in which rabbits are reared at high densities, facilitate the spread of pathogens and the occurrence of disease outbreaks due to stressed and immunosuppressed animals  was provided in powder form from Biogenkoji Research Institute, Kirishima, Japan.2.2The experimental procedure was approved by the Faculty of Agriculture, Kafrelsheikh University, Egypt (07–2018). The study was investigated by performing a feeding trial for eight weeks, followed by a digestibility trial for 14 days. In total, 48 APRI line weanling rabbits at 5 weeks of age were obtained from the Animal Production Research Institute, Sakha Station, Kafr El‐Sheikh, Egypt, and divided randomly into four groups . All weanling rabbits were approximately equal in live body weight at the beginning of the experiment (855 ± 5.53 g).A. awamori to pass through a 5‐mm sieve   was daily ground to guarantee the viability of Rabbits with the same weight were kept in batteries (60 × 50 × 35 cm). Feed and water were added ad libitum from 5 to 13 weeks of age via fodder and automatic nipple drinkers. The rabbits were subjected to a 16:8 hr light: dark photoperiod regime. Weight gain and feed conversion ratio parameters were calculated according to the following formulae:Weight gain (g) = W2‐W1, Feed conversion ratio = weight of feed consumed (g)/ body weight gain (g), where W1 is the initial weight and W2 is the final body weight.2.3g for 15 min at 4°C), and then maintained at −20°C prior to biochemical analysis. Total protein, albumin, glucose, catalase (CAT), urea, creatinine and malonaldehyde (MDA) were determined using commercial kits  according to the manufacturer's instructions. Serum activities of aspartate aminotransferase and alanine aminotransferase were determined  of 13 weeks of age were randomly selected, anesthetized   were housed in metabolism cages and fed the test diets for 14 days for the digestibility trial and faeces were collected for 4 days as a collection period  and 100 µl of blood. The tubes were mixed and incubated at 37°C for 30 min, after which the mixture was centrifuged at 112 g for 5 min. The supernatant was discarded leaving a droplet into which the sediment was re‐suspended. Smears were prepared from the deposit, dried in the air, fixed with methyl alcohol and stained with Giemsa stain. In total, 100 heterophils were examined and the number of heterophils ingesting Candida was counted and expressed as a percentage.Phagocytosis of polymorphonuclear cells using n et al. . In a pl2.6For microscopic studies, samples from the small intestines of three rabbits per group were removed immediately after the rabbits had been euthanized by using 10% neutral formalin. After fixation, portions of tissue were dehydrated in increasing ethanol concentrations, cleared in xylene, embedded in paraffin blocks and sectioned to a thickness of ~5 μm. The sections were then deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol. Finally, they were stained with haematoxylin and eosin for histopathological examination  . The feed conversion ratio was decreased in rabbits fed A. awamori at 100 mg/kg followed by the level of 50 mg/kg; however, the differences from rabbits fed 150 mg per kg were not apparent with the other A. awamori‐enriched diets. No significant differences were observed between the feed intake of control rabbits and those fed with enriched diets (p > .05).Rabbits fed with a diet enriched with 5) Table . Further3.2A. awamori‐enriched diets at 100 or 150 mg per kg diet had significantly higher protein, lipid and fibre digestibility coefficients relative to control animals (p < .05) (Table A. awamori supplementation (p > .05). Rabbits fed varying levels of A. awamori also had significantly higher intestinal villi measurements (height and thickness) (P ˂ 0.05)  Table . Digesti3.3A. awamori showed no significant differences with the exception of RBC, haemoglobin, glucose and total protein (Tables A. awamori per kg diet (P ˂ 0.05), as did blood total protein  (Table The blood indices of rabbits fed n Tables  and 5. R3.4A. awamori‐enriched diets presented significantly higher phagocytic activity (p < .05) , but not 50 mg (p > .05), A. awamori per kg diet relative to the control  Figure . Additiol Figure .3.5A. awamori are presented in Figure A. awamori regardless of the dose (P ˂ 0.05).The concentration of MDA and CAT activity of rabbits fed varying levels of 4A. awamori improved the growth, intestinal morphology, immune responses and antioxidative responses of growing rabbits.Probiotics have attracted the attention of nutritionists because of their beneficial influence on animals; they can improve innate immune response, growth and feed digestibility  are unbalanced as the oxidative damage of cultured species is directly related to the quality of diet (Apel & Hirt, y et al.  reported5A. awamori appears to provide benefits to growing rabbits’ performance without any indications of negative physiological impacts. Indeed, the addition of Aspergillus to their diet reduced the damaging effects of oxidative stress and improved the rabbits’ immune status. Thus, A. awamori can be considered as a probiotic candidate for use with growing rabbits. Specifically, we recommend that 100–150 mg A. awamori per kg of rabbit feed is applied to the diet of growing rabbits as a feed supplement.Based on our data on rabbit growth, feed efficiency, intestine histology and nutrient digestibility, The authors have declared no conflict of interest.Mahmoud H. El‐Deep: Conceptualization; Data curation; Formal analysis; Investigation; Methodology. Mahmoud Dawood: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Visualization; Writing‐original draft; Writing‐review & editing. Mohamed H. Assar: Conceptualization; Formal analysis; Investigation; Methodology; Supervision. Bilal Ahamad Paray: Conceptualization; Funding acquisition; Methodology; Visualization; Writing‐original draft; Writing‐review & editing.https://publons.com/publon/10.1002/vms3.345.The peer review history for this article is available at


Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5′-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4 Acute myeloid leukemia (AML) is a clonal disease of hematopoietic precursor cells. Despite some improvements in treatment and outcome parameters over the past decades, the prognosis of the disease is still dismal. High risk of relapse despite initial complete remission is the major reason for poor survival rates. This is due to chemorefractory residual leukemic stem cells remaining after intensive chemotherapy . HematopRIG-I-like receptor ligands have been used as a promising strategy for the treatment of solid malignancies including melanoma , 5, pancRIG-I was first identified as a gene induced by retinoic acid in a promyelocytic leukemia cell line  and its PDX AML cells were established as described previously  and a de6 GFP expressing C1498 AML cells (C1498-GFP) via tail vein injection. ppp-RNA was complexed with in vivo-jetPEI in order to protect it from nuclease digestion and enable cytoplasmic delivery of ppp-RNA. Fifty micrograms of complexed ppp-RNA were administered intravenously on days 3, 7, 10, and 14 after tumor inoculation  vs untreated (n = 5), p = 0.002), lungs (3.6% vs 68.8% GFP+ cells for ppp-RNA treated (n = 3) vs untreated mice (n = 5), p < 0.001), ovaries  vs untreated mice (n = 4), p = 0.004), and spleens (0.03% vs 12.6% GFP+ cells for ppp-RNA treated (n = 3) vs untreated mice (n = 5), p = 0.009).To asses the in vivo efficacy of RIG-I-based immunotherapy of AML we utilized the C1498 model, a murine AML cell line on C57BL/6 background classified as acute myelomonocytic leukemia , which wp < 0.0001 for ppp-RNA treated (n = 16) versus untreated mice (n = 16)), ppp-RNA treatment led to long-term remission in 3 out of 16 (19%) of the treated animals, demonstrating the potential of systemic RIG-I activation in the therapy of AML.We next evaluated the effects of ppp-RNA treatment on the survival of the mice. C1498-GFP AML-bearing mice were treated as described above and animals were sacrificed at signs of disease such as weight loss, ascites, apathy and/or development of visible tumors and survival data were plotted in a Kaplan–Meier survival curve Fig. . Beyond Since the C1498-GFP AML cell line resists transfection with most commercially available RNA transfection reagents in vitro and is thus difficult to target directly with ppp-RNA, it was of great interest to us to narrow down the mechanisms responsible for the observed tumor rejection. In order to discriminate between a direct cytotoxic effect of ppp-RNA on the tumor cells and immune cell-mediated responses, immune incompetent NSG mice lacking T, B, and NK cells were inoculated with C1498-GFP AML cells. ppp-RNA treatment was given on days 3, 6, 9, and 12. Both treated and untreated animals died on day 13 Fig.  and no s+, CD8+ T cells, CD19+ B or NK1.1+ NK cells  and CD4+ T cell (p = 0.018) depletion resulted in significant loss of therapeutic efficacy  and NK1.1 (p = 0.376) depletion remained without significant effect compared to the respective isotype controls . Of note, no long-term survival was observed in Mavs−/− mice in the treated group.In Mavs−/− mice, intact RIG-I signaling via MAVS in the host seems to be essential particularly for repeated IFN induction and long-term survival in ppp-RNA treated animals.The results demonstrate that ppp-RNA induced tumor rejection in this AML model is mediated by, but not limited to effects of type I IFN release. Despite CXCL10 levels being comparable after the first ppp-RNA treatment in WT and n = 7 for AML surviving vs. n = 10 for tumor-naive mice, p < 0.0001; Fig. Next, we evaluated if a long-lasting immunological memory was established in ppp-RNA-treated mice that had survived the AML challenge. Surviving mice were rechallenged with C1498-GFP AML cells on day 85–110 after the first AML inoculation and compared to tumor-inoculated control animals. Survivor mice withstood the AML rechallenge in all cases  an observation not seen in the group treated with both, ppp-RNA and anti-PD-1.In vitro, we observed strong upregulation of PD-L1 on C1498-GFP AML cells upon stimulation with IFN gamma  and as a trend in bone marrow  in ppp-RNA treated compared to untreated mice. Human CD3+ T cells were found at higher numbers in the treatment group in both compartments , suggesting enhanced immune cell expansion after treatment.We approached the potential of ppp-RNA-based immunotherapy for clinical translation by testing a genetically diverse panel of five human AML cell lines  and five patient-derived (PDX) AML blasts  7, which is known to sense single-stranded RNA and may be activated by ppp-RNA. In fact, previous studies show that repeated systemic activation of TLR7 leads to desensitization of the receptor [Mavs−/− mice compared to WT mice . DLI is a procedure that was initially established for relapsed chronic myeloid leukemia after SCT. Recent studies indicate a beneficial effect of DLI in patients suffering from relapsed high-risk AML after SCT , 40. To In summary, ppp-RNA treatment induces an immune cell-mediated response against disseminated AML, leads to long-term survival and establishes an immunological memory protective against leukemia relapse. In addition, treatment with ppp-RNA sensitizes tumors to therapeutic immune checkpoint blockade using an anti-PD-1 antibody.Supplementary figures 1–5Supplementary methodsSupplementary tables


Myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) are the major causes of lethal viral diseases in the European rabbit. In 2010, a new RHDV genotype (RHDV2) emerged in the field that had limited cross-protection with the classical RHDV (RHDV1). For optimal protection of rabbits and preventing spread of disease, a vaccine providing protection against all three key viruses would be ideal. Therefore, a novel trivalent myxoma vectored RHDV vaccine (Nobivac Myxo-RHD PLUS) was developed similar to the existing bivalent myxoma vectored RHDV vaccine Nobivac Myxo-RHD. The new vaccine contains the Myxo-RHDV1 strain already included in Nobivac Myxo-RHD and a similarly produced Myxo-RHDV2 strain. This paper describes several key safety and efficacy studies conducted for European licensing purposes. Nobivac Myxo-RHD PLUS showed to be safe for use in rabbits from five weeks of age onwards, including pregnant rabbits, and did not spread from vaccinated rabbits to in-contact controls. Furthermore, protection to RHDV1 and RHDV2 was demonstrated by challenge, while the serological response to MV was similar to that after vaccination with Nobivac Myxo-RHD. Therefore, routine vaccination with Nobivac Myxo-RHD PLUS can prevent the kept rabbit population from these major viral diseases. Two major viral diseases affecting the European rabbit are myxomatosis and rabbit haemorrhagic disease (RHD). Leporipoxvirus, and originated in the New World. While myxoma virus only causes mild disease in its natural host, the American rabbit, it causes serious disease and mortality in the European rabbit. However, it was not until the 1950s after being deliberately released in Australia and France that the virus became endemic [Myxomatosis is caused by myxoma virus, a member of the poxvirus family, genus  endemic ,3,4. Aft endemic . Besides endemic . During Lagovirus of the Caliciviridae family. RHDV transmission can be direct, via oral, nasal, conjunctival or parenteral routes or indirectly via fomites or insect vectors [RHD is caused by RHD virus (RHDV), a member of the genus  vectors . RHDV sp vectors . RHDV wa vectors . In 2010 vectors . Followi vectors , RHD vir vectors ,8. Furth vectors ,9,10. No vectors .The most effective method of preventing disease due to myxoma virus and RHDV and limiting outbreaks is vaccination. Over time various vaccines have been developed to protect against myxomatosis and RHD. Myxoma virus vaccines are live vaccines based on attenuated myxoma virus strains or the related Shope fibroma virus ,11. RHDVTherefore, a vaccine was developed that would provide protection against myxomatosis and both genotypes of RHDV. This vaccine was based on the existing live recombinant attenuated myxoma virus vectored RHDV1 vaccine, which already has an extensive proven efficacy and safety from years of use in the field. Here, we describe a number of safety and efficacy studies, in both commercial and pet rabbits, with the novel trivalent recombinant attenuated myxoma virus vectored RHDV1 and RHDV2 vaccine Nobivac Myxo-RHD PLUS. This vaccine was licensed in the EU in 2019.Studies were performed in 5-week-old specific pathogen free (SPF) New Zealand White (NZW) rabbits or SPF Dutch Belted (DB) rabbits  and 9-week-old SPF NZW rabbits or pregnant adult SPF NZW rabbits. NZW rabbits were obtained from Envigo, Huntingdon, UK, and DB rabbits were obtained from Covance, Denver, PA, USA. The SPF status of the animals and screening of blood samples at day of vaccination confirmed all animals were seronegative for myxoma virus and rabbit haemorrhagic disease virus (RHDV).These studies were performed in compliance with the Dutch Experiments on Animals Act 2014 with approval of the MSD Animal Health Ethics Review Committees and Dutch Central Authority for Scientific Procedures on Animals  or in compliance with the UK Animals Scientific Procedures Act 1986 . After RHDV challenge rabbits were continuously monitored up to 72 h or until all controls succumbed to be able to immediately euthanize rabbits that showed signs of acute malaise to reduce animal suffering.A newly updated recombinant live vector vaccine was used in the studies: Nobivac Myxo-RHD PLUS . This vaccine is a combination of two live recombinant myxoma-vectored RHD viruses which were constructed as described previously . In shorAll the RHD viruses used for challenge and serology testing were obtained from liver homogenate of RHDV-infected rabbits and titrated in rabbits, as RHDV does not grow in cell culture. As a representative of RHDV1, the RHDV Ascot strain was used, which was obtained from the Veterinary Laboratories Agency, Weybridge, UK . Due to The myxoma virus  used for serology was similar to that previously described .The design of studies 1 and 2 was based on the European Pharmacopoeia criteria for myxomatosis vaccine (live) for rabbits  and for Seventeen 5-week-old SPF NZW rabbits (11 vaccinated rabbits and 6 control rabbits) were used in study 1. Eighteen 5-week-old SPF DB rabbits (12 vaccinated rabbits and 6 control rabbits) were used in study 2. In each study rabbits were randomly divided into 2 groups and control rabbits were co-housed with vaccinated rabbits in cages. 6.8 focus forming units (FFU) per virus strain per rabbit) of Nobivac Myxo-RHD PLUS vaccine reconstituted in 0.5 mL Nobivac Solvent, while the controls were inoculated with 0.5 mL Nobivac Solvent (placebo) . At 21 days post vaccination (d.p.v.) vaccinated rabbits were revaccinated with a single maximum dose (≥105.8 focus forming units (FFU) per virus strain per rabbit) of Nobivac Myxo-RHD PLUS vaccine, while the controls were inoculated with Nobivac Solvent. Before each inoculation, the injection site was shaved and was kept free of fur by repeated shaving to allow better monitoring of local reactions. Clinical monitoring of local reactions occurred daily for 2 weeks after each vaccination. Body temperature was monitored in accordance with the guideline for myxomatosis vaccine indicated above using a transponder . Blood was sampled at day 0, day 21 and day 35 from all rabbits. At the end of the study (day 35) the injection sites were macroscopically examined for local reactions and tissue samples were taken from both injection sites of all vaccinates and 2 randomly selected controls.At day 0 rabbits in the vaccine group were vaccinated subcutaneously (s.c.) with a 10-fold overdose  of Nobivac Myxo-RHD PLUS reconstituted in 0.5 mL Nobivac Solvent and each rabbit of group 3 with 0.5 mL of Nobivac Solvent (placebo). At day 14 (day 21 after mating), each rabbit in group 2 was vaccinated s.c. with a maximum single dose of Nobivac Myxo-RHD PLUS reconstituted in 0.5 mL Nobivac Solvent and each rabbit of group 4 with 0.5 mL of Nobivac Solvent (placebo). Clinical monitoring, including local reactions, occurred daily until the end of study, and body temperature was monitored in accordance with the guideline for evaluation of safety of veterinary vaccines and immunosera indicated above using a transponder. Blood was sampled at day 0 and at parturition date + 1 day (end of study) of the adult rabbits. Following parturition, the numbers of kittens and live kittens and relative size of kittens were recorded.At day 0 of the study (day 7 after mating), each rabbit in group 1 was vaccinated s.c. with a maximum single dose (≥10For the design of studies 4 and 5 the European Pharmacopoeia criteria for rabbit haemorrhagic disease vaccine (inactivated)  was usedSixty SPF NZW rabbits were randomly divided into 8 groups, and each vaccinated group was floor housed together with a control group. Groups 1, 3, 5 and 7 comprised 10 rabbits each, and groups 2, 4, 6 and 8 comprised 5 rabbits each and served as unvaccinated controls. Groups 3 and 4 comprised 9-week-old rabbits and all other groups comprised 5-week-old rabbits at time of vaccination. Groups 3 and 4 comprised older animals because susceptibility to classical RHDV is age dependent. To evaluate vaccine efficacy in 5-week-old animals and an onset of immunity of 3 weeks, both 5- and 9-week-old rabbits had to be used . 3.0 FFU per virus strain per rabbit) of Nobivac Myxo-RHD PLUS vaccine reconstituted in 0.5 mL Nobivac Solvent, while the controls were inoculated with 0.5 mL Nobivac Solvent (placebo). At 3 weeks post vaccination rabbits were challenged with RHDV: groups 3 and 4 with RHDV1 Ascot, groups 5 and 6 with RHDV2 Spain and groups 7 and 8 with RHDV2 Italy2. At 7 weeks post vaccination rabbits in groups 1 and 2 were challenged with RHDV1 Ascot. Monitoring of clinical signs of RHDV infection occurred for 14 days after challenge. Blood was sampled from all rabbits before vaccination and before challenge.Rabbits in groups 1, 3, 5 and 7 were vaccinated s.c. with a single minimum antigen dose  of Nobivac Myxo-RHD PLUS vaccine reconstituted in 0.5 mL Nobivac Solvent, while the controls were inoculated with 0.5 mL Nobivac Solvent (placebo). At 3 weeks post vaccination rabbits were challenged with RHDV: groups 3 and 4 with RHDV1 Ascot and groups 5 and 6 with RHDV2 Italy2. At 7 weeks post vaccination rabbits in groups 1 and 2 were challenged with RHDV1 Ascot. Monitoring of clinical signs of RHDV infection occurred for 14 days after challenge. Blood was sampled from all rabbits before vaccination and before challenge.Rabbits in groups 1, 3 and 5 were vaccinated s.c. with a single minimum antigen dose , 100 µL intranasally (50 µL in each nostril) and 200 µL orally.After optimizing the challenge model in negative control rabbits in order to induce 100% mortality, the following dilutions were used in the studies. For challenge material a 1/50 (for RHDV1 Ascot) or 1/100 (for both RHDV2 strains) dilution of 40% Collected blood samples were transported to the laboratory for serum separation and stored at ≤−15 °C until testing. Tissue samples of injection sites stored in buffered formalin  were transported to the laboratory for histological analysis and stored at ambient temperature until testing.2) was regarded as negative. Antibodies to RHDV were measured using a haemagglutination inhibition (HI) assay as previously described [2) was regarded as negative. Serum was analysed for antibodies to myxoma virus, RHDV1 and RHDV2. Antibodies to myxoma virus were measured using an immunofluorescent test as previously described . A titreescribed ,13, as nAt the end of studies 1 and 2, after necropsy of the rabbits, the injection sites of all vaccinated rabbits and of two randomly selected control rabbits were macroscopically and histologically analysed. Macroscopic analysis included presence, shape and colour of a local reaction, presence of vaccine residues, oedema, consistency of the tissue and abscess formation on the injection site. Tissue samples taken from these injection sites were fixed in buffered formalin, processed and embedded in paraffin wax . Subsequent processing, staining with H&E  and analysis of the samples for abnormalities by a pathologist was performed.For studies 1 and 2 clinical monitoring and gross examination of the injection sites for local reactions was conducted by trained personnel who observed the animals at day −1, 0, 4 h, 7, 8, 10, 13/14, 15/16, 17/18, 21, 21 + 4 h and daily from day 22 until day 35. Observations included swelling of eyelids, nose, anus, mouth, external genitalia and ear base, ocular discharge, abnormal breathing, reduced appetite and abnormal attitude. Body temperature was measured at the same time of day at day −3, −2, −1, 0, 4 h, 1, 2, 3, 4, 21, 21 + 4 h, 22, 23, 24 and 25.For study 3, clinical monitoring was conducted by trained personnel who observed the animals daily until the end of the study. Clinical observation was performed visually to reduce stress to the animal which may affect the course of pregnancy. Observations included ocular discharge, abnormal breathing, reduced appetite and abnormal attitude. Body temperature was measured at the same time of day at day −3, −2, −1 and 0 for all animals, at 4 h, day 1, 2, 3 and 4 for animals in groups 1 and 3 and at 14, 14 + 4 h, 15, 16, 17 and 18 for animals in groups 2 and 4.For studies 4 and 5, clinical monitoring was conducted by trained personnel who observed the animals every two hours from 1 day up to 72 h post challenge or until all control rabbits were euthanized due to clinical signs caused by RHDV challenge, after which clinical observation continued once daily until the end of the study. Observations included abnormal breathing, abnormal attitude, position of the ears and mobility.For study 3, all pregnant rabbits were monitored twice a day starting 5 days before the expected delivery date. Following parturition, the offspring of all rabbits were observed until 1 day after parturition. Observations included duration of gestation, number of does aborting, number of kittens born and, after parturition, the number of live kittens and the relative size of kittens . In addition, any deformities in the kittens were recorded.p < 0.05 is considered significant. As baseline, the body temperature at day 0 per rabbit was used for analysis. In addition, vaccine virus spread to control rabbits housed together with vaccinated rabbits was analysed using a RHDV HI assay. Spreading is considered to have occurred when RHDV1 and RHDV2 HI titres are >4 (log2) in control rabbits.To access vaccine safety, clinical signs (body temperature) between vaccinated and control rabbits were compared based on a repeated measures ANOVA model with the baseline temperature (on day 0 before vaccination) as a covariate in the model. A difference of p < 0.05 is considered significant. Descriptive statistics was used on gestation period, number of does aborting, litter size and number of still births. Results were compared between vaccinated and control rabbits inoculated in the same term of pregnancy.To access vaccine safety during pregnancy, clinical signs (body temperature) were compared based on a repeated measures ANOVA model with the baseline temperature (on day 0 before vaccination) as a covariate in the model. A difference of To access vaccine efficacy against RHDV1 and RHDV2, the mortality data was summarized descriptively using frequency tables, mean, standard deviations and graphs where applicable. The Fisher’s exact test was used to compare the proportion of rabbits that survived the challenge, at 5% level of significance.During clinical monitoring no clinical signs of myxomatosis were observed in any of the rabbits in either safety study (data not shown). Small, transient swellings up to 1.5 cm diameter and lasting up to 7 days were seen at the injection site in many cases following vaccination. These could be slightly larger (up to 2 cm diameter) and more persistent (up to 9 days) following vaccination at 10× overdose. Thickened skin could also be palpated at the majority of injection sites for up to 10 days. Furthermore, following overdose vaccination, a slight swelling of the local lymph node could be palpated for 1 day  compared to the controls. This observed increase in body temperature is considered not biologically relevant and in accordance with the Ph.Eur. monograph for live myxomatosis vaccines. After repeated vaccination, the observed maximum increase was respectively 0.3 °C and 0.2 °C in the vaccinates and controls and no significant difference between vaccinates and controls was observed (p = 0.2040).No significant difference in body temperature between vaccinated and control NZW rabbits was observed . In vaccSerological data showed that all rabbits were seronegative for myxoma virus and RHDV at the start of the study. After vaccination, all vaccinated rabbits were seropositive for both myxoma virus and RHDV. With regard to virus shedding, none of the in-contact control rabbits that were housed together with the vaccinated rabbits became seropositive to either myxoma virus or RHDV Table S4No clinical signs of myxomatosis were observed in any of the rabbits during this study. In addition, no significant difference in body temperature was observed between vaccinated and control animals in both terms of the pregnancy . Analysis of the gestation length and litter size revealed no differences between vaccinates and controls . FurtherSusceptibility to RHDV1 is age dependent while susceptibility to RHDV2 is not. To show an onset of immunity of 3 weeks for RHDV1 and susceptibility to vaccination at 5 weeks, rabbits were therefore vaccinated at two different ages. One group (group 3) was vaccinated at 9 weeks of age and challenged with RHDV1 3 weeks after vaccination, and another group (group 1) was vaccinated at 5 weeks of age and challenged with RHDV1 7 weeks after vaccination. By challenging at 12 rather than 8 weeks of age ensures the animal was susceptible to RHDV1 infection.All rabbits were seronegative for myxoma virus and RHDV at the start of the study. All NZW and DB control rabbits remained seronegative for myxoma virus and RHDV until day of challenge. All, except three, vaccinated NZW rabbits were seropositive for myxoma virus and RHDV after vaccination. The exceptions were one rabbit from group 1  and two rabbits from group 7 . These three rabbits were seronegative to all three vaccine components , and therefore, it was concluded that they had most likely not been correctly vaccinated. All vaccinated DB rabbits were seropositive for myxoma virus and RHDV after vaccination .After RHDV challenges, all NZW and DB controls showed severe clinical signs indicative of RHDV infection and died within 120 h after RHDV challenge, independent of the challenge strain. All vaccinated NZW rabbits showed no clinical signs of RHDV infection and survived the challenge, except the three rabbits that showed no serological response. These three rabbits showed similar clinical signs of RHDV infection as the unvaccinated controls and died within 120 h after RHDV challenge. All vaccinated DB rabbits showed no clinical signs of RHDV infection and survived the challenge. Statistical analysis showed a significant difference in mortality after RHDV challenge between vaccinated and control rabbits within the same challenge group for all challenge groups for both NZW and DB rabbits .Two major viral diseases affecting the rabbit population are RHD and myxomatosis. Since 2010, a new RHDV genotype has emerged (RHDV2), which at first was not as pathogenic as classical RHDV (RHDV1) but was able to affect both kittens and adults. However, over time the pathogenicity of RHDV2 viruses increased matching that of RHDV1, and nowadays, RHDV2 is replacing RHDV1 in the field ,8,10. AsHere, we demonstrate a novel trivalent myxoma virus vectored RHDV vaccine providing protection against disease caused by myxoma virus and both genotypes of RHDV that is produced using in vitro culturing. For this purpose the commercial vaccine strain of Nobivac Myxo-RHD (Myxo-RHD1) was used, because it has an extensive proven safety profile and efficacy profile against myxomatosis and RHD1 from years of use in the field, provides protection for a full 12 months following one initial dose and can be produced using in vitro culture. The Myxo-RHDV1 strain is composed of an attenuated strain of myxoma virus in which the VP60 gene of RHDV1 is inserted using homologous recombination. As a consequence of this insertion, two myxoma virus genes playing a role in viral virulence (MGF and ML11) have been removed, resulting in further attenuation of the myxoma virus ,19 and, As there has been discussion as to whether safety and efficacy results in a laboratory breed rabbit (NZW) is representative for a pet breed rabbit, a vaccine safety study and vaccine efficacy study were done in both a laboratory breed and pet breed rabbit. There were no differences in results between NZW and DB rabbits in either the vaccine safety or efficacy study using Nobivac Myxo-RHD PLUS. From this we can conclude that for Nobivac Myxo-RHD PLUS both breeds are suitable to prove vaccine safety and efficacy.In line with EU guidelines vaccine safety was assessed by vaccinating rabbits with a 10-fold overdose and revaccinating with a single maximum commercial dose 3 weeks later. Minor local reactions (which were visible because the injection sites were kept free of fur by repeated shaving) and a minor increase in body temperature were observed in vaccinated rabbits. However, these observations were in line with vaccine safety regulations as indicated by the Ph.Eur. monograph for live myxomatosis vaccines . FurtherTo determine vaccine safety during pregnancy, in line with EU guidelines, pregnant rabbits were vaccinated with a maximum commercial dose during either early or late gestation. Vaccination did not affect gestation length or litter size, nor did vaccination result in any signs of disease, abortions, deformities or differences in the size of the progeny. Two kittens were stillborn, but this was likely due to dystocia and unrelated to vaccination. In addition, three rabbits, of which one was an unvaccinated control rabbit, proved not to be pregnant. The other two rabbits were vaccinated in the first or second term of pregnancy, respectively. However, after post mortem examination no abnormalities and normal follicular development of the ovaries were found, indicating the lack of pregnancy is unlikely related to vaccination and more likely related to the overall 90% pregnancy success rate of the supplier. From these data we can conclude that the results are in line with EU guidelines for the evaluation of safety of veterinary vaccines and immunosera , and theIn line with EU guidelines, vaccine efficacy was assessed by vaccinating rabbits with a single minimum commercial dose and challenging with RHDV 3 weeks later. Due to the variable pathogenicity of RHDV2 strains ,8,10, raIn conclusion, these vaccine registration studies with a novel trivalent myxoma virus vectored RHDV vaccine (Nobivac Myxo-RHD PLUS) confirm that this vaccine is safe for use in rabbits from 5 weeks of age onwards, including pregnant rabbits. The studies also confirm that a single dose of the vaccine provides protection against disease caused by myxoma virus, RHDV1 and RHDV2 from 3 weeks until 12 months post vaccination . As an additional welfare benefit, this vaccine is produced in vitro and not in live rabbits.


This review explores the origin of Ca2+ oscillations and their role in the control of mitochondrial respiration, bioenergetics, and mitochondrial oxidant stress.Degeneration of substantia nigra (SN) dopaminergic (DAergic) neurons is responsible for the core motor deficits of Parkinson’s disease (PD). These neurons are autonomous pacemakers that have large cytosolic Ca These oscillations play a key role in helping the neurons meet their bioenergetic needs, but they are also linked to cellular stress and vulnerability with aging and PD . Generally speaking, there are three classes of plasma membrane (PM) proteins that underlie these transients. One class is formed by voltage-dependent Ca2+ permeable ion channels. These channels vary in their voltage-dependence, location, and kinetics, and are accordingly classified as L-type (Cav1.1–1.4), N-type (Cav2.1), P/Q-type (Cav2.2), R-type (Cav2.3), and T-type (Cav3.3)  transients triggered by these PM proteins are controlled by Ca2+ buffering proteins—proteins endowed with Ca2+ binding sites  is 1–2 mM, cytosolic [Ca2+] is generally maintained at nanomolar levels (approximately 100 nM) by pumps and exchangers that extrude Ca2+ across the plasma membrane (PM) or into intracellular stores. The PM is endowed with plasma membrane Ca2+ ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs) that expel Ca2+. PMCAs pump Ca2+ to the extracellular space by using adenosine triphosphate (ATP), while NCX takes advantage of the Na+ gradient created by the Na+/K+ ATPase to extrude Ca2+ to the extracellular space. Given their differences in affinity for Ca2+, it is likely that basal cytosolic [Ca2+] is largely governed by the PMCA and the NCX is engaged by activity that pushes local [Ca2+] higher, as during repetitive spiking  is maintained by the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs) that move Ca2+ from the cytosol. SERCAs serve to terminate cytosolic transients induced by PM processes and to counteract constitutive “leak” of Ca2+ from the ER itself. The ER is richly invested with Ca2+ buffer proteins  that differ from cytosolic buffers in their affinity and capacity to help stabilize the high (µM) luminal ER [Ca2+]. Ca2+ release from the ER is mediated by the inositol trisphosphate (IP3) receptor (IP3R) and the ryanodine receptor (RyR). IP3R is gated by IP3 generated by phospholipase C in response to the activation of GPCRs. The primary agonist of RyR is Ca2+ itself; thus, cytosolic Ca2+ transients can trigger Ca2+-induced Ca2+ release” (CICR) from the ER  is high (~10–20 µM). In physiological situations, this concentration is achieved inside neurons only in microdomains where diffusion is restricted  gets too high  transients and are largely spared in PD —like that achieved in SN DAergic neuron dendrites—also activates proteases known as calpains, which cleave a variety of cellular proteins ; another is the excessive production of ROS by mitochondria. Both unintended consequences can have deleterious consequences over time  needs to escape predators or hunt for food and ultimately survive to mate. Only later in life, past reproductive age, this design may have negative consequences [Caver time . This siequences ,386. Theequences . As a coequences ,389,390.A fundamental question is then whether alleviating mitochondria oxidant stress could safely prevent or alleviate the progression of PD. Many of the early attempts at disease modification in PD have targeted mitochondrial ROS signaling, but all of these have failed to show efficacy ,250,251.2+ channels that could be targeted in PD. For example, DAergic neurons derived from induced pluripotent stem cells from familial PD patients are protected from rotenone toxicity upon Cav3 channel inhibition [2+ channels could enhance lysosomal exocytosis and diminish αSYN accumulation [As outlined above, there may be other Cahibition , while khibition . Negativhibition  could demulation . However


In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation. Chromosome numbers are halved in germ cells during meiosis as a single round of pre-meiotic DNA replication is followed by two rounds of chromosome segregations. Orderly chromosome segregation in the first meiotic division mechanistically requires conjoining of homologous copies (homologues) of each chromosome by the formation of physical linkages in the first meiotic prophase (reviewed in ). In mosChromatin is organized into DNA loops that emanate from linear proteinaceous chromosome cores, called chromosome axes, in meiosis . DSBs foPre-DSB recombinosomes form contemporaneously with the assembly of the axis shortly before the first DSBs appear at the start of meiosis. Despite a hypothesized essential role in enabling DSBs, pre-DSB recombinosomes do not colocalize with single-stranded DNA ends that result from DSBs . It is tWhereas pre-DSB recombinosomes are distributed across all axes at the start of meiosis in the leptotene stage, they are restricted to unsynapsed axes as homologues pair and SCs starts to form in zygotene ,16. ThisIt appears that SC formation is not the only mechanism that disrupts pre-DSB recombinosomes to terminate SPO11 activity after the completion of homologue pairing. Whereas full synapsis of autosomes marks the onset of the pachytene stage in spermatocytes, heterologous sex chromosomes remain unsynapsed apart from their short homologous pseudoautosomal regions (PARs). Curiously, pre-DSB recombinosome components are depleted from unsynapsed axes shortly after the onset of pachytene by an unknown mechanism ,17. ThusIn summary, the spatiotemporal control of pre-DSB recombinosome components exhibit several features that support the restriction of SPO11 activity to regions of chromatin and stages of meiotic prophase where DSBs are productive for homology search. Yet, the mechanisms of pre-DSB recombinosome control are poorly understood. Given the central role of IHO1 in DSB formation and pre-DSB recombinosome assembly, we examined the characteristics and upstream factors of IHO1 regulation. Unexpectedly, we found three distinct negative feedbacks that inhibit IHO1 accumulation on chromosome axes in response to DSBs. Further, we found that not only ATM, but also the other two major DDR PIKKs, ATR and PRKDC, are involved in the DSB-induced disruption of the DSB machinery, albeit their roles are separable from ATM in these feedback mechanisms.Iho1  BSA in PBS for 30 min, then slides were incubated with primary antibodies diluted in blocking solution either for 3 h at room temperature or overnight at 4°C. Subsequently, slides were washed (3 × 10 min) with PBS and incubated with secondary antibodies at room temperature for 90 min. Finally, slides were washed (3 × 10 min) with PBS and mounted with Slowfade gold antifade mounting media with or without DAPI (Invitrogen).Testes were collected in PBS pH 7.4 and fixed for 40 min at room temperature in 4% formaldehyde, 100 mM sodium phosphate pH 7.4, 0.1% Triton-X. After fixation, testes were washed three times in PBS and incubated in 30% sucrose, 0.02% sodium azide overnight at 4°C. Afterwards, testes were frozen in OCT on dry ice and stored at –80°C until sectioned. After cryo-sectioning, 7 μm sections were dried onto glass slides, incubated in cold methanol for 10 min at –20°C and subsequently in cold acetone for 1 min at –20°C. Sections were finally washed in PBS and blocked with PBS pH 7.4, 10% goat serum, 0.05% tween and 0.05% Triton-X. Slides were incubated with primary antibodies diluted in blocking solution overnight at 4°C. Subsequently, slides were washed (3 × 10 min) with PBS 0.01% Tween and incubated with secondary antibodies at room temperature for 90 min. Finally, slides were washed (3 × 10 min) with 0.01% Tween and mounted with Slowfade gold antifade mounting media with DAPI (Invitrogen).Nuclear spreads were staged based on axis development, which was assessed through detection of SYCP3 on meiocyte spreads. Briefly, punctate SYCP3 staining corresponds to preleptotene, short stretches of axis correspond to leptotene (most axis stretches shorter than 5× the widths of axes), relatively long and incomplete stretches of axes corresponds to early-zygotene and complete axes corresponds to late-zygotene and pachytene stages. Late zygotene and early pachytene could be distinguished unambiguously only in wild type where SC formation is efficient. In wild type, the completion of SC marks the start of pachytene. To identify sub-stages in wild-type pachytene spermatocytes , we usedTo stage the epithelial cycle of mouse seminiferous tubules, we used criteria that were described earlier ,44. Spec2.Testis organ culture protocol was carried out as described earlier  with minImageJ was used to process images for immunofluorescence signal measurements ,47. PartImageJ was used to process images for immunofluorescence signal measurements and R was used to analyse acquired data . In the IHO1, RAD51 and DMC1 signals were scaled and centered in each axis fragment by the deduction of the mean and division by the standard deviation. Auto-correlation and cross-correlation functions were computed using the R function ccf. For each experiment and for each fragment we computed:P-values are computed using a t-test (t-test function in R) for each given distance where each sample is given by the value of P-values are indicated in the corresponding figure legends. Experiment quantifications and conclusions in the manuscript are based on results that were reproduced in at least two independent experiments and at least two mice of each genotype. All comparisons were made between datasets obtained from animals that were either littermates or age matched.Graphs were prepared using GraphPad Prism 5 and all types of the statistical tests and Transgenic mouse strains, analysis scripts and pipelines used in this study are available from the authors upon request.According to current models of DSB formation, pre-DSB recombinosome-rich sections of axis accumulate DSBs. In turn, DSBs inhibit further DSB formation in their vicinity possibly by promoting a local disruption of pre-DSB recombinosomes . IHO1 acx > 5)  = –0.16054 and P = 5.67E–23, or RAD51, g(x) = –0.16592 and P = 8.76E–30, in the same pixel positions (x = 0). The negative cross-correlation was gradually lost with increasing distance and, curiously, turned into a weak but significant positive cross-correlation beyond 5-pixel distances (x>5)  and negative-auto-correlations of IHO1, RAD51 and DMC1 at a distance x > 5. With further increase of the distance (x > 15) the IHO1-DMC1 and IHO1-RAD51 cross-correlations approached 0.It is not straightforward to quantify and interpret the relationship between IHO1 and DMC1/RAD51 levels along axes. IHO1-rich axis sections do not inevitably accumulate DSBs because the axial IHO1 platform does not always associate with pre DSB recombinosomes . Further) Figure . This ne) Figure . In cont) Figure . These pThese data suggest that RAD51 and DMC1 peaks occur preferentially within IHO1 valleys and that there is a 5–10-pixel offset (0.3–0.65 μm) between peaks of IHO1 signals relative to DMC1 or RAD51 peaks. Thus, the observed patterns conform with the hypothesis that DSBs form in sections of axis where pre-recombinosome components accumulate, but after their formation, DSBs may promote the local depletion of pre-DSB recombinosome components from the axis. The observations are also formally consistent with additional non-exclusive possibilities. DSBs may form next to, and not within, IHO1-rich axis sections, or DSBs may form in IHO1-rich regions, but resultant resected DNA ends associate preferentially with nearby IHO1-poor axis regions.−/−Spo11 spermatocytes by γ-irradiation and assayed DSBs 1 and 24 h later. In line with an earlier study  or distinct positions (i ≠ j). This indicates that irradiation-induced DNA ends do not associate preferentially with IHO1-poor regions  = –0.23051, P = 6.07E–77). This observation supports the hypothesis that DSBs and/or single-stranded DNA-ends resulting from DSBs promote local depletion of IHO1 from axis, which may contribute to a negative feedback mechanism that prevents formation of DSBs in each other′s proximity.To test these alternatives, we aimed to analyse the relationship between axial distributions of DSB foci and IHO1 in an experimental system where DSB formation is inducible and does not depend on IHO1. These criteria are fulfilled by γ-irradiation induced DSBs. Whereas there may be differences in the processing of SPO11-induced and γ-irradiation-induced DSBs in meiosis, both γ-irradiation- and SPO11-induced DSBs produce DNA ends that associate with chromosome axes and recruit RAD51 and DMC1 . These per study  that dets Figure . γ-irrads Figure . Importa−/−Atm spermatocytes have pleiotropic phenotypes including chromosome axis fragmentation and an abnormal chromatin-wide hyperaccumulation of a marker of unrepaired DSBs, phospho-histone H2AX (Ser139), also called γH2AX  = –0.13533 and P = 1.38E–17, IHO1-RAD51 g(0) = –0.11507 and P = 1.91E-16) in −/− Spo11+/−Atm spermatocytes resembling +/−Spo11 or wild-type spermatocytes  = 0.022522 and P = 0.338988, IHO1-RAD51 g(0) = 0.012415 and P = 0.260504), suggesting an important role for ATR in IHO1 depletion in the vicinity of DSBs  = 0.09151 and P = 1.01E–05, IHO1-RAD51 g(0) = 0.07206 and P = 9.39E-11) along the axis. The hypomorphic scid mutation of Prkdc . ATR-degn3-Cre,  and effign3-Cre, ,51. Nevegn3-Cre, ,51, whicgn3-Cre, , and it s Figure . Furtherof Prkdc ,38. Thessev/sevTrip13 in (−/−Trip13), resembling wild type . We wondered if IHO1 depletion from all axes of −/−Trip13 spermatocytes was analogous to the disappearance of IHO1 from all chromosome axes, including axes of unsynapsed XY chromosomes, upon pachytene entry in wild type  and −/−Trip13 (45%) mice  and SC-defective −/−Sycp1 (43%) mice , impairribed by ) or an aribed by )   late zygotene-early pachytene spermatocytes in the DSB-defective e Figure  and B. I11YF/YF)  and mice11YF/YF)  and B (511YF/YF) . These o11YF/YF) .Most DSBs depend on the β isoform of SPO11, but a second isoform, SPO11α, is required for a late wave of DSBs that enables efficient pairing between the pseudoautosomal regions (PARs) of sex chromosomes in spermatocytes at the zygotene to pachytene transition ,56. Intr−/−Spo11 mice that express the β form of SPO11 from a transgene, hereafter called SPO11 β-only   with a ransgene , which eoter see . Therefos Figure .n = 164 cells) spermatocytes, IHO1 was never detected on axes beyond mid pachytene in fl/−Spo11p-CreIho1 mice  and DSB markers (DMC1 and RPA)  and autosomal flares of γH2AX disappeared after spermatocytes transitioned from early to late pachytene in both fl/+Spo11p-CreIho1 and fl/−Spo11p-CreIho1 mice. Thus, the repair of DSBs appears efficient in fl/−Spo11p-CreIho1 mice. MLH1 focus numbers were also similar in fl/+Spo11p-CreIho1 and fl/−Spo11p-CreIho1 mice, suggesting that IHO1 presence in pachytene is dispensable for crossover formation  and showed no obvious abnormality in testis cellularity  nor increasing DSB activity (−/− Spo11+/−Atm) nor randomizing distribution of DSBs (irradiation induced DSBs in −/−Spo11 meiocytes) result in loss of negative correlation between IHO1 and DMC1/RAD51. Therefore, we favor the hypothesis that ATR enables IHO1 depletion in the vicinity of DSBs. ATM seems to play only a small secondary role in IHO1 regulation, and this role is detectable only in the absence of ATR. In contrast, absence of ATM, and not the absence of ATR, results in a strongly elevated density of pre-DSB recombinosomes on unsynapsed axes. ATM may destabilize pre-DSB recombinosomes in the vicinity of DSBs, or, alternatively, ATM may inhibit pre-DSB recombinosome assembly and/or maintenance not only locally but across the nucleus. Regardless, ATM but not ATR limits bulk pre-DSB recombinosome numbers. These observations suggest that DSBs limit the formation and/or stability of pre-DSB recombinosomes by both ATM- and ATR-dependent mechanisms, but with distinct consequences for DSB formation.Surprisingly, we found that ATR deficiency leads to a loss of negative correlation between the positions of IHO1 and DMC1/RAD51 foci on axis. Whereas ATR loss decreases RAD51/DMC1 focus numbers ,51, we cATM is activated by blunt ended or shortly resected DNA ends shortly after DSB formation. In contrast, ATR activation requires single-stranded DNA ends that emerge only later due to ATM-promoted DNA-end resection . Hence, DSBs are not only suppressed in the vicinity of previously formed DSBs but also in chromosomal regions where homologues stably paired and synapsed. Suppression of DSBs in synapsed regions is accompanied by, and probably requires, the depletion of pre-DSB recombinosome components from chromosome axes. Whereas DDR PIKKs were not required for IHO1 depletion from synapsed axes, the TRIP13 AAA+ ATPase was required. TRIP13 remodeling promotes the removal of HORMAD1 from synapsed axes, and HORMAD1 is a key anchor of IHO1 on axes. Hence, it is likely that TRIP13 enables efficient depletion of IHO1 and pre-DSB recombinosomes by remodeling HORMAD1.However, TRIP13-mediated HORMAD1 remodeling is not the only mechanism by which synapsis controls DSBs. Albeit with reduced efficiency, MEI4-REC114-IHO1-containing pre-DSB recombinosomes still form in the absence of HORMAD1. The HORMAD1-independent pre-DSB recombinosomes also disappear from synapsed regions revealing a HORMAD1-independent mechanism that disrupts the DSB machinery in synapsed regions .Whereas the HORMAD1-independent suppression of DSBs may involve a possible remodeling of pre-DSB recombinosome components by TRIP13, we favor the involvement of a TRIP13-independent mechanism. Albeit with lower efficiency than in wild-type cells, IHO1 levels are moderately lowered and pre-DSB recombinosomes are disrupted along synapsed axes in TRIP13-deficient cells. These observations suggest that the SC promotes axial depletion of IHO1 and pre-DSB recombinosomes by an as yet uncharacterized TRIP13-independent mechanism next to the TRIP13-mediated remodeling of HORMAD1. Despite this redundancy, TRIP13-dependent depletion of pre-DSB recombinosomes may be critical for DSB suppression in synapsed regions, as early recombination intermediates, which may result from inappropriately persisting DSB-activity, were observed on synapsed axes in TRIP13-deficient spermatocytes ,61.Prophase stages that are permissive for pre-DSB recombinosome assembly and DSB formation are characterized by IHO1 presence on chromosome axes. In spermatocytes, IHO1 and pre-DSB recombinosomes are depleted not only from synapsed autosomes but also from the unsynapsed axes of heterologous sex chromosomes at the onset of pachytene. Thus, SPO11 activity is expected to cease on all axes including unsynapsed sex chromosomes after all homologues synapsed. We hypothesize that if DSB activity was sustained on unsynapsed sex chromosomes beyond zygotene, it could lead to increased mutation load in sex chromosome-linked genes and/or meiotic arrest due to DNA-damage checkpoint activation. Hence, a shutdown of the DSB machinery on unsynapsed axes at the onset of pachytene may confer benefits in the heterogametic sex of organisms that have heterologous sex chromosomes.de novo synthesis of IHO1 from mid pachytene.Interestingly, whereas IHO1 disappears from chromatin at the onset of pachytene, MEI4, REC114 and ANKRD31 persist in aggregates in PARs of sex chromosomes and PAR-like regions of three autosomes well into, but not beyond, early pachytene ,20. ThusWhereas the molecular mechanism of IHO1 depletion remains unclear, our analysis revealed the relationship between IHO1 depletion in pachytene and preceding key events in recombination. Importantly, we found that the stage-dependent depletion of IHO1 from all axes did not require the completion of synapsis between homologues in spermatocytes. This observation highlights important properties of the zygotene-pachytene transition. According to the standard definition of prophase stages, late zygotene and early pachytene are stages where axes are fully formed, and synapsis is either incomplete or complete between homologues, respectively. It was, however, poorly understood if the zygotene-pachytene transition represented a significant change in the cellular state of meiocytes beyond the status of the SC. Depletion of IHO1 at the onset of pachytene suggests that the zygotene-pachytene transition is also marked by a loss of DSB competence in wild type. Importantly, a similar timing of IHO1 depletion in wild type and diverse synapsis-defective mutants strongly suggests that the loss of DSB competence and the underlying transition between zygotene-like and pachytene-like cellular states are independent of SC completion. Thus, it is reasonable to conclude that a pachytene-like cellular state exists in SC and DSB repair-defective mutants even if late zygotene and early pachytene stages cannot be distinguished based on axis and SC morphology in these mutants.Thus, while SC completion and the zygotene-pachytene transition co-occur in wild type, there seems to be no mechanistic or checkpoint-based coupling between SC formation and zygotene-pachytene transition, as detected by IHO1 depletion. By way of exclusion, we conclude that, surprisingly, an unknown timing mechanism ensures that DSBs are suppressed at the same time as SCs are completed at pachytene onset in wild type.Unexpectedly, we discovered that stage-dependent loss of IHO1 and pre-DSB recombinosomes depended on DSB formation and the activity of all three DDR PIKKs. Thus, DSBs activate a previously unanticipated feedback mechanism to suppress DSB activity genome-wide at the onset of pachytene, where meiocytes are expected to complete SC formation. DSB formation may be required not only for stage-dependent loss of DSB competence, but also for other, as yet unidentified features of the zygotene-pachytene transition. Thus, meiocytes may fail to progress beyond a zygotene-like cellular state in the absence of DSBs. Regardless, both of these scenarios invoke a checkpoint-like regulatory circuit that makes the pachytene-associated shutdown of DSBs dependent on prior recombination initiation in meiocytes.Notably, this regulatory circuit also effects a temporal separation between DSB formation and the quality control of DSB repair and homologue synapsis. Whereas DSB formation is terminated at pachytene onset, unrepaired DSBs and asynapsis block prophase progression only in mid pachytene. Thus, there is time for the last DSBs to promote completion of synapsis in late synapsing regions before the mid pachytene checkpoint eliminates asynaptic spermatocytes. Further, the time between zygotene and mid pachytene is likely sufficient for the processing of the last DSBs, thereby preventing newly-forming DSBs from triggering a spermatogenic block in mid pachytene.The data supporting the findings of this study are available within the paper. The source data underlying both main and supplementary figures are provided as a Source Data file.gkab082_Supplemental_FilesClick here for additional data file.


Objective: The present study aimed to determine the prognostic value of HOXA cluster antisense RNA2 (HOXA-AS2) in acute myeloid leukemia (AML), and to explore its potential molecular mechanisms. We also screening of potential drugs targeting HOXA-AS2 in AML.Methods: The level 3 raw genome-wide RNA sequencing dataset of AML was download from The Cancer Genome Atlas (TCGA) Data Portal, and the potential molecular mechanisms and drugs prediction of HOXA-AS2 in AML were explored using multiple bioinformatics analysis approaches.Results: TCGA AML cohort dataset indicated that HOXA-AS2 was significantly up-regulated in AML bone marrow tissues, and high HOXA-AS2 expression was related to poor overall survival . Functional enrichment of differentially expressed genes (DEGs) suggested that the difference in prognosis between AML patients with high- and low-HOXA-AS2 expression may be due to differences in biological processes and pathways, including cell adhesion, angiogenesis, mitogen-activated protein kinase, cell differentiation, and other biological processes, and phosphatidylinositol 3 kinase-protein kinase B and Wnt signaling pathways. We also screened out three potential HOXA-AS2-targeted therapeutic drugs for AML, megestrol, carmustine, and cefoxitin, based on these DEGs. Functional enrichment analysis of HOXA-AS2-co-expressed genes revealed that HOXA-AS2 may act a part in AML by regulating nuclear factor-κB transcription factor activity, DNA methylation, angiogenesis, apoptosis, cell migration, Toll-like receptor 4, and Wnt signaling pathways.Conclusion: Our findings suggest that HOXA-AS2 is up-regulated in the bone marrow in patients with AML, and may serve as a novel prognostic biomarker for AML. Acute myeloid leukemia (AML) is a clonal malignant disease of the hematopoietic system Long non-coding RNAs (lncRNAs) play an important role in many biological activities, such as dose-compensation, epigenetic regulation, cell cycle regulation, and cell differentiation regulation, and are also associated with many diseases, including cancers and AML. LncRNA mainly functions via its secondary structure and binds to proteins, resulting in chromatin remodeling and affecting transcription factor function. In addition, lncRNAs can also bind to microRNAs, indirectly affecting mRNA expression, or can bind directly to mRNAs, affecting mRNA translation, shearing, and degradation processes. Numerous studies have shown that the lncRNA HOXA-AS2 plays an oncogenic role in a variety of cancers https://portal.gdc.cancer.gov/) EdgeR was used to normalized the RNA-seq dataset The level 3 raw RNA sequencing (RNA-seq) dataset of AML bone marrow tissues and the clinical prognostic parameters for 151 patients with AML were got from TCGA (http://gepia.cancer-pku.cn/index.html), based on the TCGA dataset and the GTEx  projects survivalROC package  using the R platform Box plots of HOXA-AS2 expression between bone marrow tissues of AML patients and healthy subjects were derived from the Gene Expression Profiling Interactive Analysis (GEPIA) website (edgeR package. Genes that met the following criteria were considered to be DEGs: |log2 fold change (FC)|>1, P value <0.05, and false discovery rate (FDR) value <0.05. These DEGs were then subjected to functional enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery v6.8  http://string.embl.de/) http://genemania.org/) https://portals.broadinstitute.org/cmap/) analysis of these DEGs P value <0.05 were considered as potential targeted therapeutic drugs for AML patients with high HOXA-AS2 expression. We also performed a survival analysis of these DEGs.We further explored the molecular mechanisms underlying the prognostic differences between AML patients with different HOXA-AS2 expression levels by screening differentially expressed genes (DEGs) using the  cor function through the R platform. PCGs significantly correlated with the HOXA-AS2 expression  were considered to be co-expressed with HOXA-AS2 in AML bone marrow tissue LncRNAs act by regulating related protein-coding genes. Screening genes co-expressed with HOXA-AS2 in AML bone marrow tissues by enrichment analysis will thus help us to understand the molecular mechanism of HOXA-AS2 in AML. We therefore screened protein-coding genes (PCGs) co-expressed with HOXA-AS2 in the whole genome RNA-seq dataset using theedgeR and GSEA were carried out according to the Benjamini-Hochberg approach P value <0.05 was considered significant. SPSS version 20.0  and R 3.5.0 (https://www.r-project.org/) were used for all statistical analyses.FDR in Figure ). Survival analysis demonstrated that high expression of HOXA-AS2 was notably related to poorer OS in patients with AML  . Time-dependent ROC curve analysis confirmed that HOXA-AS2 expression showed a high accuracy for predicting AML 5-year survival, with an area under the curve of 0.729 .We obtained box plots of HOXA-AS2 expression in bone marrow tissues of AML patients and healthy subjects using the GEPIA website, and showed that expression levels were significantly up-regulated in AML patients . A heat map of the DEGs was shown in Figure S1, as well as the volcano plot was shown in Figure 2. We subjected these DEGs to functional enrichment analysis to explore the potential mechanisms responsible for the prognostic difference between AML patients with low- and high-HOXA-AS2-expression levels. Gene ontology (GO) term analysis performed by DAVID v6.8 indicated that these DEGs were prominently related to cell adhesion, cell-cell signaling, cell migration involved in sprouting angiogenesis, single organismal cell-cell adhesion, positive regulation of mitogen-activated protein kinase (MAPK) activity, cell differentiation, positive regulation of extracellular regulated protein kinase (ERK) 1 and ERK2 cascade, integrin-mediated signaling pathway, positive regulation of MAPK cascade, cytokine receptor activity, and the epoxygenase P450 pathway (Table S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that these DEGs were prominently related to extracellular matrix (ECM)-receptor interactions, phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway, focal adhesion, pathways in cancer, cell-adhesion molecules (CAMs), cytokine-cytokine receptor interactions, calcium, cyclic adenosine monophosphate (cAMP), and Wnt signaling pathways (Table S3). BiNGO enrichment analysis also suggested that these DEGs were prominently related to cell differentiation, cell adhesion, cell migration, angiogenesis, cell proliferation, regulation of the Notch signaling pathway, regulation of the cAMP biosynthetic process, regulation of cell growth, the integrin-mediated signaling pathway, positive regulation of cell division, and tumor necrosis factor receptor activity (Table S4).We screened DEGs in a whole-genome RNA-seq dataset of bone marrow tissues from AML patients with different HOXA-AS2 expression levels using the Figure 3). In contrast, GSEA analysis using the c2  gene set revealed gene signatures related to up-regulated in pediatric AML with mutated NPM1, AML with internal tandem duplications (IDT) in fms-related tyrosine kinase 3 (FLT3), targets of nucleoporin 98 (NUP98)-homeobox A9 (HOXA9) fusion, AML cluster, erythroid differentiation, and response to oxidized phospholipids . All the above pathways enriched by GSEA were related to AML.We also investigated the potential mechanisms responsible for the prognostic difference between low- and high-HOXA-AS2-expressing groups using the GSEA approach. GSEA analysis using the c5  gene set revealed that definitive hemopoiesis biological processes may be involved in the prognostic difference between these two groups . The soft threshold distribution plot and module clustering plot of WGCNA analysis are shown in . The highest degree DEGs in the WGCNA network were PCDH17 and RNF182, with a degree of 55. These analyses demonstrated that these DEG had complex co-expression interaction relationships. We also performed a CMap analysis to predict potential HOXA-AS2-targeted therapeutics for AML. Using CMap analysis, we screened three small-molecule drugs, megestrol, carmustine, and cefoxitin, which could be used as targeted therapy for HOXA-AS2 in AML. PubChem (https://pubchem.ncbi.nlm.nih.gov/) provides the chemical structures of these targeted drugs Figure ) and the detailed results of the CMap analysis are summarized in Figure . We then used the online search tool for interactions of chemicals  Figure 9). By matching the drug-related genes and DEGs, four DEGs appeared in these three drug regulatory networks. We therefore concluded that carmustine may play a role in AML patients with high HOXA-AS2 expression levels by regulating these four DEGs: glial cell derived neurotrophic factor (GDNF), catalase (CAT), nitric oxide synthase 2 (NOS2), and dickkopf WNT signaling pathway inhibitor 1 (DKK1). By analyzing the connectivities of these four genes in the WGCNA network, we found that the DKK1 gene had a high degree of 50 in the WGCNA network, with high connectivity and regulation ability. However, the remaining three genes had lower degrees in the network, suggesting that carmustine was likely to exert its therapeutic effect in AML mainly by regulating DKK1.We further investigated the interactions among these DEGs using the WGCNA, STRING, and GeneMANIA approaches to construct interaction regulatory networks (Table S5). The top ten significantly prognostic DEGs were as follows : phosphodiesterase 3B (PDE3B), synaptotagmin-like 4 (SYTL4), dendrocyte-expressed seven transmembrane protein (DCSTAMP), Rho guanine nucleotide exchange factor 35 (ARHGEF35), methyltransferase-like 7B (METTL7B), thyrotropin-releasing hormone (TRH), UL16-binding protein 3 (ULBP3), ATPase phospholipid-transporting 10B (ATP10B), 5-hydroxytryptamine receptor 7 (HTR7), and dedicator of cytokinesis 1 (DOCK1).We also carried out a survival analysis to determine the prognostic values of these DEGs. There were 324 DEGs were prominently related to OS of AML in the present study . All the co-expressed PCGs were significantly associated with HOXA-AS2, with a Pearson correlation coefficient |r|>0.3 and P<0.05 (Table S6). GO term annotation suggested that these co-expressed PCGs participated in the following biological processes: peroxisome, definitive hemopoiesis, positive regulation of nuclear factor (NF)-κB transcription factor activity, DNA methylation, angiogenesis, intrinsic apoptotic signaling pathway in response to oxidative stress, intracellular signal transduction, Toll-like receptor 4 signaling pathway, and cell migration (Table S7). KEGG annotation suggested that these co-expressed PCGs were involved to the peroxisome, Wnt signaling pathway, and pathways in cancer (Table S8). BINGO enrichment analysis of these HOXA-AS2-co-expressed PCGs produced no meaningful results. Furthermore, because of the strict set parameters for WGCNA, we were unable to use the HOXA-AS2-co-expressed PCGs to construct a highly connected WGCNA regulatory network. However, we constructed a co-expression regulatory network of these HOXA-AS2-co-expressed PCGs using the STRING and GeneMANIA online tools, which demonstrated the complex interactions between these PCGs. The regulatory networks are shown in Figures 12 and 13. Survival analysis of these HOXA-AS2-co-expressed PCGs identified 282 genes that were prominently related to OS in AML patients. The top ten significantly prognostic HOXA-AS2-co-expressed PCGs were as follows: PDE3B, spermatogenesis-associated 9 (SPATA9), serine and arginine rich splicing factor 12 (SRSF12), Ras and Rab interactor-like (RINL), ARHGEF35, centrosomal protein 170 (CEP170), METTL7B, triggering receptor expressed on myeloid cells-like 2 (TREML2), ULBP3, and cyclin D2 (CCND2) . Four of the top ten significantly prognostic HOXA-AS2-co-expressed PCGs overlapped with the top ten significantly prognostic DEGs: PDE3B, ARHGEF35, METTL7B, and ULBP3.Analysis of the PCGs co-expressed with HOXA-AS2 may reflect its function in AML. We detected 903 PCGs co-expressed with HOXA-AS2 in AML bone marrow tissues using the Pearson correlation coefficient for genome-wide co-expression analysis, of which 621 were positively correlated PCGs, while 282 were negatively correlated PCGs  via the miR-520c-3p/glypican-3 axis in hepatocellular carcinoma Bacteroides fragilis in mice Of these three HOXA-AS2-targeted drugs used in AML, carmustine is a broad-spectrum anticancer drug with good curative effect in patients with Hodgkin's disease in vivo and in vitro experiments are needed to confirm these findings. Third, the small-molecule HOXA-AS2-targeted drugs screened using the AML whole genome dataset and CMap tool need further experimental verification. Despite these limitations, the present study found that HOXA-AS2 could be a prognostic indicator for AML. In addition, we also used a genome-wide dataset of AML bone marrow tissues to explore the molecular mechanisms of HOXA-AS2 in AML, and screened out three potential small-molecule targeted drugs. These findings thus provide a theoretical basis for further investigations of the functions and mechanisms of HOXA-AS2 in AML.This study had some limitations. This was a single-cohort study based on the TCGA dataset, and additional independent cohorts are required to verify our results. Second, although we performed functional enrichment analyses to investigate the potential molecular mechanisms of HOXA-AS2 in AML using multiple bioinformatics tools, Our study provides the first evidence for the use of HOXA-AS2 as a prognostic marker for AML, with high HOXA-AS2 expression related to a poor prognosis in AML patients. Our findings also suggested that HOXA-AS2 levels may be significantly up-regulated in the bone marrow tissues of AML patients compared with healthy subjects. Using multiple bioinformatics analysis tools, we determined the potential molecular mechanisms of HOXA-AS2 in AML, and screened out three small-molecule drugs  as potential HOXA-AS2-targeted therapeutic agents in AML patients. We also identified multiple genes associated with AML prognosis. However, further studies were required to verify these findings.Supplementary figures.Click here for additional data file.Supplementary tables.Click here for additional data file.


Acute myelogenous leukemia (AML) is a common pediatric malignancy in children younger than 15 years old. Although the overall survival (OS) has been improved in recent years, the mechanisms of AML remain largely unknown. Hence, the purpose of this study is to explore the differentially methylated genes and to investigate the underlying mechanism in AML initiation and progression based on the bioinformatic analysis.Methylation array data and gene expression data were obtained from TARGET Data Matrix. The consensus clustering analysis was performed using ConsensusClusterPlus R package. The global DNA methylation was analyzed using methylationArrayAnalysis R package and differentially methylated genes (DMGs), and differentially expressed genes (DEGs) were identified using Limma R package. Besides, the biological function was analyzed using clusterProfiler R package. The correlation between DMGs and DEGs was determined using psych R package. Moreover, the correlation between DMGs and AML was assessed using varElect online tool. And the overall survival and progression-free survival were analyzed using survival R package.All AML samples in this study were divided into three clusters at k = 3. Based on consensus clustering, we identified 1,146 CpGs, including 40 hypermethylated and 1,106 hypomethylated CpGs in AML. Besides, a total 529 DEGs were identified, including 270 upregulated and 259 downregulated DEGs in AML. The function analysis showed that DEGs significantly enriched in AML related biological process. Moreover, the correlation between DMGs and DEGs indicated that seven DMGs directly interacted with AML. CD34, HOXA7, and CD96 showed the strongest correlation with AML. Further, we explored three CpG sites cg03583857, cg26511321, cg04039397 of CD34, HOXA7, and CD96 which acted as the clinical prognostic biomarkers.Our study identified three novel methylated genes in AML and also explored the mechanism of methylated genes in AML. Our finding may provide novel potential prognostic markers for AML. Acute myelogenous leukemia (AML) is a common pediatric hematologic cancer in children younger than 15 years . In the Recent researches have demonstrated epigenetic modifications act a critical role in AML through altering the transcriptional activation of oncogenes or tumor suppressors –8. DNA mAlthough numerous studies have illustrated the regulatory effects of DNA methylation in AML, research for prognostic biomarkers based on DNA methylated genes is still lacking. Hence, we conduct an integrative analysis of methylation and transcription in AML based on bioinformatics and identify differentially methylated genes in AML. Furthermore, we analyzed the correlation between differentially methylated genes and AM and explored the DNA methylation-related prognostic biomarkers for AML patients.β)-value which ranged from 0 to 1. β-value represented the ratio of the methylated allele frequency and unmethylated allele frequency. And the expression microarray data was normalized using quantile normalization methods.DNA methylation data and gene expression profilers were obtained from 219 paired AML samples and normal samples in TARGET Data Matrix. DNA methylation data was carried out with Illumina HumanMethylation27K array, and gene expression profiles were carried out with Affymetrix Gene ST. The methylation levels were assessed using beta  plots were illustrated based on k-value. And empirical cumulative distribution function (CDF) plots revealed the consensus distributions for each k. The k at approximate maximum distribution indicated maximum stability cluster structure. The delta area plot showed the delta area score (y-axis); the delta area score determines the stability of clustering. Moreover, the item tracking plot exhibited the consensus cluster of items (columns) at each k (rows) to identify the stability of clustering. Cluster-consensus plot showed the cluster-consensus value at different k values; high cluster-consensus value indicated low stability of clustering. Item-consensus plot is the average consensus value between an item and members of a consensus cluster. An item corresponded to multiple item-consensus values with different ks.β| ≥0.2 and adjusted P-value <0.05. And DNA methylation regions (DMRs) were analyzed with a cutoff value no. CpGs ≥3, |meandiff| ≥0.2, and fisher (P) >0.05.The principal component analysis (PCA) was used to analysis the sample bias in this study. And then, DNA methylation analysis was performed using methylationArrayAnalysis R package. The probes containing single nucleotide polymorphisms (SNPs), probes located in chromosome X, and more than 10% missing values in the probes were excluded in this study. The differentially DNA methylation probes (DMPs) were predicated with a cutoff value |ΔLimma R package was used to determine the DEGs between cluster 2 and cluster 3 in AML. The cutoff value was |log2 (fold change) | ≥ 1 and P-value < 0.05. The volcano plot of DEGs was drawn using ggplot R package, and heatmap of DEGs was drawn using pheatmap R package.The biological function was analyzed by GO and KEGG pathway enrichment analysis using clusterProfiler R package with a threshold P-value <0.05. GO annotation included biological process (BP), molecular function (MF), and cellular component (CC).Correlation between DMGs and DEGs was calculated by Pearson correlation analysis using psych R package with a cutoff value correlation ≥0.5 and adjusted P-value <0.05. Moreover, the protein–protein interaction (PPI) network of hypomethylated-high expression genes and hypermethylated-low expression genes was constructed and visualized using Cytoscape.The correlation between DMGs and AML was analyzed using varElect online tool, and high score indicated the strong correlation.β-value.The overall survival (OS) and progression-free survival (PFS) were analyzed by Kaplan–Meier (KM) survival plots using survival R package based on The DNA methylation array data of 219 AML samples was obtained from TARGET Data Matrix and normalized using exclusion criteria. Then, the consensus clustering was performed using ConsensusClusterPlus R package; the CDF showed the lowest rangeability at consensus index 0.2–0.6 with k = 3  results showed the clustering exhibited unbiased in the group; it indicated gene expression of samples was consistent and repeatable  of candidate methylated genes was investigated by Cox regression analysis. The results showed AUC values of cg03583857 is 0.775  family, which is a type-I transmembrane sialomucins and acts as the marker of hematopoietic stem cells and vascular (Abnormal expression of HOX gene is a common feature in AML . Howeverde novo AML (CD96 is an immune related gene which acts as an immune related marker in AML , high exnovo AML . It has In summary, we performed an integrative analysis of multi-omics in AML and identified the differential DNA methylated genes such as CD34, HOXA7, and CD96 and explored DNA methylation and clinical implication of CD34, HOXA7, and CD96 in AML.https://ocg.cancer.gov/programs/target/data-matrix.Publicly available datasets were analyzed in this study. This data can be found here: TZ and JZ contributed to the conception of the study. YX and CC contributed significantly to analysis and manuscript preparation. JZ performed the data analyses and wrote the manuscript. WG, CZ, XC, LG, CC, and YX helped perform the analysis with constructive discussions. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China [Grant number 81771589], the Key Project of Tianjin Health Care Professionals [Grant number 16KG166] and the Program of Tianjin Science and Technology Plan [Grant number 18ZXDBSY00170].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.