The in situ chemical oxidative method of polymerization was used for synthesizing PTh and PTh/gC3N4 nanocomposite. Fourier transform infrared spectroscopy (FT-IR), X-rays diffraction (XRD), thermo-gravimetric analysis (TGA), transmittance electron microscopy (TEM) as well as scanning electron microscopy (SEM) analysis were used for confirmation of the structure along with morphology of the PTh and PTh/gC3N4 nanocomposite. The thermal stability of DC electrical conductivity of PTh and PTh/gC3N4 nanocomposite were tested under isothermal and cyclic ageing condition. The sensing response of PTh and PTh/gC3N4 nanocomposite as a function of DC electrical conductivity were recorded in petrol and ethanol atmosphere. The sensing response of PTh/g-C3N4 nanocomposite in petrol atmosphere was 6.1 times higher than that of PTh with lower detection limit to 0.005 v/v% of ethanol prepared in n-hexane.The automobile vehicles must be operated on fuel containing no more than 10% ethanol. Use of fuel having more than 10% ethanol may cause engine malfunction, starting and running issues, and material degradation. These negative impacts could cause irreversible damage to the vehicles. Therefore, ethanol mixing in petrol should be controlled below 10% level. The current work is the first to report sensing of ethanol mixing in petrol with reference to the variation in the DC electrical conductivity of polythiophene/graphitic-carbon nitride (PTh/gC Conductive polythiophene/graphitic-carbon nitride nanocomposite for the detection of ethanol mixing in petrol. Furthermore, ethanol has a greater octane rating than gasoline,1 indicating that ethanol–gasoline blends have a higher octane rating than regular gasoline. However, according to a recent analysis, ethanol–gasoline blends increase vehicle emissions of volatile organic compounds (VOCs), predominantly acetaldehydes compounds, which act as precursors to tropospheric ozone in urban areas.2 These outcomes have caused authorities to reconsider their previous support for the use of ethanol–gasoline mixes in major cities, where a high tropospheric ozone concentration is the primary environmental concern. Use of fuel containing more than 10% ethanol may cause materials degradation, starting and operating issues, and automobile engine malfunction. Automobiles can sustain irreparable damage as a result of these negative effects. Ethanol increases the danger of groundwater along with soil pollution, owing to a rise in tank corrosion, decreasing the interfacial tension between NAPL–water and lastly by preventing biodegradation and increasing the contaminant solubility. Besides its adverse effect, ethanol blending in petrol is used extensively all over of the world therefore selectively and rapidly detection to monitoring the percentage of ethanol in petrol is highly needed.3,4Ethanol, as a fuel produced mostly from plants like sugarcane and maize, is a desirable alternative to gasoline for reducing reliance on fossil fuels and lowering CO5–18 Due to its extraordinary functionalities through regulated charge transfer process, adjustable electrical conductivities, and self-resistivity changes upon exposure to various gases, conducting polymers have long been sought after as chemiresistor materials.16–21 Due to its extraordinary functionalities through regulated charge transfer process, adjustable electrical conductivities along with self-resistivity which alters when exposed to variety of gases, CPs have for a long time been used as chemiresistor.9–13 Polythiophene (PTh) being chemically, thermally and environmentally highly durable is amongst the most researched CPs. PTh and its derivatives stood out among gas sensors owing to their distinctive doping and de-doping mechanisms that produce a change in electrical conductivity when exposed to various chemicals and gases.22–33Many prospective materials have been studied for their behavior in chemical sensing throughout the years, as well as the sensing mechanisms that underlie the unique interactions of vapours, chemicals as well as gases with materials that are used in sensing applications. The development of chemical/gas/vapour sensors has recently focused heavily on carbon nanomaterials, metal oxide semiconductors, conductive polymers (CPs) and their nanocomposites.3N4, and MoS2 among these materials because of their remarkable capacity as sensing materials, supercapacitors, and hydrogen evolution reactions.34–46 Taking inspiration from the aforementioned properties, here we described our efforts in developing polythiophene (PTh) and polythiophene/graphitic-carbon nitride (PTh/g-C3N4) nanocomposites for ethanol sensing in petrol. This is, too are awareness, the principal attempt which employs a sensor based on PTh/g-C3N4 nanocomposite for detecting ethanol in petrol at ambient conditions. We have also examined the sensing performance of PTh/g-C3N4 nanocomposite at different v/v% of ethanol in n-hexane.2-Dimensional nanomaterials have captured a significant deal of attention due to its several uses in the past ten years and their extremely high surface area to volume ratio. Researchers have been fascinated by MXene, graphene, g-C2.2.1.Thiophene (E. Merck), melamine (Fisher Scientific), chloroform (Fisher Scientific), anhydrous ferric chloride (CDH), methanol (E. Merck) and acetone (E. Merck) were used in their original state. Double distilled water (DDW) was used throughout the experiments.2.2.3N4 powder was made by heating melamine with heating rate 15 °C min−1 upto 550 °C for 3.5 h in an alumina crucible. Then the vessel containing g-C3N4 powder was cooled to room temperature for further processing.47The light-yellow g-C2.3.3N4 nanocomposites were prepared by the in situ chemical oxidative polymerization. The first step was dissolving thiophene (2 mL) in chloroform (40 mL), followed by ultrasonication for 25 minutes. Additionally, another solution of a known amount of g-C3N4 (20%) was prepared by adding it to chloroform (60 mL) and ultrasonically sonicated for 30 minutes. The resulting solution containing the g-C3N4 sheets was then poured in the thiophene solution and this mixture was then ultrasonicated for 110 minutes. The thiophene monomers got absorbed on the g-C3N4 sheets during ultrasonication. Following this, a separate solution of 16.24 g (100 mmol) ferric chloride in chloroform (100 mL) was made by stirring for 20 minutes to obtain a homogeneous mixture. Now, for polymerizing thiophene monomers on the surface of g-C3N4 sheets, suspension of FeCl3 was added dropwise in thiophene and g-C3N4 mixture followed by constant stirring on magnetic stirrer. After 24 h of stirring, the as-prepared PTh/g-C3N4 nanocomposite was collected by Buchner suction funnel after draining of the residual in the solution and then washed numerous times using methanol, subsequently by double distilled water and lastly by acetone. Amid washing with methanol, the colour of the material changed dark brown from deep black. The nanocomposites were lastly dried in a vacuum oven at 60 °C for 24 hours. Finally, using a mortar and pestle, these items were ground into a fine powder. An analogous procedure was used to produce the pure PTh nanoparticles.PTh and PTh/g-C2.4.3N4 nanocomposites was performed by FT-IR (PerkinElmer 1725 instrument on KBr pellets), XRD (Bruker D8 diffractometer with Cu Kα radiation at 1.5418 Å), TGA {PerkinElmer (Pyris Diamond) instrument}, SEM {JEOL, JSM, 6510-LV (Japan)}, and TEM {JEM 2100, JEOL (Japan)} techniques respectively.21–25,30–33The characterization of PTh and PTh/g-CV, S, W and I represent the voltage (V), probe spacing (cm), the pellet-thickness (cm), current (A) and σ represents the DC electrical conductivity (S cm−1) respectively.22,23 250 mg of each sample was pelletised at room temperature using a hydraulic pressure instrument which operated at a pressure of 70 kN for 3 min.21–25,30–33DC electrical conductivities and sensing experiments were performed by four-in-line probe instrument attached with the PID controlled oven manufactured by Scientific Equipment, Roorkee, India. The thermal stability as a function of DC electrical conductivity under isothermal and cyclic ageing environments was evaluated for all the nanocomposites. The equation which was utilized for calculating the conductivity was as follows:3.3.1.3N4, and PTh/g-C3N4 are presented in −1 may appear as a result of O–H stretching frequency of water molecules. The peaks at 1640 cm−1 and 1345 cm−1 could possibly be due to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C, and C–C stretching mode of vibration in the polythiophene rings respectively. The peaks at 1207 cm−1 and 1056 cm−1 may be because of the bending vibration in PTh. The peak around 780 cm−1 reveals the out of plane C–H bond bending in polythiophene. The peak at 645 cm−1 is because of the bending mode of vibration of the C–S bond of the thiophene ring. The C–S–C ring deformation mode is the cause of the band at 510 cm−1.22–25 In the g-C3N4 spectrum, multiple pinnacles between 1225–1630 cm−1 may be due to symmetrical C–N stretching vibration and the pinnacle at 802 cm−1 may be traced as dissymmetrical mode of stretching of C–N and CN as well triazine units of the aromatic rings.48,49 In PTh/g-C3N4, it is found that on mixing of PTh in g-C3N4, the shoulder shifted to 1246 to 1647 cm−1, affirming that the thiophene monomer gets interacted with g-C3N4. The water molecules which may be trapped by the PTh/g-C3N4 nanocomposite and the amine groups present on g-C3N4 showing a merged peak at around 3200 cm−1. The peak seen at 675 cm−1 may be because of the C–S bending vibrations while all other characteristics peaks of polythiophene get merged with the g-C3N4.The FT-IR spectra of PTh, g-C3.2.3N4, PTh and PTh/g-C3N4 nanocomposite. The appeared peaks at 2θ = 27.25° and 2θ = 12.87° in g-C3N4 spectra are in accordance with the (002) and (001) Brag reflections, originated from the 2D structure of g-C3N4.48,49 In PTh spectra a wide hump is observed at about 2θ = 16–27°, indicating an amorphous form of PTh.22–25 In case of PTh/g-C3N4 nanocomposite a broad bump around 2θ = 15–25° and a sharp peak at 2θ = 27.48° are observed, illustrating that PTh and g-C3N4 are present in the nanocomposite. The low intensities peaks in PTh/g-C3N4 nanocomposite clarifying that the amorphous PTh matrix might have shadowed the high intensity peaks of g-C3N4, which could be further understood by SEM and TEM studies of PTh/g-C3N4 nanocomposite. The shift in the diffraction peak, suggested that an interaction existed between g-C3N4 and PTh in PTh/g-C3N4 nanocomposite.3.3.3N4 {3N4 {3N4 appeared in some sheet like form as shown in 3N4 nanocomposite is evident from 3N4 nanosheets. For the PTh/g-C3N4 nanocomposite, PTh is well mounted on g-C3N4 nanosheets, resulting in the morphology becoming flaky and sheet-like. Absence of free g-C3N4 nanosheets are visible in the PTh/g-C3N4 nanocomposite, it is possible to conclude that PTh effectively covers g-C3N4 nanosheets.The morphologies of PTh {3.4.3N4. Here in this image, it can be clearly observed that there is sheet of g-C3N4 upon which PTh has been successfully deposited and masked upon the entire surface of the g-C3N4 sheet. The monomers of thiophene polymerize over the large surface of g-C3N4 providing an effective π-conjugated system which promotes excessive transit of the charge carriers, which the reason for PTh/g-C3N4 showing higher sensing performance towards ethanol.3.5.3N4 and PTh/g-C3N4 nanocomposite is depicted in 3N4, the initial weight loss was found from 50 to 120 °C due to loss of water molecules. Subsequently, the oxidative thermal degradation of g-C3N4 is started at around 510 °C. While the PTh found to be least thermally stable among all of the samples. The initial loss of weight in PTh may be as a result of vaporization of water molecules followed by further degradation started around at 275 °C. In case of PTh/g-C3N4 nanocomposite, after losing of water molecules at around 80 to 105 °C, the final degradation started from 350 °C which is higher than that of PTh but lower than g-C3N4, suggesting the higher thermal stability of PTh/g-C3N4 nanocomposite than that of PTh, which may be a result of the electronic interaction taking place amid PTh and g-C3N4 nanosheets.Thermo-gravimetric analysis of PTh, g-C4.3N4 and PTh/g-C3N4 nanocomposite were calculated by a 4-in-line probe method and marked as 8.326 × 10−4 S cm−1, 5.8273 × 10−5 S cm−1 and 6.4327 × 10−4 S cm−1 respectively as shown in 3N4 sheets, the electrical conductivity will increase by generating more polarons on PTh as depicted in 3N4 in PTh may be due the insulating nature of g-C3N4. However, the fall in DC electrical conductivity was not as much and it is still found in the range of PTh, might be due to the more dominating effect of generated polarons in PTh than insulating g-C3N4 in tug of war between them. After all, despite an insignificant loss of electrical conductivity in PTh/g-C3N4 nanocomposite on incorporation of g-C3N4 in PTh, it provides a high surface area in nanocomposite by the formation of effective arrangement of the π-conjugated system of PTh on the large g-C3N4 surface area.4.1.3N4 were examined, as shown in The stability as a function of DC electrical conductivity retaining capacity under isothermal ageing conditions of the nanocomposites PTh and PTh/g-Cσtr,) at constant temperature was:σt and σ0 is represent the DC electrical conductivities at time ‘t’ and ‘0’, respectively.50–53The equation applied for calculating the relative DC electrical conductivity  strongly suggesting presence of an electronic interaction between PTh and g-C3N4, which resists the damaging of its conducting channels and loss of the doping agent. These outcomes displayed that PTh/g-C3N4 could be an auspicious candidate in various electronic applications even at 110 °C.It is clearly noticeable from 4.2.3N4 nanocomposite were also examined for their stability as a function of DC electrical conductivity performance under cyclic ageing conditions, as shown in PTh and PTh/g-Cσr) was:σT and σ50 is representative of the DC electrical conductivities at temperatures T and 50 °C.50–53The equation applied for calculating the relative DC electrical conductivity (3N4), which may be attributed to the high movability of polarons at elevated temperatures. For PTh {3N4 displayed continuous rise in conductivity with each cycle without losing its conductivity, signifying the presence of electronic interaction between PTh and g-C3N4.The DC electrical conductivity of both the samples were recorded by four consecutive cycles with continuously rise in temperature up to 130 °C. These findings revealed that DC electrical conductivity increased steadily in each cycle and followed a same pattern in both of the samples  is calculated by the following equation:σi and Δσ are representative of the initial DC electrical conductivity as well as change in DC electrical conductivity during complete exposure of gas respectively.33,50,51The % sensing response  in petrol interacts with the polarons of PTh, disrupting the conductivity channels by impeding the movement of some polarons of PTh, causing decline in DC electrical conductivity. As PTh pellet exposed to ambient air, the relative DC electrical conductivity began to increase and came back to its initial value within 95 s and then flattened may be as a reason of complete desorption of ethanol from the PTh surface. Similarly for the PTh/g-C3N4 nanocomposite, the relative DC electrical conductivity declined by exposing the pellet of PTh/g-C3N4 nanocomposite to petrol and reverted back to its initial value within 80 s in air. The sensing response of PTh and PTh/g-C3N4 nanocomposite were found to be 15.55% and 94.73% respectively. The sensing response calculated in PTh/g-C3N4 nanocomposite was 6.1 times higher than that of PTh may be due to the high adsorption–desorption on PTh/g-C3N4 nanocomposite surface. Thus, incorporation of g-C3N4 in PTh, creating high surface area with proper advancing in conducting channels of PTh for adsorption and desorption of analyte gases. The sensing response of PTh/g-C3N4 toward ethanol in petrol as well as at different volume percentage  of ethanol in n-hexane were also determined that is 95.3345%, 90.152%, 74.242%, 59.091%, 42.424%, 28.788%, 19.697%, 18.188% and 15.152% respectively as shown in Sensing of ethanol in petrol was studied by examining the performance of relative DC electrical conductivity of PTh and PTh/g-C5.2.3N4 nanocomposite's sensing response towards ethanol in petrol, the DC electrical conductivity performance of PTh/g-C3N4 nanocomposite was also recorded for different volume percentage of ethanol in n-hexane as shown in 3N4 nanocomposite increases, may be due to high interaction of ethanol with the polarons of PTh/g-C3N4 nanocomposite and gets saturated after few seconds. While in ambient air, conductivity completely reverted to its initial value by simple desorption of ethanol from PTh/g-C3N4 nanocomposite surface.In order to justify the PTh/g-C5.3.3N4 nanocomposite were tested for their rapid adsorption and desorption of analyte by observing the variation in relative DC electrical conductivity in environment of petrol for 30 s followed by ambient air for the next 30 s consecutively for three 60 s cycles as shown in 3N4 nanocomposite the variation in relative DC electrical conductivity was more superior to that of PTh, which may be due to more adsorption–desorption of analyte on PTh/g-C3N4 nanocomposite surface than that of PTh.The PTh and PTh/g-C5.4.3N4 nanocomposite sensing response toward ethanol mixing in petrol, 0.5% ethanol in n-hexane, n-hexane, n-pentane, iso-octane, n-heptane, benzene, styrene at room temperature (27 °C) are displayed in 3N4 nanocomposite toward ethanol may be due its availability of lone pairs of oxygen which interacts with polarons of PTh, causing significant loss of DC electrical conductivity (high sensing response). While for the other analytes (hydrocarbons) tested, the sensing responses were negligible in comparison of ethanol may be of their non-polar nature. Thus the lower the polarity/free lone pairs of electrons in the analyte, lower the conductivity change occurred in PTh/g-C3N4 nanocomposite, makes ethanol highly selective in comparison of different hydrocarbon compounds.High selectivity is an essential criterion for a gas sensor in order to qualify as reliable and applicable. The PTh/g-C5.5.3N4 nanocomposite is explained by the DC electrical conductivity behaviour on simple adsorption and desorption mechanism of analyte at room temperature, as depicted in 3N4 nanocomposite, graphitic-carbon nitride get interacted with pi-bonds of thiophene rings in polythiophene and form a nanocomposite providing high surfaced platform for sensing of analytes. It is already known that petrol is comprises of majority of hydrocarbons and some ethanol mixing to raise its octane number. The hydrocarbon molecules in petrol being nonpolar at ambient temperature, do not interact with the polarons of polythiophene in PTh/g-C3N4 nanocomposite. Ethanol is predicted as the only candidate that could be accountable for the variation in the conductivity of PTh/g-C3N4 nanocomposite upon exposure to petrol vapors. In presence of petrol atmosphere, the lone pairs of ethanol interact with the polarons of PTh/g-C3N4 nanocomposite, which in turn hindered the movement of polarons, eventually declination of DC electrical conductivity with time. While in ambient air condition, the ethanol molecules and petrol gets desorbed from the sensor surface, resulting in reverting its conductivity to it's about initial values, indicating complete desorption of ethanol molecules mixed in petrol. Thus, the above mechanism indicates that polarons mobility is controlled by the simple adsorption and desorption of ethanol on PTh/g-C3N4 nanocomposite surface.Sensing mechanism of PTh/g-C6.3N4 nanocomposite, characterized and studied for their stability of DC electrical conductivity and sensing response toward ethanol mixing in petrol. The stable nature of DC electrical conductivity of PTh/g-C3N4 nanocomposite under accelerated isothermal and cyclic ageing condition were demonstrated and found significantly higher than that of PTh. Also the PTh/g-C3N4 nanocomposite showed rapid response and high reversibility towards ethanol mixing in petrol and ethanol prepared in n-hexane with a detection limit of 0.005%. The sensing response in PTh/g-C3N4 nanocomposite towards ethanol mixing in petrol was 6.1 times higher than that of PTh at room temperature. In addition of excellent sensing response, this research found that PTh/g-C3N4 nanocomposite might be used as a semiconducting material in a variety of electronic and electrical devices at high temperature conditions.In this paper, we synthesized PTh and PTh/g-CThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


NR2F6  is known to be an orphan nuclear receptor that has been characterized as an intracellular immune checkpoint in effector T cells and, therefore, may control tumor development and growth. The prognostic impact of NR2F6 in endometrial cancers is evaluated in this study.Expression analysis of NR2F6 in 142 endometrial cancer patients was performed by immunohistochemistry of primary paraffin‑embedded tumor samples. Staining intensity of positive tumor cells was automatically assessed semi-quantitatively, and results were correlated with clinicopathological characteristics and survival.p = 0.022). The estimated PFS differed by 63 months (152 months (95% CI 135.7–168.4) vs. 88.3 months (95% CI 68.5–108.0), p = 0.002). Furthermore, we found significant associations between NR2F6 positivity, MMR status, and PD1 status. A multivariate analysis suggests NR2F6 to be an independent factor influencing the OS (p = 0.03).Forty five of 116 evaluable samples (38.8%) showed an overexpression of NR2F6. This leads to an improvement of the overall survival (OS) and progression-free survival (PFS). In NR2F6-positive patients, the estimated mean OS was 156.9 months  143.1–170.7) compared to 106.2 months in NR2F6-negative patients (95% CI 86.2–126.3; In this study, we could demonstrate that there is a longer progression-free and overall survival for NR2F6-positive patients with endometrial cancer. We conclude that NR2F6 might play an essential role in endometrial cancers. Further studies are required to validate its prognostic impact.The online version contains supplementary material available at 10.1007/s00432-023-04632-2. The orphan nuclear receptor NR2F6  has been recently shown to be part of the cellular, innate immune system. NR2F6 is member of the COUP-TF  and is a regulatory intranuclear receptor, which binds directly to DNA, including a control function of gene regulation and cell differentiation  via binding the PD1-receptor. Klepsch et al. found evidence that PD1-inhibition and down-regulation of NR2F6 in tumor-infiltrating lymphocytes strongly act in synergistic manners  has been shown to be associated with a better survival in cancer patients . Exclusion criteria were no surgical treatment, the absence of patient consent, and the absence of adequate tumor tissue. One hundred and forty two patients finally met the inclusion criteria , the region of tumor cells was marked, and these samples then arranged to tissue microarrays (TMAs). Each TMA consisted of up to 60 1 mmThe NR2F6 antibody  was used to evaluate the NR2F6 status of each tumor sample. Furthermore, the Estrogen receptor und Progesterone receptor were analyzed using anti-Estrogen Receptor (SP1) Rabbit Monoclonal and anti-Progesterone Receptor (1E2) Rabbit Monoclonal . For each staining, the IView DAB Detection Kit was used on a Ventana Bench Mark automated staining system .The stained TMAs were then scanned using the Ventana iScan HT scanner . The samples were computerized and semi-automatically analyzed using the Definiens Tissue Studio software . NR2F6 overexpression is defined with respect to the mean of the NR2F6 staining intensity.p-value below or equal to 0.05 was considered significant.Statistical analyses were performed using IBM SPSS Statistics for Windows . The immunohistochemical staining intensity has been correlated to clinicopathological characteristics and survival data. We hypothesized that NR2F6 overexpression correlates to a worse survival of the patients. We used the 2 (SD). The majority of the patients was classified in tumor stages FIGO I or II. 62.7% of the patients received a systematic lymphadenectomy, but only 14 patients (9.9%) finally had lymph node metastases. According to the pathologic reports, 84.5% of the tumors had an endometrioid histology and 60.6% of the patients were surgically treated by laparoscopy.Mean age of all patients was 67.5 ± 12.2 years, the mean BMI amounted to 30.1 ± 8.4 kg/mBaseline characteristics of all patients are shown in Table The clinical data were then analyzed dependent on the NR2F6 status. Failed staining lead to finally 117 tumor samples that could be evaluated. There was no significant difference between the NR2F6-positive and negative groups concerning the age, BMI, lymph node status, Ki-67 and FIGO stage. The p53-status, Grading and the histology were also not associated to NR2F6 expression. Only MMR status was associated to the NR2F6 expression as there were more patients with a NR2F6-negative tumor and MMR proficiency compared to NR2F6-positive tumors, as depicted in Table Immunohistochemical staining showed a wide variation of staining intensity. As shown in Figs. To analyze NR2F6 expression, the staining intensity was documented for all samples. Regarding gynecological cancers, there are no valid data concerning the evaluation or meaning of NR2F6 staining. Consequently, there were no validated thresholds available. We defined the mean of staining intensity to be the threshold for negativity and positivity. See distribution of NR2F6 staining intensity in Fig. p = 0.003). Moreover, PD1-negative tumors are more often NR2F6 negative than NR2F6 positive (p = 0.02).We compared different subgroups according to immunohistochemical tumor properties using the p = 0.022, Fig. To evaluate differences in the overall and progression-free survival between NR2F6-negative and NR2F6-positive tumors, the Kaplan–Meier and log-rank tests were used. Patients with NR2F6 expression had a significantly longer overall survival than those without NR2F6 expression. The estimated mean OS was 156.9 months  143.1–170.7) compared to 106.2 months in NR2F6-negative patients  vs. 88.3 months (95% CI 68.5–108.0), p = 0.05 (OS) and 0.025 (PFS), respectively).We performed a subgroup analysis in order to describe the impact of NR2F6 in the most common molecular subgroups of endometrial cancer, i.d. MMRd and p53. Concerning the survival data of p53-positive tumors depending on NR2F6 expression, there were very few patients showing a p53-aberrant tumor and NR2F6 expression. In general, patients with both a p53 wild type and NR2F6 expression showed the longest overall and progression-free survival in this subgroup analysis . The statistical analysis of the subgroup analysis is constrained by the small sample size. Nevertheless, NR2F6 overexpression shows a better survival in MMR-deficient patients .The multivariate Cox regression could be performed in 92 and 93 tumor samples, as their information concerning overall or progression-free survival status and the examined factors was complete. The analysis suggests NR2F6 expression as an independent prognostic marker concerning the overall survival  results in different studies. A validation of all immunohistochemical parameters is urgently needed.To determine a threshold for the NR2F6 staining intensity, we chose the mean staining intensity, as previously described. Additionally, we analyzed all results using the classification described by Klapper et al. , constitKlepsch et al. showed that an inhibition of NR2F6 in T cells may increase the efficacy of PD-L1-targeting immune checkpoint therapies in mice Below is the link to the electronic supplementary material.


N6-methyladenosine (m6A) modification is the most abundant reversible methylation modification in eukaryotes, and it is reportedly closely associated with a variety of cancers progression, including colorectal cancer (CRC). This study showed that activated lipid metabolism and glycolysis play vital roles in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection.Protein and mRNA levels of FTO and ALKBH5 were measured using western blot and qRT-PCR. The effects of FTO and ALKBH5 on cell proliferation were examined using CCK-8, colony formation, and EdU assays, and the effects on cell migration and invasion were tested using a transwell assay. m6A RNA immunoprecipitation (MeRIP) and RNA-seq was used to explore downstream target gene. RIP was performed to verify the interaction between m6A and HK2. The function of FTO and ALKBH5 in vivo was determined by xenograft in nude mice.In this study, FTO and ALKBH5 were significantly down-regulated in CRC patients and cells both in vivo and in vitro in a high-fat environment. Moreover, FTO and ALKBH5 over-expression hampered cell proliferation both in vitro and in vivo. Conversely, FTO and ALKBH5 knockdown accelerated the malignant biological behaviors of CRC cells. The mechanism of action of FTO and ALKBH5 involves joint regulation of HK2, a key enzyme in glycolysis, which was identified by RNA sequencing and MeRIP-seq. Furthermore, reduced expression of FTO and ALKBH5 jointly activated the FOXO signaling pathway, which led to enhanced proliferation ability in CRC cells. IGF2BP2, as a m6A reader, positively regulated HK2 mRNA in m6A dependent manner. Additionally, down-regulation of FTO/ALKBH5 increased METTL3 and decreased METTL14 levels, further promoting CRC progression.In conclusion, our study revealed the FTO-ALKBH5/IGF2BP2/HK2/FOXO1 axis as a mechanism of aberrant m6A modification and glycolysis regulation in CRC.The online version contains supplementary material available at 10.1186/s13578-023-01100-9. Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies worldwide . The proN6-methyladenosine (m6A) is the most pervasive internal modification form of eukaryotic mRNA and is implicated in gene dysregulation through the methylation of the adenosine base at the nitrogen-6 position . m6A-medDemethylases such as obesity-associated protein (FTO) and alkylation repair homolog protein 5 (ALKBH5) regulate methylation reversal, whereby their implications in carcinogenesis have been reported in multiple cancers . Hu et aCommon features of cancer include high levels of glycolysis and lactic acid formation under aerobic conditions. The accumulation of glucose in tumor tissue provides a massive source of energy for tumor progression . GlycolyIn this study, we discovered that the demethylases FTO and ALKBH5 were down-regulated in CRC, especially in obese patients. Functional assays indicated an anti-cancer role of FTO and ALKBH5 in CRC. The mechanism of action of FTO and ALKBH5 involved the glycolysis via regulation of HK2 expression in an m6A-dependent manner by IGF2BP2. Moreover, FTO and ALKBH5 negatively regulated METTL3 and positively regulated METTL14 expression levels, enhancing their role as tumor suppressors of CRC.2 at 37 °C.Thirty-six pairs of frozen tissues were obtained from patients diagnosed with CRC who also underwent surgical excision at the Jiangsu Province Hospital. All protocols were reviewed and approved by the Ethics Committee of Nanjing Medical University. SW620 and HCT116 cell lines were purchased from the Chinese Academy of Science  and respectively cultured in a Leibovitz’s L-15 medium  and RPMI-1640 medium . DLD1, RKO and NCM460 cells were gifts from Changhong Miao of Fudan University Shanghai Cancer Center. Cell culture mediums were supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin (Yeasen) in a humidified incubator with 5% COALKBH5, FTO, METTL3, METTL14, WTAP, HK2 and IGF2BPs knockdowns in addition to over-expression plasmids were synthesized by Shanghai Genomeditech Biotech Co. Ltd. 293 T cells were used for lentivirus packaging with PEIMAX treatment . After infection with a concentrated virus and 5 μg/ml polybrene (Genomeditech) for 48 h, stably transfected cells were screened through treatment with 5 μg/mL puromycin (Yeasen). Western blotting was performed to verify the efficiency of transfection efficiency. All shRNA targets were listed in Additional file 3 CRC cells were seeded in 96-well plates and counted 72 h in the respective media with 10% FBS. Cell proliferation was measured at 450 nm absorbance by adding 10 μL in CCK-8 (Yeasen) after 2 h. For the colony formation assay, 1 × 104 cells were seeded in 6-well plates and cultured in respective media with 10% FBS for 7 days. Images were then captured after the cells were fixed with 4% formaldehyde for 30 min and stained with 2% crystal violet (Yeasen) for another 30 min. For the EdU assay, cells seeded in 96-well plates were cultured with 50 μM EDU for 2 h and then fixed with 4% paraformaldehyde. The cells were then incubated with 1 × Apollo reaction cocktail  for 30 min after permeabilization with 0.5% Triton-X. After DNA staining with Hoechst33342, the images were finally visualized using a fluorescence microscope .For the cell counting kit 8 (CCK-8) assay, 5 × 104 cells per insert. The inserts were placed into 24-well plates containing 800 μL of 30% FBS medium. After culturing for 48 h, the cells on the upper side of the filter were fixed with 4% formaldehyde for 30 min and stained with 2% crystal violet for another 30 min. The cells on the upper side were then wiped with swabs, while the cells on the lower side were imaged randomly under a microscope.Cells were suspended in a medium without FBS and seeded in 8 μm pore inserts  at a density of 2 × 10® III SuperMix plus (Yeasen) according to the manufacturer’s protocols. qRT-PCR was finally performed with a Roche machine using SYBR Green PCR master mix (Yeasen). GAPDH was used as the internal reference. The qPCR primers used in this study are listed in Additional file Total RNA was extracted from cells using TRIzol reagent , and cDNA was synthesized using 4xHifairCells were lysed in NP40 buffer containing protease and phenylmethanesulfonyl fluoride  for 30 min and then boiled with 1 × loading buffer at 105 °C for 10 min. The extracted proteins were separated using sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto a nitrocellulose filter membrane. The membranes were blocked using 8% nonfat milk for 2 h and then washed three times with Tris-Buffered Saline Tween-20 buffer. The blocked membranes were incubated with primary antibodies at 4 ℃ overnight, followed by respective secondary antibodies (1:5000) at room temperature for 2 h. Antibody information is listed in Additional file N6-methyladenosine RNA methylation was quantified using an m6A RNA methylation quantification kit. Briefly, 300 ng of RNA were bound to wells and then incubated with capture antibody for 1 h. The mixture was then incubated with detection antibodies for 30 min and reacted with an enhancer solution for another 30 min at room temperature, the reaction signal was read using a microplate spectrophotometer at 450 nm.Cells were lysed with RIP lysis buffer and then incubated with specific antibodies at 4 ℃ for 2 h. The mixture was then incubated with protein A/G magnetic beads at 4 °C overnight. The bound RNA was separated from the beads by washing with RIP buffer solution. The eluted RNA was finally extracted and reverse-transcribed into cDNA for further analysis using qRT-PCR.Human CRC and paired adjacent normal tissues were soaked in paraffin and fixed with 4% formaldehyde. After cutting and dewaxing, the slices were incubated in citric acid antigen retrieval buffer and blocked with 3% BSA blocking buffer, followed by incubation with primary antibodies at 4 ℃ overnight. Next, slices were incubated with secondary antibodies for 1 h at room temperature after being washed three times. The DAB color-developing solution was added and hematoxylin staining was performed. Finally, the slices were dehydrated and images were taken randomly by scanning.For glucose assay, the cell supernatant was collected, 20ul of the sample to be tested was added to each well, and 100ul of the working solution of the substrate was added and incubated at 37 ℃ for 5 min to read the OD value A1. Then each well was added with enzymic antibody working solution of 20ul, and the reaction plate was placed at 37 ℃ for 15 min after shaking and mixing. The absorption value A2 was measured at 550 nm with enzymic marker within 30 min. All OD values are calculated by subtracting A1 from A2. For ATP level assay, the culture solution was removed, and the lysate was added in the ratio of 200 µl to each well of the 6-well plate. The lysate was blown repeatedly to fully lysate the cells. After lysate, the supernatant was taken for subsequent determination after centrifugation at 12000 g at 4 ℃ for 5 min. Then, 100 µl of ATP detection solution was added to each well and placed it at room temperature for 3–5 min, so that all the background ATP is consumed, thus reducing the background. A sample of 20 µl was then added to the test hole and the value was measured with a chemiluminescence instrument.6 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4–5 weeks). The animal procedures were approved by the Animal Care and Use Committee of Nanjing Medical University. Five weeks after injection, the tumors were excised from the mice and the weight, width (W), and length (L) were measured. The volume (V) of each tumor was calculated using the formula: V = (W2 × L/2).For the mouse xenograft model, 2 × 10All experiments were independently repeated at least three times. The means between groups were compared using unpaired or paired student’s t-tests. All data were presented in the form of mean ± SD and p < 0.05 was considered statistically significant.To evaluate the expression of FTO and ALKBH5 in human CRC samples, immunohistochemistry (IHC) staining was used to visualize the results of the tissue microarrays (TMAs) that contained 36 samples Fig. A. The poTo examine the effect of FTO expression on CRC growth, stably transfected cell lines with FTO knockdown and over-expression were constructed using lentiviral transfection technology. The efficiency of FTO knockdown and over-expression was verified by western blotting  and m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) in CRC cells with stable FTO over-expression, ALKBH5 over-expression, and control cells. The differential mRNA and differential m6A peaks modified genes are shown in volcano plots Fig. A, B. ThrThe stable cell lines of FTO and ALKBH5 knockdown and over-expression were verified by RT-PCR  and found IGF2BP family proteins may affect the m6A modification of HK2. To validate our hypothesis, we silenced IGF2BP1, IGF2BP2, and IGF2BP3 respectively in HCT116 cells, and QPCR indicated that only IGF2BP2 down-regulation led to HK2 decrease  The FTO protein expression levels were detected by western blot. (B-E) CCK-8 and EdU  results of FTO over-expression in HCT116 and SW620 cells.  Colony formation results of FTO over-expression in HCT116 and SW620 cells, together with its statistical chart. (H-J) Migration and invasion results of FTO over-expression in HCT116 cells and SW620 cells, together with its statistical chart . **p < 0.01,***p < 0.001.Additional file 2: Figure S2. Up-regulation of ALKBH5 hinders cell proliferation, migration, and invasion of CRC (A) Western blot analysis of transfection efficiency of over-expression ALKBH5 in HCT116 and SW620 cells. (B-G) The proliferation ability of ALKBH5 over-expression in HCT116 and SW620 cells was evaluated by CCK-8, colony formation, and EdU assays . (H-J) The cell migration and invasion activities of HCT116 and SW620 cells with ALKBH5 over-expression were assessed by transwell assays . **p < 0.01; ***p < 0.001.Additional file 3: Figure S3. HK2 inhibitor 2DG restrains oncogenic effect induced by FTO/ALKBH5 silence (A) Western blot showing HK2 and FOXO1 protein levels in treated with FTO/ALKBH5 knockdown and 2DG in HCT116 cells. (B) CCK-8 indicated the ability of proliferation in FTO/ALKBH5 deficient CRC cells was rescued by 2DG treatment. (C-D) Colony formation was performed in FTO/ALKBH5 knockdown and 2DG treated HCT116 cells. (E–G) Analysis of cell migration and invasion while treated with 2DG in FTO/ALKBH5 deficient HCT116 cells .*p < 0.05; **p < 0.01; ***p < 0.001.Additional file 4: Figure S4. HK2 inhibitor 2DG decreases cell proliferation, migration and invasion in vitro (A) HK2 and FOXO1 protein levels were detected by western blot in HCT116 cells. (B-D) CCK8 and colony formation assay was performed to indicated effect of 2DG in proliferation in HCT116 cells. (E–F) Transwell assay showing 2DG’s effect on cell migration and invasion in HCT116 cells . (G) Western blot indicated HK2 and FOXO1 protein level after METTL3 / METTL14 silence. ***p < 0.001.Additional file 5: Figure S5. FTO and ALKBH5 inhibits glucose metabolism but does not affect cell apoptosis. RIP-QPCR showing HK2 m6A level in FTO/ALKBH5 knockdown or over-expression cells. (B) Glucose levels were detected in FTO/ALKBH5 knockdown or over-expression cells of HCT116. (C) ATP levels were mesured in FTO/ALKBH5 silence or up-regulation cells of HCT116. (D) Flow cytometry of FTO/ALKBH5 over-expression by FITC/PI staining. (E) Tunel staining was performed in tumor tissues of FTO/ALKBH5 over-expression. (F) Western blot assay was performed in FTO/ALKBH5 over-expression for BCL2 and Caspase3 proteins. **p < 0.01; ***p < 0.001.Additional file 6: Table S1. Short hairpin targets. Table S2. Primers of genes. Table S3. Antibody information.


We use single-crystalX-ray diffraction to confirm the presence of longer-range intercalantsuperstructure and employ polarization-, temperature-, and magneticfield-dependent Raman measurements to examine both the symmetry ofemergent phonon modes in the intercalated material and potential magnetoelasticcoupling. Magnetometry measurements further indicate a correlationbetween the onset of magnetic ordering and the relative degree ofintercalant superlattice formation. These results show Raman spectroscopyto be an expedient, local probe for mapping intercalant ordering inthis class of magnetic materials.Transition-metal dichalcogenides (TMDs) intercalatedwith magneticions serve as a promising materials platform for developing next-generation,spin-based electronic technologies. In these materials, one can accessa rich magnetic phase space depending on the choice of intercalant,host lattice, and relative stoichiometry. The distribution of theseintercalant ions across given crystals, however, is less well defined—particularlyaway from ideal packing stoichiometries—and a convenient probeto assess potential longer-range ordering of intercalants is lacking.Here, we demonstrate that confocal Raman spectroscopy is a powerfultool for mapping the onset of intercalant superlattice formation inFe-intercalated NbSe Understanding the origin of this behavior, therefore,requires a deeper understanding of the lattice dynamics in these systems.In addition, designing systems with analogous switching behavior closerto room temperature is desirable for any technological application.In this regard, the family of selenide analogues, FexNbSe2, are especially promising candidates sincethese compounds may exhibit higher magnetic ordering temperaturescompared to the sulfides due to the higher spin–orbit couplingin heavier selenide compounds.12 However, these selenides remainunderexplored relative to the sulfides and their transport and switchingbehavior have yet to be investigated.Magnetically intercalated transition-metaldichalcogenides (TMDs)represent a promising class of emergent quantum materials for ultralow-powerapplications based on the manipulation of electron spin.2 Ramanscattering has been shown to be sensitive to the structure of intercalantsuperlattices within this family of magnetically intercalated TMDs.13 In particular, for several TxMS2 compounds , prominentRaman-active phonon modes emerge for x = 0.25 and0.33, which are completely absent in the unintercalated host lattice.13 The onset of these modes coincides with the observation, via electrondiffraction, of well-ordered 2a0 ×2a0 and √3a0 × √3a0 superlatticesfor x = 0.25 and 0.33, respectively (where a0 is the in-plane lattice constant for the hostlattice).3 The correlation between theseRaman modes and the underlying intercalant superlattice establishesRaman spectroscopy as a powerful and convenient tool to probe superlatticestructure on the mesoscopic scale. While the Raman activity has beeninvestigated for a small subset of these intercalated TMDs, particularlythe sulfides, substantially less is known about the selenide variantsand, in particular, for off-stoichiometric concentrations away from x = 0.25. Notably, the aforementioned switching behaviorin FexNbS2 has been shown tobe most pronounced when the compound is slightly off-stoichiometricfrom x = 1/3, hinting that disorder in the intercalantsuperlattices of these compounds may play a role in the coupling betweenelectrical and magnetic orders.9 Here, we investigate how the Raman response evolves as a functionof iron concentration in FexNbSe2, for 0.14 ≤ x < 0.25, finding a correlationbetween superlattice formation and iron occupancy. We also probe themagnetic behavior of these compounds and observe a maximum Néeltemperature (TN) around 130 K as x approaches 0.25. Furthermore, we conduct polarization-,temperature-, and magnetic field-dependent Raman measurements to discernthe symmetry of the observed phonon modes and investigate possiblemagnetoelastic couplings in this system.A key ingredient thatdirects the nature of magnetic ordering inthese materials more generally is the distribution of transition-metalintercalants within the host lattice.xNbSe2 were grown via chemical vapor transport withiodine as the transport agent. Elemental powders of Fe (99.9% AlfaAesar), Nb , and Se  were loaded in a 0.5:1:2 stoichiometric ratio, along with1–2 mg cm–3 iodine, and sealed in evacuatedquartz ampoules. The ampoules were loaded in a horizontal two-zonefurnace with the hot end temperature set to 1100 °C and the coldend set to 1000 °C, ramping at ∼1 °C min–1. The gradient was held for 9 days, after which the hot end was cooledto 900 °C and subsequently kept 100 °C lower than the coldend to minimize condensation of iodine vapor on crystals in the coldend. The cold end temperature was then lowered to 700 °C, thegradient was held for 2 days, and then the power to the furnace wasturned off. After cooling to room temperature, large, thin platelets were found dispersedacross the ampoule. The crystals were washed several times in eitheracetonitrile or ethanol to remove any excess iodine on the surface.Single crystals of Fe–1. Samples were measured in backscattering configuration using a ×100objective (N.A. 0.8) and measured with a laser power < 2 mW toavoid local heating effects. Measurements were performed using 10s acquisition time and 10 accumulations, and all spectra were acquiredwith an 1800 groove mm–1 grating.Confocal Ramanspectra were collected on a Horiba LabRAM HR Evolution spectrometerwith a 633 nm laser excitation source and the corresponding ultralow-frequency(ULF) notch filters, with an edge cutoff ∼10 cm–1 grating) coupled to a liquid-nitrogen-cooledCCD detector was used to collect the Raman data. The excitation wavelengthwas 632.8601 nm from a helium neon laser, and the spectra were collectedin the 180° backscattering configuration. Raman spectra as afunction of temperature and magnetic field were collected using anattoDRY cryostat , where the sample was zero-field-cooledand studied with a magnetic field-compatible objective . Ultra-broadband polarizers and achromatic half-wave plateswere used to select and control polarization, including correctingfor Faraday rotation in the objective under applied magnetic field.The laser power was ∼1.5 mW to avoid local heating, and integrationtimes were ∼20 min.A triple-grating Raman spectrometer  and energy-dispersive X-ray spectroscopy (EDX) were conductedon an FEI Quanta SEM with an Oxford EDX detector using 20 keV acceleratingvoltage and 4 nA beam current.xNbSe2 (>1 mm × 1 mm) with relatively flat surfaces wereselectedfor single-crystal X-ray diffraction (SCXRD). The x = 0.23 crystal was measured on a Rigaku XtaLab P200 diffractometerwith Mo Kα radiation at 293 K. Data reduction and scaling andempirical absorption correction were performed in CrysAlis Pro. UsingOlex2, the structure was solved with the SHELXT structure solutionprogram using Intrinsic Phasing and refined against F2 on all data by full-matrix least squares using the SHELXLrefinement package.16 The x = 0.14 and x = 0.19 crystals were measured on a Bruker AXS D8 Venture diffractometerunder the same conditions, and the structures were solved within theaccompanying Bruker APEX4 software program.Suitable single crystals of Fe0.14NbSe2 was mechanicallyexfoliated with scotch tape and transferred onto SiO2/Sisubstrates. Exfoliated flakes with thicknesses < 200 nm were identifiedusing optical microscopy and atomic force microscopy. Target flakeswere picked up with a poly(bisphenol-A-carbonate)(PC)/poly(dimethylsiloxane) (PDMS) stamp and transferred onto a 200-nm-thickamorphous Si3N4 membrane with 2-μm holesfor transmission electron microscopy (TEM) imaging. Selected areaelectron diffraction (SAED) was performed on an FEI TitanX TEM operatedat 80 kV, and data were collected along the [0001] zone axis of theFe0.14NbSe2 flakes. The obtained patterns correspondto a 720 nm sample region, defined using a 40 μm diameter SAEDaperture.A single crystal of Fe–6 emu. Crystals weremounted in polypropylene straws on crosses of cotton thread with thefield parallel to the c axis.DC magnetization measurementsas a function of temperature and applied field were carried out ina Quantum Design Physical Property Measurement System (PPMS) Dynacoolwith a 12 T magnet using the Vibrating Sample Magnetometer (VSM) option,with a detection limit of 10c crystallographic axis),(iii) fast cooling to 20 K above the target temperature (at 10 K min–1), (iv) slow cooling to the target temperature (at1 K min–1), (v) holding the sample in 1 T fieldfor 1 h, and (vi) setting the field to 0 T and measuring the remanentmagnetization in the sample over set time.Thermoremanentmagnetization (TRM) measurements were conducted in the Quantum DesignPPMS with VSM option using the following protocol: (i) warming thesample to 400 K in zero magnetic field, (ii) setting the field to1 T . Therange of Fe concentrations focused on in this work spanned from 0.14≤ x ≤ 0.23, highlighting samples withvarying amounts of Fe deficiency relative to the stoichiometric amountof x = 0.25—which is associated with formingan ideal 2a0 × 2a0 superlattice. Single-crystal X-ray diffraction (SCXRD)data were obtained for single crystals at x = 0.14,0.19, and 0.23 (P63/mmc. The out-of-plane lattice constant c was found to be 12.6011(9), 12.6174(8), and 12.6498(8)Å for x = 0.14, 0.19, and 0.23, respectively,which are all slightly larger than that for 2H-NbSe2 (c0 = 12.547(3) Å), suggestinga very minor expansion upon Fe intercalation.17 The in-plane lattice constant a, b for the x = 0.23 crystal was found to be 6.9151(4)Å, which is close to twice that of native 2H-NbSe2 (a0 = 3.4425(5) Å),and, similarly, the a, b latticeconstant for the x = 0.19 sample was determined tobe 6.9046(3) Å. For each of these two stoichiometries, the Feintercalants occupy octahedral interstitial sites between the NbSe2 layers (Wyckoff position 2a), forming anordered 2a0 × 2a0 superlattice Å, and no superlattice was observed from SCXRD, as the Fe intercalantswere best fit, on-average, to be weakly distributed among all octahedralsites. This global picture, however, does not preclude the possibilityof Fe intercalants locally ordering on shorter length scales. Therefore,to confirm whether the x = 0.14 sample exhibits morelocal superlattice formation, selected area electron diffraction (SAED)data were taken on exfoliated flakes from the same single crystal and zero-field-cooled(ZFC) traces. The Fe0.14NbSe2 sample, on theother hand, exhibits paramagnetic behavior down to approximately 20K followed by a broad transition below 20 K. There is also a substantialbifurcation in FC vs ZFC behavior for Fe0.14NbSe2 (12 Thermoremanent magnetization (TRM) measurements—whichare sensitive to glassy dynamics20—weresubsequently conducted on the Fe0.14NbSe2 sampleto probe for such glassy behavior . The onset of a nonzero remanent magnetization for temperatures≤ 10 K, with a concomitant slow decay in magnetization signalover time, corroborates the presence of a spin-glass-like phase below20 K.20 Overall, the lack of robust AFM orderingfor Fe0.14NbSe2 likely stems from a higher degreeof structural disorder, consistent with the SCXRD measurements, whichwould disrupt the uniformity of the magnetic exchange interactionsamong the spin-bearing Fe intercalants.To evaluatethe effect of Fe content on the magnetic ordering within this intercalationregime, magnetic susceptibility as a function of temperature was measuredupon warming for the same representative Fecrystals 2. As sho.14NbSe2 2c, which0.23NbSe2 crystal was found to be significantlyweaker than the respective out-of-plane magnetic susceptibility, signalingstrong magnetocrystalline anisotropy (MCA) for the out-of-plane c direction in this material. This MCA effect arises dueto unquenched orbital angular momentum, stemming from the unevenlyoccupied eg set in the d-electron configuration of octahedralhigh-spin Fe2+, and is consistent with other Fe-intercalatedTMDs.10 The high-spinFe2+ configuration is a valid approximation of the spinstate of the Fe intercalants in this material because the expectedeffective moment for octahedral high-spin Fe2+ (μeff = 4.90μB) matches closely with the experimentalμeff = 5.11μB calculated for theFe0.23NbSe2 sample, which was obtained basedon the slope of a linear fit to the paramagnetic regime within therespective inverse susceptibility (1/χ) vs temperature plot at 130 K. This trend in increasing ordering temperature correlateswith the increased degree of intercalant superlattice formation, basedon SCXRD, as Fe content increases, which is sensible given that awell-ordered intercalant superstructure should give rise to more uniformand robust magnetic exchange interactions between magnetic ions.Regarding other magnetic properties of these compounds,the in-planemagnetic susceptibility for the Fe crystalS2 was foxNbSe2 samples (H-NbSe2 (x = 0), which has Dh64 point group symmetry, the optical phonons at the Brillouin zone(BZ) center have the following irreducible representation: Γ= A1g + 2A2u + 2B2g + B1u + E1g + 2E2g + 2E1u + E2u.25 Of these, there are four Raman-active modes, namely, A1g + E1g + 2E2g. In Raman backscattering configurationalong the ZZ direction, in which these measurementswere conducted, the E1g mode should not be detected becausethe E1g Raman tensor (R1gE) for the P63/mmc space group does notcontain nonzero polarizability matrix elements for this sample geometry—therebyleaving only A1g and E2g modes to be observed.26 Therefore, for x = 0, the sharpmode at 28 cm–1 was assigned to the well-known interlayerE2g shear mode for 2H-NbSe2 and the peak at 228 cm–1 was assigned to the correspondingintralayer A1g mode.28 Another intralayer E2g mode is expected for 2H-NbSe2 at 237cm–1 though this feature was not readily apparentwith 633 nm excitation; however, upon switching to 532 nm excitation,this peak was later detected —consistent with prior literature comparing Raman scatteringof 2H-NbSe2 across different excitationwavelengths.27 The broad feature centeredaround 192 cm–1 for x = 0 was attributedto a soft phonon mode, related to charge–density wave interactions,which arises from a second-order Raman scattering process of two Brillouinzone-edge longitudinal acoustic phonons.31Raman scattering studieswere performed to further characterize and differentiate the structuralproperties between Fe samples 3. For thH-NbSe2 host lattice. The interlayer shear mode for NbSe2 at 28 cm–1 disappears with Fe intercalation,which can be attributed to Fe intercalants forming covalent bondswith the NbSe2 host lattice, thus precluding the NbSe2 layers—originally held together by weak van der Waals(vdW) forces—from exhibiting any strong interlayer shearingmotions. Most significantly, a series of new phonon modes emerge inthe intercalated compounds in the spectral region below 150 cm–1 as well as adjacent to the host lattice NbSe2 A1g Raman peak. As the Fe concentration increases,the new modes between 70 and 150 cm–1  can now be folded onto the zone-center Γ point of the superlatticeBZ (ΓSL).25 This new crystalsymmetry, therefore, folds these optical phonons with originally highcrystal momenta onto a new symmetry point with lower crystal momentum,thereby allowing incident photons with very little inherent momentumto interact with and scatter off these excited vibrational modes.25 Notably, the modes in this energy region appear more well resolvedas Fe concentration increases—similar to the evolution of thesuperlattice-related features below 150 cm–1—whichwould correlate with a progressively higher degree of intercalantordering and thus a higher degree of potential zone-folding effects.Aside from the new superlattice featuresbelow 150 cm2-related A1g mode upon Fe intercalation,with the A1g peak shifting from 228.0 cm–1 for x = 0–224.5 cm–1 for x = 0.14. This frequency red shift could be due to weakenedpolarizability around the Nb–Se vibrational mode as electrondensity gets redistributed away from Nb–Se bonds toward someFe–Se covalency upon initial Fe intercalation. Interestingly,this A1g peak blue-shifts back toward higher frequenciesas more Fe is introduced, shifting from 224.5 cm–1 for x = 0.14–226.0 cm–1 at x = 0.23. This diverging effect could stem froman increasingly higher filling level of the host lattice conductionband, which is primarily of Nb dz2 character,2 thereby restoringsome electron density around Nb–Se vibrational modes. A similarfrequency blue shift is observed for the Fe–Se-related modesabove 240 cm–1 as x increases from x = 0.14 to x = 0.23, which may also berationalized by a stiffening of the Fe–Se-related spring constantdue to increased charge transfer from intercalated Fe2+ ions to the Nb–Se host layer.33In regard to other changes, there is an additional redshiftingof the NbSe0.23NbSe2 system, polarized Raman measurementswere carried out by measuring Raman intensity as a function of rotationangle between incident and scattered light polarization vectors  expected at the zone-center ΓSL point are: Γacoustic = A2u + E1u and Γoptical = 4A1g + A1u + 2A2g + 5A2u + 5B1g + 2B1u + B2g + 5B2u + 5E1g + 7E1u + 7E2g + 6E2u.38 Based on the optical phononspresent, the expected Raman-active modes are 4A1g + 5E1g + 7E2g.38 As in the unpolarized Raman experiment, the backscatteringmeasurement geometry along the ZZ direction precludesthe detection of the E1g modes for materials with thisspace group symmetry.38To betterunderstand the symmetry of emergent Raman modes in the intercalatedFe vectors 4. Two li1g and E2g Ramanmodes, the relativeorientation between the incident and scattered light polarizationvectors determines which modes should appear most intensely in Ramanscattering at a given angle. In parallel configuration, in which thepolarization directions of the incident and scattered light are thesame  and the relativeangle between them is 0°, only modes that contain a nonzero polarizabilitymatrix element for αxx and αyy in the corresponding Raman tensors willbe detected. Therefore, when in this sampling configuration, bothtypes of modes should be observed, as the respective Raman tensors(R1gA and R2gE) under P63/mmc symmetry satisfy this criterion.38 Similarly, in cross-configuration, in which the relativepolarization vectors are orthogonal (90°) to one another , only phonon modes that containa nonzero polarizability matrix element for αxy and αyx should appear stronglyin Raman scattering. Consequently, only E2g modes shouldpersist in this sampling geometry because only the E2g tensor(R2gE) obeys this conditionunder P63/mmc symmetry.38For the A1g or E2g character of the emergentRaman modes in the intercalatedsystems. For Fe0.23NbSe2, all A1g and E2g modes appear together in parallel configuration(0°), consistent with the confocal Raman experiment. However,as the relative angle between the incident and scattered polarizationvectors changes from 0 to 90°, we observe the attenuation ofthe features at 125, 226, and 263 cm–1 and the prevalenceof the remaining modes at 97, 248, and 253 cm–1.Therefore, based on the governing polarization selection rules, theformer set of phonon modes can be ascribed to A1g symmetrywhile the latter can be assigned to E2g symmetry. Thereis some remaining intensity in the spectral region for the superlatticemodes around 125 cm–1 under cross-polarization,which could be due to a convolution of A1g and E2g modes pertaining to possibly nearly degenerate out-of-plane andin-plane intercalant superlattice vibrations.13Operating under this framework, we candiscern the A0.23NbSe2 crystal. Thetemperature-dependent Raman spectra, for both parallel and cross configurations causing a Zeeman splitting amongdegenerate magnon normal modes, leading to two magnon peaks symmetricallyoffset above and below the original peak energy in the Raman spectrum.46 In addition, unlike systems with strong magnetoelastic couplingsuch as FePS3, which undergoes significant changes in theunit cell and Brillouin zone upon magnetic ordering and, concomitantly,exhibits emergent phonon modes due to zone-folding effects,42 there does not appear to be such a symmetry effect in Fe0.23NbSe2 when it orders antiferromagnetically. This lackof an effect below the Néel temperature may be due to Fe0.23NbSe2 not undergoing a change in symmetry tothe superlattice Brillouin zone above and below the magnetic transition,as is the case with FePS3.40 As such, at least within this intercalation regime for x < 0.25, there is no observation of significant magnetoelasticeffects as probed by Raman scattering. Future experimental work onmore Fe-rich intercalation compounds in the x = 0.33regime, which possess a different space group symmetry and intercalantsuperlattice structure compared to the x = 0.25 analogues,may reveal whether such systems are more amenable to hosting suchphenomena—potentially due to more compatible zone-folding effectsupon magnetic ordering from the interplay between crystal symmetryand relative antiferromagnetic spin configurations.Magnetic field-dependent Raman spectra were acquiredat 1.6 K,with applied field along the magnetic easy axis, to probe the presenceof antiferromagnetic magnons as well as strong magnetoelastic couplingat large in the crystal. We do not observe any signatures of antiferromagneticmagnons, which would be characterized by an applied magnetic field(xNbSe2 for off-stoichiometriciron amounts x = 0.14, 0.19, and 0.23 suggest theemergence of intercalantsuperlattice formation in iron-deficient samples as low as x = 0.19. These superlattices can be conveniently probedby confocal Raman scattering, and the symmetry of these new modescan be distinguished by angle-polarized Raman spectroscopy. The natureof these superlattices in Fe-deficient compounds between 0.19 ≤ x ≤ 0.23 is likely based on a progressively lessdefective 2a0 × 2a0 superstructure approaching the ideal packing at x = 0.25. In Raman scattering, three intercalant superlatticemodes emerge at 97, 113, and 125 cm–1 at x = 0.19 and become more intense and well resolved as Feconcentration increases to x = 0.23. Future studieswill aim to map out the Raman-active modes for higher intercalationamounts in the FexNbSe2 family,namely, for 0.25 ≤ x ≤ 0.33 and for x > 0.33, in which a crossover to a noncentrosymmetric P6322 space group is expected and thus a √3a0 × √3a0 superlattice should be observed.33 Assessing the extentof magnetoelastic coupling in the intercalation regime near x = 0.33 would also lend valuable insight as to whetherfundamentally new phonons can emerge from zone-folding and coupledirectly with the spin ordering in the system. Furthermore, examininghow the modes for a different superlattice prevail within FexNbSe2 will play a key role in screeningfor different superlattice formation within a given crystal, providinga road map for probing and exploiting local disorder and coexistingstructural domains within this magnetically intercalated TMD family.Overall, the structural and magnetic propertiesof Fe


Candida albicans is an opportunistic human pathogen that colonizes the gastrointestinal and genitourinary tracts of healthy individuals. C. albicans yeast cells can switch to filamentous forms. On biotic and abiotic surfaces, the planktonic free-floating yeast cells often form biofilms, a multi-drug-resistant three-dimensional community of yeast and filamentous cells. While alterations in gene expression patterns during planktonic to biofilm growth transitions in C. albicans have been studied, the underlying molecular mechanisms largely remain unexplored. Previously, we identified a histone H3 variant (H3VCTG), which acts as a negative regulator of biofilm growth in C. albicans. In the current study, we performed genome-wide profiling of H3VCTG nucleosomes in C. albicans planktonic cells and found them to be enriched at promoter regions. In planktonic cells, H3VCTG-enriched regions are mostly devoid of histone H3 post-translational modifications that allow active transcription, thus strengthening the role of H3VCTG as a negative regulator of biofilm formation. By combining genome-wide transcriptional alterations, nucleosome positioning (MNase-seq), and DNA accessibility (ATAC-seq) assays, we show a significant reduction in the total number of nucleosomes in biofilm cells as compared to planktonic cells indicating a more open chromatin state during biofilm growth. Finally, we propose that H3VCTG-nucleosome eviction at promoters of biofilm-relevant genes in biofilm-grown cells contributes to achieve the open chromatin state by facilitating easy promoter access of master regulators (activators and repressors) for modulation of gene expression observed during growth phase transitions.Candida albicans lives as a commensal in most healthy humans but can cause superficial skin infections to life-threatening systemic infections. C. albicans also forms biofilms on biotic and abiotic surfaces. Biofilm cells are difficult to treat and highly resistant to antifungals. A specific set of genes is differentially regulated in biofilm cells as compared to free-floating planktonic cells of C. albicans. In this study, we addressed how a variant histone H3VCTG, a previously identified negative regulator of biofilm formation, modulates gene expression changes. By providing compelling evidence, we show that biased eviction of H3VCTG nucleosomes at the promoters of biofilm-relevant genes facilitates the accessibility of both transcription activators and repressors to modulate gene expression. Our study is a comprehensive investigation of genome-wide nucleosome occupancy in both planktonic and biofilm states, which reveals transition to an open chromatin landscape during biofilm mode of growth in C. albicans, a medically relevant pathogen. Candida albicans is one of the most prominent fungal species in the gut microbiome of healthy individuals and lives as a commensal on the mucosal membranes of the human reproductive system, gastrointestinal tract, urinary tract, mouth, skin, and other organs  genes was found to be significantly higher as compared to the canonical histone H3 in cells grown in planktonic condition. Indeed, when cells were shifted from planktonic to biofilm mode of growth, the occupancy of H3VCTG consequently reduced at these promoter regions. Furthermore, absence of H3VCTG resulted in an altered expression of genes responsible for biofilm formation, even in planktonic cells  followed by +1, +2, +3, etc., nucleosomes positioned after the TSS. The nucleosome-depleted region (NDR) is located between the −1 and +1 nucleosomes and serves as the site for recruitment of transcriptional machinery. It has been observed that the width of NDR is narrower and more confined during inactive transcription but expands and becomes wider during active transcription  which aid in the functional segregation of the genome . MNase is both an endo- and exonuclease which can readily cleave free linker DNA but not the DNA sequences bound to a protein (Next-Generation Sequencing (NGS) to identify genome-wide nucleosome positioning in a population of cells; this technique is known as MNase followed by high-throughput sequencing (MNase-seq) (assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) assist in precisely predicting the open chromatin regions of the genome, not bound by histones or non-histone proteins  . Alternaproteins . BrieflyC. albicans. High-throughput sequencing methodologies were used to determine the nucleosome occupancy, open chromatin regions, and gene expression alterations when cells were grown either in planktonic yeast form or induced to produce biofilms. We observed a significant reduction in the total number of nucleosomes, resulting in a more open chromatin landscape in biofilm cells compared to planktonic cells. Strikingly, we observed that gene regulation is modulated by the eviction of a previously identified histone H3 variant (H3VCTG), a repressor of biofilm growth in C. albicans.In this study, we sought to address the role of nucleosome occupancy in the context of planktonic to biofilm morphological transitions in CTG nucleosomes in planktonic cells as compared to H3 nucleosomes at promoters of a set of BR genes (MNase digestion followed by chromatin immunoprecipitation and next-generation sequencing (MNase ChIP-seq) for H3VCTG by tagging both alleles of the HHT1 gene with a V5-epitope in planktonic cells of C. albicans  start site for each of these genes and plotted  analysis of two biological replicates of the wild-type C. albicans SC5314 strain grown in either planktonic or biofilm conditions  were found to have an altered expression based on log2(fold change) values of >1 and <−1, with a false discovery rate of 5% in our analysis  (ALS2)   histone proteins in C. albicans.To define the state of chromatin at promoters of BR genes, we performed the nditions . Approxianalysis . Out of lm cells  were found CPH1) , 23, cel) (ALS2) , and extd SAP10) , 26 haviand TPK1 –29. StCTG in the planktonic cells and its effect on gene expression, we generated average nucleosome occupancy profiles focusing on the promoter regions of BR genes. Since the TSS data were only available for 737 upregulated BR genes and 694 downregulated BR genes, we performed further analysis using this set of 1,431 out of 1,628 differentially regulated genes. Remarkably, biased enrichment of H3VCTG nucleosomes was evident at promoters of genes with altered expression across the two conditions as compared to genes with unaltered expression  and acetylated at the ninth lysine residue (H3K9ac) for this analysis along with RNA polymerase II (RNA pol II)  . To deteic cells  into thric cells . RNA pol H3 PTMs . Genes u H3 PTMs . In concC. albicans SC5314 cells were grown under planktonic conditions , which further confirmed that promoter regions are largely devoid of nucleosomes at varying extent . As expeC. albicans modulated by nine master regulators, viz., Bcr1, Tec1, Ndt80, Efg1, Rob1, Brg1, Gal4, Rfx1, and Flo8 (transcription factor binding sites (TFBS) across the two modes of C. albicans growth, we obtained the data for binding sites of six of these master regulators in biofilm cells from a previously published report  for presence of H3VCTG nucleosomes and observed an increased deposition of H3VCTG at these TFBS in planktonic cells  of BR genes that undergo nucleosome eviction , further exerting its previously established role as the negative regulator of biofilm formation. By integrating the multi-omics approach, we investigated the impact of nucleosome positioning on gene expression modulation upon biofilm induction and characterized the chromatin architecture at promoters of BR genes. Our findings revealed a decrease in nucleosome occupancy and presence of an open chromatin state at promoters of BR genes in biofilm cells. Importantly, we demonstrated that the increased chromatin accessibility facilitates binding of transcriptional modifiers (activators and repressors), thus exerting control over gene expression. Additionally, we observed preferential eviction of H3VCTG nucleosomes from promoters of BR genes in biofilm cells. Altogether, we uncovered the mechanism by which H3VCTG helps in achieving a specific growth mode, whether planktonic or biofilm state, by its preferential binding at promoters of BR genes.In this study, we performed genome-wide identification of H3VC. albicans  was amplified using 6791V5-CFP and 6791V5-CRP primers and cloned in pBS-NAT  (HHT1-V5-NAT fragment from pRA01 using primer pairs 6791V52LFP and RA1. Transformants were selected for nourseothricin (100 µg/mL) resistance and confirmed by Southern blotting. Oligos used for strain construction are listed in The C-terminal of H3V pBS-NAT  plasmid -V5-HIS)  was perfC. albicans cells was grown overnight in YPD and harvested by centrifugation at 3,800 rpm for 5 min. Cells were washed with autoclaved water and resuspended in 200-µL extraction buffer. Cells were lysed by vortexing with 0.3 g of acid-washed glass beads and 200 µL of phenol:chloroform:isoamyl alcohol (25:24:1). Next, 200 µL of 1× Tris-EDTA (TE) solution was added to the tubes followed by centrifugation at 13,000 rpm for 10 min. The supernatant was collected, and DNA was precipitated by 100% ethanol at −20°C for 1 h. The tubes were centrifuged at 13,000 rpm, and the pellet obtained was washed with 70% ethanol. The pellet was air dried and resuspended in 30 µL of 1× TE and 1-µL RNase A (10 mg/mL).Ten-milliliter culture of C. albicans strains were grown overnight in YPD, and 6 × 107 cells were harvested by centrifugation at 3,800 rpm for 5 min. Cells were washed with 1 mL of autoclaved water and resuspended in 400 µL of 12.5% trichloroacetic acid solution. The cell suspension was frozen at −20°C overnight. Next day, the cells were thawed on ice and pelleted at 13,000 rpm, 4°C for 10 min. The pellet was washed in 80% acetone solution followed by pelleting at 13,000 rpm, 4°C for 10 min. After two rounds of washing, the pellet was air dried and resuspended in 50 µL of lysis buffer (1% SDS + 0.1 N NaOH) with 1× loading dye. The lysate was incubated in a boiling water bath for 10 min. Proteins were separated by electrophoresis on a 12% SDS-PAGE gel and blotted onto a nitrocellulose membrane  in a semi-dry apparatus (Bio-Rad). The blotted membranes were blocked by 5% skim milk containing PBS (pH 7.4) for 30 min at room temperature. The membranes were then incubated with 1:5,000 dilution of anti-V5-antibodies  or 1:5,000 dilution of anti-PSTAIRE antibody  in 2.5% skim milk in PBS at 4°C overnight with constant shaking. Next, the membrane was washed thrice with PBST (0.1% Tween-20 in 1× PBS) solution. Anti-mouse HRP-conjugated antibodies  were added at a dilution of 1:5,000 in 2.5% skim milk PBS solution and incubated for 1–2 h at 4°C on constant shaking. The membranes were then washed thrice with the PBST solution. Signals were detected using the chemiluminescence method.α32P-dCTP using a random primer labeling kit (BRIT-LCK-102). Fifty-nanogram probe DNA in an appropriate volume of autoclaved water was boiled for 5 min and chilled immediately. Next, 5-µL random primer buffer, 5-µL random primer, 12-µL dNTP cocktail except dCTP, 4-µL α32P-dCTP, and 2-µL Klenow polymerase were added to the reaction mixture followed by incubation at 37°C for 40 min. The reaction was stopped by 2-µL 0.5 M EDTA, and volume was made up to 100 µL by adding 1× TE solution. The pre-hybridized blot was then incubated with the radiolabeled probe for 16 h at 65°C. The blot was washed by wash buffer I  twice for 30 min each at 65°C and wash buffer II  thrice for 20 min each at 65°C. The membrane was exposed to Phosphor imager film, and the image was captured by Phosphorimager.RA107 strains were confirmed using Southern hybridization. Genomic DNA was isolated and quantified using Quantity One software (Bio-Rad). Six to eight micrograms of DNA was digested and separated on an agarose gel. The gel was sequentially treated with 0.5 N HCl for acid nicking of DNA at room temperature for 10 min. This was followed by denaturation and neutralization of DNA using denaturation buffer (0.5 N NaOH and 1.5 N NaCl) and neutralization buffer , respectively, for 30 min each at room temperature. Acid-nicked DNA was transferred by capillary action onto a positively charged nylon membrane  in 10× saline-sodium citrate (SSC) buffer at room temperature for 12–16 h. Following transfer, the membrane was washed with 2× SSC for 15 min, dried, and crosslinked by exposure to UV for 5 min in Genelinker . The blot was pre-hybridized in 10-mL 1× Southern hybridization buffer  at 65°C for 2–4 h. The specific probe amplified using 6791SUSFP/6791USNAT1RP primers was radiolabeled with Primers designed for quantitative PCRs (qPCRs)  for 4–12 h before the experiment and washed with 2-mL autoclaved 1× PBS solution. A total of 0.5 OD equivalent cells/mL were added from the overnight culture, and the volume was made up to 3 mL with YPDU media. The polystyrene plate was incubated at 37°C with agitation at 100 rpm for a period of 1.5 h (adherence step). Floating cells and excess media were removed, and wells were washed with 1× PBS solution. Three milliliters of fresh YPDU media was added to the wells and incubated at 37°C for 48 h at 40 rpm agitation (maturation step). The biofilm cells were scrapped, and the content of each well was transferred to a 50-mL tube for downstream processing of the samples.C. albicans cells, 100 OD equivalent cells were grown in planktonic and biofilm conditions and harvested. Cells were crosslinked with formaldehyde  for 15 min at room temperature (RT). The reaction was stopped using 125 mM glycine and incubated at RT for 5 min. Fifty OD cells were washed with autoclaved water and treated with resuspension buffer (0.5-mL β-mercaptoethanol and 9.5-mL water) at 30°C for 30 min at 180 rpm. Cells were washed once with 5-mL S-buffer  and resuspended in the same buffer. A 10 µg/mL of Zymoylase 20T  was used for spheroplasting the cells and incubated at 30°C at 60 rpm for 45 min for planktonic cultures and 90 min for cells grown in the biofilm condition. After 90% spheroplasting was achieved, spheroplasts were washed twice with ice-cold 5-mL S-buffer at 4,000 rpm at 4°C. Ten milliliters of lysis buffer  was mixed thoroughly at high speed using a magnetic bead for 5 min at 4°C. Out of the 10-mL lysis buffer, 2 mL was used to resuspend the spheroplasts. Clumps were carefully removed to make the suspension consistent. Spheroplasts were poured into the remaining lysis buffer in a drop-wise manner, and stirring was performed at a minimal speed for 15 min at 4°C. The suspension was poured into a fresh 50-mL falcon and gently vortexed for 3 min at 4°C. Glycerol-Ficoll cushion  was prepared and poured into a 50-mL oakridge tube. This was pre-chilled before usage. The spheroplasts were laid on top of the Glycerol-Ficoll cushion and centrifuged at 12,500 rpm for 40 min at 4°C. The supernatant was gently decanted, and the pellet (nuclei) fraction was gently resuspended in 1-mL ice-cold PC buffer .For nuclei isolation from 3 and 1% SDS) and incubating at 65°C overnight. This was followed by Proteinase K treatment by adding 10 mM EDTA (pH 8.0), 4 mM Tris-Cl buffer (pH 6.8), and 2 µL of 20 mg/mL of Proteinase K enzyme. Incubation was performed at 37°C for 2 h. Phenol:chloroform:isoamyl alcohol was added to the solution in a 1:1 ratio and vortexed vigorously, followed by centrifugation at 13,500 rpm for 15 min at RT. The aqueous layer was gently aspirated in a fresh microfuge tube. This was followed by DNA precipitation by adding 1 µL of glycogen, 70 µL of 3 M Na-acetate, and one volume of absolute ethanol and stored at −20°C overnight. Centrifugation was performed at 14,000 rpm for 30 min at 4°C. The DNA pellet was washed using 70% ethanol and resuspended in 18 µL of water. Two microliters of RNase A (10 mg/mL) was added for degrading the RNA molecules, and the chromatin integrity was checked on 2% agarose gel. The isolated DNA fragments were then used for semi-quantitative and qPCR analysis.MNase digestion and chromatin extraction were performed as previously described  with somFor the chromatin immunoprecipitation (ChIP) assay, the nuclei were isolated, and MNase digestion was performed as mentioned previously. The MNase reaction was stopped using 2 mM of EGTA and 2 mM EDTA, and the reaction mix was incubated on ice for 15 min. The chromatin extraction was carried out by passing the MNase-digested nuclei fraction through a 24-gauge needle five times and a 30-gauge needle three times. This was followed by centrifugation at 13,000 rpm for 30 min at 4°C. The supernatant was aliquoted in a fresh microfuge tube, and 100 µL of the fraction was aliquoted as the input sample (processing for de-crosslinking mentioned below). The rest of the sample was divided into two fractions: IP (+ antibody) and mock (− antibody). To the IP fraction, 3 µL of anti-V5 antibody  was added, and the tubes were incubated at 4°C overnight on rotation. A 30-µL of Protein-A beads  was added to IP and mock samples and incubated again at 4°C for 8–12 h on rotation. The beads were then washed twice with 1 mL each of low salt, high salt, LiCl wash buffers, and 1× TE solution. The samples were rotated in a rotaspin for 15 min at RT for every wash. After washing, the DNA was eluted from the beads twice using 250 µL of elution buffer. The samples for elution were incubated at 65°C for 5 min, rotated for 15 min at RT, and collected by centrifugation. The samples were processed for de-crosslinking by adding 20 µL of 5 M NaCl in the elution buffer to each tube and incubated at 65°C overnight. This was followed by the removal of proteins by adding 10 µL of 0.5 M EDTA, 20 µL of 1 M Tris-Cl (pH 6.8), and 2 µL of Proteinase K (20 mg/mL). This reaction mix was incubated at 37°C for 2 h. After incubation, samples were treated with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) mix, and the aqueous phase was extracted by centrifugation. DNA was precipitated by the addition of one-tenth volume of 3 M Na-acetate, 1-µL glycogen , and 1-mL absolute ethanol and incubated at −20°C for at least 12 h. The samples were centrifuged at 14,000 rpm for 45 min at 4°C followed by washing with ice-cold 70% ethanol. Seventy percent ethanol was decanted, and the DNA pellets were air dried. The input sample was resuspended in 25 µL of autoclaved MilliQ water and 5 µL of 10 mg/mL RNase A. The resuspended input sample was incubated at 37°C for 15 min before usage. The IP samples were resuspended in 20-µL MilliQ water. The ChIP was confirmed using RS219/RS220 primers and sent for sequencing.C. albicans wild-type cells SC5314 grown in planktonic and biofilm conditions. The isolated nuclei were counted using a hemocytometer, and 105 nuclei equivalent were spun down at 4,000 rpm for 5 min at 4°C. The supernatant was carefully removed using a pipette, and the nuclei pellet was resuspended in transposition mix as follows: 5 µL of 2× TD Tagment DNA buffer , 1 µL of TDE1 enzyme , and 4 µL of water. The tagmentation reaction was carried out for 60 min at 37°C, and 10 µL of water was added post-incubation. For enrichment of tagmented DNA, PCR was set using 27.5 µL of KAPA2G Robust HotStart ReadyMix  and 2.5 µL of 25 µM each of Ad1_moMX/Ad2.1 primer pair. The PCR was carried out for seven cycles as follows: 72°C for 5 min, 98°C for 30 s, 98°C for 10 s, 63°C for 30 s, and 72°C for 60 s. The enriched tagmented DNA was size selected in two steps for <600 bp and >50 bp fragments using AMPure SPRI beads . The DNA fragments were finally eluted in 20-µL 10 mM Tris-Cl (pH 8.0) and sent for Illumina sequencing.For the ATAC assay, the protocol was modified from the published literature , 37. NucC. albicans SC5314 cells grown under planktonic conditions were obtained from a previous study using the accession number GSE21960  using bowtie2 software  of C. albicans genome was used from a previous publication  using bowtie2 software  of 5% by using the Benjamin-Hochberg method. Only the genes showing a q-value <0.05 and log2(fold change) of >1.0 and <−1.0 were considered significantly upregulated or downregulated, respectively.Raw values for microarray analysis were obtained from a previous publication . The staCTG MNase ChIP-seq raw reads were generated in-house from cells grown in planktonic condition. The raw reads were trimmed using the fastp tool  for downstream analysis. The log2 ratio of IP/Input was obtained using the bamCompare option from deepTools (CTG MNase ChIP-seq (input and IP files) using the bamCoverage option from deepTools software (CTG-containing nucleosomes for eviction analysis were called using nucleR (The RNA polymerase II, H3K4me3, and H3K9ac ChIP-seq data sets were obtained from a previous publication . The H3Vstp tool . The trisoftware . This wasoftware . PCR andsoftware . The DNAeepTools  softwaresoftware . H3VCTG-g nucleR  as mentiP-values ≥0.05 were considered nonsignificant. Precise P-values of the corresponding figures are mentioned in the figure panels or the figure legends, if significant. All statistical analyses were conducted using GraphPad Prism software (The statistical significance of differences was calculated as mentioned in the figure legends. software .


Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.The online version contains supplementary material available at 10.1186/s12974-023-02852-4. Flavivirus in the family Flaviviridae, is a zoonotic mosquito-borne pathogen that causes an acute inflammatory disease in brain ). The relative enrichment levels indicate the fold changes over the IgG control. The following primers were used in the ChIP assays: Rnf19a forward, GCTGCCCTCAGACTGTCATAC; Rnf19a reverse, TGTCTCCCTTCTGTTGGGGT; Cd2ap forward, CCCTGGGCCAAGCATATTCA; Cd2ap reverse, AGGATCCTGTTGCAGTGTCG; Ipo11 forward, GCATCTCTGAAACGTTATC; Ipo11 reverse, CACTGCAAAGGCTGCCCACC.ChIP assays followed the reported protocol by Giaimo and Huang. In brief, BV2 cells in 10-cm tissue culture dishes were infected with JEV. At 24 hpi, cells were harvested and cross-linked with 1% formaldehyde for 15 min at room temperature. The cross-linking reaction was stopped by addition of 125 mM glycine  for 5 min, and cells were chilled on ice. Then pellet cells by centrifugation for 5 min at 4 °C and ~ 271 × g and wash with 1 × phosphate-buffered saline (PBS). The cell pellet was resuspended in 1 × SDS lysis buffer  and incubated on ice for 10 min. The lysates were sonicated for 2 min to fragment DNA to an average length of 400–800 bp, and subsequently diluted to tenfold with 1 × ChIP buffer . Cellular debris was spun down, and the supernatant was diluted tenfold with 1 × ChIP dilution buffer  and incubated with 5 μg of antibodies against H3K27me3 or control rabbit IgG antibody, with rotation overnight at 4 °C. Protein A/G beads (Invitrogen) were pre-blocked with 0.5 mg/mL BSA and 0.125 mg/mL calf thymus DNA for 1 h at 4 °C, and then added to the lysate–antibody mixture for another incubation for 2 h at 4 °C. The protein A-Sepharose beads were recovered by centrifugation (1280 × g for 4 min) and washed with 1 mL of each of the following wash buffers in the following order: (i) once with low-salt immune complex wash buffer ; (ii) once with high-salt immune complex wash buffer ; (iii) once with LiCl buffer ; and (iv) twice with TE buffer . Immunoprecipitated protein–DNA complexes were eluted twice with fresh elution buffer  according to the instructions of the manufacturer’s protocol and the paired-end 150-bp sequencing was conducted by MGISEQ-2000RS (BGI). The low-quality reads and adaptor sequences were removed by Trimmomatic (v0.39) software. Then the clean reads were mapped to the mouse mm10 genome by bowtie2 (2.3.4) with the default parameters. Samtools (v1.7) was used to filter out the unmatched reads and the read duplicates were removed by Picard tools (v2.18) was used to remove the duplicated reads. MACS2 (v2.2) was utilized to call peaks and the differential peaks were found by the R packages DiffBind (v3.0.9). Differential ChIP-seq peaks were selected based on the following criteria: |log2 fold change|≥ 1 and FDR ≤ 0.05. The heatmap and volcano plots were generated using the R package pheatmap (v1.0.12) and ggplot2 (v3.2.1) for visualizing the different genes. The peaks were annotated by ChIPseeker (v1.26) for gene category analysis and Clusterprofiler (v3.12.0) for gene function annotation such as KEGG and GO analysis.The viability of cultured cells was detected using the Cell Titer-Glo One Solution Assay kit (Promega) according to the manufacturer’s instruction. The quantification of luminescence signals was calculated based on the reported method .HEK-293T cells were transfected with the indicated combinations of plasmids and collected 48 h after transfection. Cells were lysed for 30 min with 500 μL lysis buffer containing protease inhibitor. A part of cell lysates (40 μL) were subject to immunoblot assay to detect the expression of target proteins, and the rest of cell lysates were incubated with IgG and protein A + G agarose to remove nonspecific reaction. The supernatant after centrifugation was incubated with DDDDK-Tag antibody and protein A + G agarose overnight at 4 °C. The unbound proteins and antibodies in agarose beads were washed by PBS five times and eluted with 2 × SDS loading buffer by boiling for 5 min. Proteins isolated from the beads and the cell lysates were subjected to immunoblot assay.To analyze the effect of Rnf19a on the ubiquitination of RIG-I in transfected cells, HEK-293T cells were transfected with pCAGGS-Rnf19a, pCAGGS-Flag-RIG-I and HA-tagged ubiquitin. The proteasome inhibitor MG132 was added 24 h after transfection for 4 h, and the remaining steps are roughly the same as the co-immunoprecipitation steps described above.HEK-293T cells were seeded in a 48-well plate and transfected with the indicated plasmid and a luciferase reporter plasmid luc-NF-κB together with pRL-TK (Renilla luciferase plasmid) as a control reporter vector. At 48 h post-transfection, the cells were disrupted with lysis buffer (Promega), and luciferase activity was measured by the Dual-Luciferase Reporter Assay (Promega) on a Synergy 2 microplate reader (BioTek). The results were calculated by normalization of firefly luciferase activity to Renilla luciferase activity.t-test for two groups.All results are expressed as mean ± SEM. Statistical analyses were performed by using the GraphPad Prism 6 software. Statistical significance was determined with a two-sided unpaired To study the effect of JEV infection on H3K27me3 modification in microglia, BV2 mouse microglial cell line and primary mouse microglial cells were infected with JEV at MOI of 5. The productive infection of JEV in both cells was determined by plaque assay  and subjected to plaque assay and immunoblotting assay. The results showed that JEV efficiently replicated in the mouse brain   BV2 cell line was generated by using CRISPR–Cas9 approach Fig. E. It wasTo explore whether H3K27me3 modification promotes JEV-induced inflammatory response via inhibiting Rnf19a expression, Rnf19a KD and NC BV2 cells were treated with EZH2 inhibitor, following JEV infection, and the mRNA abundance of pro-inflammatory cytokines was determined by qRT-PCR. An inhibition of the downregulation of inflammatory cytokines caused by GSK343 treatment after JEV infection was observed in Rnf19a KD cells  E3 ligases which has been reported to be involved in inflammation regulation, cancers, spermiogenesis, neurogenesis, synaptic plasticity and contextual fear memory –42, was Our research has also observed an intriguing phenomenon that Rnf19a is capable of degrading RIG-I through both the proteasome and lysosome pathways. Previous reports have indicated that ubiquitin ligases, such as Nedd4, can also facilitate protein degradation through the endosomal–lysosomal pathway . AdditioRIG-I, as a cellular pattern recognition receptor, has been known to play a decisive role in recognizing genomic RNAs of flavivirus and thus inducing the production of interferon which may restrict the viral replication . HoweverThis study highlights the potential for regulating the neuroinflammatory response caused by JEV infection through H3K27me3 modification in microglial cells. The research also found that increased H3K27me3 modification during JEV infection leads to downregulation of Rnf19a, which acts as a negative regulator of inflammation by degrading RIG-I. These findings provided a fresh perspective on the mechanism behind JEV-induced overactivation of neuroinflammation, viewed through an epigenetic lens, and suggest a potential therapeutic target for Japanese encephalitis.Additional file 1: Figure S1. JEV multiplication kinetics in BV2 cells, primary microglial cells and BALB/c mouse brains. BV2 cells (A) and primary mouse microglial cells (B) were infected with JEV at an MOI of 5. At 6, 12, 18, 24 and 30 hpi, viral titers were measured by plaque assay. (C) 6-week-old BALB/c mice (n = 9) were intracerebrally injected with 200 PFU of JEV P3 strain in 20 μL DMEM or equal amount of DMEM (mock infection). Mouse brain tissues were collected at 1, 3 and 5 dpi and subjected to plaque assay. Each dot represents a single mouse.Additional file 2: Figure S2. JEV productively infects cortex and hypothalamus regions in mouse brain. 6-week-old BALB/c mice (n = 9) were intracerebrally injected with 200 PFU of JEV P3 strain in 20 μL DMEM or equal amount of DMEM (mock infection). Mice brain tissues were collected at 1, 3 and 5 dpi and subjected to IHC assay by using anti-JEV E monoclonal antibody. JEV E positive cells appear red while nucleus is purple. Scale bar, 200 μm.Additional file 3: Figure S3. Cell viability determination of EZH2 knockdown and Rnf19a knockdown BV2 cells.Additional file 4: Figure S4. Effect of EZH2 and Rnf19a knockdown on production of pro-inflammatory cytokines in supernatant of JEV-infected cells. EZH2 (A) or Rnf19a (B) knockdown (KD) or negative control (NC) BV2 cells were infected with JEV at an MOI of 5. The cell-supernatant was harvested at 36 hpi and the concentrations of TNF-α, CCL5 and IL-6 were determined by ELISA kits. Data are expressed as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Additional file 5: Figure S5. Effect of EZH2 knockdown, GSK343 and Rnf19a knockdown on JEV replication in BV2 cells and primary mouse microglial cells. (A) EZH2 KD and NC BV2 cells were infected with JEV at an MOI of 5. The viral titers were measured by plaque assay at 6, 12, 18, 24 and 30 hpi. (B) Primary microglial cells were treated with GSK343 (10 μM) following JEV infection at an MOI of 5. The viral titers were measured by plaque assay at 6, 12, 18, 24 and 30 hpi. (C) Rnf19a KD and NC BV2 cells were infected with JEV at an MOI of 5. The viral titers were measured by plaque assay at 6, 12, 18, 24 and 30 hpi.Additional file 6: Figure S6. Cell viability determination of GSK343-treated in BV2 cells (A) and primary mouse microglial cells (B).Additional file 7: S7.