As we tune the SHG signal onto these resonances by varying the laser energy, the SHG amplitude is enhanced by several orders of magnitude. In the resonant case the bilayer SHG signal reaches amplitudes comparable to the off-resonant signal from a monolayer. In applied electric fields the interlayer exciton energies can be tuned due to their in-built electric dipole via the Stark effect. As a result the interlayer exciton degeneracy is lifted and the bilayer SHG response is further enhanced by an additional two orders of magnitude, well reproduced by our model calculations. Since interlayer exciton transitions are highly tunable also by choosing twist angle and material combination our results open up new approaches for designing the SHG response of layered materials.Second-harmonic generation (SHG) is a non-linear optical process, where two photons coherently combine into one photon of twice their energy. Efficient SHG occurs for crystals with broken inversion symmetry, such as transition metal dichalcogenide monolayers. Here we show tuning of non-linear optical processes in an inversion symmetric crystal. This tunability is based on the unique properties of bilayer MoS 2 - an inversion-symmetric crystal - mediated by interlayer excitons.Efficient second-harmonic generation (SHG) occurs for crystals with broken inversion symmetry, such as transition metal dichalcogenide monolayers. Here the authors show SHG tuning in bilayer MoS Nonlinear optics is based on the interaction of photons within the crystal. This can give rise, among other processes, to second-harmonic generation (SHG) where two photons of identical energy combine into one photon of twice this energy4. Currently existing applications in photonics and laser physics make use of this process in bulk crystals for example using potassium dihydrogen phosphate (KDP) or lithium niobate (LiNbO3)5.Nonlinear optical spectroscopy of atomically thin crystals gives crucial insights into their physical properties and investigates potential applications8 and also for applications of a large class of materials such as II-VI and III-V semiconductors12, nanotubes13, magnetic- and nonmagnetic layered materials20, perovskites and antiferromagnetic oxides22.SHG is vital both as a spectroscopic tool5. This dependence on symmetry makes nonlinear optics in atomically thin crystals such as graphene a very rich field of research29. Although pristine graphene is an inversion-symmetric crystal, the crystal symmetry or symmetry of the electronic states is potentially very sensitive to charges or single molecules in the layer vicinity30, an imbalance in population of valley states31, or electric currents32. However, graphene is a gapless material, so light-matter interaction cannot be controlled to the same extent as in atom-thin semiconductors based on transition metal dichalcogenides (TMD).The occurrence of SHG in a crystal is directly linked to its symmetry, where crystals with broken inversion symmetry can exhibit SHG and crystals which are inversion symmetric can typically not2 and WSe2 have broken inversion symmetry and show intrinsically strong SHG signals34. Excitons are strongly bound in these materials35 and govern optical processes also at room temperature, contrary to the model semiconductor GaAs10. This is crucial for nonlinear optics as excitons resonantly enhance light-matter interaction by several orders of magnitude35. As a result, in monolayers the excitonic contribution to SHG can be orders of magnitude higher than the intrinsic contribution from the crystal which exists also off-resonance38. An intrinsic challenge for exploiting SHG signals from nanostructures is the overall small signal, despite the giant response of a monolayer per unit thickness40. Hence, in the interest of both fundamental physics and applications, we need to uncover the exact origins of SHG in layered semiconducting materials to enable further tuning and amplification.TMD monolayers such as MoS2 bilayers with 2H stacking: Varying the laser excitation energy allows us to address resonantly not only intralayer and but also interlayer exciton states with large oscillator strength41, which makes MoS2 bilayers particularly interesting. At these specific energies we generate SHG signals in MoS2 bilayers which are (i) comparable in amplitude to off-resonance SHG monolayer signals and (ii) several orders of magnitude larger than reported in the literature for TMD bilayers with 2H stacking43. The prominent role of interlayer excitons in MoS2 bilayers allows for an efficient SHG tuning mechanism: in applied electric fields, we lift the degeneracy of the interlayer exciton states through the Stark effect. This results in an additional enhancement of the SHG signal at the interlayer exciton resonance by two orders of magnitude, surpassing the response of an ungated monolayer at this energy. We uncover the excitonic origin of the highly tunable SHG response through the linear polarization dependence of the SHG signal with respect to the armchair direction of the sample. We compare bilayer results with measurements in monolayers. We uncover variable SHG enhancement in trilayers located in the same van der Waals stacks. We present a model analysis of the emergence of the strong SHG in the bilayer, taking into account the impact of electric fields perpendicular to the bilayer as well as different sources of asymmetry.In this work we demonstrate strong, tunable SHG based on exciton resonances in nominally inversion-symmetric MoS2 have been uncovered41. We aim to distinguish the possible SHG generation that comes from a weak, intrinsic, crystal-related contribution from the possible excitonic contribution by using a tunable laser source. This allows comparing off-resonance with on-resonance experiments.Very recently, details of the rich excitonic resonances in bilayer MoS2 bilayer deposited on 90 nm of SiO2 on top of a Si substrate, a very typical sample configuration (see Mtethods and Supplementary Information for details in the fabrication process). The bilayer sample (as well as the trilayer sample below) has been directly exfoliated from a 2H bulk crystal, i.e., without manual stacking individual layers. We ensure therefore 60° twist angle (and not 0° as for 3R stacking) between adjacent layers, as confirmed by measuring the energy separation of the A–B excitons and observing the interlayer exciton44. We use a pulsed Ti:Sapphire laser source coupled to an optical parametric oscillator  and we scan twice the laser energy (i.e. 2 × EL) over an energy range that corresponds to the main optical transitions in the MoS2 bilayers, namely the interlayer and intralayer exciton resonances48.For the experimental results shown in Fig. T = 4 K in vacuum in a confocal microscope (excitation/detection spot diameter of the order of the wavelength), see supplement and7 for details. We plot in Fig. EL = 0.9 and 1.13 eV in steps of ≈6 meV. Each SHG spectrum is a single peak that shifts in energy as we vary the excitation laser energy EL. The spectral width of the SHG signal is limited by the laser pulse duration (ps).Experiments are performed at a temperature of EL) is tuned below the intralayer A energy . But surprisingly for a crystal with inversion center, as we increase the laser energy, we see clear resonances in the SHG response, i.e. a strong variation in SHG intensity as a function of laser energy EL in Fig. We start with the off-resonance case: we do not detect any SHG signal within our typical integration time (2 minutes and excitation power of a few mW) when twice the laser energy  and interlayer (IE) exciton energies44.Comparing SHG spectra and white light reflectivity, we deduce that the maxima in the SHG intensity occur when 2 × 2 layers from Fig. The surprisingly strong SHG signal could suggest a symmetry breaking in our nonencapsulated sample. A possible reason can be a drastically different dielectric environment experienced by top and bottom MoS2 bilayer samples encapsulated in high quality hexagonal BN49. This more symmetric dielectric environment52 eliminates potential sources of symmetry breaking. In addition, we also exclude the possibility of adsorbates or molecules at the sample surface in encapsulated samples which might impact the measurements as in surface SHG studies53 and suggested in graphene30. In Fig. EL using a smaller step size of ≈3 meV between two adjacent SHG spectra. The SHG signal coming from hBN is several orders of magnitude weaker than from the MoS2 bilayer, see33 and our data in the supplement. Our experiments in Fig. 2 bilayers in a far more symmetric dielectric environment show SHG at the exciton resonances. Comparing the signal strength for the A-exciton resonance, we record an SHG signal about an order of magnitude weaker for the hBN-encapsulated sample  is below the A-intralayer exciton resonance for monolayers and bilayers. We directly compare the measured SHG signal at this laser energy for monolayers and bilayers, see peaks in Fig. EL = 1.005 eV twice the laser energy 2 × EL is in resonance with the interlayer IE transition of the bilayer resulting in a strong amplification of the SHG signal while being nonresonant for a monolayer, see comparison in Fig. Previous reports on SHG in MoS41 that can potentially enhance SHG. For the measurements in Fig. L2) and in the outer layers (AL1 and AL3) respectively, between 1.9 and 1.92 eV. Local maxima around 1.98 eV can be attributed to interlayer excitons, with B-exciton contributions around 2.1 eV. From our measurements, we conclude that the trilayer combines the high intrinsic SHG response of the monolayer with the tunability (see below) of the interlayer exciton-mediated SHG. The exciton transitions for mono- and trilayers are identified and labeled in more detail by comparing with white light reflectivity41 shown in the supplement.Although SHG signals in trilayers higher than in bilayers have been reported at fixed laser energy, very little is known of the energy dependence of the nonlinear susceptibility of the trilayer. The trilayer crystal has no inversion center, as the monolayer, and sizeable nonresonant SHG can be expected. In addition, there exist several intralayer and interlayer exciton resonancesL2 is redshifted compared to the A-exciton resonance for the outer layers L1 and L3. Because of this shift, these contributions do not cancel each other as could be expected from symmetry if they would be at the same energy. The A-exciton of the monolayer gives about 1500 counts  or top (IE2) layer, see sketch in Fig. Fz lifts the degeneracy of IE1 and IE2 as their static, permanent dipoles point in opposite directions. This leads to a Stark shift to lower and higher energy, respectively41. This lifting of the degeneracy can be seen in Fig. 1 and IE2 of roughly 10 meV for Fz = 0.13 MV/cm. In Fig. Fz = 0, 0.07 and 0.13 MV/cm, respectively. In addition to the increase in SHG amplitude with Fz, we therefore observe that the spectral range for which amplification is observed is widened. This broadening is the consequence of the energy splitting between IE1 and IE2 transitions in an applied electric Fz. The broadening can also be seen directly in Fig. 66 and will be further discussed below.We now turn our attention to the spectral range over which the SHG enhancement occurs. In a 2H homobilayer at 2. Here the SHG signal is very sensitive to the electric field, being one of the most striking experimental observations. Further we briefly address other contributions to the SHG. The detailed theory is presented in the supplement.We first focus on the model considerations for SHG mediated by the interlayer excitons in bilayer MoSDh3 which lacks an inversion center and allows for the SHG. An ideal homobilayer in absence of external fields has an inversion symmetry and is described by the Dd3 point group where the SHG is forbidden. Qualitatively, this is because the constituent monolayers in the bilayer are rotated by an angle of π with respect to each other in 2H stacking68, and the electric field at a double frequency generated in one monolayer is compensated by the contribution of the other, Fig. A TMD monolayer is described by the noncentrosymmetric point group F perpendicular to the monolayers breaks the symmetry and enables the SHG described by a phenomenological relationEω is the incident field and Pω2 is the polarization induced at a double frequency, x and y are the main in-plane axes of the structure. Since the bilayer is centrosymmetric, the nonlinear susceptibility χ in Eq. (χ(−Fz) = −χ(Fz). This relation reflects the three-fold symmetry of the bilayer where the quadratic components of the vector component ExEy transform as vector components Px, Py, see ref. 37 for details. Polarization dependence of the SHG predicted by Eq. :C2 is the combination of the interband momentum matrix element, electron charge, free-electron mass and the band gap, ΦB:1s(0) is the envelope function of the B:1s exciton at the coinciding electron and hole coordinates, T is the hole tunneling matrix element between the layers, Δ = EB − EIE is the energy splitting between the IE and B-excitons (which are mixed due to the hole tunneling47), and ΓIE is the damping of the IE resonance. As demonstrated in previous works60, the IE exciton optical activity is mainly due to the mixing with B-excitons, thus, in Eq. .Intralayer excitons show negligible Stark shifts in applied fieldsses Fig. a. The sePω2 is maximized when twice the laser energy is resonant with an indirect exciton state as 2 × EL = 2ℏω = EIE. Second, the intensity of the SHG scales as Fz. So both experimental observations strongly support our analysis.Two important conclusions can be drawn from Eqs.  and 2)::2): Firsmentclass2pt{minims and B-excitons. A weaker effect of the electric field F on the A-excitons can be readily understood taking into account the fact that the A-excitons are mixed, e.g., with the interlayer B-excitons via hole tunneling. Corresponding energy distance Fz.Importantly, the experiment demonstrates also the SHG on other excitonic species in bilayers, including weakly field-dependent effect on intralayer A-excitons, Fig. Fz = 0. Possible origin of SHG in this case could be the quadrupolar or magneto-dipolar SHG, where akin to graphene case27, the effect is related to the light wavevector qz at the normal incidence. The symmetry analysis again yields Eq.  phase difference of ϕ ~ qza where a is the interlayer distance in the bilayer yields an imbalance of the contributions of individual monolayers and results in the resonant contributions at A- and B-excitons similar to calculated in37 for monolayers, but being by a factor of ϕ ~ 10−2…10−3 smaller (see supplement). Our estimates show, however, that this effect is too small to account on its own for the surprising experimental findings which demonstrate that the resonant susceptibilities of the bilayer are roughly one order of magnitude smaller than those of the monolayer.We now discuss the enhancement of the SHG at the intralayer exciton resonances in the nominally centrosymmetric situation when elds Eq.  with χ ∝Fz by Fz + F0 where F0 is the normal component of the build-in field. In this case at Fz = −F0 the effectively symmetric situation can be realized. While the measured dependence of the SHG intensity for IE, Fig. Fz ≈ − 0.035 MV/cm, the SHG intensity measured at the A-exciton resonance does not significantly drop in this electric field range. Alternatively, we may suppose that the build-in field is inhomogeneous in the sample plane within our detection spot and the contributions of the A-exciton and IE states come from slightly different nanoscopic regions.Thus, we arrive at the conclusion that, despite relatively symmetric environment of our bilayer sample, the structure lacks an inversion center. Possible options are (i) small built-in electric fields and (ii) inequivalence of the intralayer excitons in monolayers forming a bilayer. Option (i) results in the replacement of 51. In this situation, the IE is mainly activated by the electric field, while intralayer excitons are less sensitive to Fz (see supplement). Inhomogeneous broadening effects due to disorder, impurities, etc. are inevitable and inhomogeneities on the order of one micrometer can occur even in hBN-encapsulated samples52. For the in-plane disorder the crucial parameter is the ratio between the size of the inhomogeneity, l, and the laser spot diameter, δ. For l ≪ δ, inhomogeneous broadening effects will effectively cancel out and the structure will effectively maintain the space inversion. However, for l ≳ δ, the inhomogeneities will lift the inversion symmetry, thus a contribution of one monolayer will dominate over the other and SHG occurs. It is important to note that, the asymmetry of the hBN environment due to the different thicknesses of the top and bottom layers and ‘vertical’ disorder caused, e.g., by the fluctuations of the top layer thickness, can result in the SHG at Fz = 0 at the A-exciton, e.g., due to the difference of the intralayer A-exciton energies and their nonradiative dampings. We stress that the six-fold symmetry of the SHG signal (see supplement) clearly rules out, e.g., an in-plane asymmetry due to the strain or in-plane fields20.Option (ii) implies that the energies of A- and B-excitons, their nonradiative broadening, or their oscillator strengths are inequivalent in the two monolayers that form the bilayer. This may be due to the dielectric disorder, which, although suppressed, can still be present in state-of-the-art, hBN-encapsulated samples2 bilayers that can surpass the off-resonance SHG signal in monolayers. Drastic enhancement of the bilayer SHG amplitude is observed when twice the laser energy is in resonance with the excitonic transitions. The SHG signal outside exciton resonances remains orders of magnitude smaller than the monolayer signal, indicating a very small intrinsic χ value from broken inversion symmetry. At the interlayer exciton resonance, we tune the SHG signal by over an order of magnitude in electric fields applied perpendicular to the layer and demonstrate that the spectral width of the SHG resonance increases. With our model calculations, we relate the SHG in applied electric fields to the Stark splitting of the interlayer exciton and its mixing with intralayer excitons, which results in a quadratic dependence of the SHG amplitude on the applied electric field.In summary, by varying excitation laser energy, dielectric environment, and applied electric fields we show strong and tunable exciton-mediated SHG in 2H MoS266. Very importantly it has been shown recently that several heterobilayer systems host interlayer excitons with static electric dipoles and high optical oscillator strength, for example, MoSe2/WS2 with hybridized conduction states62, WSe2/WS2 with hybridized valence states63, MoS2/WS2 with electrically tunable valence state hybridization64 and similar predictions of electronic state hybridization for specific twist angles in MoTe2/MoSe265. It can be expected that also in these and other systems SHG can also be efficiently tuned via the Stark effect, as demonstrated here for homobilayer MoS2.Our scheme for tuning SHG based on interlayer excitons with a permanent static electric dipole can be applied to a variety of other systems with strong interlayer exciton resonances, such as homobilayer MoSe2/Si substrates were cleaned for 10 min in aceton and isopropanol using an ultrasonication bath and were subsequently exposed in oxygen plasma for 60 s. Bulk 2H MoS2 (2D Semiconductors) was first exfoliated on Nitto Denko tape and the exfoliated areas were attached on a polydimethylsiloxane (PDMS) stamp, supported by a microscope glass slide. Monolayers, 2H bilayers, and trilayers were identified based on the optical contrast under an optical microscope prior to transfer on the SiO2/Si substrate. For the hBN-encapsulated samples, hBN flakes were first exfoliated on a Nitto Denko tape from high-quality bulk crystals while the same PDMS-assisted transfer process on SiO2/Si substrates was followed. A staircase sample of monolayers, 2H bilayers and trilayers was susbequently transferred and capped in hBN. Between every transfer step, annealing at 150 °C was applied for 60 min. Optical images of hBN encapsulated but also bare MoS2 in SiO2 are shown in 2, 130 nm hBN, FLG, few-layered (≈15 nm) hBN, 2L MoS2, few-layered hBN (≈20 nm), and FLG again. The bottom and top FLG are in contact with gold (Au) electrodes to apply a potential difference and generate an electric field perpendicular to the structure. The precise sequence of the complete stack from bottom to top includes hBN/FLG/hBN/2H-MoS2/hBN/FLG. A schematic representation of the device is shown in The 90-nm-thick SiOT = 4 K to 300 K). For SHG measurements, we use ps pulses, generated by a tunable OPO synchronously pumped by a mode-locked Ti:sapphire laser. SHG signal is collected in reflection geometry. A combination of linear polarizers and halfwave plates allows the control of excitation and detection polarization for the polarization-resolved measurements with the setup sketched in T = 4.2 K using a microscope objective (NA = 0.8). The position of the sample with respect to the focus can be adjusted with cryogenic nanopositioners. The reflected light from the sample is sent to a spectrometer with a 150 grooves per millimeter grating. The spectra are recorded by a liquid-nitrogen cooled charged coupled device (CCD) array. For low temperature white light reflectance measurements a white light source; a halogen lamp is used with a stabilized power, focused initially on a pin-hole that is imaged on the sample. The emitted and/or reflected light was dispersed in a spectrometer and detected by a Si-CCD camera. The excitation/detection spot diameter is 1 μm, i.e., smaller than the typical size of the homobilayers. We obtained differential reflectivity from reflectivity spectra as (RML − Rsub)/Rsub, where RML is the intensity reflection coefficient of the sample with the MoS2 layer and Rsub is the reflection coefficient of the hBN/SiO2 stack.Optical spectroscopy is performed in a home-built micro-spectroscopy setup assembled around a closed-cycle, low vibration helium cryostat with a temperature controller (Supplementary informationPeer Review File


Botryosphaerella sudetica KNUA107, isolated from Ulleung Island, South Korea, as a bioresource and analyzed the effects of mixotrophic cultivation on its bioresource production efficiency. Internal transcribed spacer (ITS) , ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) regions were used for molecular identification and phylogenetic analysis. B. sudetica KNUA107 had a strong relationship with the green algae of Botryococcus and Botryosphaerella genera, which are colony-forming species, and was also associated with members of the Neochloris genus. To improve biomass productivity, we tested mixotrophic cultivation conditions using several organic carbon sources. Glucose supplementation stimulated B. sudetica KNUA107 growth and reduced the time needed to reach the stationary phase. In addition, the colony size was 1.5–2.0 times larger with glucose than in photoautotrophic cultures, and settleability improved in proportion to colony size. The total lipid content and biomass productivity were also higher in cultures supplemented with glucose. Among the lipid components, saturated fatty acids and monounsaturated fatty acids had the highest proportion. Our study suggests that B. sudetica KNUA107, which has enhanced efficiency in biomass production and lipid components under mixotrophic cultivation, has high potential as a bioresource.In this study, we characterized the potential of colony-forming green algae,  Green algae are photosynthetic organisms used as bioresources . They arPediastrum genus) and others form colonies that connect single cells   . Multice genera) . High se genera) , 2015. H genera) . Continu genera) .Although green algae are photosynthetic organisms, they can metabolize organic and inorganic carbons . SeveralBotryosphaerella sudetica KNUA107, isolated from Ulleung Island, as bioresources for bioenergy production. To enhance bioresource production, we used mixotrophic cultures supplemented with different organic carbon sources and screened their effect on algal growth. Settleability and lipid contents under various mixotrophic culture conditions were measured and analyzed. Based on these analyses, we identified an organic carbon source that enhanced bioresource productivity and confirmed the value of B. sudetica KNUA107 as a bioresource for bioenergy production.In this study, we evaluated the suitability of the colony-forming green alga, B. sudetica KNUA107 strain was assigned the GenBank accession number MW683220 in the NCBI database. B. sudetica KNUA107 grew the vegetative cell in the form of single cells and multicellular colonies in the liquid medium, and the colonies were irregular and appeared to be tightly bound between cells , followed by Botryococcus sp. (92%) and N. aquatica (91%). Previous data indicated that some members of these three green algae genera have colony-forming abilities and morphological characteristics similar to those of B. sudetica KNUA107  was further determined by phylogenetic analysis. ML trees were constructed based on ITS, rbcl, and tufa sequences of B. sudetica KNUA107 and several green algal species . Hence, B. sudetica KNUA107 had the closest phylogenetic relationship with B. sudetica. Additionally, B. sudetica KNUA107 has a colony-forming ability similar to previously studied B. sudetica algae  and confirmed that it is related to B. sudetica. We evaluated biomass and lipid productivity to demonstrate its potential as a bioresource for bioenergy production. Furthermore, we applied mixotrophic cultivation, which promotes bioenergy production in microalgae, to B. sudetica KNUA107. Several mixotrophic conditions were applied to the culture to investigate their effect on biomass productivity. Glucose was the only added carbon source that increased biomass in high-density cultures, increasing growth rate and settleability. Moreover, the glucose mixotrophic condition increased B. sudetica KNUA107 content in lipids and their saturated components. Therefore, B. sudetica KNUA107 is a suitable strain to produce bioresources under mixotrophic conditions. Our research elucidates the impact of organic carbon sources on biomass productivity and suggests the potential of colony-forming green algae as an industrially useful bioresource.In this study, we isolated a colony-forming green algae strain at Ulleung Island (–2 s–1), and with a 16:8 h light:dark cycle. The culture was rotated at 160 rpm in an orbital shaker . After a 2-week incubation, the algal sample was streaked onto a BG11 (pH 7.4) agar plate. Subsequently, single colonies grown were aseptically transferred to fresh BG11 agar plates. The transferring process was repeated until a colony consisting of one species was aseptically obtained  in April 2017. The sample was transferred to the laboratory and inoculated in 150 mL of BG11 medium (pH 7.4) in a 250 mL flask . The inoobtained .B. sudetica KNUA107 strains, cells that were incubated for 2 weeks in 150 mL of BG11 were collected by centrifugation at 4000 rpm for 10 min and washed twice with sterile distilled water. The collected algal cells were resuspended in 400 μL DNA extraction buffer  in 2 mL microfuge tubes. The suspended samples were vortexed for 5 min after adding 8 μL RNAse A  and sterile glass beads and heated at 65°C for 30 min. Cellular debris was removed by centrifugation at 13,000 rpm for 10 min, and 400 μL of the supernatant was transferred into a new tube. DNA was purified with a DNA purification kit  according to the manufacturer’s protocol. The internal transcribed spacer (ITS), ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) were used as marker genes   . DNA sequencing was conducted at the Genotech facility . The sequence information of previously reported related algal strains (with similar sequences) was retrieved from NCBI; B. sudetica KNUA107 collected from Ulleung islands received the GenBank accession number MW683220. The selected sequences were aligned using the MEGA (version 7.0) software package to analyze the phylogenetic relationship between algal strain  , and theWCGC-3′) . The ampl strain . Based ol strain .B. sudetica KNUA107 culture was prepared for photoautotrophic and mixotrophic cultivation. The algal cells were cultured with shaking at 160 rpm in an illuminated incubation room at 25°C. The prepared algal cells were diluted to approximately 156.67 ± 20.81 mg/L. BG11 medium was used for photoautotrophic cultivation  and sediment (1 mL). The measurements were performed for 20 min per set, with three sets per culture condition.Colony size, a phenotype associated with settleability, was measured , 2015 in530) was measured in mixtures with sufficient reaction, and the total lipid content was calculated based on the measured value  and at the end (30-day-old) of the cultivation period by centrifugation at 4,000 rpm for 30 min. The collected cells were then freeze-dried. The sulfo-phospho-vanillin colorimetric method was used to determine the total lipid content . Canola ed value .Lipids were extracted and analyzed by gas chromatography/mass spectrometry (GC/MS) to determine their composition and content . Freeze-The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/H-SYu: investigation and writing—original draft. Y-SK: investigation. H-SYu and Y-SK: conceptualization, data curation, formal analysis, and methodology. H-SYu: supervision. Y-SK and H-SYo: funding acquisition. H-SYu, Y-SK, and H-SYo: writing—review and editing. All authors contributed to the article and approved the submitted version.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.


ATG10 is one of the autophagy core genes involved in the autophagosome formation. We hypothesize that SNPs located in regulatory regions of the ATG10 gene could predispose individuals to AML development. We therefore genotyped three SNPs within the ATG10 locus. We identified the ATG10rs3734114 as a potential risk factor for developing AML, whereas the ATG10rs1864182 was associated with decreased risk. These findings highlight ATG10 as a key regulator of susceptibility to AML. Furthermore, we believe that ATG10 SNPs could be exploited in the clinical setting as an AML prevention strategy.Acute myeloid leukemia (AML) is a hematological neoplasm with a very poor survival rate. To date, diagnostic tools to monitor individuals at higher risk of developing AML are scarce. Single nucleotide polymorphisms (SNPs) have emerged as good candidates for disease prevention. AML is characterized by altered autophagy, a vital mechanism to remove and recycle unnecessary or dysfunctional cellular components. ATG10 gene, central for the autophagosome formation. We screened a multicenter cohort involving 309 AML patients and 356 healthy subjects for three ATG10 SNPs: rs1864182T>G, rs1864183C>T and rs3734114T>C. The functional consequences of the ATG10 SNPs in its canonical function were investigated in vitro using peripheral blood mononuclear cells from a cohort of 46 healthy individuals. Logistic regression analysis adjusted for age and gender revealed that patients carrying the ATG10rs1864182G allele showed a significantly decreased risk of developing AML , whereas patients carrying the homozygous ATG10rs3734114C allele had a significantly increased risk of developing AML . Functional analysis showed that individuals carrying the ATG10rs1864182G allele had decreased autophagy when compared to homozygous major allele carriers. Our results uncover the potential of screening for ATG10 genetic variants in AML prevention strategies, in particular for subjects carrying other AML risk factors such as elderly individuals with clonal hematopoiesis of indeterminate potential.Acute myeloid leukemia (AML) is the most common acute leukemia, characterized by a heterogeneous genetic landscape contributing, among others, to the occurrence of metabolic reprogramming. Autophagy, a key player on metabolism, plays an essential role in AML. Here, we examined the association of three potentially functional genetic polymorphisms in the  Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an average age of disease diagnosis above 65 years old . It is aSTAT3rs9909659 could be used to predict the treatment outcome to chemotherapy with daunorubicin and cytarabine . Q. QATG10 ATG10 isoforms described, the study of the ATG10 mRNA levels was performed using a set of primers allowing for the amplification of a region common to all isoforms .p62 is a multifunctional protein involved in different pathways, including autophagy and the proteasomal degradation of ubiquitinated proteins. p62 is an autophagy substrate and like LC3-II has been extensively used as a reporter of autophagy activity . Alteratmulation K, withoumulation L. DecreaATG10rs1864182G and to evaluate its impact on the proteasomal degradation, we evaluated the total ubiquitination profile by immunoblot analysis . The existence of a familiar or personal medical history , the incidence of a primary cancer undergoing chemotherapy, and the presence of clonal hematopoiesis of indeterminate potential (CHIP) are well-known risk factors for AML development. Therefore, the development of new strategies for risk stratification of individuals at higher risk will be important to place them on the medical radar.AML is a severe disease with a rapid progression and a high fatality rate, particularly in the elderly. AML affects 3 to 4 persons per 100,000 per year  [ATG10 SNPs with AML and to explore the functional implications of these SNPs on autophagy. We described the association of ATG10rs1864182G with a decreased probability of developing AML. ATG10rs1864182 was also shown to serve as biomarker for primary or acquired resistance to chemotherapy when using gefitinib -TKI drug) in advanced lung adenocarcinoma patients with EGFR mutations [ATG10rs1864182 in the resistance to chemotherapy may be a useful future biomarker for tailoring AML treatments.After studying the impact of different SNPs on autophagy and other-related processes , in this cancers . Genetic cancers . The ATGt cancer  and melat cancer . However (NSCLC) . To the utations . StudyinATG10rs3734114 was recently associated with increased brain metastasis in NSCLC patients [ATG10rs3734114CT/CC genotypes had an increased cumulative brain metastasis hazard of 46% compared to 13% in patients with the TT genotype [ATG10rs3734114C/C with a higher risk of developing AML, suggesting that the ATG10rs3734114C/C variant could be a risk factor for AML.patients . The stugenotype . In our ATG10rs1864183 and AML. Nevertheless, its association with the development of pharyngeal cancer has been previously described [ATG10rs1864182 and ATG10rs3734114 as risk biomarkers for AML.Finally, in our study, we did not find any association between the escribed . AltogetATG10rs1864182 and ATG10rs3734114 was observed when analyzing the LD between the two alleles. This indicates that the SNPs are not mutually exclusive, even though they have opposite association with AML. Because of the low probability of ATG10rs3734114, we do not have enough subjects to evaluate which effect (protective or risk) prevails in case of co-occurrence, but it would be interesting to address this in the future. It would also be interesting to explore association between these two SNPs and other described SNPs impacting AML [Random association between ting AML .ATG10rs1864182G and ATG10rs3734114C/C with AML, we explored which potential impact those sequence variations might have on mRNA expression and protein function in healthy individuals. Although we were able to detect alterations in the ATG10 protein, with decreased levels of the long isoform in the presence of ATG10rs1864182G, the amount of ATG10 transcripts was not altered. It is important to note that the primers used in our study amplify all the isoforms and do not allow a selective quantification of the different ATG10 transcripts. A putative differential expression between transcripts could justify why ATG10rs1864182 is described to act as an expression quantitative trait locus across multiple tissues, including whole blood, in the Genotype-Tissue Expression (GTEx) project [After flagging the association of  project .ATG10 long isoform in ATG10rs1864182G carriers were associated with the reduction of autophagic flux, its canonical function, in agreement with previous observations [ATG10 immunoblot profile (ATG10 isoforms. Although not described in literature, we hypothesize that these other detected bands could correspond to yet unknown ATG10 isoforms or even ATG10 isoforms that underwent post-translational modifications, which may alter their mobility in the SDS-Page gel. This hypothesis should be explored further in future studies.The decreased protein levels of rvations . Given trvations , and in rvations ,54,55, t profile , we deteATG10rs3734114C/C, due to the low number of individuals in the cohort, no conclusions can be drawn with respect to mRNA levels, protein levels and autophagy. For this reason, an increased number of homozygous mutants for ATG10rs3734114 are needed, or alternatively an in vitro approach using gene editing could be adopted.Regarding ATG10rs1864182, the ATG10rs3734114 is characterized by a missense mutation resulting in a codon for a different amino acid. These mutations can potentially change the functional role of ATG10 in autophagy and/or in other non-canonical processes in which it may be involved. Hopefully, with advances in artificial intelligence tools uncovering the 3D shapes of proteins such as AlphaFold [ATG10 isoforms is involved in non-canonical roles [As with lphaFold , accuratal roles  affectinATG10rs1864182G and a lower-risk of developing AML, which is accompanied by an impairment of ATG10’s autophagy function. In addition, we describe an increased risk of developing AML in individuals carrying the ATG10rs3734114C/C. Our work reveals new mechanisms by which genetic variations in ATG10 may coordinate the development of AML. The gathered evidence could be further exploited in prevention strategies or screening protocols of subjects carrying other risk factors for AML, such as CHIP individuals.In conclusion, we found an association between


In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.The calcium ion (Ca The control of intracellular Ca2+ signals is dynamic. Overall, fluctuations in Ca2+ concentrations are modulated by an influx and/or efflux promoted by membrane channels, such as store-operated calcium channels (SOCEs), plasma membrane Ca2+-ATPase (PMCA) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. Generally, this orchestration in the Ca2+ concentration follows a signaling pathway that obeys the order: stimuli, G-protein coupled receptor, phospholipase C (PLC) activation, mobilization of phosphatidylinositol 4,5-biphosphate P2), production of inositol 1,4,5-phosphate (IP3), IP3 recognition by IP3-sensitive receptor channels (IP3Rs) in the endoplasmic reticulum (ER) and downstream Ca2+ cascade , Ca2+ has to cross several barriers to reach the parasite, which include the red blood cell membrane (RBCM) and parasitophorous vacuole membrane (PVM) (Cane (PVM) .2+ fluctuations in Plasmodium species are very complex and demand the support of intracellular Ca2+ storage. For example, gametocytes or schizont fractions from Plasmodium chabaudi infected RBCs (iRBCs) present 10-20 times more Ca2+ than uninfected RBCs. Moreover, it has been observed that this ion concentrates in parasite storage compartments -sensitive channel ‐mediated protein export across the PV and out into the RBC cytosol by conditional knockdown approach, significantly reduced Ca2+ permeability in iRBCs  was reported in the parasitophorous vacuole (PV) required for proper parasite development (2+ sensor yellow cameleon (YC)-Nano, dynamic measurement of intracellular Ca2+ in different life stages of P. falciparum shows significant fluctuations throughout the parasite development: ring (~370 nM), trophozoite (~30 nM), schizont (~310 nM), merozoite (~950 nM), and gametocyte  stages  stages .2+ fluctuations have now begun to be identified, although it is still a subject under debate. For example, cytoplasmic Ca2+ increase may be related to potassium (K+) availability, especially when parasites are faced with an abrupt change from high to low K+ concentration. Exposition of P. falciparum merozoites to an ionic environment with a low K+ concentration  increases the levels of cytosolic Ca2+  pathway in ecretion . Nonetheecretion . Moreovesurvival .2+ levels. For example, melatonin, which appears as a critical signal controlling synchronous maturation of Plasmodium in vivo, triggers an increase in Ca2+ cytoplasmic concentration through Ca2+ release from intracellular stores by an IP3-dependent pathway activation  and dihydroartemisinin (dART) was ~2.3-fold weaker than those observed between human SERCA and dART, indicating that dART do not inhibit PfSERCA pump activity, refuting the initial conclusion , which stock Ca2+ in an acidic environment  and vacuolar-H+-ATPase (V-ATPase), can use PPi and ATP, respectively, to pump protons toward the lumen of acidocalcisomes, providing acidification of the structures, supporting Ca2+-storage maintenance in these organelles , causing an increase in cytosolic Ca2+ levels , present in the food vacuole membrane (FVM), is apparently very important to balance these processes  in the parasite  and a vacuolar-type H+-pyrophosphatase (TgVP1) are localized in these organelles  was also revealed, highlighting evidence of an acidic environment for Ca2+ maintenance  from P. falciparum in the Xenopus laevis oocystes has been shown to cause Ca2+ uptake after the alkalinization of the intracellular environment, suggesting that the out- or inward-directed Ca2+ proton movement is pH-dependent , essential to ookinete forms and parasite transmission to the mosquito in vivo, but not essential to the erythrocytic stages of P. berghei. A pbcax disrupted strain revealed a stage-specific role of this transporter for Plasmodium survival  identified 103 potential proteins with EF-hand motifs. Nonetheless, this number is undoubtedly overestimated due to the divergence of the EF-hand motif and some rifins. Without rifins, 43 proteins containing EF-hands were recorded in P. falciparum (2+-dependent protein kinases (CDPK) family  family .P. falciparum (PfCaM) is localized diffusely in the cytoplasm during mature stages of the intraerythrocytic cycle and at the apical pole end of merozoites within the ductule of rhoptries , that contain four EF-hand motifs. This group of proteins are involved in parasite cell division at centrosome-like structures, probably in a Ca2+-dependent manner  are known in mammals. P. falciparum protein kinase 2 (PfPK2) is the unique homolog of human functional CaMK that phosphorylates its substrate in a Ca2+- and CaM-dependent manner, and it is expressed during invasion , which are not present in humans. Canonical CDPKs have four EF-hand Ca2+-binding domains attached to the C-terminus of a catalytic kinase domain that shows high homology with CaMK. While CaMKs can self-inhibit through a C-terminal helix, CDPKs are regulated by their Ca2+-binding domains. In these cases, CDPKs undergo structural and conformational changes, promoting the regulation of other proteins by phosphorylation , with correspondent orthologs in plants, but not in animals or fungi . The PfCDPK1 inhibitor, purfalcamine, blocked SERA5 phosphorylation, leading to the blockage of merozoite egress  showed prominently reduced activity compared to wild-type parasites. This lower activity seems to be compensated by PKG, influencing the up-regulation of transcription levels of CDPK5 and CDPK6 in the CDPK1 T145M mutant parasites , suggestarasites . Some rearasites . PfCDPK1 by cAMP . Additioe egress . Despitee egress . Neverthe egress .Plasmodium. In all rodent and some other malaria species, the cdpk2 gene is lacking , such as hepatocytes, involving the induction of the circumsporozoite protein (CSP) cleavage upon contact with hepatocytes . As prev PfCDPK1 . Still, rphology . DespiteP. falciparum genome by in silico analysis , also known as STPP 2B or PP3, is a heterodimeric complex containing a catalytic subunit (CNA) and a regulatory subunit (CNB) . CN is cs target .Plasmodium spp. is required for host cell attachment and invasion in a receptor-dependent pathway distinct from the AMA1-RON2  system but with some degree of functional overlap  contains EF-hands and IQ (the first two amino acids of the motif: Ile and Gln) calmodulin-binding motifs but, in contrast, is monomeric. The CaM-binding motif was found to inhibit phosphatase activity in psis PP7 . PP7 is psis PP7 . These opsis PP7 .2+ signaling and phosphatases are focused on calcineurin. However, other phosphatases without Ca2+-binding motifs could be affected by Ca2+ signaling through their protein partners possessing these motifs. According to STRING prediction and Gene Ontology analyses, amongst the 67 candidate phosphatases identified in P. falciparum, ten potentially interact with proteins involved in Ca2+ signaling: putative acid phosphatase (PF3D7_0918000), putative protein phosphatase 2C (PF3D7_0829100), putative 4-nitrophenylphosphatase (PF3D7_0715000), putative protein phosphatase (PF3D7_0802800), putative RNA lariat debranching enzyme (PF3D7_1340600), putative acid phosphatase (PF3D7_1403900), hypothetical protein (PF3D7_1464600), hypothetical protein (PF3D7_1469200), protein phosphatase 2C (PF3D7_0410300) and putative phosphoesterase (PF3D7_1206000)  . Howeveralarials .P. falciparum reticulocyte binding-like protein 1 (PfRh1) performs a role in initial sensing of Ca2+ followed by signal transduction, causing erythrocyte binding antigen-175 (EBA-175) release from microneme and allowing tight junction formation  has been proposed as a protein to potentially connect this motor complex to the IMC membrane. It is also involved in gliding and invasion processes in a Ca2+-dependent manner. PfIMC1l interacts directly with Ca2+ and its interaction with actin is enhanced in the presence of this ion  and the CDPK4 substrate SOC6 (PBANKA_070770), involved in IMC biogenesis  and P. falciparum reticulocyte binding-like protein 2b (PfRh2b) (P. berghei rhoptry neck protein 11 (PbRON11) in sporozoites reduced attachment and motility, leading to the impairment of the infection in the mosquito salivary gland and hepatocyte cells. This protein contains putative EF-hand domains and might act by controlling rhoptry protein secretion in a Ca2+-dependent manner  results in a cytoplasmic lowering of Ca(PfRh2b) . Rhoptry(PfRh2b) . The rept manner .2+ with parasite egress was previously reported . Conditional knockdown of ICM1 revealed an essential role in Ca2+ mobilization to initiate both Plasmodium gametogenesis and merozoite egress  and PKG activation, essential for the protein discharge of secretory organelles, known to support this process . Correlareported . Events  by Ca2+ . More ree egress . Additiol stores .P. falciparum phosphodiesterase β (PfPDEβ) leads to a dramatic reduction in schizont cAMP and cGMP hydrolytic activity, resulting in elevated cAMP levels and inappropriate cAMP-induced increased phosphorylation of PKA substrates. In addition, PKA seem to assume a compensatory role with PKG, in order to phosphorylate P. falciparum myosin A (PfMyoA), an important component of the so-called glideosome, a parasite complex involved in host cell invasion, in PfPDEβ mutants, bypassing the need for PKG activity by elevated cAMP levels upon Ca2+ signaling, possibly by PKA action   and an ionophore, ionomycin, did not change the amounts of cytosolic Ca2+ in knockdown parasites bearing a glucosamine-inducible ribozyme gene (PfERC-glmS) from ER or neutral Ca2+ storages, suggesting that the availability of Ca2+ from different sources does not change upon knockdown of PfERC. Moreover, PfERC is required for the complete maturation of the aspartic protease plasmepsin X (PMX) in a Ca2+-dependent manner, which is required to cleave the subtilisin-like protease (SUB1) , has been remarkably underexplored, considering that proteins from this family are widely found from protozoans to mammals . A membeprotein) . This pre (SUB1) .2+ importance for SUB1 discharge and proteolytic cascade events have been reported. Chelation of intracellular Ca2+ in P. falciparum schizonts blocks the SUB1 discharge from merozoite exonemes into PV, resulting in a decrease of SERA5 proteolytic cleavage and harming PVM rupture and merozoite egress  in a temperature-dependent process  in hepatocytes and intracellular Ca2+ elevation, essential to EEF development (Ca process . It has e stages . Sporozoelopment . The intPlasmodium species contain distinctive features when compared to other eukaryotes. Such characteristics define its phylum or genus, in which attachment to the host cell, motility, invasion and egress are essential for survival and dissemination. Since Ca2+ signaling regulates important and specific Plasmodium cellular processes such as microneme secretion, attachment, gliding motility, invasion and egress, actors involved in these pathways, which are regulated by this ion, could be considered potential drug targets. Striking progress to achieve a broader understanding of Ca2+ signaling in Plasmodium has been made, including the potential involvement of host compounds in Ca2+ uptake, such as K+, Na+, ionic strength and melatonin (elatonin .2+ uptaking by iRBCs, together with transport through PVM and the parasite cellular membrane. Moreover, IP3R or alternative functional protein identification in Plasmodium would be an important breakthrough to explore Ca2+ mobilization and storage, as well as backstage actors which support those processes as promising therapeutic targets. Actually, a plethora of Plasmodium proteins with standard and unusual Ca2+ binding motifs, which are known or suspected to be involved in Ca2+ signaling, could be explored to this end. This also includes proteins without Ca2+-binding motifs acting as indirect effectors.However, several gaps in understanding remain in these organisms, covering mechanisms involved in increased Cain vivo protein-partners, such as BioID and APEX-2 proximity-labelling techniques (2+ ratiometric techniques coupled to imaging reporters (2+ in the backstage of malaria parasite biology and drug screening assay design.The association of classical techniques employed to study permeability, protein channels and pumps, together with more recent high-throughput approaches is a promise to fulfill these gaps. Mass spectrometry-based proteomics , includichniques , metabolchniques  and new eporters  are examAll authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Properties of nanolayers can substantially differ from those of bulky materials, in part due to pronounced interface effects. It is known that combinations of layers of heavy and ferromagnetic metals leads to the appearance of specific spin textures induced by interface-induced Dzyaloshinskyi–Moria interaction (DMI), which attracts much interest and requires further studies. In this paper, we study magneto-optical effects in two- and three-layer films composed of a few nanometer thick Co layer adjacent to nanofilms of non-magnetic materials . For experimental studies of the interface magnetization-induced effects, we used the optical second harmonic generation (SHG) technique known for its high sensitivity to the symmetry breaking. We found that the structural asymmetry leads to the increase of the averaged SHG intensity, as well as to the magnetic field-induced effects in SHG. Moreover, by choosing the proper geometry of the experiment, we excluded the most studied linear in magnetization SHG contributions and, thus, succeeded in studying higher order in magnetization and non-local magnetic effects. We revealed odd in magnetization SHG effects consistent with the phenomenological description involving inhomogeneous (gradient) magnetization distribution at interfaces and found them quite pronounced, so that they should be necessarily taken into account when analyzing the non-linear magneto-optical response of nanostructures. Structuring of a medium at nanoscale can give rise to new effects in their optical and magnetic response ,2. It isThe technique of optical second harmonic generation (SHG) is known for its extremely high sensitivity to the state of surfaces and buried interfaces, including morphology, crystallographic symmetry, magnetization state, etc., ,11. At tThe films were fabricated by magnetron sputtering of metal or dielectric targets on quartz or glass substrates using a high vacuum magnetron AJA 2200 multichamber system  operating at the basic pressure of 10ed films .As the fundamental beam in non-linear optical studies we used the radiation of a Ti:Sapphire laser with the wavelength of 820 nm, pulse duration of 20–80 fs, repetition rate of 80 MHz and the mean power of 50 mW focused on the sample into a spot of about 30 μm in diameter. The pump radiation was incident on the sample at the angle of 45Magneto-optical effects in the SHG response were studied in two geometries: (i) longitudinal non-linear magneto-optical Kerr effect, when the magnetic field-induced rotation of the polarization plane of the SHG wave with respect to initially p-polarization is detected, and (ii) “forbidden” effect, when the longitudinal magnetic field is applied and the magnetization-induced changes of p-polarized SHG was detected. It is worth to remind that the latter effect should vanish for homogeneously magnetized media . For stuFirst, the morphology of all the films was studied by atomic force microscopy; it was shown that the mean roughness of the surface is less than 2 nm . At the All the films reveal pronounced magneto-optical longitudinal effect, i.e., induced rotation of the SHG polarization plane that appears as the SHG magnetic contrast Concerning the “forbidden” SHG effect that consists in modulation of the p-polarized SHG intensity by applied longitudinal magnetic field, the observed magnetic contrast in all cases does not exceed a few percent see . Among tAmong three-layer Co-containing films, only asymmetric Pt(3)/Co(3)/W(3) shows detectable M is the magnetization, Let us discuss the consequences of the observed effects in the SHG response of studied films of different compositions and in the considered experimental geometries. First it is necessary to remind that due to the inversion symmetry of all the constituent materials used for the fabrication of the films, SHG sources in the electric dipole approximation are attributed to symmetry breaking at the interfaces. This is valid as well in the case of magnetic field induced terms of the non-linear susceptibility that determine magneto-optical effects at the SHG wavelength. Following the description presented in ,19, the M non-linear optical effects, while the last terms appear if the magnetization distribution is inhomogeneous. It is worth noting that The first term in  describeAccording to the symmetry analysis first presented in , longituterfaces ,20. If sIf considering multilayer films, the strongest magnetic field induced effect in the SHG response is observed only in the case of non-equivalent magnetic interfaces, i.e., Pt/Co and Co/W, coexist in the same structure, while for symmetric ones like Co/Pt/Co or Pt/Co/Pt it is within the experimental error. Strong MOKE-SHG effect for asymmetric three-layer films was reported recently for the transmission geometry , while oSimilar conclusion on the role of interfaces in the appearance of MOKE-SHG effects in Co-containing films stems from the analysis of the value of the “forbidden” effect in various structures. First of all it is necessary to remind that for the case of longitudinal magnetic field only induced rotation of the SHG polarization plane is expected for homogeneously magnetized surfaces, so the intensity of the p-polarized SHG detected in the experiment should remain unchanged . This contensity ,19. At tshown in ,19. One Summing up, we studied experimentally the appearance of magneto-optical effects accompanying second harmonic generation in two- and three-layer films containing ferromagnetic cobalt layers adjacent to different non-magnetic specimen. Besides common longitudinal magneto-optical SHG effect consisting in the magnetic field induced SHG polarization plane rotation, extra one forbidden for homogeneous ferromagnetic interface is observed in a number of layered structures. A set of experiments performed in two- and multilayered films containing ferromagnetic layers shows that this SHG effect is the most pronounced if (i) the structure contains interfaces between ferromagnetic (Co) and heavy metals , and (ii) is enhanced if the Co-containing multilayer is squeezed between different heavy metals (Pt/Co/W). As a qualitative explanation of the “forbidden” effect in the intensity of the p-polarized SHG induced by the longitudinal magnetic field we suggest inhomogeneous magnetization distribution in the direction perpendicular to the films’ surface.


Even though allogeneic stem cell transplantation is the most intensive treatment for acute myeloid leukemia (AML), chemo-resistant leukemia relapse is still one of the most common causes of death for these patients, as is transplant-related mortality, i.e., graft versus host disease, infections, and organ damage. These relapse patients are not always candidates for additional intensive therapy or re-transplantation, and many of them have decreased quality of life and shortened expected survival. The efficiency of azacitidine for treatment of posttransplant AML relapse has been documented in several clinical trials. Valproic acid is an antiepileptic fatty acid that exerts antileukemic activity through histone deacetylase inhibition. The combination of valproic acid and all-trans retinoic acid (ATRA) is well tolerated even by unfit or elderly AML patients, and low-toxicity chemotherapy  can be added to this combination. The triple combination of azacitidine, valproic acid, and ATRA may therefore represent a low-intensity and low-toxicity alternative for these patients. In the present review, we review and discuss the general experience with valproic acid/ATRA in AML therapy and we discuss its possible use in low-intensity/toxicity treatment of post-allotransplant AML relapse. Our discussion is further illustrated by four case reports where combined treatments with sequential azacitidine/hydroxyurea, valproic acid, and ATRA were used. Acute myeloid leukemia (AML) is an aggressive malignancy that can only be cured by intensive chemotherapy possibly followed by autologous or allogeneic stem cell transplantation ,2. The mRelapse is a common cause of death after potentially curative intensive conventional chemotherapy, and a relatively large group of patients relapse even after the most intensive treatment including allogeneic stem cell transplantation ,2. ThereSeveral experimental and clinical studies have investigated the effects of ATRA combined with valproic acid in the treatment of AML. Important characteristics of the two drugs are summarized in Valproic acid is a fatty acid that is used as an antiepileptic drug; it also has anti-AML effects probably due to its function as a histone deacetylase inhibitor and a systemic metabolic modulator  3,5]. I,5. I3,5]RUNX1-RUNX1T1 fusion oncogene  translocation) suggest that valproic acid upregulates histone acetylation in the p21 promoter region, leading to increased p21 expression, suppressed phosphorylation of the retinoblastoma protein, and finally induction of G0/G1 arrest [RUNX1-RUNX1T1 fusion [RASSF1A expression) may be an antileukemic mechanism especially in patients with normal and adverse karyotype [The antileukemic effects of valproic acid wer investigated in several previous studies. Studies in a murine xenograft model based on the Kasumi-1 AML cell line with the 1 arrest . This isi.e., t8; transloc1 fusion . Howeveraryotype . Second,aryotype . Third, aryotype . Howeveraryotype , and suc+ hematopoietic cells showed only growth enhancement for intermediate concentrations but no antiproliferative effect with higher concentrations. Thus, AML patients are heterogeneous with regard to the effects of valproic acid on their leukemia cells, and this heterogeneity may be caused by differences in the balance between genes that mediate chemosensitivity versus chemoresistance, as described above.The studies described above suggest that the antileukemic effects of valproic acid are mediated through several molecular mechanisms that possibly differ between patients. A previous in vitro study investigated patient heterogeneity with regard to the functional effects of valproic acid and three other HDAC inhibitors on primary AML cells . Two maiDNMT3A)  together with several genes involved in G-protein coupled receptor signaling, and the combination decreased the protein levels of Gata-2, NFκB p65, and Bcl-2 in AML cells [Several studies have investigated the effects of valproic acid on AML cells derived from patients receiving valproic acid treatment. First, valproic acid monotherapy with serum concentrations corresponding to the accepted therapeutic level can induce hyperacetylation of histones H3 and H4, signs of myelomonocytic differentiation, and hematological improvement according to the response criteria for myelodysplastic syndromes (MDS) . These rML cells . Third, ML cells  comparedML cells , and valML cells .The effect of in vivo therapy based on ATRA plus valproic acid on the proteomic and phosphoproteomic profiles of human AML cells was investigated in a recent study . This inExperimental studies of human and murine hematopoietic stem cells show that valproic acid increases their proliferation and self-renewal capacity . The druThe effect of valproic acid on AML-associated angiogenesis has been investigated in a murine xenograft model using the Kasumi-1 AML cell line . ValproiT cells. Valproic acid has strong antiproliferative but not proapoptotic effects on T cells in the presence of cytarabine, and it can also alter the expression of the activation markers CD38 and CD69 as well as the release of FasL, heat shock protein (HSP)90, and various cytokines . The lev+ T cell proliferation and IFN-γ production; and increases the proportion of anti-inflammatory IL-10-positive T cells [+CD4+ alloreactive T cells and the granzyme B expression by CD8+ T cells were both decreased when these cells were cocultured with dendritic cells that were previously exposed to valproic acid. Finally, HDAC inhibition can reduce CD1a expression together with the capacity of chemokine-induced migration, immunostimulatory capacity, and cytokine release in monocyte-derived dendritic cells [Dendritic cells. Valproic acid inhibits the release of IFN-α, TNFα, and IL-6 by plasmacytoid dendritic cells; reduces their expression of costimulatory molecules as well as their capacity to promote CD4 T cells . The dru T cells . Further T cells . These lic cells . These lMonocytes/macrophages. Low levels of valproic acid alter the differentiation of human monocyte-derived macrophages and their expressions of CXCL8/IL-8, IL-1β, IL-6, TNFα, and IL-10 (but not IL-12), whereas higher valproic acid levels reduce the release of cytokines in general . FurtherMesenchymal stem/stromal cells (MSCs). Valproic acid increases the glycolytic, respiratory, and T-cell suppressive capacity of MSCs ; increasValproic acid affects several immunocompetent cells, and immunosuppressive effects seem to be most common. The effects for various cell types are described more in detail below.To conclude, valproic acid can modulate the function of several immunocompetent/immunomodulatory cells, but it is difficult to know whether this immunomodulation is important for direct  or indirect  antileukemic effects of various immunocompetent cell subsets in vivo.Valproic acid alters the serum levels of many different soluble mediators , includiValproic acid therapy alters the systemic serum metabolic profiles of AML patients : it had The effects of ATRA and other vitamin A derivatives in non-APL AML have been reviewed in several recent excellent reviews ,55,56,57NPM1 mutations with FLT3-wild type (wt) [Experimental studies suggest that the antileukemic effect of ATRA varies between patients ,58, and ype (wt) . Anotherype (wt) . Howeverype (wt) ,63. Furtype (wt) . It shouype (wt) ,65, and RUNX1-RUNX1T1 translocation seems to confer ATRA resistance [t AML. The sistance , althougsistance .FLT3-ITD. Studies in an animal model of FLT3-ITD/NPM1c-driven AML suggest that ATRA has an antileukemic effect in this AML model through effects on leukemic stem cells, but this pharmacological effect is counteracted by experimental expression of the stem cell-associated transcription factor EVI1 [tor EVI1 .NPM1 mutations. Experimental studies suggest that ATRA can lead to degradation of mutated NPM1 with re-localization of NPM1-wt encoded by the second gene and therefore can induce cell cycle arrest, differentiation, and increased sensitivity to conventional cytotoxic drugs [ic drugs ,69. Thesic drugs ,60.IDH1 mutations. Experimental studies suggest that patients with IDH1 mutations are more sensitive to ATRA [ to ATRA .MN1 expression. MN1 encodes a transcription cofactor that is a member of the RAR/RXR complex, and high expression is associated with resistance to ATRA [High  to ATRA .EVI1 expression. This gene encodes a stem cell-associated transcription factor that is downregulated during hematopoietic differentiation. High expression is observed for 10% of AML patients and is associated with an adverse prognosis [EVI1 expression, but the effect is weak or absent in AML derived from progenitor cell transformation and shows low EVI1 expression [High rognosis ,71,72,73rognosis ,75,76, arognosis , therebyrognosis . These apression . Howeverpression .Several studies suggest that the effects of ATRA on primary non-APL AML cells vary between patients. The following factors seem to be associated with susceptibility to ATRA:Additional pharmacological factors may also influence the effect of ATRA. First, bone marrow stromal cells can degrade ATRA and therefore limit the antileukemic effects . Second,ATRA has several effects on normal hematopoiesis and is important both for granulopoiesis and erythropoiesis. A detailed review of these effects is outside the scope of this article, but a detailed discussion with additional references can be found in recent reviews ,84,85. AThe role of vitamin A in immunoregulation has been reviewed in several recent articles ,93,94,95The conclusion from several previous reviews is that the effects of vitamin A on immunoregulation and immunocompetent cells will depend on the dose, the tissue, and the biological context of the immune response ,93,94. R+ T cells, and a decreased proportion of donor CD4+ T cells in marrow grafts leads to reduced incidence and severity of GVHD [+ and CD8+ T cells in the gut mucosa, leading to an exacerbation of colonic GVHD with increased mortality [The effects of ATRA on immunocompetent cells will be of particular importance in allotransplant recipients because immune-mediated antileukemic activity contributes to the overall antileukemic effect of this strategy but especially because immune-mediated complications contribute to the posttransplant non-relapse mortality . ATRA is of GVHD . This stortality . Retinoiortality .p = 0.0008). The same was true for treatment-related mortality , and bloodstream infections were increased in patients with low vitamin A levels  [The results from animal studies are in contrast with the observations from two studies in humans. A study of 114 consecutive pediatric allotransplant recipients demonstrated low levels of vitamin A at day +30 posttransplant to be associated with increased gastrointestinal GVHD at day +100  . Surpris = 0.03) . Another = 0.03) .Taken together, these observations suggest that ATRA therapy after allotransplantation influences the immunological functions of the stem cell recipients, but possible effects with regard to the development of GVHD are difficult to predict and further studies are needed.ATRA alters the systemic  metabolic profiles in AML patients, especially the levels of several amino acid and lipid metabolites, but these effects are relatively weak compared with valproic acid . ATRA haThe results described previously in A recent study investigated the in vivo effects of ATRA alone and ATRA/valproic acid-based therapy on the proteomic and phosphoproteomic profiles of primary human AML cells . The resEven though both ATRA and valproic acid have effects on transcriptional regulation and gene expression, we regard their effects on protein levels and posttranscriptional protein modulation  to be of particular importance because several new AML therapies target various proteins  or protein modifications . In our opinion, proteomic/phosphoproteomic studies are therefore an important part of the scientific basis for the design of future pharmacological combinations.Only a minority of patients (approximately 25–35%) respond to this treatment.Most responders show stabilized disease when using the MDS response criteria see . Excepti9/L to stabilization at or above 20–50 × 109/L.The most common response is stabilized/increased peripheral blood platelet counts, and for most patients, the duration of such responses is 2–5 months, although exceptional patients without complete hematological remission have responses that last for 12–18 months. The improvement in platelet counts can be from pretreatment levels below 10 × 10Responses can even be seen for patients with high-risk disease  ,37 and fToxic effects are usually dose-dependent and reversible, and the most common toxicities are gastrointestinal side effects and fatigue.Several studies have investigated the effects of valproic acid monotherapy or combination therapy including ATRA or low-dose chemotherapy for elderly or unfit AML patients, including patients with high-risk or relapsed AML ,119,120.Thus, the valproic acid plus ATRA combination has clinically relevant antileukemic effects with acceptable toxicity in human AML, but the effect is usually limited unless low-toxicity chemotherapy is added.Experimental studies suggest that the HDAC inhibitor panobinostat downregulates the expression of the transcription factor E2F1 and therefore suppresses the expressions of BRCA1, CHK1, and Rad51; this leads to an increased susceptibility to both cytarabine and daunorubicin-induced apoptosis . StudiesOnly a few early clinical studies of panobinostat in AML have been published. First, it has been combined with intensive chemotherapy both for younger patients  and eldeTo the best of our knowledge, there are no reports on the use of valproic acid + ATRA + azacitidine in allotransplant recipients. We therefore established a protocol where AML patients were included after written informed consent; the study was performed in accordance with the Helsinki declaration and approved by the regional ethics committee  and The Norwegian Medicines Agency . Collection of the clinical data was registered by official Norwegian authorities , and the study was registered in public databases . All patients had non-APL variants of AML diagnosed in accordance with the WHO criteria . The dia2 twice daily was given for the first 14 days of each cycle (day 1–14); this therapeutic strategy has previously been used for AML-stabilizing treatment [This phase II study intended to include at least 10 patients. The scientific basis for the design of the study is described in reatment ,37,136. reatment . Finallyreatment ,37,138. 9/L from the nadir level, with this difference being >20% of the nadir value. Second, increased neutrophil counts were defined as (i) an increase from <0.5 to >1.0 × 109/L or, (ii) in the case of a higher pretreatment level, at least a doubling of the original value. Finally, increased reticulocytes were defined as (i) a doubling of the count or normalization of initially decreased levels. These effects had to be detected in at least two independent samples, and the duration of stabilization was defined as the time from first significantly altered levels until the first cell count below the pretreatment level.There are no generally accepted response criteria for patients receiving AML-stabilizing treatment. In our opinion, one should use the generally accepted criteria for complete hematological remission, i.e., <5% blasts in the bone marrow together with normalization of peripheral blood cell counts . In prevSeveral studies have investigated the combination of hypomethylating agents and valproic acid in AML ,141,142.9/L. His platelet counts later increased to stable levels above 40–50 × 109/L  and his peripheral blood blast counts were stable, but he still required regular erythrocyte transfusions. The median valproic acid serum level was 395 μmol/L (range 299–512 μmol/L) until he was admitted to a hospital by the end of the third cycle when he had signs of AML progression with musculoskeletal pains, increasing peripheral blood blast counts and severe thrombocytopenia. Progression occurred after 94 days of treatment, and he died 29 days later.The patient was a 22-year-old male with an early second AML relapse (complex karyotype) four months after re-transplantation for the first posttransplant relapse . His ECOThe patient was a 57-year-old female who was allotransplanted for AML in second complete remission with a matched unrelated donor. She developed acute GVHD in the skin early after transplantation but required only topical steroid treatment. AML relapse was diagnosed 71 days after transplantation . CyclospThe patient was a 63-year-old male who developed secondary AML after treatment for non-Hodgkin’s lymphoma. He achieved complete hematological remission after the second induction cycle and was transplanted with reduced intensity conditioning (RIC). AML relapse was diagnosed less than four months posttransplant. Skin GVHD was diagnosed before the relapse and was treated with oral prednisone 30 mg daily in addition to topical steroids and continued cyclosporine therapy. Cyclosporine treatment was stopped when relapse was diagnosed, whereas steroid use was gradually reduced. The patient developed severe diarrhea three weeks after the start of the study treatment. Despite increased steroid treatment, the symptoms did not improve; he could not receive oral nutrition/medication and was removed from the study 46 days after inclusion. He died 21 days later due to AML progression, severe gastrointestinal GVHD, and pneumonia.9/L with mainly blast cells and severe thrombocytopenia <10 × 109/L. Her platelets increased to 12 during the first cycle; at the same time, her leukocytes were 22.6 × 109/L with neutrophils 5.9 × 109/L. After the second azacitidine treatment, the dose of hydroxyurea was gradually increased to 1500 mg daily. The situation was stable with no signs of GVHD, total leukocytes in the range of 20–30 × 109/L, normalized neutrophils 5.9 × 109/L, but platelets <10 × 109/L. She required erythrocyte transfusions every 2–3 weeks from inclusion until disease progression was diagnosed 98 days after start of the treatment. She died 30 days later.A 39-year-old female was diagnosed with atypical chronic myeloid leukemia (aCML). She received RIC conditioning and was transplanted from a HLA identical sibling. There was no acute GVHD, but she relapsed 20 months posttransplant with AML. At the start of treatment, she had 30% leukemic blasts in the bone marrow. She received azacitidine and hydroxyurea according to the protocol together with valproic acid . Pretransplant peripheral blood cell counts showed total leukocytes 6.8 × 10All of our patients received a combination of sequential azacitidine/hydroxyurea, continuous valproic acid, and intermittent ATRA therapy. We emphasize that one should be very careful when making conclusions based on our case reports, but some suggestions can be made. First, especially case report 1 but also case report 4 suggest that our low-toxicity treatment stabilized the clinical status and could be administered while the patient was at home. Second, an acceptable quality of life with this outpatient treatment was then possible. Finally, the treatment probably did not induce GVHD, and this is in accordance with the previous studies of valproic acid effects on immunocompetent cells where immunosuppressive effects seem to dominate (see above). It is also in accordance with a previous pediatric study describing higher frequency of acute GVHD in patients with low vitamin A levels compared with higher levels . HoweverTo the best of our knowledge, no other data are available for such a combination treatment for posttransplant AML relapse, but a clinical study of low-dose treatment with the hypomethylating agent decitabine in combination with the alternative HDAC inhibitor panobinostat alone was recently published . This trVarious strategies for posttransplant prophylaxis and treatment of AML relapse are summarized in As emphasized in a recent review , most clThe initial immunomodulatory strategy is to enhance the graft-versus-leukemia effect by reducing or ending immunosuppressive treatment. If significant GVHD or GVL effects do not occur after this intervention, DLI may be used to trigger GVL . SeveralIntensive chemotherapy can be considered as a possible strategy especially for young patients that are fit enough to tolerate such treatments; if patients achieve complete remission, an allogeneic re-transplantation may be considered, but this decision should be individualized and should be based on careful evaluation of the risks of severe toxicity versus the possibility of maintained remission and cure ,143. ThiPatients receiving the best supportive care alone for AML spend 25–30% of their time in hospital and approximately 15% of their time attending clinic appointments . Many ofTwo studies suggest that elderly/unfit AML patients treated with hypomethylating agents show improved QoL compared with patients receiving only supportive care ,159, butRelatively few studies of health-related QoL are available for allotransplanted patients. One study investigated 115 patients during the first year posttransplant ; 72% of Leukemia relapse is a common cause of death in allotransplanted AML patients. The risk of relapse may possibly be reduced by additional posttransplant chemotherapy, e.g., kinase inhibitors , low-dosThe efficiency of hypomethylating agents and especially azacitidine as an AML-stabilizing treatment for posttransplant AML relapse was documented in several clinical studies  135,137,151,152.Valproic acid as well as several other HDAC inhibitors are now considered for the treatment of AML, but even the newer inhibitors seem to have a limited antileukemic effect when used as monotherapy ,187,188.As described in Checkpoint inhibitors are now being tried in the treatment of AML . This thTo answer our initial question, in our opinion, valproic acid and ATRA should be further investigated for the treatment of post-allotransplant AML relapse. It is a unique combination with antileukemic effects that may be mediated both through systemic metabolic modulation and through direct effects on the leukemic cells. However, neither of the two drugs should be used as monotherapy but should be used as a part of a low-toxicity AML-stabilizing treatment, especially for unfit patients, e.g., patients with reduced posttransplant quality of life. It should also be emphasized that reducing the risk of relapse through posttransplant antileukemic pharmacotherapy and/or DLI needs to be further explored ,143,195


Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects. Already at low disease burden, AML is marked by severe hematopoietic failure, which is the major cause of death. Additionally, despite intensive chemotherapeutic protocols, high relapse rates and primary resistance frequently occur, requiring further treatment intensification, including allogeneic hematopoietic stem cell transplantation1. However, many elderly AML patients are not eligible for intensive treatment protocols and therefore show disappointingly low overall survival rates with few patients surviving beyond five years of follow-up2. A major obstacle in AML therapy is persistence of quiescent leukemia-initiating cells, which are protected in their bone marrow niche3. Therefore, uncovering the microenvironmental regulation of AML cells and defining, ideally less toxic, niche-targeted therapies is of utmost need to make leukemia cells more vulnerable to genotoxic drugs and efficiently eradicate residual leukemia cells. Recently, we have gained increasing insight how AML cells are regulated in the bone marrow niche in murine models and humans6. AML infiltration induced a significant release of inflammatory cytokines in the human bone marrow, which proved to accelerate leukemogenesis7. The transmembrane glycoprotein CD38 appears to regulate cytokine release, adhesion, and cellular migration toward sites of inflammation8. CD38 is widely expressed on immune cells and its expression level depends on the maturation and activation state9. Besides, most AML blasts show high CD38 expression without obvious correlation with cytomorphological and genetic characteristics10. Indeed, CD38 monoclonal antibodies have shown high efficacies in multiple myeloma at different lines of therapy, which is exerted through both antibody-mediated cytotoxicity, as well as modulation of immune surveillance in the niche12. Notably, the first-in-class human IgG1κ anti-CD38 antibody daratumumab showed, throughout a number of clinical studies, a beneficial spectrum of side effects. Therefore, inhibition of CD38 in AML appears to be an appealing concept. Here, we show that CD38 inhibition leads to significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, with modest cell autonomous cytotoxic activity. Daratumumab significantly induces antibody dependent phagocytosis in AML and shows interference with AML cell trafficking in vivo in a xenograft transplantation model.Acute myeloid leukemia (AML) is the most common acute leukemia in adults with increasing incidence with rising age7. We could confirm that co-culture with stroma cells led to an increased expansion of AML cells . Further, we did not observe any substantial cytotoxic effects of daratumumab on healthy peripheral blood mononuclear cells . Further, similar cytostatic effects were observed when AML cells were treated with isatuximab, another anti-CD38 antibody .To test if CD38 inhibition affects leukemia growth in AML, we applied the monoclonal antibody daratumumab on a series of different AML cell lines in mono-culture, as well as in co-culture to elaborate putative niche-mediated effects of daratumumab. AML cell lines were cultured with endothelial cells  or MS-5 fibroblasts, which have been shown before to significantly support AML cell line growthlls Fig. a. Daratulls Fig. b,c. Appllls Fig. b. The an13. In fact, treatment with 0.1 µM ATRA led to a 25-fold higher CD38 expression in AML cells independent of the culture condition, while healthy control cells only showed a three to sixfold increased expression  in OCI-AML3 and Kasumi-1 cells as the most drug-responsive AML cell lines, which were preincubated with calcein-AM and treated with increasing concentrations of daratumumab or isotype control. Here, daratumumab did not induce significant cell death in the presence of normal human serum (10%) , adhesion  and angiogenesis-related factors  in mono- or co-cultured cells treated with either ATRA, daratumumab or both. However, we did not observe any differences in mRNA levels neither in AML cells nor endothelial cells co-cultured with either AML or control peripheral blood mononuclear cells after treatment with daratumumab (Supplementary S5a-b). In conclusion, the microenvironment-mediated anti-leukemic activity of daratumumab is not due to interference with mitochondrial transfer nor in the expression of adhesion-, migration- and angiogenesis-related factors in vitro.As human CD38 was shown to bind to CD31, which is highly expressed on endothelial cells, we assumed a putative role in interference with adhesion and circulation of leukemia cells7. Given the robust upregulation of CD38 expression in AML upon ATRA treatment . In 3 out of 5 experiments we observed a striking decrease in human AML cells in peripheral blood by -30%, while overall no significant reduction in leukemia burden was observed in bone marrow and spleen  were purified using Lymphoprep™ . Patient characteristics are summarized in Supplemental Table 1.−/− (NSG) mice were bred and used in the animal care facility at the University Hospital Essen. Human mononuclear cells derived from untreated AML patients were depleted from CD3+ cells using 2 µl OKT3/1 × 106 cells  and intravenously transplanted into NSG mice. Bone marrow and spleen cells were harvested from leukemic mice and used for secondary transplantation without prior conditioning. Leukemic mice were equally randomized according to their peripheral blood leukemia burden to each treatment group. Daratumumab (8 mg/kg) or IgG1 control antibody were given once weekly intraperitoneally for 4 weeks, while all-trans-retinoic acid (ATRA) was administered at 10 mg/kg intraperitoneally for 4 consecutive days per week for a total of 4 weeks , 24 h later mice were transplanted with primary human AML cells. After 16 h, mice were sacrificed and leukemic infiltration in bone marrow, spleen and peripheral blood was assessed by flow cytometry.NSG mice were treated with daratumumab or IgGAll data are shown as mean ± SEM. Shapiro–Wilk test was applied as a test of normality. In case of normality, Student´s t test was applied for comparisons between two groups. Mann–Whitney U test and Wilcoxon signed-rank test were applied for nonparametric unpaired and paired analyses. Multiple comparisons were calculated using the one-way ANOVA test. Analyses were performed with GraphPad Prism software . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Supplementary Information.