Alaria angusta and protolinckiosides A (PL1), B (PL2), and linckoside L1 (L1) of the starfish Protoreaster lincki using a 3D cell culture model. The 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), soft agar, 3D spheroids invasion, and Western blotting assays were performed to determine the effect and mechanism of the action of investigated compounds or their combinations on proliferation, colony formation, and the invasion of 3D HCT 116 spheroids. AaLs, PL1, PL2, and L1 individually inhibited viability, colony growth, and the invasion of 3D HCT 116 spheroids in a variable degree with greater activity of linckoside L1. AaLs in combination with L1 exerted synergism of a combined anticancer effect through the inactivation of protein kinase B (AKT) kinase and, consequently, the induction of apoptosis via the regulation of proapoptotic/antiapoptotic proteins balance. The obtained data about the efficacy of the combined anticancer effect of a laminaran derivative of brown algae and polyhydroxysteroid glycosides of starfish open up prospects for the development of new therapeutic approaches for colorectal cancer treatment.Colorectal cancer is one of the most frequent types of malignancy in the world. The search for new approaches of increasing the efficacy of cancer therapy is relevant. This work was aimed to study individual, combined anticancer effects, and molecular mechanism of action of sulfated laminaran AaLs of the brown alga  Oncological diseases are one of the main causes of human mortality in the modern world, and the economic and financial burden for cancer research is increasing . ColorecMarine natural compounds such as polysaccharides from brown algae and steroidal compounds from starfish are of constant interest due to their extremely broad spectrum of biological action. Neutral water-soluble 1,3;1,6-β-D-glucans of brown algae (laminarans) and their chemical derivatives possess immunomodulatory, antiproliferative, and radioprotective activities . To dateEscherichia coli [Natural steroidal compounds represent a large class of secondary metabolites found in various terrestrial and marine organisms, in particular, in marine invertebrates. Mainly, the interest in these compounds is explained by their various biological activities, including anticancer, antiviral, anti-inflammatory, analgesic, hemolytic, hypotensive, neuroprotective, and immunomodulatory properties ,18,19,20hia coli .We hypothesized that the combination of the unique biological properties of the metabolites of brown algae and starfish will contribute to the development of highly effective approaches for the treatment of advanced colorectal cancer.Alaria angusta and polyhydroxysteroid glycosides from the starfish Protoreaster lincki with focus on the proliferation, colony growth, and invasion of 3D spheroids of colorectal carcinoma HCT 116 cells.The aim of the present work was to investigate the individual and combined anticancer effects and molecular mechanism of the action of sulfated laminaran from the brown alga A. angusta was isolated according to a modified method described in recent work [A. angusta were determined using high-performance liquid chromatography (HPLC), chemical , and 13C NMR spectroscopy  [Alaria marginata [Saccharina cichorioides and from Saccharina japonica [Saccharina gurjanovae [Coccophora langsdorfii [Sargassum duplicatum [Sargassum fusiforme [Laminaran from the brown alga ent work . To remotroscopy . It was troscopy . The lamyotales) , Alaria arginata , Saccharjaponica , Saccharrjanovae   has a 3β,4β,6β,7α,8,15α,16β,26-octahydroxysteroid nucleus, protolinckioside B (PL2) has a 3β,5,6β,8,15α,24-hexahydroxysteroid nucleus, and linckoside L1 (L1) has a 3β,4β,6β,8,15α,24-hexahydroxysteroid nucleus. The monosaccharide residues in PL1 and L1 are the 2-O-methyl-β-D-xylopyranose attached to C-3 of the steroid nucleus, and in PL2—the α-L-arabinofuranose attached to C-24 of the side chain of the steroid aglycone .Currently, the two-dimensional cell culture (2D culture) is a widely used in vitro model in cell biology. The success of this model is due to the property of most types of animal and human cells to proliferate in special culture media and form homogeneous colonies. However, the cell growth of a monolayer culture in many parameters does not reflect the true picture of tumor growth in a living organism, where interactions not only between the cells of the tumor, but also with the surrounding extracellular matrix, represented in its core by connective tissue cells and collagen cells, are of great importance in its progression. The three-dimensional cell culture, represented by spheroids (3D spheroids), is the most effective system that is as close as possible in properties and organization to a tumor in organisms, which is currently used for screening potential anticancer drugs .50) was not achieved in the concentration range up to 200 μM even in 3 days of treatment  by 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. PL1 at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM was found to reduce the cell viability by 17%, 19%, 15%, 18%, 31%, and 32%, respectively, but the concentration of compounds causing 50% inhibition of the spheroids’ viability ) on the proliferation of different types of human cancer cells. Thus, it was shown that laminaran (5 mg/mL) from the brown alga L. digitata inhibited the proliferation of colorectal cancer cells HT-29 by 40% without affecting the growth of normal intestinal epithelial cells EC-6 [L. digitata, reduced the growth rate of PC-3 human prostate cells, arrested the cell cycle in the S phase, and induced apoptosis in cancer cells through the regulation of the expression level of PTEN and P271kip1 proteins [L. digitata was obtained and its antiproliferative activity was determined. The sulfated laminaran (at a dose of 1.6 mg/mL) was found to have a stronger inhibitory effect on the proliferation of Lovo cancer cells than the native polysaccharide [S. cichorioides, S. japonica, and Fucus evanescens, as well as their sulfated derivatives, was studied by our scientific group [F. evanescens was shown to have more pronounced anticancer activity among sulfated laminarans from S. cichorioides and S. japonica and effectively inhibited proliferation, colony formation, and migration of triple-negative breast cancer cell line MDA-MB-231 [The sulfated laminaran’s derivative AaLs exhibited a moderate antiproliferative effect against 3D HCT 116 spheroids growth at a concentration range from 400 to 1600 μg/mL with ICectively D. Severaproteins . Ji Y.B.proteins . Additioccharide . The autic group . It was A-MB-231 .Dictyota dichotoma with X-ray radiation, where the radiosensitizing activity of sulfated laminaran was observed at low concentrations of 10, 20, and 40 µg/mL [50  A. The inPL1 (12.5 μM) in combination with AaLs  inhibited the growth of the colony of 3D HCT 116 spheroids by 16%, 22%, and 23%, respectively, compared with the individual action of PL1 C,D. The There are various invasive assays in the literature, most of which differ in the composition of the polymer matrix that forms an artificial barrier. In this work, a commercial preparation of ECM gel  was used to create a polymer matrix, which is a standardized mixture of the extracellular matrix proteins obtained from Engelbreth-Holm-Swarm murine sarcoma.The 3D HCT 116 spheroids were shown to significantly invade into the polymer matrix after 72 h of their incubation A,F. PL1 Since linckoside L1 demonstrated the highest individual anti-invasive activity among the investigated polyhydroxysteroid glycosides, it was chosen for the study of the combined effect with a sulfated derivative of laminaran AaLs. It was found that the treatment of 3D HCT 116 spheroids by L1 (12.5 μM) in combination with AaLs  led to the reduction of the invasion area of the spheroids by 16%, 26%, and 44%, respectively, compared to the area of spheroids treated by L1 individually A,B. It wA. angusta and linckoside L1 from P. lincki is associated with the regulation of the activity of AKT kinase, and the major players of apoptosis, namely proapoptotic (Bax), antiapoptotic (Bcl-XL) proteins, and the executioner of apoptosis—caspase 3 and cleaved caspase 3. As shown in the A. angusta and polyhydroxysteroid glycosides from the starfish P. lincki.Metastasis is a consequence of tumor progression based on the disturbance of survival/apoptosis balance in cancer cells . One of In conclusion, we confirmed that the sulfation of laminaran allows increasing its anticancer activity in vitro compared to the activity of native laminaran. Additionally, it was determined that the in vitro anticancer activity of polyhydroxysteroid glycosides PL1 and L1 was more pronounced than the PL2 one in the model of 3D HCT 116 spheroids. PL1 and L1 have similar structural features: 3β,4β-hydroxyl groups in ring A of the steroidal nucleus and the 2-O-methyl-β-D-xylopyranose residue attached to C-3 of the steroid nucleus, while compound PL2 has another ring A of the steroidal nucleus and the α-L-arabinofuranose residue attached to C-24 of the side chain of the steroid aglycone. Probably, indicated above, the structural features of PL1 and L1 contributed to their significant anticancer activity.A. angusta with linckoside L1 from the starfish P. lincki inhibits colorectal cancer progression in vitro, and it is much more effective than the individual effect of these compounds at low, nontoxic concentrations. Moreover, this study, for the first time, provides strong evidence that AaLs in combination with L1 suppressed the proliferation, colony formation, and invasion of 3D HCT 116 spheroids via blocking Akt kinase activity and triggered the apoptosis of colorectal carcinoma cells.The combination of sulfated derivative of laminaran AaLs from the brown alga It enriches our understanding of the molecular mechanism of the anticancer action of polysaccharide derivatives of brown algae and polyhydroxysteroid glycosides of starfish, opening up prospects for the development of new approaches for therapy of advanced colorectal cancer in the future. We hope that the obtained data will be of great interest, particularly to researchers working on marine natural compounds and oncology.A. angusta (Aa) was collected from Simushir Island in August 2016, Sea of Okhotsk during a cruise of the research vessel “Academik Oparin”. A thallus of fresh algal biomass (100 g) was washed thoroughly with tap water, powdered, and pretreated with 70% aqueous ethanol (w/v = 1:10) for 10 days. Then defatted alga was air-dried at room temperature.The sample of the alga A. angusta by the methods as described previously [The laminaran AaL was isolated from the brown alga eviously .A. angusta was modified by chlorosulfonic acid/pyridine method to obtain the sulfated laminaran AaLs as described earlier [Native laminaran AaL from  earlier .The content of carbohydrates was determined according to the method of Michel Dubois et al. . The abs2SO4 (1 mg/mL) was used as a reference standard.The content of the sulfate group was determined by the turbidimetric method after hydrolysis of the sulfated laminaran AaLs with HCl (1N) . The absThe molecular weight of sulfated laminaran AaLs was determined by size-exclusion chromatography (SEC), using an Agilent 1100 Series HPLC instrument  equipped with a refractive index detector and series-connected SEC columns, Shodex OHpak SB-805 HQ and OHpak SB-803 HQ, . Elution was performed with 0.15 M NaCl aqueous solution at 40°C, with a flow rate of 0.4 mL/min. The dextrans of 5, 10, 50, 80, 250, 410, and 670 kDa  were used as reference standards.13C NMR spectra of native and sulfated laminarans were obtained on an Avance DPX-500 NMR spectrometer  at 50 °C. The sample concentration was 15 mg of polysaccharide/mL of D2O.The P. lincki BLAINVILLE, 1830  were collected at a depth of 1–5 m in the Arabian Sea near the city of Thiruvananthapuram in India. The species identification was carried out by Dr. Padmakumar K.P. . A voucher specimen (No. 2010-2) was deposited at the G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia.Specimens of R 25.2 min), PL2 , and L1 (3.0 mg tR 8.9 min). The pbtained PL1, PL2, and L1 were identified based on their thin-layer chromatography , HPLC  nuclear magnetic resonance (NMR), and mass spectrometry (MS) data.Polyhydroxysteroid glycosides PL1, PL2, and L1 were isolated according to the scheme described earlier . In brieThe vehicle control is the cells treated with the equivalent volume of phosphate-buffered saline (PBS) or dimethyl sulfoxide  for all of the presented experiments.Sulfated laminaran AaLs was dissolved in sterile PBS, filtered by 0.22 µm membrane  to prepare stock concentrations of 20 mg/mL.Polyhydroxysteroid glycosides PL1, PL2, and L1 were dissolved in sterile DMSO to prepare stock concentrations of 40 mM.Cells were treated with serially diluted AaLs  or polyhydroxysteroid glycosides PL1, PL2, and L1  (culture medium used as diluent).® CCL-247™) were obtained from the American Type Culture Collection . HCT 116 cells were cultured in in McCoy’s 5A medium supplemented by 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. The passage number was carefully controlled and the mycoplasma contamination was monitored on a regular basis.The human colorectal carcinoma cells HCT 116 (ATCCThe 3D HCT 116 spheroids were formed by the liquid overlay technique (LOT) method with slight modifications. Briefly, to create nonadherent surfaces for the efficient spheroids’ formation, 50 µL of preheated (60 °C) agarose (1.5%) was overlaid the bottom of 96-well plates and left to solidify for 1 h at room temperature under sterile conditions.3) were inoculated in an agarose layer and cultured in 200 µL of a complete Dulbecco’s modified eagle medium (DMEM)/10% FBS medium for 96 h at 37 °C in a 5% CO2 incubator. An image of each spheroid was made with a ZOE™ Fluorescent Cell Imager . ImageJ software bundled with 64-bit Java 1.8.0_112  was used to measure the spheroid integrity, diameter, and volume.HCT 116 cells . The concentration at which a compound exerts half of its maximal inhibitory effect on cell viability (IC50) was calculated by the AAT-Bioquest® online calculator [The 3D HCT 116 spheroids were treated by replacing 100 µL of supernatant with a complete medium containing polyhydroxysteroid glycosides PL1, PL2, and L1 at 6.25–200 µM or AaLs at 400–1600 µg/mL for 24, 48, and 72 h. Then, 15 µL of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent  was added to each well with spheroids and incubated for 3 h at 37 °C in a 5% COlculator .50. Namely, spheroids were pretreated by PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The metabolic activity of the 3D spheroids after the combined treatment were measured by MTS assay as described above.To study the combined effect of PL1, PL2 or L1 with AaLs, the 3D HCT 116 spheroids were treated by compounds in fixed concentration ratios, which were chosen according to their ICn = 6) from each treatment were collected and applied onto 0.3% basal medium eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C, in a 5% CO2 incubator for 21 days, and the cell’s colonies were scored using a microscope Motic AE 20  and the ImageJ software.The 3D HCT 116 spheroids were treated by replacing 100 µL of supernatant with a complete medium containing individual compounds: polyhydroxysteroid glycosides PL1, PL2, and L1 at 12.5–50 µM and AaLs at 200–800 µg/mL for 24 h, or their combinations PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The spheroids  was made at the time points of 0, 24, 48, and 72 h with the aid of a microscope Motic AE 20. A quantitative assessment of the invasion of spheroids was carried out using the ImageJ program and was determined as the difference between the total area invaded by the cells leaving the spheroid and the area of the spheroid as described previously [After the 3D HCT 116 spheroids were formed, they were treated by replacing 100 µL of supernatant with a complete medium containing individual compounds: polhxydroxysteroid glycosides PL1, PL2, and L1 at 12.5–50 µM and AaLs at 200–800 µg/mL for 24 h, or their combinations PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The 3D HCT 116 spheroid from each treatment was collected into the 1.5 mL microcentrifuge tubes and left for sedimentation for 10 min. Then, the supernatants were aspirated, 100 µL of cooled ECM gel from an Engelbreth-Holm-Swarm murine sarcoma  was added to the spheroid and transferred to 96-well plate covered by 100 µL of ECM gel (4 °C). The spheroids were maintained at 37 °C, in a 5% COeviously .n = 9) from each treatment were collected into the 1.5 mL microcentrifuge tubes and left for sedimentation for 10 min. The spheroids were washed by PBS and then lysed by 1X lysis buffer  according to the manufacturer’s protocol. The Lysate protein was extracted and blotted (20–40 µg) as described previously by Vishchuk et al. [To determine the effect of the combined treatment with the investigated compounds on the activity of kinases and apoptotic proteins, the 3D HCT 116 spheroids were treated by combinations of PL1, PL2 or L1 at 12.5 µL and AaLs at 10, 20, and 40 µg/mL as described above in The polyhydroxysteroid glycosides/sulfated laminaran dose-effects were calculated by “Compusyn software”  using the median effect method described by Chou .t test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.All of the assays were performed in at least three independent experiments. Results are expressed as the mean ±standard deviation (SD). The Student’s


Q~33 and long propagation length > 3 μm. We introduce the graphene suspension height as an effective plasmonic tuning knob that enables in situ change of the dielectric environment and substantially modulates the plasmon wavelength, propagation length, and group velocity. Such active control of micrometer plasmon propagation facilitates near-unity-order modulation of nanoscale energy flow that serves as a plasmonic switch with an on-off ratio above 14. The suspended graphene plasmons possess long propagation length, high tunability, and controllable energy transmission simultaneously, opening up broad horizons for application in nano-photonic devices.Due to the two-dimensional character of graphene, the plasmons sustained by this material have been invariably studied in supported samples so far. The substrate provides stability for graphene but often causes undesired interactions  that compromise the quality and limit the intrinsic flexibility of graphene plasmons. Here, we demonstrate the visualization of plasmons in suspended graphene at room temperature, exhibiting high-quality factor  Graphene plasmons hold potential for infrared optoelectronic devices, but the interaction with the substrate often degrades their quality. Here, the authors report the characterization of plasmons in suspended graphene with tunable suspension height, showing enhanced quality factors and propagation lengths at room temperature. Importantly, GP properties can be dynamically tuned by changing the graphene Fermi energy through electrical gating, which finds applications ranging from light modulation to light detection and sensing11. In addition, extensive efforts have been devoted to creating passive graphene components for plasmonic circuitry, such as waveguides, resonators, and couplers12. However, the current state-of-the-art of GPs is far from real-world applications due to their short propagation length, limited tunability, and lack of energy flow controllability. This is because the performance of GPs is limited by a supporting substrate underneath graphene, which brings about extrinsic damping pathways, including dielectric losses, phonon hybridization, and impurity scattering. Such extrinsic damping comes mainly from the natural IR phonons that reside in the chosen substrates, such as SiO2 and BN6. Even at cryogenic temperatures, dielectric losses from the environment still contribute a significant damping that cannot be overcome by improving the intrinsic properties of graphene13. The dielectric permittivity and phonon hybridization also restrict the operating frequency bandwidth of GPs. Besides, when plasmons propagate across different substrates, electromagnetic energy is affected by the dramatic change of the local dielectric environment, thereby causing reflection, transmission, and radiative out-coupling associated with the plasmonic impedance mismatch between the two different dielectric environments17. Therefore, the design of a dielectric environment to address the aforementioned challenges in one system is essential to bring GPs close to real photonic devices.Graphene plasmons (GPs), the collective oscillations of Dirac-fermion electrons in doped graphene, enable subwavelength light confinement in the infrared (IR) and terahertz frequency domains19 serves as an ideal platform to avoid dielectric losses from the substrate20. Notably, suspended graphene structures have been widely explored in photodetectors, ultrafast photocurrents and terahertz generation21, visible light emission22, nanomechanical resonators23, and thermal transport devices24. Here, we demonstrate that a remarkable improvement in the quality factor and propagation length of GPs can be achieved in suitably designed suspended graphene plasmonic structures. Furthermore, a broad tunability of the plasmon dispersion, propagation length, phase and group velocities, and energy flow is demonstrated by merely in situ adjusting the suspension height, leading to a change of dielectric environment that is difficult to achieve in substrate-supported plasmonic structures. Based on the active control of plasmon micrometer propagation, we implement an effective switch for near-unity-order control of plasmonic energy.Suspended graphene with very high carrier mobility approaching ballistic transport and long carrier relaxation time2/Si substrate perforated with circular dimples of different diameters in the 30 nm–50 μm range  for several hours to achieve a high doping level by surface adsorption of gas molecules, which act as electron acceptors26. The monolayer thickness of the graphene used in this work is confirmed by the Lorentzian profiles of the 2D peak in its Raman spectra  indicates a high quality of the samples. By comparison, the G peak of suspended graphene redshifts by 2.1 cm−1, indicating unnoticeable strain (only 0.014%) induced in the carbon monolayer by our suspension structures. Besides, the Raman ratio I2D/IG decreases significantly from 5 in suspended graphene to 3 in supported graphene due to the scattering of graphene electrons caused by charged impurities from the SiO2 substrate. The doping level can be directly controlled through the gas concentration and doping time . We find that plasmons remain almost unchanged for several hours and gradually degrade after a few days due to the desorption of NO2 gas molecules. Interestingly, the plasmons can be effectively turned off by thermal enhancement of desorption in our experiments .As schematically shown in Fig. −1 range to image propagating plasmons in graphene. The IR light beam is focused on a metallic atomic force microscope (AFM) cantilever probe tip  is critical for the characterization of long-distance propagation. In previous studies, it was well established that plasmon polaritons manifest as a periodic modulation (fringes) of the observed near-field signal as a function of the tip position relative to graphene edges and other surface features3. Furthermore, s-SNOM can distinguish tip- and edge-launched plasmons9, thus providing a valuable platform for characterizing plasmon propagation, reflection, and transmission15.We use a scattering-type scanning near-field optical microscope (s-SNOM) with a tunable quantum cascade laser operating in the 892–1600 cmtip Fig. . The bacEF ≈ 0.68 eV) with an IR laser operating at λ0 = 10.87 μm (920 cm−1). In the near-field nanoscopy image shown in Fig. λp of the suspended GPs is calculated to be 938 nm from analytical RPA theory27 and numerical simulations28, a value that agrees well with the experimentally observed distance between two adjacent fringes in Fig. 5. Besides, inspection of Fig. 5. In contrast, the nanoscopy images of suspended graphene without doping are featureless  is chosen to obtain more accurately measured near-field amplitude images. Fringe patterns can be observed with a periodicity corresponding to λp/2, which is confirmed by our electromagnetic simulations  appear in Fig. λp/2-period fringes can be assigned to tip-launched plasmons reflected at the edge of the suspended area and subsequently out-coupled to radiation by the tip2. The plasmon edge-reflection is caused by the mismatch of capacitive coupling across dielectric interfaces, mainly determined by the difference in the wave vectors of plasmons between substrate-supported and suspended regions. The λp-period fringes can also be produced by edge-launched plasmons propagating to the tip and being out-coupled9. We note that the efficiency of plasmon excitation at the edges depends on the combined effect of dielectric polarization29 and the size and shape of the hole edge  can be expressed as 31, from which While in Fig. Q and LSPP as a function of Fermi energy , where the dispersion splits into three bands6. We observe that, as d increases from 0 to 453 nm, the coupling strength gradually weakens  h(x) is obtained by fitting the height profile to a parabola. Following this approach, the plasmon wavelength can be tuned over a wide range (from 400 nm to 1.2 μm) due to its strong dependence on the permittivity of the environment. Figure d, the plasmon wave vectors extracted from the measured near-field amplitude images (symbols) agree well with the calculated results (maroon curve)34. Besides the significant control obtained over the plasmon wavelength by changing the suspension height, both phase (νP = ω/q1) and group (νg = ∂ω/∂q1) velocities sharply increase with d in the d < 80 nm region, and they saturate at d ~ 200 nm, as shown in Fig. g can vary within a range of 0.42–2.5 × 107 m/s, suggesting the possibility of broadly controlling the propagation speed of information in a graphene waveguide.The elimination of interactions from the substrate renders a pure plasmonic environment, providing a unique platform for manipulating the out-of-plane interaction between intrinsic GPs and the dielectric environment. In Fig. Q and propagation length LSPP for different suspension heights d. Once graphene is detached from the substrate, the quality factor Q increases sharply due to the reduced coupling to dielectric losses from the SiO2 substrate  is confined within 100 nm above or below the graphene plane11. We observe a twofold improvement of Q when d increases to 55 nm, while a milder increase in Q takes place after d exceeds 100 nm. For the achieved maximal suspension height (d = 453 nm), the quality factor Q is enhanced by about a factor of six, and the corresponding LSPP increases by one order of magnitude (from 0.25 to ~3.0 μm) compared to the values observed in supported graphene.Modulation of the interaction between GPs and a substrate can also affect the plasmon quality factor. In Fig. 35. In Fig. d2 of a graphene bubble from −50 nm (concave shape) to +60 nm (convex) by changing the gas pressure, as revealed by AFM /(Sedge-Ssheet2)36, where Sdielectric, Sedge, Ssheet1, and Ssheet2 represent the near-field signals of the bright fringe at the air-dielectric interface, the bright fringe at the natural graphene edge, and from the background of suspended (Ssheet1) and supported (Ssheet2) graphene, respectively  at the considered Fermi energy EF = 0.4 eV  to control the reflection at the interface due to a different graphene morphology compared with the situation in which d2 < 0. When θ gradually increases from 0 to 45°, the reflectance gradually increases from 6.5 to 8.6%. However, when the graphene is below the substrate surface in the suspended region . Due to the small thickness of single-atomic-layer graphene, almost no free radiation is emitted in the scattering process under these conditions 37. By analogy to electronic devices, we can define the ratio between the maximum and minimum energies passing across the interface as the switch ratio of plasmonic devices38; the observed switch ratio reaches >14 by in situ changing the shape of suspended graphene. Simultaneously, the plasmon wavelengths are very different at the two sides  can be excited in suspended monolayer graphene with an ultrahigh doping level (up to 0.9 eV) introduced through gas-molecule adsorption. Importantly, a high-quality factor (Q = 33) and a long propagation length (LSPP > 3 μm) in plasmons evolving in tailored suspended graphene at room temperature are achieved by eliminating interactions with a substrate. In addition, suspended graphene provides a unique clean plasmonic platform that grants us better understanding and tuning of the interaction physics of the extrinsic environment with GPs. More precisely, by in situ changing the suspension height of graphene, we implement an efficient manipulation of the plasmon wavelength, propagation length, and phase/group velocity. We further present an effective route to fully control the transmission of plasmonic energy at the air-dielectric hole-edge interface by applying electromechanical concepts to plasmonic devices, in which the suspended graphene can be deformed by either introducing a gate voltage or varying gas pressure. Furthermore, we discuss how the deformation can be tailored to create a plasmonic switch (by gas) and transistor (by gating) to achieve near-unity-order manipulation of plasmonic energy flow. The combination of micrometer propagation and energy manipulation can be applied to design other advanced devices, such as plasmonic resonators, filters, and modulators, which are pivotal to control energy flow in ultra-compact integrated plasmonic circuits.To conclude, we demonstrate that high-frequency propagating plasmons (~1400 cm2/500 μm Si substrate using 100 kV electron-beam lithography (EBL)  on ~350 nm of ZEP520A electron-beam lithography resist. The hole arrays were etched by C4F8 and SF6 gases . Then, the resist layer was dissolved with butanone and the whole wafer was cleaned with isopropyl alcohol. For holes that were deeper than 300 nm, we used plasma-enhanced chemical vapor deposition to grow a 300 nm-thick layer of SiO2 in the hole to cover the silicon substrate. The remaining residues on the silicon oxide surface were removed by oxygen plasma cleaning with 5 Pa and 80 W for 20 min. Graphite flakes were then mechanically deposited onto the substrate. Optical microscopy was used to identify monolayer graphene sheets, and their layer numbers were further confirmed by Raman spectroscopy. To control the shape of suspended graphene, the samples were placed into the chamber with N2 to create a pressure difference (ΔP) between the inside and outside of the suspended graphene membrane. ΔP was utilized as a controlling load to change the suspension height based on the well-established gas diffusion method according to ΔP = (PaV0/(V0 + Vs))-Pe39, where Pa and Pe are the applied and external atmosphere pressures; and V0 and Vs are the volumes of the hole and suspended graphene bubble, respectively. A schematic diagram of the gas diffusion process is shown in Fig. P = k E tk is a dimensionless coefficient, E is Young’s modulus, t is the thickness of the graphene layer, d2 is the maximum deflection, and r is the circular radius of the suspended graphene region. Our experimental parameters for bubble filling are shown in Supplementary Fig. 2 gas molecules to tune the Fermi energy of graphene. HNO3 vapor was also used to dope the graphene and achieve higher carrier density. We verified that heating the doped samples produced a large decrease in doping.Circular holes with sizes ranging from 30 nm to 50 μm were patterned on a 300 nm SiO−1). The spot sizes of the Mid-IR beam under the AFM tip were made ~30 μm in lateral size to cover the large area of suspended graphene, thus facilitating tip and edge launching of the GPs explored in this work. The probes were commercially available AFM tips metalized and having an apex radius of ~25 nm (Nanoworld). The tip-tapping frequency and amplitudes were ~270 kHz and ~30–50 nm, respectively. The near-field amplitude images were obtained from the third-order demodulated harmonic of the near-field amplitude, which resulted in massive suppression of background noise.Near-field results were measured using a scattering SNOM setup (Neaspec GmbH) equipped with wavelength-tunable lasers  at a light wavelength of 10.87 μm are ε(SiO2) = 3.8 and ε(Si) = 8.9, respectively40. For suspended graphene, we have T. Under the conditions kBT ≪ kBT ≪ |EF | , the conductivity σ can be modeled in the local limit of the RPA41 and decomposed into intraband and interband contributions as e is the electron charge, and F = c/300 and the carrier mobility μ.The dispersion relation of GPs can be obtained from the poles in the imaginary part of the Fresnel reflection coefficient tg = 1 nm and an effective dielectric function ε = 1 + I tg). For the near-field image simulations in Fig. θ was determined by the suspension height d2 through a function fitted from experimental measurements , transmitted (GP moving to the right), or out-coupled as a free-space IR photon. When the suspended graphene was above the substrate surface , the structure of suspended graphene was matched through a simple deflection profile as a result of the competition between the elastic energy of the deformed suspended graphene and the uniform force associated with gas pressure, as well as the adhesion energy (between graphene and the substrate), which can be fitted as 42, where a = 1.25 μm is the radius of the hole. We set the center point of suspended graphene to x being along the radial direction). The relationship between the contact angle θ and the height a): tan(θ) = 2a, where θ is in the [0, π/2) range. Inelastic losses of GPs were not considered throughout the process, and the graphene thickness was assumed to be 1 nm. The reflectance R at the interface was defined as the ratio of the reflected GP energy (propagating to the left) to the incident GP energy (propagating from the left to the interface): In the simulations of GP transmission in Fig. Supplementary Information


Saccharomyces cerevisiae as an assay to identify TDP-43 genetic interactions. Specifically, we transformed human TDP-43 cDNAs of wild-type or disease-associated mutants (M337V and Q331K) en masse into 4653 homozygous diploid yeast deletion mutants and then used next-generation sequencing readouts of growth to identify yeast toxicity modifiers. Genetic interaction analysis provided a global view of TDP-43 pathways, some of which are known to be involved in cellular metabolic processes. Selected putative loci with the potential of genetic interactions with TDP-43 were assessed for associations with neurotoxicity and inflammatory activation of astrocytes. The pharmacological inhibition of succinate dehydrogenase flavoprotein subunit A (SDHA) and voltage-dependent anion-selective channel 3 (VDAC3) suppressed TDP-43-induced expression of proinflammatory cytokines in astrocytes, indicating the critical roles played by SDHA and VDAC3 in TDP-43 pathways during inflammatory activation of astrocytes and neuroinflammation. Thus, the findings of our TDP-43 genetic interaction screen provide a global landscape of TDP-43 pathways and may help improve our understanding of the roles of glia and neuroinflammation in ALS and FTD pathogenesis.Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 has been implicated in numerous aspects of the mRNA life cycle, as well as in cell toxicity and neuroinflammation. In this study, we used the toxicity of the TDP-43 expression in  TARDBP gene in humans, is involved in transcriptional repression and splicing and stability of RNA -2, 5-diphenyltetrazolium bromide (MTT) assay, cells were treated with various reagents for a designated period. After treatment, MTT  was added to the cells, which were then incubated for 2 h at 37 °C in a 5% COMitochondrial reactive oxygen species (ROS) levels were measured as previously described . GFP-traMitochondrial membrane potential in live cells was assessed using the tetramethylrhodamine ethyl ester perchlorate (TMRE)  probe as described previously . For livGapdh) and β-actin (Actb)) for the quantification of gene expression, as previously described [−ΔΔCt method was used to calculate the relative differences in gene expression [Tnf, Il1b, Gapdh, and Actb were as follows: Tnf: 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ (forward), 5′-ACT TGG GCA GAT TGA CCT CAG-3′ (reverse); Il1b: 5′-AGT TGC CTT CTT GGG ACT GA-3′ (forward), 5′-TCC ACG ATT TCC CAG AGA AC-3′ (reverse); Gapdh: 5′-TGG GCT ACA CTH AHC ACC AG-3′ (forward), 5′-GGG TGT CGC TGT TGA AGT CA-3′ (reverse); Actb: 5′-ATC CGT AAA GAC CTC TAT GC-3′ (forward), 5′-AAC GCA GCT CAG TAA CAG TC-3′ (reverse).RNA was extracted from cells using TRIzol reagent , and RNA was “cleaned up” using a RNeasy Mini Kit  according to the manufacturer’s instructions. Using 100 ng of RNA, cDNA was synthesized at 37 °C for 120 min with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (RT-PCR)  was perfescribed ,42. The pression . The priThe levels of IL-1β and TNF-α in culture media were quantified using an enzyme-linked immunosorbent assay (ELISA) kit . ELISA assays were conducted in 96-well plates using the media and methods stipulated in the manufacturer’s instructions. For the standards, mouse recombinant proteins were used at concentrations of 10–2500 pg/mL. IL-1β and TNF-α protein levels were normalized to the total protein content of the astrocyte culture media samples. All measurements were obtained from duplicated assays.Cultured cells were lysed in a triple-detergent lysis buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.02% sodium azide, 0.1% NaDodSO4 (SDS), 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Protein concentrations in cell lysates were determined using a protein assay kit . Each protein sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before being blotted and incubated overnight at 4 °C using the following primary antibodies: anti-TDP-43  and anti-β-actin . Afterward, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and detected using enhanced chemiluminescent (ECL) solution.p-values < 0.05 were considered statistically significant. Sample sizes for experiments were chosen to ensure adequate statistical power on the basis of G*power 3.1 software [Two independent transformants were tested in the yeast spotting assays. Cell viability experiments were performed in eight sister wells , which were not repeated. The number of sister wells was also described in other experiments. Different treatments were compared with Student’s t-test or two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test using SPSS software . software .S. cerevisiae as an assay to identify TDP-43 genetic interactions, we found that overexpression of human TDP-43 cDNAs, WT, or disease-associated mutants (M337V and Q331K), caused toxicity in S. cerevisiae, as indicated by a lower number of visible spots when compared with the control vector transformation , theThe relative abundance of each yeast barcode is a proxy for the differential growth of the corresponding deletion strain, which is based on a positive correlation between the barcode number and growth of a specific yeast strain—higher barcode numbers show higher growth in a specific yeast strain . This syOne overlapping gene (SDHC) was identified as a toxicity suppressor between TDP-43 WT and M337V , whereashttp://inparanoid.sbc.su.se, accessed on January 2017) [http://genemania.org, accessed on December 2020) [In the yeast validation assay, we found 13 yeast gene deletions that had suppressing effects on TDP-43 toxicity  based onry 2017) , and theer 2020) . A TDP-4Co-expression indicates when two genes are linked if their expression levels are similar across conditions or they are expressed together in a gene expression study. Similarly, two genes are defined to be co-localized if they are both expressed in the same tissue or if their gene products are both identified in the same cellular location. The GeneMania plugin from Cytoscape was used to build the interaction network for common pathways that might regulate such functional interactions ,35.According to the DAVID analysis, the nodes of the network were enriched for several functional groups  as follows: tricarboxylic acid cycle, protein import into peroxisome matrix, cellular metabolic process, endosomal transport, organic substance transport, transcytosis, regulation of protein localization to cell surface, cytokine secretion, regulation of protein localization, cellular response to stress, and mitotic cell cycle. Moreover, GO term analysis indicated a functional enrichment for respiratory electron-transport chain, nucleotide-excision-repair complex, coated pit, cellular metabolic process, and organic substance transport, among others .Among the 13 interaction loci of TDP-43 tested in the yeast spot assays, 3 genes  with strong suppressing effects on TDP-43 toxicity were selected for further evaluation. One gene showing strong suppressing effects was chosen for each group .Commercially available pharmacological inhibitors of SDHA , HSP90AB1 (geldanamycin), and VDAC3 (TRO19622) were used for functional evaluation of the TDP-43 genetic-interacting loci in N2a neuroblastoma cells. N2a is a neuroblastoma cell line that has been used in many TDP-43 studies, particularly for the assessment of TDP-43-induced neurotoxicity and associated molecular mechanisms in vitro ,57,58. WWe targeted SDHA, HSP90AB1, and VDAC3 using their pharmacological inhibitors  to investigate the functional role of TDP-43 genetic interaction modifiers in astrocytic phenotypes in vitro. The optimal concentration of inhibitors for primary astrocyte cultures was first determined , and an Finally, we investigated the effects of TDP-43 and pharmacological inhibition of SDHA, HSP90AB1, and VDAC3 on mitochondrial membrane potential and ROS levels in astrocytes. Transfection of astrocytes with WT and mutant (M337V and Q331K) TDP-43 was found to reduce mitochondrial membrane potential and increase levels of mitochondrial ROS in astrocytes . PharmacTDP-43 genetic interaction analyses and pharmacological validation experiments indicate that TDP-43 and its interactors SDHA and VDAC3 play important roles in the inflammatory activation of astrocytes; consequently, they may also be associated with the pathological features of ALS and FTD.Our findings from TDP-43 gene in this study. Transformation of human TDP-43 cDNAs of WT or disease-associated mutants (M337V and Q331K) into a 4653 homozygous diploid yeast deletion set and subsequent barcode sequencing identified 13 yeast toxicity suppressors with human orthologs. Our findings reveal a multitude of known co-localization and co-expression genes, as well as novel TDP-43 genetic interaction loci and related genes that warrant further study. In our network analysis, co-expression of genes only indicates that they could be functional at the same time or in the same space—it does not imply functional linkage, they may function antagonistically or collaboratively for the same process or they could be functioning in two completely unrelated processes. Thus, the co-expression or co-localization in the current network require further investigation to demonstrate a functional relationship. GO analysis of TDP-43 genetic interaction loci has demonstrated that there is an enrichment of genes related to cellular metabolic process  genes  and prot process . SupportTDP-43 genetic interactions revealed a global view of TDP-43 pathways, some of which are involved in cellular metabolic processes and organic substance transport. We also found that SDHA, HSP90AB1, and VDAC3 are the major interacting genetic loci of TDP-43, and these three interacting loci showed strong toxicity-suppressing effects and were therefore further investigated in relation to their inflammatory activation of astrocytes and neurotoxicity. Previously, it was reported that both neurons [TDP-43 has previously been implicated in various aspects of the mRNA life cycle , cell to neurons ,70,71 an neurons ,72,73 sh neurons ,75: in s neurons ,77,78. A neurons . InteresTDP-43 in astrocytes induces neurodegeneration via a PTP1B-mediated inflammatory response [Recent findings, particularly in ALS models ,79, haveresponse . NeverthCytoplasmic aggregation of TDP-43 is a common phenotype observed in majority of ALS cases  and has Our study demonstrates that pharmacological inhibition of SDHA and VDAC3 substantially attenuates WT or M337V mutant TDP-43-induced inflammatory activation of astrocytes, as characterized by the expression of TNF-α and IL-1β. However, inhibition of SDHA or VDAC3 does not alter Q331K mutant-induced astrocytic activation. We, therefore, suggest that WT or M337V mutant TDP-43-induced inflammatory activation of astrocytes is mediated by SDHA or VDAC3 pathways, and that they potentially play roles in neuroinflammation associated with the pathogenesis of ALS ,22,83. HTDP-43 showing suppressive effects on TDP-43 toxicity, which have not been reported previously. Functional analysis provides evidence for the role of these TDP-43-interacting loci, especially SDHA and VDAC3, in inflammatory activation of astrocytes and concurrent neuroinflammation in the pathogenesis of ALS and FTD.Our studies found 13 novel genetic interaction loci of


Immersion in forest environments was shown to produce beneficial effects to human health, in particular psychophysical relaxation, leading to its growing recognition as a form of integrative medicine. However, limited evidence exists about the statistical significance of the effects and their association with external and environmental variables and personal characteristics. This experimental study aimed to substantiate the very concept of forest therapy by means of the analysis of the significance of its effects on the mood states of anxiety, depression, anger and confusion. Seven forest therapy sessions were performed in remote areas and a control one in an urban park, with participants allowed to attend only one session, resulting in 162 psychological self-assessment questionnaires administered before and after each session. Meteorological comfort, the concentration of volatile organic compounds in the forest atmosphere and environmental coherence were identified as likely important external and environmental variables. Under certain conditions, forest therapy sessions performed in remote sites were shown to outperform the control session, at least for anxiety, anger and confusion. A quantitative analysis of the association of the outcomes with personal sociodemographic characteristics revealed that only sporting habits and age were significantly associated with the outcomes for certain psychological domains. The direct exposure to forest environments was attributed a wide range of straight benefits to human health. Such benefits cover primarily the psychological sphere , cognitive processes, social life  and spiritual wellbeing ,2,3,4. OAs pointed out by a recent comprehensive review, the impact on the psychophysical well-being of humans by immersion in forest environments was by far the most studied and revealed statistically significant and sometimes intense results . HoweverIndividual benefits can turn into benefits for the society as a whole due to cost savings for healthcare systems, insurance and safety, as well as gains in productivity. A recent study showed that the economic value of natural protected areas, assessed on the basis of the mental health of visitors, amounts to about 8% of the global gross domestic product, i.e., around USD 6 trillion . Such anbright cells and activation of various immune cell subsets was observed in late spring, while a slight activating and a balancing effect regarding TIM-3, an inhibitory immune checkpoint molecule, were observed in winter, thus suggesting positive forest healing effects throughout the year [The preventive and healing effects directly delivered by forest environments have been widely documented in the scientific literature, and, in most cases, concern experiences of free walking or meditation in the absence of appreciable physical exercise . The benthe year .The forest healing effects can be wide-spectrum, such as in the case of the immune system , long-laAn additive effect (constantly growing benefits) deriving from the regular repetition of sessions was observed, which was larger in the case of subjects with depressive tendencies . A recenForest experiences, where professionals assist participants according to structured protocols, often based on psychotherapy, meditation and mindfulness practices , showed The benefits of forest immersion are mediated by all human senses. Viewing repetitive natural structures , such as trees, and hearing the typical sounds of forests convey the perception of a relaxing and unchallenging environment, in which it is much easier to adapt and increase one’s own perceptive fluidity ,23, evenSmell plays a key role through the inhalation of biogenic volatile organic compounds (BVOC) emitted by plants and soil in the forest atmosphere, in particular, certain terpenes endowed with antioxidant and anti-inflammatory activities, as well as beneficial to psychological and cognitive processes . First oThe benefits and potential of forest therapy were officially recognized by the United Nations in the framework of the green recovery from the COVID-19 pandemic . The maiDespite the growing scientific evidence, many countries, especially in the Western world, lag behind in the rigorous identification of sites and trails for forest therapy practices, including preliminary selection and qualification based on actual functionality  according to structured programs of activities. First, the following consolidated evidence was seldom incorporated: while exposure to most greenspaces allows for improvements in mood and wellbeing, specific and significant preventive effects with regard to the risk of depression were attributed only to natural environments, in particular forested ones, remote from urban areas ,36. ThisSecond, especially in the Western world, forest therapy experiences in remote forest areas involving large numbers of participants, according to structured and repeatable programs and including control sessions in urban settings, were seldom performed, let alone the search for the main determinants of health outcomes that would allow the generalization of qualification criteria. Despite recent improvements in the methodological rigor, the recommendations issued in a comprehensive review published in 2017 are still relevant .The main motivation of this study was to quantitatively assess the effectiveness of a few forest therapy sessions performed in remote areas on the improvement of certain mood states, i.e., anxiety, depression, anger and confusion, compared with a control session, thus substantiating the very concept of forest therapy. Another aim was to contribute to the preliminary identification of the main determinants of the outcomes of forest therapy sessions, taking advantage of the different environments involved, the relatively large number of participants and the representativeness in terms of sociodemographic characteristics.The main hypotheses of this study were: (1) forest therapy carried out in remote forest areas is more effective than in urban parks; (2) forest therapy  is more effective than forest bathing (free); (3) different environmental conditions, either structural or variable, and personal characteristics, affect the outcome of forest therapy sessions.The Italian Alpine Club and the Institute of Bioeconomy of the National Research Council arranged seven forest therapy sessions from August to October 2020. All sessions, named FTS1 to FTS7, were performed in Italy at remote forest areas . All sessions had a duration of 3.5 ± 0.5 h, along paths with total lengths not exceeding 4 km and uphill gradients smaller than 150 m.A further control session, named FTSC, was performed in June 2021, in an urban park with an area of about 4 hectares, located downtown in the city of Florence, Italy  and surrounded by buildings and trafficked roads.3 to 20,000 μg/m3. The PID was equipped with a pump, sucking in ambient air at a rate of 220 mL/min, and an ultraviolet, 10.6 eV lamp, allowing the ionization of organic substances present in the sampled air, including all the monoterpenes (MTs) found in biogenic VOCs (BVOCs). The resulting electric current flowing between two electrodes was measured, amplified and transformed into a concentration level of the ionized substance or group of substances. TVOC concentration could be considered representative for BVOC concentration, although clearly higher due to the presence of VOCs other than MTs, at least at forest sites far from anthropogenic pollution sources where the change in TVOC concentration is mainly driven by plants’ emissions, as explained in a previous study [During each session, the concentration of total volatile organic compounds (TVOCs) was continuously collected by means of a portable (0.72 kg) photoionization detector , with detection limits from 1 μg/mus study . FinallyThe first session (FTS1), which can be classifiable as forest bathing, was performed without professional guidance, suggesting to participants to relax in the woods during two stops lasting between 20 and 30 min each, and providing scientific information about the surrounding environment (botany and geology) and the sessions meaning for one’s own health. All the other sessions were performed with professional guidance, according to the method of conducting illustrated in Volunteer participants were enrolled and insured by local sections of the Italian Alpine Club following information spread by means of standard channels . No selection was applied except for age (>18 years) and the requirement of being healthy. Participants were allowed to attend only one session, to prevent any contribution from the possible persistence of the effects. Overall, 207 participants were recruited, returning 162 valid self-assessment questionnaires, the nature of which is described below in Limited to participants that returned valid questionnaires and relevant demographic data, there were more female (84) than male (50) participants, the age group 41–60 was the most represented for both genders  and the place of residence of more than 59% of participants was outside towns with more than 40,000 inhabitants. p = 0.044; weight: p < 0.01). Moreover, chi-square test matrix for homogeneity among categorical variables was performed for the entire study population for sex, study degree, job, smoking and residence variables, resulting in homogeneity only for sex variable (p = 0.182). Ryan–Joiner test for normality was performed for height and weight variables for the entire study population, resulting in a non-normal distribution  questionnaire and listened to a short speech. Such time could have helped relieving transport-related stress. The POMS questionnaire was completely anonymous and included information about gender, age, place of residence, job and habits such as smoking and sports. The POMS questionnaire consisted of 34 questions about moods perceived at that precise moment, recommended for the analysis of psychological outcomes of forest therapy sessions  and deri®, partly based on mindfulness practices, in particular on the Mindfulness-Based Stress Reduction (MBSR) program developed since 1982 [Except for session FTS1, and for session FTSC that was carried out according to a different and simplified method of conducting referred to in Mindfulness was shown to reduce pain in a wide range of clinical and non-clinical problems , improveIn the forest environment, mindfulness can be practiced by means of walking mind-fully, using breathing as the center of attention, observing the surrounding environment with acceptance and creating a deep connection with nature using the five senses. Walking meditation is one of MBSR’s main practices, while walking in forests can lower blood pressure and reduce the concentration level of cortisol , resulting in relaxation ,48. WalkConscious and focused use of all human senses—sight, hearing, smell, touch and taste—is another milestone of the MBSR program as a means to increase the awareness of the experience and amplify the effects of forest immersion. Through the perception of the surroundings, senses provide orientation, rooting into the present an interpretation of our experiences ,51. IndeMoreover, in the conduction of the forest therapy sessions, a method of communication was adopted, which alternated metaphorical speeches with literal verbalizations in order to stimulate increased emotional involvement in the participants . In the In the context of forest therapy sessions, the attention of the participants to the present moment was stimulated in order to promote a more effective immersion, in addition through the conscious use of the five senses, the breath and the movement, looking for the deep connection with the surrounding environment. Both mindfulness practices and metaphorical discourses contributed to this purpose, as they could anticipate or deepen the proposed experience at an imaginative and evocative level and prepare neural maps for what will be physically practiced. Some neuroimaging studies showed that metaphors activated cortical areas corresponding to the suggested action; for example, the expression “she grabbed the idea” could activate the motor cortex .Generating a sensation of coherence between the internal dimension (feeling) and the external dimension . The sense of coherence , in accordance with temperament and emotional intelligence, seems predictive of the individual’s ability to cope with stressful situations ;Facilitating processes of introspection and decoding of the feelings and emotions of the participants to feed a condition of conscious presence;Promoting processes of identification with the elements of nature, which are in dynamic balance and can offer more effective learning and working models ;Stimulating the transformative activity of any dysfunctional aspects detected .During the structured forest therapy sessions , touching wood and trees and, when possible, tasting products of the undergrowth;6.Meditating with the trees: Choosing a specific tree as the most faithful mirror of oneself and approaching it with the right attitude and intentions, towards the identification with the tree and its qualities such as resilience, vitality and projection towards the sky and the light.The method of conducting the forest therapy sessions was structured in six main steps, briefly:Data reported on the reduced POMS questionnaires described in Pre-post average percentage differences of the outcomes for each considered mood state were computed for each session based on data stored in Microsoft Excel worksheets. Then, Minitab 19 was used for further statistical analyses. One-way ANOVA was preferred since the samples were small in number and with quasi-normal distributions. For non-normal samples, the Mann–Whitney test was used. The Ryan–Joiner test was used to test for normality of continuous variables, while the chi-square test was used to test for homogeneity among categorical variables. An ANOVA mixed-effects model was used to test for associations between pre and post changes of each POMS domain and sociodemographic features.p = 0.141), depression , anger  and confusion . In order to ensure that the comparison among different sessions would not be affected by statistically significant differences in initial mood states , a one-way ANOVA test was performed on the pre-intervention POMS questionnaires  across ap values. The control group had a limited numerosity; thus, the observed trends toward a reduction in any of the mood states might become statistically significant with a larger sample, especially for anxiety (p = 0.191).The differences in scores for any mood states before and after forest therapy sessions were tested; in particular, data were aggregated for intervention groups (FTS1 to FTS7), while FTSC was tested separately. A one-way ANOVA was performed to test for pre–post differences in the aggregated intervention groups (FTS1 to FTS7) and in the control group (FTSC), resulting in a statistically significant reduction in each POMS domain score for the aggregated intervention groups, while the control group resulted non-significant for each POMS domain. The possible specific effects due to the remoteness of forest areas and the conditions under which such effects could arise were investigated by means of the comparison of outcomes  of each intervention group (FTS1 to FTS7) with the corresponding outcomes from the control group in the urban park (FTSC).p < 0.05). For anxiety, only one outlier was removed from session FTS7. For depression, one outlier was removed from each of the following sessions: FTS1, FTS2 and FTS6. For anger, one outlier was removed from each of the following sessions: FTS1, FTS2, FTS5, FTS6, FTS7 and FTSC. For confusion, only one outlier was removed from session FTS1.First, Grubb’s test for outliers was performed on the changes-from-baseline for all mood states derived from the answers to the POMS questionnaires  in orderp < 0.05).Next, a Ryan–Joiner test for normality was performed for the change-from-baseline of each mood state derived from every forest therapy session. p < 0.05) or close to significantly (0.05 ≤ p < 0.10). The latter were added because the limited number of participants to certain forest therapy sessions might have hindered the achievement of statistical significance.A one-way ANOVA test was applied for comparing normal samples, while a Mann–Whitney test was applied when the comparison involved at least one non-normal sample. Sessions FTS1, FTS2 and FTS5 showed no specific additional benefit compared to the control session FTSC. Session FTS3 showed an improvement in anger significantly higher than FTSC and comparative improvements in depression and confusion close to significance. Session FTS4 again showed an improvement in anger significantly higher than FTSC and a comparative improvement in confusion close to significance. Session FTS6 showed improvements in anxiety and confusion significantly higher than FTSC and a comparative improvement in anger close to significance. Finally, session FTS7 showed an improvement in anxiety significantly higher than FTSC and a comparative improvement in anger close to significance.A mixed model ANOVA was applied to check for any statistically significant association among the collected sociodemographic traits of the participants in the forest therapy sessions, excluding the control session FTSC, and the respective changes-from-baseline for any mood state. The variance estimation method was the restricted maximum likelihood and the Kenward–Roger method was used for testing fixed effects. In total, 128 questionnaires were considered, reporting complete sets of sociodemographic information. p = 0.035), in particular, the absence thereof (p = 0.020) with a negative coefficient, i.e., anxiety decreased significantly for people who never played sports. Changes in depression were significantly associated with age (p = 0.031), with a positive coefficient, as well as with sport (0.038), in particular, the absence thereof (p = 0.041), with a negative coefficient. Changes in anger were significantly associated with sports (p = 0.011), in particular, the absence thereof (p = 0.002), with a negative coefficient. Changes in confusion were not significantly associated with any considered factors, although the association with age was close to significance (p = 0.058) with a positive coefficient.The results showed a statistically significant association of the change in anxiety with sport activity  were significant, suggesting that remote forests, where sessions FTS1 to FTS7 were performed, could bring specific benefits compared to urban forests. However, a main limitation of this study was the small numerosity of the control group , which might have hampered the significance of the respective outcomes.In order to try to overcome the above-mentioned limitation and provide further insight into the factors affecting the outcomes of forest therapy sessions, the outcomes from each of the intervention groups were separately compared with the corresponding outcomes from the control group. As presented in Excluding FTS1, which was conducted non-professionally, and FTS5, which was affected by a small numerosity and relatively harsh weather conditions, what peculiar characteristics could underlie the additional and significant benefits deriving from certain forest therapy sessions performed in remote sites?Based on Based on the available data, essentially on the performances of FTS6 compared with all the other sessions and of FTS3 compared to FTS4, the hypothesis could be advanced that the concentration of TVOCs is an effective factor affecting the performance of professionally conducted forest therapy sessions in remote forests. This can be regarded as an original experimental result of this study.However, the matter is further complicated by partially contrasting results about the systemic absorption of MTs, which represent most of the BVOCs emitted by plants, which, in turn, are likely to contribute substantially to the observed concentration of TVOCs. To the best knowledge of the authors, only two studies dealt with this important topic. In 2015, Japanese scholars found that the serum concentration of α-pinene, but not of other monoterpenes, increased by several folds after walking in a conifer forest for one hour . In 2021Although very important and innovative, both studies ,67 were Despite lower levels of weather comfort and concentration of TVOCs, FTS7 generated few significant results compared with the control session FTSC. Conversely, a very good weather comfort and concentration levels of TVOCs substantially higher than in FTS7 and only slightly lower than in FTS4 did not allow session FTS2 to achieve any significant result in comparison to FTSC, underperforming in comparison to all the other sessions. The hypothesis could be advanced that water streams and water bodies play an important role, as already arisen in previous studies, for example, in a controlled experiment using virtual reality . HoweverIt is further hypothesized that the presence of water streams or water bodies is very important, but only if appropriate to the specific environment. In fact, FTS6 outperformed all the other sessions even in the absence of those elements, but one could not expect their presence in a typically dry Mediterranean forest. Conversely, FTS2 underperformed all the other sessions, and one would expect to find water streams in a mountainous environment dominated by Scots pine. It remains unknown whether substantially higher concentration levels of TVOCs at the site of FTS2 could compensate for a possibly less efficient environment: ongoing research at that site might help clarify the issue. Indeed, FTS2 was performed in late summer, while, for Scots pine forests, it was found that young sprouts in late spring and early summer emit far more BVOCs than mature needles in late summer .Overall, the coherence among the main natural elements, sometimes called environmental coherence , could pSession FTS1, which produced no significant outcome compared to FTSC, was performed in the same site as FTS6, although with a remarkably lower level of weather comfort (temperature of 30 °C compared to 15 °C in FTS6) and according to a non-professional method. FTS1 enjoyed the highest average and peak concentration of TVOCs across all forest therapy sessions. Although the comparison between FTS1 and FTS6 could lead to hypothesize an important role for the method of conducting forest therapy sessions, with professional guidance more effective in producing significant outcomes, the large difference in weather comfort jeopardizes this hypothesis. Further experiments will be needed in order to gain insight into this matter.Based on The reasons why anxiety was significantly only reduced in FTS6 and FTS7 in comparison to the control are hard to assess. Possibly, they have to do with the forest environments characterizing FTS6 and FTS7 , which could be regarded as more relaxing, or less aggressive, than in FTS3 and FTS4 , but further ongoing field trials will hopefully clarify this issue.Overall, anger appears as the most affected psychological domain, having been significantly reduced in comparison to the control in FTS3 and FTS4 and close to significantly in FTS6 and FTS7. Possibly, items used to build the anger index were representative of acute conditions and could have been more easily reduced by means of forest therapy sessions performed at remote locations. Finally, confusion was significantly reduced compared to the control in FTS6 and close to significantly in FTS3 and FTS4. Beyond environmental coherence, the concentration level of TVOCs, which was substantially lower in FTS7 compared to FTS3, FTS4 and FTS6, could have contributed to the result.Concerning the association of the psychological outcomes from intervention sessions (FTS1 to FTS7) with the sociodemographic traits presented in Age was the other trait showing some association in particular with depression (significant) and confusion (very close to significant). The benefits for depression and confusion increased with age, which could be ascribed either to increasing trait levels of such indices with age or to a greater sensitivity of older people to forest immersion. Again, further research will help clarify this issue.It is interesting to note that no other sociodemographic characteristics were significantly associated with the results of this study: neither biometric features such as height and weight, nor sex, job, education, residence and smoking habits. This evidence could mean that forest therapy can be successfully applied to most people, irrespective of their conditions.A recent systematic review on the effects of nature walks on states of anxiety and depression evidenced clear and significant effects on anxiety but somewhat inconclusive effects on depression , in agreResearch was performed in 2018 based on a 3-day structured forest therapy session in a semitropical forest in Taiwan, mainly aimed at checking the effects on creativity and involving 21 participants  across all age groups  . The anaIn a recent study performed in Italy over healthcare staff during the COVID-19 pandemic, 77 participants filled the same reduced POMS questionnaires used in this study, before and after short breaks (20–30 min) in nature  . The resIn a study performed in Taiwan, 128 middle-aged to elderly subjects were recruited  to attend structured forest therapy sessions focused on the activation of the senses . Each seA study was performed in Poland on young adults  according to a forest therapy program structured over two days (indoor and in forest) . The anaAnother study was performed again in Poland on young adults based on structured 30-min walks . The anaIn a study performed in South Korea, 38 university students with a mean age of around 22 years, with 24 males and 14 females, were assigned in equal numbers to a forest therapy group or a control group . The expIn the opinion of the authors, it would be useful to re-examine some of the above-cited studies based on appropriate and standardized statistical methods in order to build a really useful international database concerning the performance of forest therapy and the determinants of the respective outcomes.A few important limitations affected this study. All the forest therapy sessions lasted 3.5 ± 0.5 h, thus, the dose effect could not be investigated. All professionally conducted forest therapy sessions in remote forest areas followed the same program, thus the effect of different programs could not be investigated. In this respect, a recent Korean study performed with elderly individuals showed that forest therapy programs focused on Qigong or active walking produced specific neuropsychological and electrophysiological benefits, both beneficial for preventing dementia and relieving related health problems . MoreoveParticipants in the forest therapy sessions discussed in this study were allowed to attend only one session, thus the frequency effect could not be investigated. Neither the psychological trait conditions nor the follow-up conditions of the participants were collected; thus, the possible persistence of effects could not be investigated either. Another limitation was that participants were not asked about their personal frequentation of forest or green areas, even if the collected information about personal sporting habits might give some clues to this effect. The latter limitation could have introduced a bias in terms of persistence of effects for some sessions. Moreover, further information on the duration and intensity of sport, understood here as a session of physical activity, would be useful to refine the analysis.The administration of the POMS questionnaires to the participants allowed collecting instantaneously perceived mood states, not corroborated by any measure on physiological parameters. Only admittedly healthy participants were recruited, thus the effects on individuals affected by psychological or physiological disorders could not be assessed. Moreover, no health screening of participants was performed before the forest therapy sessions, thus it cannot be excluded that some participants were affected at least by acute physiological, psychological or psychiatric illnesses, or by alcohol and drug abuse.Although representative enough of different outdoor forest environments in central and northern Italy, the study sites could not be considered representative of all the possible environments; for example, chestnut forests, widespread in mid-mountain areas in the Italian Apennines, were not investigated, along with coastal Mediterranean forests. Moreover, elements other than forest species and few other natural features  were not considered, such as artificial elements . Although large enough in comparison with other experiences reported in the literature, the investigated sample  could not allow the disentangling of all the possible factors affecting the considered psychological outcomes, as well as adequately representing all the relevant age groups, genders and other sociodemographic characteristics. In particular, the control session FTSC suffered from a relatively scarce number of participants. Finally, only TVOCs concentration was measured, while individual monoterpenes are known to potentially produce different and specialized health effects , which cIt is planned to overcome most of the above-listed limitations in the ongoing research across many other sites by using many more participants and including larger control groups in urban settings across different seasons and performing more comprehensive measures such as sociodemographic, psychological (trait and state) and physiological measures, as well as measuring the relative and absolute concentrations of individual BVOCs and MTs. In addition to extending the performance database, the continuation of the study aims at more rigorously identifying the factors affecting the observed healing effects and further linking the medical and forestry research, as recommended in a recent study .Another limitation concerns the considered health domains, which were focused only on mood states. Within the psychological domain, for example, cognitive processes were shown to receive distinct benefits from forest therapy ,71 and wBased on the results obtained in this study and within its limitations, the following preliminary guidelines concerning the short-term amelioration of psychological indices can be advanced. Meteorological comfort, environmental coherence and concentration levels of TVOCs appear quite important in the determination of the outcomes, especially contributing to specific additional benefits brought by forest therapy sessions performed in remote forest areas. Contrary to anxiety, anger and confusion, the depression domain seems not to receive specific benefits from the remoteness of the forest site. Older people could benefit more from forest therapy sessions, especially for depression and confusion. People abstaining from sporting activities, and likely from regular contact with nature, could benefit more from forest therapy sessions, especially for anxiety, depression and anger. These preliminary guidelines could help to optimize forest therapy sessions, as well as to plan suitable sessions targeted to specific groups of participants, for example, those belonging to a given age group or following certain habits, or aimed at improving specific psychological indexes.The mobilization of financial resources and the introduction of specific legislative elements aimed at promoting, standardizing and regulating forest therapy programs were deemed essential to the effective management of the forest ecosystems towards human health . More spFurther important tasks concern the certification of sites based on structural and environmental characteristics and direct functionality , as well as the certification of professional providers of services connected to forest therapy . In the The results achieved in this study confirm, reinforce and expand the knowledge about the healing effects of forests by means of structured forest therapy sessions. The short-term reductions in the levels of anxiety, depression, anger and confusion were all significant for forest therapy sessions performed in remote areas and taken altogether, contrary to a control forest therapy session performed in an urban park.Specific effects of remoteness, in comparison to the control session, arose under certain conditions with anxiety, anger and confusion but not for depression. Some characteristics of the site appeared to affect the outcomes, especially the level of meteorological comfort, the concentration of TVOCs and the environmental coherence, as well as personal characteristics of the participants, such as sporting habits and age.Despite several limitations, a few preliminary technical guidelines were issued, which could help to identify, design and optimize forest therapy stations and sessions also targeted to specific groups of participants or improving specific psychological conditions.


Extrinsic factors and genetic predisposition contribute to the etiology of sarcoidosis, converging in a phenotype of altered immune response associated with multisystemic inflammatory granulomatous tissue infiltration. Immunological reconstitution after hematopoietic stem cell transplantation (HSCT) may represent a unique window for the pathogenesis of the disease. We describe the incidence, clinicopathological features, and HLA associations of sarcoidosis after HSCT in a single-center cohort of patients, together with data from previously published cases.We retrospectively analyzed clinical characteristics and HLA haplotypes from allogeneic  or autologous (auto) HSCT patients from January 2001 through May 2021 at the University Medicine Goettingen (UMG), and data from previously published cases.A total number of 19 patients was identified. These included 4 patients from our center  and 15 patients from the literature review. Thirteen patients had received an allo HSCT, and six patients had received an auto HSCT. Sarcoidosis occurred after a median interval of 20  and 7 (after auto HSCT) months, respectively. The predominant HLA allele associated with sarcoidosis was HLA DRB1*03:01. Sarcoidosis involved the respiratory tract in 15 patients , and it was associated with graft-versus-host disease in 7 of 13 patients receiving allo HSCT. None of the donors or patients had a history of sarcoidosis before transplantation. Disease manifestations resolved with standard glucocorticoid treatment without long-term sequelae.Sarcoidosis may occur at low frequency during reconstitution of the immune system after HSCT. HLA allele associations reflect the associations observed in the general population, particularly with DRB1*03:01. Further insights into the interplay between Tcell reconstitution and the development of sarcoidosis could also provide novel approaches to an improved understanding of the pathogenesis in sarcoidosis. The diagnosis of sarcoidosis was assumed based on non-caseating epithelioid granulomas on histologic examination and exclusion of other causes of granulomas. Data retrieved from the literature were reported as stated in the original publications.Case reports and case series describing sarcoidosis following HSCT were searched using the MeSH terms “stem cell transplantation” and “sarcoidosis” in Medline/Pubmed. Gender, ethnicity, hematologic disease, prior history of sarcoidosis in the donor, remission status before HSCT, donor type, HLA status, conditioning regimen, interval to the onset of sarcoidosis after HSCT, imaging findings, pulmonary function tests (PFTs), involvement of non-pulmonary organs, evidence of graft-versus-host disease (GvHD), and response to glucocorticoids (GC) were extracted from the identified case reports or case series, when available.Phenotypes and genotypes are reported as recommended by the WHO Nomenclature Committee for Factors of the HLA System, using four digits in the UMG cohort. HLA phenotypes and genotypes of patients previously reported in the literature are reported as stated in the original publication. Specifically, high-resolution HLA typing was performed by sequence-based typing  according to the standards for histocompatibility and immunogenetics testing of the European Federation for Immunogenetics (EFI) at our center . All 10 ®  and GraphPad Prism® Version 9.1 . Contingency tables and Fisher’s exact test as well as unpaired, two-tailed Student’s t-test were used for data analysis, as appropriate. p-values <0.05 were considered significant, and p-values <0.15 indicate trends worth reporting. Time to onset of disease (cumulative incidence) was calculated using Kaplan–Meier analysis (Log-rank test). The cutoff for age as criterion for the onset of sarcoidosis was calculated using the software X-tile . Allele frequencies of HLA alleles were calculated via direct counting  and the HLA allele frequencies in Germany and Europe  were stated as found in the classical allele frequency search , leading to a complete remission (CR) without evidence of GvHD. Thirty months later, enlarged pulmonary lymph nodes (LN) and ground glass opacities were noted on chest computed tomography (CT). A LN biopsy showed non-necrotizing granulomas, consistent with a diagnosis of sarcoidosis. Pulmonary function tests showed a reduced diffusion capacity for carbon monoxide (DLCO/SB) and forced vital capacity (FVC), compatible with a restrictive pattern. As the patient remained asymptomatic, no specific treatment was initiated. A subsequent CT showed spontaneous regression of the pulmonary changes after six months.Case #2: A 65-year-old male patient diagnosed with angioimmunoblastic T-cell lymphoma (AITL) received an allo HSCT from a MUD with no known history of sarcoidosis leading to a CR without evidence of GvHD. Eighteen months after HSCT, the patient reported dyspnea and had a restrictive pattern on PFT. A chest CT showed basal consolidations and enlarged mediastinal LN. A bronchoalveolar lavage (BAL) showed non-specific inflammation without findings suggestive of sarcoidosis. Two months later, non-caseating granulomas consistent with sarcoidosis were histologically diagnosed after biopsy of a pulmonary lesion, and the patient was started on GC therapy with a tapering regimen, eliciting a good clinical response.Case #3: A 56-year-old male patient diagnosed with acute myelogenous leukemia (M5) received an allo HSCT from a matched related donor (MRD) without a history of sarcoidosis. Subsequently, the patient suffered from cutaneous, hepatic, and intestinal GvHD. Twenty-four months after allo HSCT, the patient complained of acute abdominal and thoracic pain, which was interpreted as post-herpetic neuralgia and side effects of gastrointestinal GvHD. A chest and abdominal CT showed bilateral hilar lymphadenopathy, intrapulmonary nodules, and prominent abdominal lymph nodes. Sarcoidosis was diagnosed following biopsy of a hilar LN, and, as the patient was asymptomatic, a watch-and-wait strategy was employed. However, in the following months, the patient complained of arthralgias with shoulder pain, morning stiffness in the fingers and elbows, as well as shortness of breath following moderate physical exertion. Therefore, a GC regimen with concomitant non-steroidal antirheumatic drugs was initiated with good clinical regression of symptoms.Case #4: A 56-year-old male patient was diagnosed with AITL. Standard chemotherapy was consolidated with auto HSCT. Five months after transplantation, enlarged pulmonary LN were noted on routine chest CT, and histopathologic examination showed granulomas consistent with pulmonary sarcoidosis. Oral GC therapy was initiated leading to a remission of pulmonary nodules and LN. After tapering of GC, an increase in size of mediastinal and infradiaphragmal LN was noted, and a repeat biopsy was performed leading to the diagnosis of a relapse of the AITL. An allo HSCT from a MRD with no known history of sarcoidosis was performed. Post allo HSCT, the patient suffered from GC-sensitive cutaneous and intestinal GvHD; no relapse of sarcoidosis was noted.Overall, we observed symptomatic sarcoidosis in a total of 4 out of 1,065 and 957 patients, who had received allogeneic and autologous stem cell transplantations in the UMG transplantation program, respectively. The disease emerged within a median follow-up observation period of 22 months, resulting in an estimated incidence of 156/100,000 and 58/100,000 for patients at risk after allo and auto HSCT, respectively.An additional 15 cases of sarcoidosis following HSCT have been reported in the literature, with 5 patients having received an auto HSCT , 21, 22,n = 7) followed by leukemia (n = 4) and myelodysplastic syndrome (MDS)/myelofibrosis (MF) (n = 3). The reported underlying diseases leading to auto HSCT were breast or testicular cancer (n = 4), as well as polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes (POEMS) syndrome in one patient.The most frequently reported underlying disease leading to treatment with allo HSCT was lymphoma , fludarabine (FLU) (n = 7), and cyclophosphamide (CYC) (n = 5). Two patients each also received alemtuzumab (ALM) or antithymocyte globulin (ATG) as part of the conditioning regimen, respectively. In five patients, chemotherapy had been combined with total body irradiation (TBI). In the autologous cohort carboplatin, etoposide, BEAM, and melphalan were the most frequently applied cytostatic drugs.The most frequently applied chemotherapeutic agents in the allo HSCT cohort were busulfan (BU) . Involvement of extrapulmonary organs was present in 11/19 patients; the most common organ manifestation was skin followed by liver. Seven of the 13 allo HSCT also had evidence of GvHD; in two patients, the GvHD status was not reported. A response to GC was noted in all patients where they were given . No additional immunosuppressive therapies were used. The characteristics of the patients in both groups  are summarized in All patients had pulmonary sarcoidosis. Most patients showed changes on CT/chest radiographs  (p = 0.1438) (The median age of patients with sarcoidosis after HSCT was 50 years (range 25 to 69 years). The median time to onset of sarcoidosis post HSCT overall was 17 months (range 3 to 120 months), with patients developing sarcoidosis after allo HSCT at a median time of 20 months (range 3 to 30 months) and 7 months (range 3 to 120 months) after auto HSCT . This notion is further supported by the HLA allele associations observed for sarcoidosis post HSCT, which reiterates the associations described for sporadic sarcoidosis , 8–14 -2 and interferon (IFN)-g, and augmented macrophage tumor necrosis factor (TNF)-a production, resulting in immune response amplification -mediated immunity, which is necessary for the resolution of sarcoid granulomas, are ATG and ALM. Two of our allo HSCT patients received ATG, while it was not reported in any of the case reports. ATG, a polyclonal antibody, is used to prevent GvHD and suppress allograft rejection. Owing to its polyclonality, there are diverse effects of ATG on the immune system, among them Tcell depletion, modulation of leukocyte/endothelial interactions, apoptosis in B cells, interference with DC function and the induction of regulatory T and natural killer (NK) T cells . So far Alemtuzumab, however, has been associated with the development of sarcoidosis and there are several case reports describing the occurrence of sarcoidosis post ALM treatment in multiple sclerosis patients –38. WhilDifferent hypotheses have been proposed, aiming to explain the pathogenesis of sarcoidosis after malignancy: Firstly, it has been postulated that sarcoidosis develops as a reaction to the immunosuppressive effect of chemotherapies or to a specific chemotherapy agent, such as bleomycin, known to achieve high concentrations in LN, skin, and lung tissue .Secondly, Brincker et al. hypothesized that the appearance of sarcoidosis post malignancy may be driven by antigens derived from tumor cells leading to immunological events resulting in granuloma formation . Thirdlyvice versa, none of the patients with sarcoidosis in their pre-transplant medical history experienced sarcoidosis recurrence.In three patients the occurrence of sarcoidosis has been reported after possible transmission from the donor –25. The The immunological environment post HSCT, similar to that present in sarcoidosis pathogenesis outside of the context of HSCT, could promote the development of sarcoidosis, especially in a genetically susceptible individual with a specific HLA allele. The reported disease pathogenesis and potentially triggering factors are summarized in During immune reconstitution, higher levels of cytokines such as MCP-1, CCR1, CCR2, IL-8, and Rantes are present, leading to a tissue environment promoting the formation of non-necrotizing epithelioid granulomas –49.All allogenously transplanted patients reported previously, including those in our cohort, developed sarcoidosis after complete donor engraftment of their bone marrow, suggesting that the development of sarcoidosis in these circumstances was initiated by the donor immune system. Interestingly, it has been shown that individuals with a HLA-B8/DR3 phenotype produce higher amounts of TNF-a compared to their IFN-g generation potential , making The older age and shorter time to the onset of sarcoidosis post HSCT of the analyzed patients compared to the normal population might be explained with the progressive involution of thymic tissue during aging with a decline in T-cell output and T-cell senescence with restricted T-cell receptor repertoire diversity, leading to a slower immune reconstitution and impaired immune responses following transplantation . HoweverThe occurrence of extrapulmonary manifestations of sarcoidosis in patients with leukemia, MDS, and MF compared to lymphoma patients has been linked to malignant antigens as a sarcoidosis trigger in leukemic diseases . While tOur study has several limitations. First, sarcoidosis post HSCT is a rarely reported event, although based on observations by Bhagat et al.  and the Based on the 2022 HSCT patients that have been treated at our institution over a 20-year span and four cases of sarcoidosis occurring therein, we estimate the incidence at 156 and 58 cases per 100,000 for allo and auto HSCT, respectively, which is higher than reported by Bhagat et al.  and highThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of the University Medical Center Göttingen (protocol number 30/6/21). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.RW-K and PK designed the study. Material preparation, data collection, and analysis were performed by RW-K, JB, JK, JGR, GW, and PK. JH and WJ treated patients and edited the manuscript. The figures were created by RW-K and PK. The first draft of the manuscript was written by RW-K. All authors contributed to the article and approved the submitted version.We acknowledge support by the Open Access Publication Funds of the Göttingen University.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.