Depression is a mental disorder that significantly reduces quality of life, and the discovery of new drug targets is an urgent problem for modern neuroscience. Brain-derived neurotrophic factor (BDNF) and its receptors have been found to participate in mechanisms of depression and antidepressant drugs’ action. In this study, we focused on a less-studied truncated isoform of receptor TrkB: TrkB.T1. Initially, we noticed that the level of TrkB.T1 is low in the hippocampus of Antidepressant-Sensitive Cataleptics (ASC) mice, which are characterized by genetically determined depressive-like behavior in contrast to “normal” C57BL/6J mice. Next, overexpression of TrkB.T1 receptor in hippocampal neurons of ACS mice was induced to clarify the role of this receptor in mechanisms of depressive-like behavior. TrkB.T1 overexpression lowered BDNF protein concentration in the hippocampus. On the behavioral level, TrkB.T1 overexpression severely decreased aggression and enhanced social behavior. Additionally, this excess of receptor TrkB.T1 slightly promoted anxiety and depressive-like behavioral traits without affecting learning and memory. Our results show that this TrkB isoform participates in the control of aggression, anxiety, and depressive-like behavior and in the regulation of BDNF system functioning in ASC mice . Considering our findings, we believe that hippocampal receptor TrkB.T1 can be a drug target for the correction of behavioral pathologies. Ntrk2 transcript. These isoforms are the full-length tyrosine kinase receptor (TrkB.FL), the C-terminally truncated receptor (TrkB.T1), a C-terminally truncated Shc+ receptor (TrkB.shc), and a C-terminally truncated receptor retaining tyrosine kinase activity (TrkB-T-TK) [The Trk family of receptors includes TrkA, TrkB, and TrkC, which mediate the cellular effects of neurotrophins . TrkB iskB-T-TK) .NTR [Lacking the intracellular domain, TrkB.T1 cannot transduce signals to intracellular BDNF-initiated pathways. Nonetheless, TrkB.T1 has been found to have many functions. Having an extracellular domain identical to that of TrkB.FL, TrkB.T1 can bind BDNF with the same affinity and form homo/heterodimers with receptors TrkB.T1 and TrkB.FL . TrkB.T1NTR , which iNTR . ReceptoNTR . In the NTR . In gliaNTR .Since its discovery, studies on receptor TrkB.T1 have revealed its association with anxiety , aggress2A receptor. On the other hand, BDNF receptors have not been studied in this model yet.To discover the participation of receptor TrkB.T1 in depressive-like behavior, here we chose the Antidepressant-Sensitive Catalepsy (ASC) mouse strain: a genetic model of depression that has been developed by backcrossing predisposed-to-catalepsy CBA/Lac mice and catalepsy-prone AKR/J mice ,19. ASC Bdnf, Ntrk2, and Ngfr) and on mRNA levels of Mapk3, Creb1, and early response genes (Fos and Jun) required for long-term potentiation (LTP) induction in the hippocampus of ASC mice, which show genetically determined depressive-like behavior.In this work, we assessed the effects of TrkB.T1 overexpression in hippocampal neurons on different kinds of behavior using a wide range of behavioral tests  as well as on the expression of key genes of BDNF systems . All experimental animals were 2 months old. Behavioral tests were divided into two series, and the first one included the open field test, elevated plus maze, FST, and resident–intruder test. The second series consisted of the Morris water maze test and TST. In each test series, two groups of mice were evaluated: an experimental group (11 mice) and a control group (10 mice). An outline of the experiment is shown in In the resident–intruder test, 1.5-month-old mice of the CD1 strain (weight 25 ± 1 g) were used aside from ASC mice. Mature male mice of C57BL/6J strain  were used for comparative assessment of BDNF, TrkB, and TrkB.T1 mRNA levels. The animals were kept in the vivarium of the Institute of Cytology and Genetics SB RAS under standard laboratory conditions with a 12/12 h light/dark cycle and free access to feed and water.For fluorescence imaging, we employed three mice injected with adeno-associated virus (AAV) vector AAV-TrkB.T1-EGFP.With primers  containing recognition sites of restriction endonucleases BamHI and AgeI, a gene corresponding to TrkB.T1 was synthesized by means of murine cDNA as a template. The obtained sequence was digested with restriction endonucleases BamHI and AgeI  and ligated into the pAAV_Syn_EGFP vector to be expressed under the control of a synapsin (Syn) promoter . Sanger 2. The cells were split at 70% confluency, and the medium was refreshed every 2–3 days. HEK293FT cells were transfected with plasmids using polyethylenimine , following the manufacturer’s instructions.HEK293FT cells were subcultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% of fetal bovine serum , 1% of GlutaMAX , and 1% of a penicillin–streptomycin solution . The cells were incubated at 37 °C with 5% CO9 viral genomes per microliter).Packaging of plasmid AAV_Syn_TrkB.T1 or control plasmid pAAV_Syn_EGFP DNA into AAV capsids was performed by cotransfection of HEK-293FT cells with plasmids AAV-DJ  and pHelper  . Harvesthttp://labs.gaidi.ca/mouse-brain-atlas/?ml=-1.5&ap=-2&dv=2, accessed on 2 September 2021). Virus AAV_Syn_TrkB.T1 (AAV-TrkB.T1) or AAV_Syn_EGFP (control) was injected into the hippocampus bilaterally (1 μL per side) through a Hamilton syringe at a rate of 0.1 μL/min over a 10 min period. To minimize any drawback of the virus as the needle was removed, the needle was kept in place for further 2 min after the injection. After injections, the incision was closed with interrupted silk sutures, and the animal was placed in a warm cage.Animals were anesthetized with a solution (1 mL/kg i.p.) of 2,2,2-tribromoethanol  in 2-methyl-2-butanol  and placed in a stereotaxic frame . The skull was drilled bilaterally , thereby improving the contrast of an animal against the background. Mice were placed near the wall of the arena and allowed to freely explore the space for 5 min. Using EthoStudio software and hardware, the following parameters were automatically estimated: total distance traveled (m) and time spent at the center of the arena (%), the diameter of which was 27.5 cm. The numbers of rearings and groomings as well as average grooming duration (s) were determined manually. After each mouse, the arena was wiped with wet and dry wipes.Elevated plus maze test. The test setup consisted of four arms 30 cm long and 6 cm wide connected at a right angle and located at a height of 60 cm above the floor. One pair of arms had walls 20 cm high (closed arms), and the other pair had no walls (open arms). The arena was illuminated from above with diffused light. Mice were placed at the intersection of two arms, and movement was automatically recorded for 5 min using the EthoStudio software. The following parameters were automatically estimated: distance traveled (m), time spent in open and closed arms (%), and studied area of each arm (%). Peeks into closed arms and head dips were counted manually.The intermale aggression test. The test was performed within the resident–intruder paradigm as described previously [eviously ,26. A raTail suspension test. Mice were fixed with adhesive tape by the tail and hung on a horizontal bar at a height of 30 cm from a table surface. The number of immobilization episodes and latency time and duration of each episode were recorded using the EthoStudio software.Forced swim test. Mice were placed in a clear plastic box (15 × 25 cm) filled with water at 25 °C. After 2 min of adaptation, total immobility time and latency time of the first episode of immobility were recorded in the EthoStudio software Version 2.0 during 4 min [ng 4 min .Morris water maze test. The setup comprised a round pool 110 cm in diameter with walls 30 cm high. The pool was filled with water , and powdered milk was added to make it opaque. We tracked each animal by transmitted (inverted)-lighting techniques developed for the open field test. In the EthoStudio software, the pool was divided into four sectors. A glass round escape platform (5 cm diameter and 14.5 cm height) was located 1 cm below the water surface near the center of the lower right quadrant of the maze. A black rectangle measuring 6 × 12 cm  was glued to the pool wall closest to the platform. Mice were automatically tracked by means of the EthoStudio software.Each training period lasted 4 days, and each session contained three trials. During the training, mice were allowed to search for the platform for 60 s. At the end of each test, mice were placed on the platform and held (if necessary) there for 15 s. Measured parameters were latency to find the platform (s), distance traveled (m), and total distance to the platform (m), which was calculated as the sum of distances from the mouse to the platform at each time point. The average value of three tests was subjected to further analyses.On the fifth day, the platform was removed, mice were placed at the center of the pool, and their movements were recorded for 60 s. Time spent in the target sector (%) was estimated, and then the average value for the three trials was calculated. A statistically significant excess of time spent in the target sector over a random level (25%) indicated that the mouse remembered the location of the platform.Two days after the behavioral testing, the animals were decapitated, and hippocampi were isolated on ice, frozen in liquid nitrogen, and stored at −80 °C until subsequent procedures.2, 2 µL of mixture of forward and reverse primers, and 0.16 µL of Taq DNA polymerase, and adjusted the volume to 20 µL with deionized H2O. Two types of standard were used for quantitative analysis of mRNA levels: external and internal. An internal standard (mRNA of the rPol2 gene) was employed to make sure that the reverse transcription worked and as a basis for calculating mRNA level of the studied genes. In preliminary experiments, no differences were found in the level of rPol2 mRNA among the control and experimental groups. A series of dilutions of DNA (of a known concentration) isolated from the mouse liver served as an external standard, which made it possible to check whether the PCR worked and to determine the number of mRNA copies of the studied genes in the samples. Gene expression is presented as the ratio of cDNA of an analyzed gene to 100 copies of rPol2 cDNA [We mixed a 1 µL aliquot of cDNA with 2 µL of PCR buffer (containing SYBR green I), 2 µL of 25 mM dNTP, 2 µL of 25 mM MgClol2 cDNA . Meltingg for 15 min at 4 °C, the supernatant was collected and centrifuged at 20,000× g at 4 °C for an hour. The cytosolic fraction was collected, and the precipitate remaining in the tube was resuspended in homogenizing buffer (see above). Both fractions were stored at −80 °C. Protein concentration was assessed spectrophotometrically using the Pierce BCA Protein Assay Kit  and a NanoDrop 2000C spectrophotometer . The samples were adjusted to equal concentrations (1 mg/mL) using 2× Laemmli’s buffer and heat denatured for 10 min at 95 °C. Extracts  separated by SDS-PAGE were blotted onto a nitrocellulose membrane  with the help of a Trans-Blot Turbo Transfer System . The membranes were blocked in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST) containing 5% of nonfat dry milk (NFDM-TBST) for 1 h, washed, and then incubated with primary antibodies shown in Membrane proteins were isolated by homogenizing samples in a buffer consisting of 300 mM sucrose, 10 mM Tris-HCl pH 7.2, 1 mM EDTA, 5 mM β-mercaptoethanol, and protease inhibitors . Then, the samples were centrifuged at 500× In the fifth week after injection, mice were transcardially perfused with phosphate-buffered saline (PBS) and a 4% paraformaldehyde solution. Brains were excised, postfixed with 4% paraformaldehyde for 6 h, and immersed in 20% sucrose in PBS for 2 days. Using a cryostat , 14 µm slices were obtained, and finally mounted in an antiquenching medium with DAPI  followed by examination under a Zeiss AxioImager2 microscope with 10× and 40× air-immersion objectives. The mice used for the imaging were not subjected to the behavioral testing.t test if the data were normally distributed. Nonparametric Mann–Whitney U test was utilized when data were non-normally distributed. The results of the Morris water maze test were subjected to repeated measures analysis of variance (ANOVA) with Fisher’s test post hoc comparison. Analysis of covariance (ANCOVA) was applied to determine whether immobility in the tail suspension test and social activity in the resident–intruder test depended on reduced locomotor activity in the open field test.All data were tested for normality of distribution by Shapiro–Wilk, Kolmogorov–Smirnov, D’Agostino and Pearson, and Anderson–Darling tests. Gene expression data and the results of open field, elevated plus maze, tail suspension, forced swim, and resident–intruder tests were processed by Student’s p = 0.004) was reduced in the hippocampus of ASC mice, in agreement with reports on reduced BDNF expression in patients with major depressive disorder [p = 0.81), the TrkB.T1 mRNA level proved to be downregulated (t(10) = 3.08, p = 0.01) in the hippocampus of ASC mice compared to C57BL/6J mice  in the hippocampus of “depressive” (ASC) and “nondepressive” (C57BL/6J) mice. We revealed that the BDNF mRNA level (t(12) = 3.54, disorder . Of note/6J mice A. As farp < 0.0001) and protein levels (t(15) = 3.37, p = 0.004; To identify the role of hippocampal TrkB.T1 in mechanisms underlying depressive-like behavior of ASC mice, we induced TrkB.T1 overexpression by injecting the hippocampi with a recombinant AAV carrying the gene expressing TrkB.T1. Five weeks after the injection, we performed behavioral testing, and next confirmed TrkB.T1 overexpression in the hippocampus both at mRNA (t(18) = 5.22, p = 0.02) (p = 0.75); the same was true for the time spent in open arms (t(18) = 1.25, p = 0.40).Mice with TrkB.T1 overexpression spent significantly less time at the center of the arena in the open field test (t(19) = 2.48,  = 0.02) . This fip = 0.003). On the other hand, the elevated plus maze did not reveal any differences in exploratory activity, assessed by means of peeks (t(18)= 0.83, p = 0.41), latency to the first peek  and the number of head dips   = 3.46,  = 0.98) .p = 0.05). This result was confirmed by the elevated plus maze test (t(18) = 2.26, p = 0.036).Mice with TrkB.T1 overexpression also showed a strong tendency toward shorter distance traveled in the open field test (t(19) = 2.00, p = 0.28), explored area in closed arms of the elevated plus maze , explored area in open arms , and grooming parameters , duration , and latency time of the first grooming episode (t(19) = 0.16, p = 0.16)).Other parameters analyzed in both tests were not affected by TrkB.T1 overexpression: explored area in the open field test  in the tail suspension test (p = 0.59) and the number of immobility episodes (t(21) = 0.51, p = 0.61) did not differ between the two groups. To investigate whether immobility in the tail suspension test depends on diminished locomotor activity, ANCOVA was performed with total traveled distance in the open field test being a continuous predictor of immobility in the tail suspension test. In this case, AAV-TrkB.T1-EGFP–injected mice still demonstrated a significantly higher level of immobility in the tail suspension test as compared to corresponding control animals .Mice with TrkB.T1 overexpression showed longer immobility time as compared to the control (t(21) = 2.61, ion test . Neverthp = 0.70), latency to the first immobility episode (t(16) = 1.73, p = 0.10), and the number of immobility episodes (t(16) = 0.34, p = 0.10)), we assumed that the TrkB.T1-induced prodepressive effect is too weak to be detected by both tests.Because the influence of receptor TrkB.T1 on immobility was not confirmed in the forced swim test (immobility duration (t(14) = 0.54, p = 0.0004; p = 0.0003; p < 0.0001; p = 0.004). The number of social contacts (t(18) = 0.23, p = 0.63) was not significantly affected by TrkB.T1 overexpression. At the same time, latency to the first social contact (t(18) = 2.00, p = 0.06) manifested a strong decreasing trend in the experimental group. In ANCOVA with traveled distance as a continuous predictor, we also detected a significant impact of TrkB.T1 overexpression on the number , duration , and latency time  of aggressive contacts and on the duration of social interactions .The resident–intruder test revealed that TrkB.T1 overexpression significantly diminishes the number of aggressive contacts , distance traveled by a mouse (p < 0.01 and p < 0.05 for groups AAV-EGFP and AAV-TrkB.T1), and cumulative distance between the mouse and the platform at each time point (p < 0.05 and p < 0.01 for groups AAV-EGFP and AAV-TrkB.T1)), indicating that both groups successfully learned the task. For each examined parameter, we found a significant effect of the training day on the distance traveled , cumulative distance , and latency to find the platform .We carried out the Morris water maze test to evaluate the effects of receptor TrkB.T1 on learning and memory . All parp ˃ 0.05), cumulative distance , and latency to find the platform ). This meant that the two groups had a similar ability to learn, and the same was true for the ability to memorize.By contrast, the influence of TrkB.T1 overexpression was not significant  and AAV-TrkB.T1 (p < 0.01)) and the influence of training was confirmed . Therefore, the effect of TrkB.T1 overexpression on spatial memory and learning was not detectable .On retention day (the test without the platform), both groups spent significantly more time in the target sector compared to the other sectors (AAV-EGFP (p = 0.02), while transcription of its gene remained unchanged (t(16) = 1.56, p = 0.13; Ntrk2 gene (mRNA (t(15) = 0.48, p = 0.63) and protein (t(17) = 1.29, p = 0.21)) was not affected by TrkB.T1 overexpression. Neither was the expression of the receptor of proBDNF, i.e., receptor p75NTR encoded by the Ngfr gene , protein (t(17) = 0.64, p = 0.52); TrkB.T1 overexpression significantly lowered the BDNF protein level in mice of the experimental group (t(17) = 2.39, p = 0.26), Mapk3 , and early response genes: Fos (t(17) = 0.61, p = 0.54) and Jun (t(17) = 0.67, p = 0.50; We did not find any impact of TrkB.T1 overexpression on mRNA levels of LTP-related genes: Creb1 (t(18) = 1.15, Our preliminary study suggests that transcription of genes of TrkB.T1 and BDNF is significantly lower in the hippocampus of ASC mice  than in “nondepressive” C57BL/6J mice. Intrigued by this novel finding, we induced hippocampal TrkB.T1 overexpression in ASC mice using an AAV-TrkB.T1 construct. Then, we analyzed behavioral patterns and assessed the expression of genes that may be responsible for changes in behavior. Overexpression of TrkB.T1 did not cause regulatory compensation in terms of receptor TrkB.FL expression in the hippocampus of the studied mice, implying that the observed behavioral and genetic alterations are TrkB.T1-induced.Open field test results showed that overexpression of TrkB.T1 receptor has a mild effect on anxiety, judging by less time spent at the center of the arena and more time spent near the wall of the arena by mice from experimental group (TrkB.T1 overexpression). To rule out a possible false positive result, we measured the explored area of the open field in both groups. Because there were no differences in this parameter between the groups, it is safe to assume that the difference in time spent at the center is actually due to a difference in anxiety rather than changes in locomotor or exploratory activity. This result, however, failed to be replicated in the elevated plus maze test: the two groups spent similar periods in closed arms, and the same was true for open arms. Head dips, which are an indicator of anxiety , also diWe noticed the decrease in the number of rearings in TrkB.T1 overexpressed mice. Given that the explored area of arena was not changed, the decrease in the number of rearings in the open field test may also be due to greater anxiety . In suppOf note, TrkB.T1 knockout mice tend to spend less time in the center of the arena in the open field test and spend significantly less time in the open arms of the elevated plus maze . Thus, TIn addition, the overexpression of TrkB.T1 reduced the distance traveled by the experimental group in the elevated plus maze. Mice with TrkB.T1 overexpression also tended to have a shorter distance traveled in the open field test. On the other hand, the locomotor activity measured by the Morris water maze test did not differ between the groups. It is noteworthy that the knockout of receptor TrkB.T1 in C57BL/6J mice does not affect their locomotor activity . TrkB.T1FST and TST are designed to assess depressive-like behavior in rodents. Our results uncovered an increase in immobility time in the TST but not in the FST during hippocampal TrkB.T1 overexpression. The discrepancy between these two closely related tests is explained by the fact that both tests, although similar in the constructs they are designed to assess, are probably different in terms of the biological substrates underlying the observed behavior . Taking Mapk3, Creb1, and early response genes (Fos and Jun) required for LTP induction [The Morris water maze test revealed that hippocampal receptor TrkB.T1 does not affect learning dynamics and spatial memory in ASC mice. These results are consistent with unchanged mRNA levels of the nduction ,39. Our nduction ,12. On tnduction . In the nduction .We found that the baseline level of BDNF in ASC mice is lower than that in “nondepressive” C57BL/6J mice. This is expected because a depressive phenotype is known to be associated with BDNF deficiency . For exaAdditionally, we noticed a dramatic effect of receptor TrkB.T1 on aggression and social behavior. Overexpression of TrkB.T1 (i) reduced the number and duration of aggressive contacts, (ii) increased latency time of the first aggressive contact, (iii) extended the duration of social contacts, and (iv) diminished latency time of the first social contact. The antiaggressive effect of TrkB.T1 overexpression along with the decline of BDNF levels is consistent with the findings of Ilchibaeva and coauthors , who havWe showed that receptor TrkB.T1 is involved in mechanisms underlying anxiety, depressive-like behavioral traits, and aggression in ASC mice, which are genetically predisposed to depressive-like behavior. We confirmed for the first time that receptor TrkB.T1 regulates the BDNF level in neuronal cells. This knowledge means that TrkB.T1 should be taken into consideration in further studies on behavioral pathologies and underlying mechanisms.


Using the hybrid electrodes, thin-film transistors (TFTs) were fabricated, which exhibited an electron mobility of ~6.7 cm2 V−1 s−1, a current on-off ratio of ~1.04 × 107, and a subthreshold voltage of ~0.122 V/decade. These electrical properties are comparable with those of TFTs fabricated using Al electrodes. This suggests the possibility of customizing flexible transparent electrodes within a carbon nanomaterial system.In this study, we prepared flexible and transparent hybrid electrodes based on an aqueous solution of non-oxidized graphene and single-walled carbon nanotubes. We used a simple halogen intercalation method to obtain high-quality graphene flakes without a redox process and prepared hybrid films using aqueous solutions of graphene, single-walled carbon nanotubes, and sodium dodecyl sulfate surfactant. The hybrid films showed excellent electrode properties, such as an optical transmittance of ≥90%, a sheet resistance of ~3.5 kΩ/sq., a flexibility of up to  Flexible transparent electrodes have attracted significant interest in the field of wearable devices, such as electronic skins, foldable photovoltaics, soft robots, and flexible displays ,19,20,21With excellent optical, electrical, and mechanical properties, one- and two-dimensional carbon nanomaterial composites are finding applications as flexible transparent electrodes. In particular, a very small amount of such materials is needed to fabricate conducting electrodes, as high aspect ratios of one-dimensional CNTs lead to low percolation thresholds. However, CNT network films have large contact resistance from the percolation of charges through their junctions ,5,6. In Generally, graphene solutions are prepared by dispersing graphene oxide (GO) using Hummers’ method and directly exfoliating graphene flakes in organic or inorganic solvents ,23,24,25In this study, we developed an aqueous solution process for obtaining flexible transparent electrodes based on carbon nanomaterials. Using this method, we synthesized a high-quality, non-oxidized aqueous graphene solution from highly ordered pyrolytic graphite (HOPG) via the halogen intercalation method. We also prepared a hybrid aqueous solution of non-oxidized graphene and SWNT, which exhibited good dispersity and long-term stability. We then used this solution to fabricate hybrid film electrodes. We evaluated the mechanical, electrical, and optical properties of the hybrid electrodes with various graphene to SWNT ratios and film thicknesses. Furthermore, we fabricated fully transparent indium gallium zinc oxide (IGZO) thin-film transistors (TFTs) using the hybrid electrodes.3(CH2)11OSO3Na)  were purchased from Sigma Aldrich .Highly ordered pyrolytic graphite (HOPG) (Grade SPI-2 #466HP) was purchased from SPI . Raw HiPco single-walled carbon nanotubes (R grade SWNT) were purchased from Unidym . Natural graphite (325 mesh) was purchased from Alfa Aesar . Iodine monochloride (ICl) (reagent grade 98%) and sodium dodecyl sulfate (SDS)  and ICl  of 10 mg were introduced into a glass reactor and sealed with a Teflon cap. The reactor was then immersed in a silicon oil bath at 160 °C for 54 h. After the reaction between the graphite and intercalant, the as-prepared graphite intercalation compound (GIC) was immediately placed in a quartz tube at 800 °C with 60 sccm of Ar flow for 10 min to obtain expanded graphite. Subsequently, the furnace was cooled to room temperature while argon continued to flow. Expanded graphite of 10 mg was added in 1 wt.% aqueous SDS solution of 10 mL. To disperse and break the expanded graphite, the solution was homogenized for 1 h and then bath-sonicated for 30 min, followed by slow-speed centrifugation at 2100 rpm for 20 min. Finally, the top half of the dispersion was extracted with a vacuum pipette (~8 mL) and retained for use.HOPG of 20 mg  and Raman spectroscopy  with a laser excitation wavelength of 532 nm. The thickness and optical transmittance of the graphene/SWNT electrodes were examined using a surface profiler  and a UV-vis-NIR spectrometer . The surface morphology of the graphene/SWNT electrodes was visualized using field-emission scanning electron microscopy . X-ray photoelectron spectroscopy  measurements were carried out using a monochromatic Al Kα X-ray source (1486.6 eV) to determine the chemical compositions of the graphene. The work function was measured using a Kelvin probe system . The sheet resistance was measured using a four-point probe system .2O3 (100 nm) was deposited for the top gate dielectric via atomic layer deposition (ALD) at 130 °C under 10−2 Torr followed by thermal treatment at 300 °C for 30 min under ~10−6 Torr. A transparent active layer was synthesized with spin-coating of IGZO (20 nm), followed by thermal treatment at 150 °C for 5 min and at 400 °C for 2 h. Source and drain electrodes were deposited using the graphene/SWNT solution by spray-coating followed by curing at 80 °C for 5 min. The channel regions 2 mm in width and 200 μm in length were patterned with shadow mask. Subsequently, the TFTs were soaked in ethanol at 50 °C for 5 min and then dried with N2 blowing, followed by heating in an oven at 50 °C for 5 min. For the comparison of the electrical properties of the TFTs, IGZO TFTs were fabricated using Al electrodes. The electrical properties of these devices were characterized using a semiconductor parameter analyzer  under ambient conditions.Fully transparent TFTs were fabricated on a glass substrate. For the gate electrode, ITO (150 nm) was deposited on glass by sputtering at room temperature. Subsequently, a layer of Al−1), D band (~1330 cm−1), G− band (~1540 cm−1) (or Breit–Wigner–Fano), G band (~1590 cm−1), and 2D band (~2640 cm−1) of SWNT ..−1), D  is summarized in ε = 3.6% (bending radius (R) = 1.4 mm). The sheet resistances of the SWNT- and the non-oxidized graphene/SWNT-based flexible electrodes fluctuated slightly within ±1% due to the bending strain. However, the hybrid electrodes exhibited better mechanical stability against repeated bending cycles (ε = 2.0% (R = 2.5 mm)) than the SWNT-based electrodes , current on-off ratio (Ion/Ioff), and subthreshold swing (SS) of the devices were estimated to be ~6.7 cm2 V−1 s−1, ~1.04 × 107, and ~0.122 V/decade, respectively  and high mechanical stability, as indicated by a sheet resistance of less than 1% even after 103 bending cycles at ε = 2.0% (R = 2.5 mm). Furthermore, the IGZO thin-film transistors fabricated using the hybrid electrodes exhibited device characteristics comparable with those of the TFTs fabricated using Al electrodes, with an electron mobility of ~6.7 cm2 V−1 s−1, a current on–off ratio of ~1.04 × 107, and a subthreshold voltage of ~0.122 V/decade. This demonstrates a significant step toward the application of carbon nanomaterials in flexible transparent device components for next-generation wearable devices.In this study, we prepared and investigated the properties of flexible transparent electrodes obtained using non-oxidized graphene/SWNT aqueous solutions. Owing to minimal defects in the non-oxidized graphene, 60 nm-thick hybrid film containing 30 wt.% graphene showed an optical transmittance of more than 90% and sheet resistance of ~3.5 kΩ/sq. In addition, the film exhibited excellent flexibility, with no electrical change up to


The quadratus lumborum (QL) block is an ultrasound-guided regional anesthesia technique that aims to provide analgesia to the abdomen. Previous studies show that this technique may fail to provide analgesia in the cranial abdominal wall in dogs. The QL block is a difficult technique with the risk of damaging vital structures. The objective of this study is to assess a new latero-ventral approach to the QL (LVQL), which could be easier and safer to perform than the interfascial approach (IQL) widely reported. Another objective is to evaluate the distribution of high-volume injections (0.6 mL/kg) of dye/contrast achieved by both approaches. Six thawed dog cadavers were used to randomly perform the LVQL in one hemiabdomen and the IQL in the other. Distribution of the injectate throughout the ventral branches of the spinal nerves from T10 to L4 and the sympathetic trunk (CT and dissection) was assessed. The two approaches consistently stained L1-L3 ventral branches, which is compatible with obtaining somatic analgesia in the middle and caudal abdominal wall. The administration of high-volume injections did not result in a more cranial distribution of the injectate compared to previous studies. The sympathetic trunk was more consistently stained by the IQL, therefore, the LVQL may not promote visceral desensitization of the abdomen.The QL block is a high-level locoregional anesthesia technique, which aims to provide analgesia to the abdomen. Several approaches of the QL block have been studied to find out which one allows a greater distribution of the injectate. The aim of this study is to compare the traditional interfascial QL block (IQL) with a new latero-ventral approach (LVQL). We hypothesize that this new approach could be safer and easier to perform, since the injectate is administered more superficially and further away from vital structures. Our second objective is to assess whether a higher volume of injectate (0.6 mL/kg) could reach the ventral branches of the last thoracic nerves, leading to a blockade of the cranial abdomen. Six thawed canine cadavers (12 hemiabdomens) were used for this purpose. Both approaches were performed in all cadavers. A combination of methylene blue/iopromide was administered to each hemiabdomen, randomly assigned to the LVQL or IQL. No differences were found regarding the ease of perform the LVQL with respect to IQL. The results show that both techniques reached the ventral branches from L1 to L3, although only the IQL consistently stained the sympathetic trunk (5/6 IQL vs. 1/6 LVQL). The use of a higher volume did not enhance a more cranial distribution of the injectate. The quadratus lumborum (QL) block is an ultrasound-guided regional anesthesia technique that aims to block the ventral branches of the thoracolumbar nerves and sympathetic trunk . SeveralThe QL block was first described by Blanco et al.  as a varQuadratus lumborum block is a complex technique with a significant risk of damaging abdominal structures ,18. The This study intends to assess a new approach to the QL block, in which the injectate is administered lateral and ventral to the quadratus lumborum muscle (LVQL), closer to the skin surface and further away from important abdominal structures, to increase the ease and safety of the technique. A second objective is to assess the distribution of a high volume of injectate (0.6 mL/kg), comparing the LVQL to the interfascial QL block (IQL) as described by Garbin et al. . We hypoThe Biosafety Committee in Experimentation (CBE 433/2021) and the Ethical Committee for Animal Experimentation (CEEA 740/2021) of the University of Murcia approved this essay. Six canine cadavers (12 hemiabdomens) were used in this research. Cadavers with skin or muscle lesion in the lateral aspect of the thoracolumbar area, or vertebral alterations were excluded from the study. The animals were humanely euthanized for reasons unrelated to the study and immediately frozen. The cadavers were thawed at room temperature 48 to 72 h before the procedures.−1 of a solution made up of the same amount of methylene blue  and radiopaque contrast medium iopromide  was injected in each hemiabdomen. The same researcher (Eliseo Belda) performed all the injections.Two different approaches to the QL block (LVQL and IQL) were compared. Both approaches were performed in all the dogs. The technique to be performed in each hemiabdomen was randomly assigned, which was determined by extracting a code from a sealed envelope. To perform the study, cadavers were placed in lateral recumbency and an extensive clipping of the thoracolumbar area was carried out. The injections were made under ultrasound guidance, using a linear array  and echogenic needles . A volume of 0.6 mL kgTo perform both approaches, the ultrasound probe was placed transversal to the longitudinal axis of the spine, caudal and parallel to the last rib, at the level of L1. The array was slipped dorsally and tilted down until visualization of the target structures: erector spinae muscles (ESP), external oblique muscle (EO), internal oblique muscle (IO), transverse abdominal muscle (TA), transverse process of L1 (TP), transverse abdominal muscle aponeurosis (TAp), QL muscle, psoas minor muscle (PM) and vertebral body of (VB) L1, could be achieved .Once those structures were identified, the needle was inserted in plane in a ventro-dorsal, latero-medial direction. After passing over the EO and IO, the tip of the needle was directed towards the TAP which offered slightly more resistance. The perforation of the TAP generates a “pop” sensation. Sonographic images and videos were recorded, and their quality was subjectively analyzed later  as good , moderate (QL and PM margins were not clearly identified) and poor . The visualization of the needle tip at the moment of the injection was also registered.The targeted injection site in this approach was the latero-ventral aspect of the QL, on its border with the TAP. To perform the LVQL, the needle was stopped immediately after piercing the TAP, and the dye/contrast solution injected .The injection site of this approach was the interfascial plane situated between the QL and PM . This plFollowing the administration of the injectate, cadavers were taken to a computed tomography (CT) scan  to evaluate contrast distribution. To perform the CT, cadavers were placed in sternal recumbence and 3 mm thickness slices were obtained at vertebral levels from T8 to L7. Images were subsequently reconstructed and analyzed by two radiologists .Finally, the cadavers were subjected to anatomical dissection , the technique is explained in detail elsewhere . Brieflyp < 0.05.The statistical tests were performed using SPSS, version 24.0 . A Shapiro–Wilk test was used to assess the normality of the distribution. The distribution of dye/contrast in the CT and anatomical dissection studies between the two treatments were evaluated by a Yuen’s test. The quality of the ultrasound images and visualization of the needle tip were analyzed by a Fisher’s test. Mean ± standard deviation, median (range) or numbers of animals were chosen to express the data as deemed most appropriate in each case. Data are expressed as mean ± standard deviation, median and range or number of animals, as it was considered more appropriate in each case. Differences were considered statistically significant when Cadavers weighed 27.7 ± 12.5 kg and presented a median body condition score (BCS) of 4 (3–5) out of 9.p = 0.5). The target structures were found in all cases.The LVQL technique resulted in images classified as moderate (1/6) or good (5/6) accordingly to their quality. The IQL approach produced images of moderate (3/6) or good (3/6) quality. There were no statistical differences in image quality between the studied approaches (p = 0.85). In the case where the needle tip could not be observed, the correct positioning of the needle was confirmed after feeling two “pop” sensations as described before, as well as for the expected distribution of the injectate between QL and PM during the injection. The tip of the needle was observed at the time of injection in 6/6 cases for LVQL and in 5/6 for IQL. No statistically significant differences were found between the two approaches (p = 0.74). In the LVQL, the contrast media was mainly distributed within the ventral and lateral aspects of the QL. Regarding the IQL, the contrast media was observed surrounding the QL and PM muscles, as well as the sympathetic trunk area  VB, from T13 to L7, and in the IQL, 7.5 (5–9) VB, from T12 to L7 . No statunk area . One hemp = 0,03). The ventral branches of the spinal nerves in the LVQL were stained in a more distal portion, while the IQL dyed the nerves closer to their roots (p = 0.0011)  .The results of this study do not support our initial hypothesis, as both approaches were feasible and with a similar degree of difficulty. The studied approaches (LVQL and IQL) could promote the blockade of L1–L3 ventral branches. However, despite using high volume 0.6 mL/kg) injections, neither technique was able to consistently stain the ventral branches of the last thoracic spinal nerves (T10–T13), which are responsible for the innervation of the cranial abdominal wall . Therefo mL/kg inIt was also hypothesized that because the site of injection selected for the LVQL approach was located closer to the skin surface, this approach could be easier to perform than the IQL. However, no differences were found comparing the quality of the images and the visualization of the needle tip of both techniques. The IQL could be more difficult to perform in larger and obese dogs, due to the increased distance from the probe to the injection site. Most of the cadavers included in our study presented a low to moderate BCS (4 out of 9). This fact, together with the wide clinical expertise of the researcher in charge of performing the ultrasound-guided administration of the injectate, could have biased these results. It must also be considered that, in one out of six hemiabdomens in the IQL approach, the tip of the needle could not be visualized. With this being a cadaveric study, the position of the tip of the needle was inferred from the feeling of the two “pop” sensations. This practice cannot be recommended in live animals, as it is mandatory to visualize the tip of the needle during the administration of the drugs to avoid vascular, neural or intracavitary injections. Other safety measures that should be undertaken before the administration of the drugs are an aspiration and a resistance to injection test, to avoid intravascular or intraneural injections, respectively ,21.The site of injection for the LVQL approach was selected bearing in mind its proximity with the trajectory taken by the ventral branches of the first lumbar spinal nerves. These nerves cross through the interfascial space formed by the QL and PM muscles to run along the ventral and ventro-lateral aspects of the QL, piercing the TAP . Garbin The CT study revealed a wide distribution of injectate. In several cadavers of both groups, a caudal distribution of iopromide that reached L7 was observed. These findings are similar to previous descriptions ,6. In onThe use of a larger volume of injectate than that employed by Garbin et al.  did not In our study, the LVQL approach did not consistently stain the sympathetic trunk, while the IQL presented a more constant spread from L1 to L3 levels. These results show that the LVQL approach may provide a similar analgesic effect to those reported for a TAP block ,10,12. TIn 50% of cases in the IQL group, the genitofemoral nerve was stained. This nerve is responsible for the sensitive innervation of the proximal aspect of the medial thigh, skin of pudendal region, prepuce, mammary gland, vaginal tunic and vaginal process ,27. A pr−1 per hemiabdomen was chosen. This high volume must be considered when local anesthetics are administered, in order to avoid its systemic toxicity [Our study has several limitations. The results could be biased by the small number of cadavers available. The injections were carried out mostly in lean cadavers. Probably, the quality of the images obtained from larger or obese animals would be different because the ultrasonographic appearance of important structures to guide the block could be impaired in these types of patients. As the QL block is an operator-dependent technique, the fact that the ultrasound studies were performed by a researcher with high-clinical experience could also bias the results, particularly those comparing the ease of both approaches ,3,4,5. Ttoxicity ,29,30,31In conclusion, the LVQL would be a suitable option to provide analgesia to the abdominal wall, but not to the abdominal viscera. Despite the high volume of injectate administered in this study, the injections did not consistently reach the ventral branches of the last thoracic nerves in any group. Therefore, none of these approaches may be recommended to provide analgesia for the cranial abdominal wall. More clinical trials are necessary to test the QL block in live animals, to determine its analgesic effects and the extension of the blockade at both somatic and visceral levels.


Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol. Ozone is widely used in society for disinfection, deodorization, disinfecting , and cliTo apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase retention in the affected area. We have shown the anti-inflammatory effect of ozonated macrogol ointment on human gingival fibroblasts and its osteogenic potential on osteoblasts . HoweverThe ozonated macrogol ointment was provided by Mediplus Pharma Inc., Tokyo, Japan. The preparation procedure of the ozonated macrogol ointment is below. Ozone gas was administered to concentrated glycerol  to produce 2000 ppm of ozonated glycerol using an Ozonizer . In addition, a macrogol ointment (polyethylene glycol (PEG) 400 and PEG 4000: Yoshida Pharmaceutical Co., Ltd., Tokyo, Japan) and a water-soluble ointment (polyethylene glycol) were added to increase the viscosity of ozonated glycerol for the purpose of retention. The ratio of macrogol ointment to ozonated glycerol was 1:2 . The visThe shear rate in the B-type viscometer BM II  was calculated by measuring in advance a Newtonian fluid of known viscosity (viscosity standard fluid) at different rotor speeds .4) were cultured in DMEM containing 0.5% FBS (0.5% DMEM) for 24 h. Cells were cultured with 0.05, 0.5, or 5 ppm ozonated macrogol ointment for 2 min. The ozone concentration is the final concentration for each well. The culture medium was then removed. The cells were washed with 0.5% DMEM and cultured with 0.5% DMEM containing bromodeoxyuridine (BrdU) for 24 h. The level of DNA synthesis was determined by measuring BrdU incorporation using a BrdU cell proliferation assay kit . For the MTT -2,5-diphenyl tetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) assay, cells were cultured with ozonated macrogol ointment at 0.05, 0.5, or 5 ppm in DMEM containing 10% FBS (10% DMEM) for 2 min, and the culture medium was then removed. After washing with 10% DMEM, cells were cultured with 10% DMEM for 24 h. The subsequent procedures were performed as previously described [For DNA synthesis, Saos-2 cells (1 × 10escribed . The mic4) were cultured with 0.5 ppm ozonated macrogol ointment in DMEM containing 1% FBS (1% DMEM) for 2 min. As a control, Saos-2 cells were cultured without ozonated macrogol ointment. Cells were washed and cultured with 1% DMEM. Levels of type 1 collagen in the media were measured by indirect ELISA using the biotinylated anti-type 1 collagen antibody  and samples (360 µg/mL protein). ELISA was performed as described in the user manual of a CytoSet kit  [For collagen production, Saos-2 cells  . Cells f4) were cultured with a 0.5 ppm ozone macrogol ointment for 2 min. The culture medium was then removed. Cells were washed with 0.5% DMEM and cultured with 10% DMEM for 24 h. Cells were lysed with 0.05% Triton X-100, and the ALP activity of the lysates was measured using a LabAssay ALP kit . Protein concentrations of cell lysates were also measured using a BCA protein assay kit. ALP activity was normalized to the protein content of the cell lysates. The microplate was used on a 96-well plate. Three wells were used for one sample to be analyzed. Samples were measured by the Microplate Reader model 550 using a wavelength of 405 nm. The analysis was performed three times for the same experiment.Saos-2 cells (1 × 10t-test (measurement of ALP activity and ELISA for collagen production) using the StatMate software . Differences were considered significant at p < 0.05.Quantitative data were statistically analyzed using either a one-way analysis of variance (ANOVA) followed by Tukey’s test (DNA synthesis and MTT assays) or the Student’s The viscosity of the ozonated glycerol and the ozonated macrogol ointment were 1499 and 3000 mPa·s, respectively. The viscosity of the ozonated macrogol ointment was about two times greater than that of the ozonated glycerol.After 24 h of incubation, the ozonated macrogol ointment treatment increased Saos-2 proliferation in a dose-dependent manner . OzonateGiven the results of the proliferation assays, we further evaluated the effect of 0.5 ppm ozonated macrogol ointment on type 1 collagen production and ALP activity by Saos-2 cells. The medium containing ozonated macrogol ointment effectively increased type 1 collagen production  and ALP Ozonated glycerol has low viscosity and moderate fluidity because its main ingredient is glycerol. On the other hand, ozonated macrogol ointment is an ointment made by mixing ozonated glycerol with PEG 400 and PEG 4000, and has high viscosity and retention at the affected site. The viscosity of the ozonated macrogol ointment was about two times higher than that of the ozonated glycerol. The present study showed that although high concentrations of ozonated glycerol ointment, such as ozonated glycerol , also deType 1 collagen and ALP are widely known as markers secreted in the early phases of bone formation . In thisIn summary, we compared the ozonated glycerol and macrogol ointment. The effect of ozonated macrogol ointment on type 1 collagen production and ALP activity in Saos-2 cells was investigated. The results indicated that ozonated macrogol ointment controls the cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers as well as ozonated glycerol. Currently, we shed light on the functional studies of ozonated macrogol ointment on long-term effects in terms of cellular damage toward clinical application.


Radiation-induced damage (RID) after radiotherapy (RT) of primary brain tumors and metastases can be challenging to clinico-radiographically distinguish from tumor progression. RID includes pseudoprogression and radiation necrosis; the latter being irreversible and often associated with severe symptoms. While histopathology constitutes the diagnostic gold standard, biopsy-controlled clinical studies investigating RID remain limited. Whether certain brain areas are potentially more vulnerable to RID remains an area of active investigation. Here, we analyze histopathologically confirmed cases of RID in relation to the temporal and spatial dose distribution.2) and the biologically effective dose (BED2) for normal brain tissue (α/β ratio of 2 Gy) and analyzed the spatial and temporal distribution using frequency maps.Histopathologically confirmed cases of RID after photon-based RT for primary or secondary central nervous system malignancies were included. Demographic, clinical, and dosimetric data were collected from patient records and treatment planning systems. We calculated the equivalent dose in 2 Gy fractions (EQD22 60 Gy) to a large planning target volume (PTV) (median 203.9 ccm) before diagnosis of RID. Despite the low EQD22 and BED2, three patients with an accelerated hyperfractionated RT developed RID. In contrast, brain metastases patients  were often treated with two or more RT courses and with radiosurgery or fractionated stereotactic RT, resulting in a higher cumulative EQD22 (median 162.4 Gy), to a small PTV (median 6.7 ccm). All (n = 34) RID lesions occurred within the PTV of at least one of the preceding RT courses. RID in the high-grade glioma group showed a frontotemporal distribution pattern, whereas, in metastatic patients, RID was observed throughout the brain with highest density in the parietal lobe. The cumulative EQD22 was significantly lower in RID lesions that involved the subventricular zone (SVZ) than in lesions without SVZ involvement .Thirty-three patients were identified. High-grade glioma patients (n = 18) mostly received one normofractionated RT series (median cumulative EQD22 and BED2 in high-grade glioma patients. The anatomical location of RID corresponded to the general tumor distribution of gliomas and metastases. The SVZ might be a particularly vulnerable area.Accelerated hyperfractionated RT can lead to RID despite computationally low EQD2The online version contains supplementary material available at 10.1186/s13014-023-02385-3. RID 2) > 100 Gy [Known risk factors for radiation necrosis include higher total radiation dose, larger fraction size and the use of concurrent and/or adjuvant systemic antineoplastic therapy –8. A bra> 100 Gy .The frequent need for invasive tissue biopsy to secure the diagnosis of RID has prompted substantial research efforts directed at the identification of imaging biomarkers capable of reliably distinguishing between RID and tumor progression. As such, a variety of diagnostic procedures have been explored, including standard magnetic resonance imaging (MRI) based methods, diffusion weighted imaging (DWI), perfusion-weighted imaging (PWI), magnetic resonance spectroscopy (MRS), positron emission tomography (PET), single photon emission computed tomography (SPECT), radiomics, and combinations thereof, with varying success –18. Histradiation-induced cellular atypia can result in further diagnostic challenges [Reported histopathological characteristics of radiation necrosis include coagulative and fibrinoid necrosis, gliosis, wall thickening and hyalinization of vessels, telangiectasia and calcium deposition, located predominantly in the white matter –22. For allenges , 26. As allenges .Treatment options for radiation necrosis remain limited and include corticosteroids, bevacizumab, surgical resection, laser interstitial thermal therapy (LITT) and hyperbaric oxygen therapy (HBOT) , 29.Several reports in the literature have suggested that certain areas of the brain, such as the subventricular zone (SVZ), a neural stem cell niche located along the lateral wall of the lateral ventricles , 31, migHerein, we provide an in-depth analysis of 34 histopathologically confirmed RID lesions after brain-directed RT with a focus on radiation parameters and spatio-temporal distribution with specific consideration of the role of the SVZ. Furthermore, we explore putative differences between RID in patients with primary and secondary brain tumors, i.e., high-grade gliomas (HGGs) and brain metastases (BMs). The aim of this study was to correlate the radiation dose with the spatial and temporal occurrence of RID.Patients with histopathologically confirmed RID who had undergone photon RT between January 2006 and January 2020 for malignant primary and secondary brain tumors were included in this retrospective monocentric study. Patients who had received proton or heavy ion RT and those treated for benign lesions were excluded. Diagnostic tissue samples had to contain RID . All samples of “mixed lesions” containing areas of solid tumor were excluded.The study was approved by the local institutional review board (EA2/056/20).Demographical, clinical, and dosimetric data, as well as imaging data were collected from the patient records and the radiation treatment planning system (TPS). The time-to-RID, defined as the interval between the day of the last fraction of RT and the first occurrence of RID on MRI imaging , was noted.2, BED2).The EQD2 and biologically effective dose (BED) were calculated for RT courses in the area of RID using previously described formulas –39. An αPreoperative  gadolinium-enhanced T1-weighted 3D Magnetization Prepared Rapid Acquisition with Gradient Echoes (MPRAGE) images of RID were imported into the TPS Varian Eclipse . The extent of RID (defined as the contrast-enhancing lesion) was manually delineated for each patient. All contours were reviewed by a board-certified radiation oncologist, specialized in neuro-oncology (DK). The radiation plans of prior RT courses were co-registered to the MRI using rigid image registration. The planning target volumes (PTVs) were then transferred to the MRI of the RID. RT plans that clearly had no relevant dose deposition in the area of RID  were excluded. Radiation plans created in Brainlab iPlan RT  and Accuray Precision TPS  were exported into Varian Eclipse and then processed as described. The MRIs with the corresponding structure sets were exported in DICOM/DICOM RT format.The exported MRIs and structure sets were imported into 3D Slicer, version 5.0.3 , 42 for The transform created during the ANTs registration was then used to register the structures (RID lesions and PTVs) to the MNI152 space. The structures were converted into binary label maps, exported and then summed using the ImCalc module of Statistical Parametric Mapping 12 (SPM12), revision 7771 , 54 in MIn order to assign the RID lesions to the different lobes, the lobe atlas provided with the ICBM 2009a nonlinear symmetric template , 50 was The coverage of the RID by the PTVs of the single RT courses was calculated within 3D Slicer using the intersect function for each PTV. Furthermore, in order to assess the cases that had more than one preceding RT in the area of the RID, a combined PTV was computed within 3D Slicer and coverage of the RID was calculated accordingly.2 values between lesions with and without SVZ involvement, the Mann-Whitney U test was used. For correlation analysis, the Spearman correlation coefficient r was calculated. P values ≤ 0.05 were considered statistically significant.All statistical analyses were performed using IBM SPSS Statistics, version 28.0 . Patient characteristics for the two main subgroups, HGG and BM, were summarized using descriptive statistics. For the assessment of differences in the cumulative EQD2A total of 33 patients with a diagnosis of RID met the inclusion criteria of our study. The diagnosis that led to brain-directed photon RT was HGG for 18 (55.5%) patients. This included glioblastoma WHO grade 4 (11 patients), oligodendroglioma WHO grade 3 (4 patients) and astrocytoma WHO grade 3 (3 patients). BMs were present in 15 (45.5%) patients. One patient in the BM group developed two independent RID lesions in different lobes of the brain. This resulted in 34 RID lesions in total. The median age at resection of the RID was 56 years (interquartile range (IQR) 49–64.5 years). Details on patient and lesion characteristics are summarized in Table Pathological diagnosis was made based on surgical resection (88.2%), biopsy (8.8%) or autopsy (2.9%). In 70.6% of the lesions only RID was observed, whereas in 29.4% single tumor cells were present in addition to RID. The proportion of lesions with tumor cells present was higher in the HGG subgroup (44.4% vs. 12.5%).18F-FET PET/CT), but the lesion was resected because of progressive symptoms. One RID lesion was diagnosed by autopsy after death of the patient from systemic tumor progression.The main reasons for performing resection or biopsy were suspected tumor recurrence or progression or ambiguous results of the obtained imaging, either in combination with symptoms associated with the RID lesions (63.6% of the lesions) or without any symptoms caused by RID  and the BM subgroup (median 46.4%). The median coverage of the lesion by combined PTV, i.e., the percentage of the RID that was covered by any of the PTVs of the preceding RTs, was 97.8%, with slightly higher values in the HGG subgroup than in the BM subgroup  and among the HGG subgroup (72.2%). In the BM subgroup frontal lobe involvement was less frequent (37.5%), while still being the second most common location after the parietal lobe (43.8%). Overall, lesions in the BM group were more evenly distributed throughout the whole brain, whereas in the HGG subgroup no involvement of the occipital lobe and cerebellum was found. Only one lesion was found in the cerebellum in the BM group and none of the lesions were located in the brainstem.52.9% of the lesions involved the SVZ; the proportion was higher for the HGG subgroup (77.8%) than for the BM subgroup (25.0%). All RID lesions involved the white matter, and the vast majority (94.1%) involved the cortex as well. Only two RID lesions were exclusively located in subcortical compartments, one each in the HGG and BM subgroup, respectively. Basal ganglia were involved in 29.4% of all lesions, more frequently in the HGG than in the BM subgroup (50.0% vs. 6.3%).2 administered was 103.2 Gy for the whole cohort, with higher values in the BM subgroup (median 162.4 Gy) than in the HGG subgroup (median 60 Gy). Furthermore, higher fraction size (median 8 Gy vs. 2 Gy) and single course EQD22 (median 71.6 Gy vs. 60 Gy) were found in the BM subgroup. On the other hand, PTV volumes, excluding whole brain radiotherapy (WBRT), were smaller in the BM subgroup (median 6.7 ccm vs. 203.9 ccm). The relationship between single RT course EQD22 and PTV volume is illustrated in Fig. Table 2 > 100 Gy, except of one patient with a cumulative EQD22 of 98.1 Gy. The majority of RT courses (90.6%) were administered on five days per week, whereas one RT course was performed with only three fractions per week and four courses were performed with two fractions per day (b.i.d.), i.e., using an accelerated hyperfractionated RT (AHFRT) regimen, three of these in the HGG group and one in the BM group. The three AHFRT courses in the HGG group were used in patients that received only one RT course in the area of the RID and the EQD22 for these lesions was among the lowest in the whole study population (53.3 Gy for each lesion).The patients that received two or more RT courses all had a cumulative EQD22 values were considerably lower in RID lesions that involved the SVZ  than in lesions without SVZ involvement ; the differences in distributions were statistically significant (p = 0.01).As illustrated in Fig. 2 and the time-to-RID  = -0.095, p = 0.594).The median time-to-RID  was 5.8 months (range 0.7–29.0 months). There was no statistically significant correlation found between the cumulative EQD2In 73.5% of the lesions systemic therapies were administered during or close to (± 1 months) the RT for at least one of the RT courses in the area of the RID. The proportion of cases receiving systemic therapy was higher in the HGG subgroup (83.5%) than in the BM subgroup (56.3%). An overview of the different drugs used is provided in Additional file 2: Table The diagnosis of RID, including pseudoprogression and radiation necrosis, remains a common neuro-oncological challenge, with several important aspects to discuss. Despite its status as the diagnostic gold standard, histopathological evaluation may yield ambiguous results, especially in “mixed lesions” which include both RID and vital tumor cells and/or foci of solid tumor , 25, 57.For instance, Patrizz et al.  classifiradiation-induced cellular atypia (reminiscent of viable tumor cells) in RID lesions constitutes another confounding factor [In our study, we chose to include only cases with “pure” RID and cases with single tumor cells present, if RID accounted for most of the sample. All cases with solid tumor present in addition to RID were excluded. We deemed that this was the best compromise, as excluding all cases with tumor cells present would have led to the exclusion of almost all glioblastoma patients, but including cases with solid tumor present would have had negatively affected the diagnostic accuracy, as tumor necrosis is often present in HGG. Moreover, the potential presence of g factor , 26, cirThe main reasons for performing resection or biopsy in our study were suspected tumor recurrence or progression or ambiguous results of the obtained imaging, either in combination with or without symptoms associated with the RID lesions. Two thirds of the patients developed symptoms, similar to those commonly seen in brain tumors and in some cases severe such as hemiparesis or epileptic seizures. This highlights the current diagnostic and therapeutic challenges clinicians are confronted with in the management of RID.2 for healthy brain tissue instead, as it is better suited to reflect the differences in the fractionation schemes. Using this approach, we could not find a statistically significant correlation between the cumulative EQD22 in Gy and the time-to-RID.Regarding the temporal occurrence pattern of RID, Campos et al.  reportedAll RID lesions in our study occurred at least in part within the area of the PTV of one or more of the preceding RTs and in most cases coverage of RID by the PTVs was high. This is in line with the findings of Winter et al.  who descThe location pattern of RID in our study differed between the two subgroups. In the HGG subgroup RID lesions occurred preferentially in the frontotemporal areas of the brain, while posterior parts and the cerebellum were spared. If we compare our data to studies that examined the location frequency of glioma or glioblastoma, very similar location patterns are found –63. ThusIn the BM subgroup, RID lesions were more heterogeneously distributed throughout the whole brain in our cohort. Studies that investigated the location of BM , 65 also2 values were considerably lower in RID lesions that involved the SVZ, possibly suggesting a higher vulnerability of this region.SVZ involvement of RID lesions was highly frequently observed within the HGG subgroup of our study (77.8%), substantially more often than in the BM subgroup (25.0%). However, SVZ involvement and radiation of it is more common for gliomas, too, given the necessary safety margins and respective contouring guidelines. For example, a study from our institution comprising 200 glioblastoma patients found that 66.0% of the tumors had contact to the SVZ . Similar2 (median 60 Gy) in comparison to the BM patients. The three patients in this subgroup that received AHFRT had relatively low EQD22 (53.3 Gy) and BED2 (106.6 Gy) values. This was also noted in the Quantitative Analyses of Normal Tissue Effects in the Clinic (QUANTEC) publication by Lawrence et al. [Patients with HGG mostly received one course of normofractionated RT or AHFRT, partially with a SIB, to a large PTV (median 203.9 ccm) with a more moderate cumulative EQD22 values compared to the HGG subgroup. The median PTV size was substantially smaller (6.7 ccm).BM patients, on the other hand, received two or more RT courses in more than 80% of the cases. All patients in this subgroup received hypofractionated RT (single dose ≥2.5 Gy) in at least one of their RT courses, resulting in higher cumulative (median 162.4 Gy) and single course (median 71.6 Gy) EQD22 higher than 100 Gy, except for one patient with a cumulative EQD22 of 98.11 Gy. This finding is supported by another recently published study from our institution on the reirradiation of IDH wild type glioblastoma using moderate cumulative EQD2 values that did not find any cases of confirmed radiation necrosis [Mayer et al.  reportednecrosis .2 values compared to the BM cases, might be the more frequent involvement of the SVZ in this subgroup. In the guideline for radiation necrosis published by the DEGRO in 2022 [2 values were significantly lower in RID lesions that involved the SVZ than in lesions without SVZ involvement. There are several possible confounders, such as the much larger PTV volumes and the frequent concurrent administration of temozolomide in HGG patients that constitute inherent risk factors for RID [One possible explanation for the occurrence of RID in the HGG subgroup in spite of the lower EQD2 in 2022  it is st in 2022 –35. Van  in 2022  reported in 2022  pointed  for RID –8, but fOur study has several limitations. Data collection was carried out retrospectively and at a single institution. With 33 patients and 34 lesions included, the sample size might be regarded as relatively small. However, to our knowledge, this is still one of the largest samples of RID lesions diagnosed by histopathologic confirmation. The lack of a control group without RID in this study causes some difficulties in interpreting the data, especially regarding the location analysis. Limiting the study to patients diagnosed by histopathologic confirmation bears the risk to only include the more severe and symptomatic cases of RID that had to be resected or biopsied. Furthermore, it is possible that in the location analysis RID lesions occurring in certain areas are underrepresented, because performing resection or biopsy in these areas was not clinically feasible and/or contraindicated. However, as to date there is still no other method that can provide the same diagnostic accuracy for RID as pathologic confirmation and it is still regarded the gold standard, studies based on pathology-proven samples remain an indispensable and highly valuable tool for investigating the mechanisms leading to RID after brain-directed RT.2 and BED22. The actual risk in comparison to other fractionation schemes has yet to be evaluated. Our data supports the notion that the SVZ is potentially more vulnerable to the development of RID, given that lesions there developed after a lower cumulative EQD22. The larger irradiated volumes as well as the more frequently performed concurrent administration of temozolomide in these lesions might have to be considered as independent risk factors for RID; thus, further research on the topic should ideally address these potential confounders.In this analysis of histopathologically confirmed RID, the location of RID mostly followed the general distribution pattern of the tumors irradiated. A previously reported correlation between the radiation dose and the period between RT and the occurrence of the RID could not be confirmed. High-grade glioma patients that receive twice-daily AHFRT schemes might develop RID despite a presumably lower EQD2Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


We examined internet searches on psoriasis in Germany and found that in weeks with high search volume, mean temperature and humidity were lower and sunshine level was higher compared to weeks with low search volume. A connection between psoriasis severity and weather has long been suspected. Cold, dry weather has been noted anecdotally to exacerbate symptoms, whereas sunshine improves the condition . PsoriasIn this study, we examined internet searches related to psoriasis in Germany, looking at whether patterns in searching were weather related. The internet search volume for the search term “psoriasis” (disease) for Germany was obtained from Google Trends . This weWeather data from the German Wetterdienst  was used. Daily data for mean temperature, percentage relative humidity, and total hours of sunshine was obtained for 6 weather stations: Berlin, Cologne, Frankfurt, Hamburg, Munich, and Stuttgart. Weekly mean values for temperature and humidity as well as total weekly hours of sunshine were calculated. A value for all of Germany was calculated using data from the 6 stations.The RSV ranged from 53 to 100. Although there was only a slight correlation between searching and specific weather features , 3D scatter plots suggested differences between weeks of high and low searching. Low search weeks blue in A occurret test: P<.001).Notable differences were apparent when comparing mean values between the lowest  and highest  search weeks. The mean temperature was higher during low rather than high search weeks , as was humidity , but sunshine hours was lower . The difference for humidity was significant (2-tailed P<.001), they were logged. Separate regressions were performed with logged values for temperature, humidity, and sunshine as dependent variables. The resulting coefficients indicated that higher temperature and humidity led to lower search volumes but more sunshine was associated with increased searching . Regression fitting suggests an RSV of 78 at 10 °C, declining to an RSV of 40 at 15 °C. At 65% humidity, an RSV of 93 is expected; when humidity rises to 80%, the RSV should drop to 47. With 40 hours of sunshine per week, an RSV of 68 is expected.These trends were further explored using linear regression. Since the search data were not normally distributed (Shapiro-Wilk W=0.96; This study revealed that weeks of high internet searching related to psoriasis were associated with cold, dry weather conditions. Differences were most apparent when comparing the highest and lowest search weeks. The possible association between high searching and more sunshine is of interest; this is contrary to anecdotal evidence but supports recent survey work , which uGoogle Trends data allowed for the examination of search volume trends over long time periods and on a weekly basis. However, only national data were available, meaning regional assessment was not possible. Local differences may exist. We assumed that internet searching is constant throughout the year, but increases in winter months may occur when people spend more time on computers due to longer, darker evenings. Future studies could control for this problem by calibrating search volume with results from nonseasonal conditions. Another assumption is that internet searching reflects the severity of psoriasis. One would expect this to be the case because people are more likely to search when their symptoms worsen and discomfort is felt. It is well known that internet searching reflects the incidence of a condition . The eff


Dirinara applanta and Parmotrema tinctorum, and compared their microbial communities to those found in the pine bark to which the lichens were attached. Our hypothesis was that the endolichenic communities would exhibit distinct diversity patterns, community structures, network structures, and specialist composition compared to the surrounding epiphytic community.Lichens represent not only the mutualism of fungal and photosynthetic partners but also are composed of microbial consortium harboring diverse fungi known as endolichenic fungi. While endolichenic fungi are known to exert a remarkable influence on lichen ecology through their crucial roles in nutrient cycling, bioprospecting and biodiversity, the enigmatic community structures of these fungal inhabitants remain shrouded in mystery, awaiting further exploration and discovery. To address knowledge gap, we conducted metabarcoding on two lichens using 18S gene amplification, Our investigation has shed light on the clear demarcation between the endolichenic and epiphytic fungal communities, as they exhibit markedly different characteristics that set them apart from each other. This research demonstrated that the endolichenic communities are less diverse as compared to the epiphytic communities. Through community similarity analysis, we observed that two endolichenic communities are more similar to each other in terms of community composition than with the adjacent epiphytic communities. Moreover, we unveiled a striking contrast in the network structures between the endolichenic and epiphytic communities, as the former displayed a more modular and less nested features that is evocative of a potent host-filtration mechanism.Through our investigation, we have discovered that lichens harbor less intricate and interconnected fungal communities compared to the neighboring epiphytic environment. These observations provide valuable insights into the metagenomic architecture of lichens and offer a tantalizing glimpse into the unique mycobiome.The online version contains supplementary material available at 10.1186/s40793-023-00531-8. Lichens are a symbiotic mutualism comprising lichen-forming fungi and their photosynthetic partners . MutualiPrevious research about the community structure of ELF found that these fungi are a diverse group with a majority belonging to the Ascomycota phylum , 16. TheDirinaria applanta and Parmotrema tinctorum, as well as the EPF community of Pinus thunbergii, using metabarcoding techniques. Precisely, our aim was to investigate fundamental inquiries regarding the distinctions between ELF and EPF communities, focusing on aspects such as diversity patterns and network architecture. To accomplish this, we compared the fungal communities across six different aspects: alpha diversity, taxonomic composition, community similarity, network structure, core microbiome, and host specialists.To gain a better understanding of the distinct structure of ELF communities, it would be valuable to conduct a comparative study between lichen communities and the surrounding environment using metabarcoding techniques. The goal of this study is to uncover the unique architecture of the ELF community in comparison to the neighboring epiphytic (EPF) community. We selected the EPF community as a suitable comparator to ELF because it is likely that the tree bark represents the original fungal pool from which the ELF community was established. This is supported by the fact that the tree bark is physically closest to the epiphytic lichen and existed prior to lichenization. To explore the differences between ELF and EPF communities, we analyzed the fungal communities within the epiphytic lichens Pinus thunderbergii bark, and the epiphytic lichen Dirinaria applanta and Parmotrema tinctorum from five sites (n = 75) on Jeju Island in the southern part of South Korea  , the five collection sites did not show significant climatic differences  as described by Yang et al. [We collected twenty five samples of g et al. .Fig. 1CoThe bark and lichen segments were homogenized, and DNA was extracted using a PowerSoil DNA isolation kit . We conducted a polymerase chain reaction (PCR) of the fungal ITS1 region  using thhttps://docs.qiime2.org).Amplicon library sequencing was performed using the Illumina MiSeq platform . Raw sequences were processed using QIIME2 v.2021.4  by demulhttps://forum.qiime2.org) and rarefied to 3,000 sequences per sample using the “phyloseq” package [The following analyses were performed using R v.3.5.3 . For the package . The subp value of the permutation test [Cws, Nestedness metric based on overlap and decreasing fill (NODF), Betweenness, Closeness, InDegree, OutDegree, and Expected influences. Cws represents the level of modularity in the network, while NODF quantifies the extent of nestedness. Betweenness and Closeness indicate vectors that contain the betweenness centrality and closeness centrality of each node, respectively. InDegree and OutDegree indicate vectors that encompass the inward and outward degrees of each node. ExpectedInfluences indicates the sum of incoming or outgoing edge weights connected to a node. Identifying the core mycobiome [p < 0.05. Chi-squared tests [2 fold change > 2 and p < 0.05.The alpha diversity indices, Chao1 richness , Fisher’ion test   and Parmotrema . For a comparative analysis of the fungal communities, a rarefaction process [Dirinaria and ten Parmotrema communities were excluded from the further analyses because they were below this cutoff values.After denoising and filtering out the host lichen fungal sequences, the ITS amplicon data yielded 571,273 total fungal sequences in the  process  was implDirinaria and Parmotrema, but not significantly different between the two ELF communities. Shannon’s diversity and evenness did not vary significantly between host types. Thus, the data suggested that fungal diversity was lower in lichens than in pine bark.The alpha diversity of the ELF and EPF communities was assessed using four diversity indices Fig. b. ResultDirinaria and Parmotrema. In contrast, Basidiomycota accounted for 27.9% of the EPF communities and were less abundant in the ELF communities. At the class level, the Ascomycota-dominant pattern diverged in the two ELF communities, with Dothideomycetes being the dominant fungal class in both communities. Sordariomycetes were more abundant in Parmotrema than in Dirinaria. The most abundant order in all fungal communities was Capnodiales.Taxonomic compositions of the ELF and EPF communities were distinguishable, with differences in Basidiomycota frequency being particularly pronounced  than to that of Parmotrema (Pi. vs. Pa.: 0.983 ± 0.030)  and diversity (Shannon)  Fig. c. The neon) Fig. d. Notables Figs. b and d. ity Fig. e. Taken Dirinaria and Parmotrema were smaller, with 8 and 17 ASVs, respectively.  to identify host specialists. We observed host-specific ELF and EPF at the genus level (shown in Fig. alTraits , showed Dirinaria communities, there were 52 EPF and 20 ELF specialists identified (Additional file Parmotrema, 64 EPF and 42 ELF specialists were observed. These findings were consistent with the LDA analysis and showed that the abundance of plant-pathogen fungi was higher in the ELF communities than in the EPF communities.To further analyze the differences between the fungal communities, pairwise comparisons were conducted using a chi-squared test Fig. b. The reWhile the diversity and structure of fungal communities within lichen thalli play a critical role in their ecological functioning , 17, thePinus bark [D. applanta [P. tinctorum [The fungal communities of EPF were found to exhibit higher richness and diversity compared to ELF communities, suggesting that epiphytic fungi might serve as a potential source of fungal diversity in lichen thalli attached to the bark. Moreover, the considerable overlap of ASVs between the EPF and ELF communities further strengthens this proposition. The structure of ELF communities appeared to be influenced by the filtering effect of lichen hosts, as their taxonomic compositions were more strongly associated with host identity than with geographical location. The occurrence of Basidiomycetes in the EPF communities was a frequent finding, corroborating previous reports on the occurrence of wood-decay fungi on nus bark . The lowapplanta  and chloinctorum , which iinctorum . CapnodiDirinaria were found to be more similar to the EPF communities than those of Parmotrema, possibly due to the physical distance between the lichens and the bark. This is consistent with a previous study by Noh et al. [P. tinctorum is relatively loosely attached to Pinus bark with its rhizine. Furthermore, this might be attributed to the similarity in physical properties of the hosts. The resemblance between Dirinaria ELF and EPF could potentially be elucidated through subsequent experimental approaches.The hypothesis of host-filtration was supported by community similarity analyses, which showed that the ELF communities of two different host lichens were more similar to each other than to the EPF communities. Notably, the ELF of h et al.  which shThe network analysis revealed that the ELF communities exhibited a more modular and less nested architecture compared to the EPF communities. A higher modularity suggests that the ELF communities are more robustly sorted out by the host lichen compared to the pine bark, as discussed by Chagnon et al. . This spDirinaria were found to have a higher level of core mycobiome overlap with the EPF communities compared to the ELF communities of Parmotrema. The core member of ELF, Pestalotiopsis, a fungus known for its role as both a plant endophyte and a pathogen was observed in the Parmotrema ELF communities. This observation was consistent with the finding that the Parmotrema ELF communities had the most diverse plant pathogen fungi, followed by the Dirinaria ELF communities and the EPF communities. These results suggest that ELF communities are similar to phyllosphere fungal communities, as previously proposed [The ELF communities tend to have a lower number of core mycobiome compared to the EPF communities. According to a previous study, the core mycobiome of ELF communities was found to be largely dominated by fungi from the Capnodiales order . In lineproposed .Our analysis compared the structure of the ELF communities with that of their neighboring environments, the EPF communities. We found that the ELF community had a less diverse, less nested, and more modular structure than the EPF community. These features suggest that the distinctive architecture of the mycobiome in lichens may be shaped by strong host-filtration processes. Additionally, we found that the ELF community exhibited similarities to phyllosphere fungal communities, with a high abundance of plant pathogen fungi. While we have provided insights into the structure of ELF communities, many questions remain unanswered, such as the origin of ELF, its ecological role, and the interactions between ELF and host lichens. Future studies are needed to explore the ecological niche of ELF.Additional file 1. Fig. S1 Community similarity ordination based on geographical distance. The ordinations are visualized in two approaches: a non-metric multidimensional scaling and b principal co-ordinates analysis based on Brat-Curtis distance. Ellipses indicate the 95% confidence interval of group variance. c PCoA plots showing ELF community variability between the two host lichens from same site. Fig. S2 Centrality indices of the fungal communities. a Betweenness and Closeness indicate vectors containing the betweenness and closeness of each node. b InDegree and OutDegree indicate vectors containing the inward and outward degree of each node. c Expected influences indicates the sums of incoming or outgoing edge weights connected to a node. Mean ± SEM, ****p < 0.001, **p < 0.01, ns not significant.


Astragalus and Danshen in IPF treatment.Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial lung disease of unknown etiology, is characterized by continuous damage to alveolar epithelial cells, abnormal repair of alveolar tissue, and alveolar wall scar formation. Currently, the recommended treatment for IPF in Western medicine is relatively limited. In contrast, traditional Chinese medicine and compound prescriptions show advantages in the diagnosis and treatment of IPF, which can be attributed to their multi-channel and multi-target characteristics and minimal side-effects. The purpose of this study was to further corroborate the effectiveness and significance of the traditional Chinese medications We performed whole-genome methylation analysis on nine rat lung tissue samples to determine the epigenetic variation between IPF and non-fibrotic lungs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and quantitative reverse transcription polymerase chain reactions.Astragalus and Salvia miltiorrhiza  act on the Isl1, forkhead box O3, and Sonic hedgehog genes via regulation at transcriptional and epigenetic levels during IPF.We identified differentially methylated regions and 105 associated key functional genes in samples related to IPF and Chinese medicine treatment. Based on the methylation levels and gene expression profiles between the Chinese medicine intervention and pulmonary fibrosis model groups, we speculated that Astragalus and Danshen in treating IPF, and suggest several promising therapeutic targets for preventing and treating IPF.These findings provide novel insights into the epigenetic regulation of IPF, indicate the effectiveness of The online version contains supplementary material available at 10.1186/s12890-023-02601-6. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. Currently, the commonly accepted pathogenesis of IPF includes continuous damage to alveolar epithelial cells, abnormal repair of alveolar tissue, and alveolar wall scar formation . The patRecent studies have found that epigenetic phenomena widely occur in pulmonary fibrosis, which opens a new avenue for its prevention and treatment. The most prevalent type of epigenetic modification in eukaryotes is deoxyribonucleic acid (DNA) methylation, which regulates gene expression by changing the activity of non-coding elements , particuAstragalus and Danshen effectively inhibited the proliferation of lung tissue fibers and matrix, intervened in myofibroblast activation, and delayed the progression of pulmonary fibrosis , Isl1, FOXO3, CCAAT enhancer binding protein beta [Cebpb], heat shock protein family A (Hsp70) member 5 [Hspa5], Shh, and BMP4) were filtered by degree value for KEGG pathway enrichment analysis. The results showed that the tumor necrosis factor (TNF), autophagy, apoptosis, cyclic adenosine monophosphate (cAMP), Hippo, phosphatidylinositol-3-kinase (PI3K)-Akt, interleukin 17 (IL-17), transforming growth factor beta (TGF-β), and FOXO signaling pathways were involved  were identified for further research. Polr2a, Isl1, Shh, and BMP4 were hypermethylated in the TRE but hypomethylated in the MOD group. FOXO3, Cebpb, and Hspa5 were hypomethylated in the TRE but hypermethylated in the MOD group.DMR-associated genes were also detected, and 105 key functional genes were identified. Several IPF treatment-related DMGs have been identified, including Isl1, an LIM homeodomain gene, plays critical roles in segmental patterning as well as cell differentiation, fate determination, and diversity production during embryogenesis [Isl1 in tumorigenesis have been extensively studied. It plays an important role in gastric cancer progression and development by regulating the expression of cyclin B1 and B2  and c-MYC [Isl1 functions as a marker for well-differentiated pancreatic neuroendocrine tumors [Isl1 in the lungs, such as regulating NK2 homeobox 1 (Nkx2.1), is necessary for lung lobation and tracheoesophageal separation [Isl1 in many diseases and developmental processes are concerning. Isl1 hypermethylation is an independent predictor of disease recurrence and progression in non-muscle invasive bladder cancer [Isl1 may also help in establishing the epigenetic memory of cardiomyocyte fate commitment [Isl1 was hypermethylated in the TRE and hypomethylated in the MOD groups, indicating that methylation levels were related to IPF treatment. The expression profiles of Isl1 between the TRE and MOD groups were consistent with methylation levels. These results indicate the effectiveness of Astragalus and Danshen in IPF treatment. Although the roles and detailed mechanisms of Isl1 in IPF treatment remain unknown, Isl1 may be speculated to be a promising therapeutic target.ogenesis –39. The nd c-MYC . In addie tumors , rhabdome tumors , and neue tumors  and alsoe tumors . The rolparation . The char cancer . Isl1 mammitment . In our FOXO3, a member of the FOXO transcription factors, mediates many biological processes, such as autophagy in skeletal muscle [FOXO3 is a tumor suppressor in many cancers, including gastric [FOXO3 in IPF fibrogenesis. Its expression is downregulated, and it is hyperphosphorylated in IPF fibroblasts. FOXO3 knockout mice showed heightened susceptibility to the lung-damaging effects of bleomycin, including increased fibrosis, loss of lung function, and higher mortality. In our study, FOXO3 was hypermethylated, and its expression was downregulated in the MOD but hypomethylated and upregulated in the TRE group. Thus, to some extent, the use of TCM can act on therapeutic targets.l muscle , neural l muscle , regulatl muscle , and pril muscle . In addi gastric , colorec gastric , breast  gastric , and lun gastric . HoweverShh signaling regulates many biological processes, such as adrenocortical development [Shh during lung development. Deletion of Shh in the lung causes failure in branching and growth of the distal lung [Shh overexpression causes epithelial and mesenchymal cell proliferation to increase, leading to an abundance of lung mesenchyme but no functioning alveoli [Shh was hypermethylated and its expression was downregulated in the TRE group but upregulated and hypomethylated in the MOD group, indicating its potential regulatory role in IPF treatment. Due to the complex composition of TCM compounds, the in-depth research on the active ingredients of compound drugs and the pathological mechanism of diseases is limited. Currently, more attention is focused on single TCMs and their effective monomer components. However, TCM has synergistic effects on compatibility. Based on the research on TCM pairs, we can carry out the research on the mechanism of multi-target, multi-component, and multi-channel synergistic effects of TCM compounds. In addition, the current research on the treatment of pulmonary fibrosis with TCM mostly focuses on the prevention and treatment of target organ damage from the perspective of intervention pathway, and less on the molecular mechanism and target of TCM on the genetic level. This study deeply explores the mechanism and target of Yiqi Huoxue herbs in the intervention of pulmonary fibrosis through genome-wide methylation. It provides evidence of Chinese medicine intervention in pulmonary fibrosis on the genetic level. However, given the inherent differences between humans and other animals, the data obtained based on animal experiments also have certain limitations, and accordingly require verification in human studies.elopment , the outelopment , and medelopment . In addital lung –61. Shh  alveoli . AccordiAstragalus and Danshen act on Isl1, FOXO3, and Shh via regulation at the transcriptional and epigenetic levels during IPF treatment. Our study not only indicates the effectiveness of Astragalus and Danshen in treating IPF but also provides various promising therapeutic targets that warrant further study.In this study, we investigated whole-genome methylation patterns in the lung tissues of the CON, MOD, and TRE groups. Using this approach, DMGs in different comparison groups were identified, and 105 key functional DMGs were used for further analysis. Based on the methylation levels and gene expression profiles between the TRE and MOD groups, it can be speculated that Below is the link to the electronic supplementary material.Supplementary Material 1