For the clinical verification of the SFN, the “Small Fiber Neuropathy Screening List” (SFN-SL) was used. A punch biopsy of the skin was performed followed by enzyme immunoassay analysis with PGP 9.5 antibodies. Up to 60% of the sarcoidosis patients reported the presence of at least one complaint, and it was possible that these complaints were associated with SFN. The most frequent complaints included dysfunctions of the cardiovascular and musculoskeletal systems and the gastrointestinal tract. A negative, statistically significant correlation between the intraepidermal nerve fiber density (IEND) and SFN-SL score was revealed. In patients with pulmonary sarcoidosis, small fiber neuropathy might develop as a result of systemic immune-mediated inflammation. The most common symptoms of this complication were dysautonomia and mild sensory dysfunction.Sarcoidosis (SC) is a granulomatous disease of an unknown origin. The most common SC-related neurological complication is a small fiber neuropathy (SFN) that is often considered to be the result of chronic inflammation and remains significantly understudied. This study aimed to identify the clinical and histological correlates of small fiber neuropathy in sarcoidosis patients. The study was performed in 2018–2019 yy and included 50 patients with pulmonary sarcoidosis ( In patients with sarcoidosis, the development of systemic inflammation and internal organ dysfunction are observed, which significantly reduces quality of life and worsens patient prognosis. One of the most common complications is considered to be small fiber neuropathy (SFN), a condition that remains significantly understudied ,2,3,4. QThe development of SFN is considered to be the result of cytokine-mediated inflammation, which is typical for various autoimmune diseases, including sarcoidosis ,6,7,8. SThe prevalence of sarcoidosis varies throughout the world. In Japan, there is 1 case per 100,000 people, while in Scandinavian countries, the prevalence of sarcoidosis is as much as 63 cases per 100,000 people. In the Russian Federation, sarcoidosis is described to have a prevalence of 22 to 47 cases per 100,000 people, depending on the region ,13,14,15Currently, there are no generally accepted criteria for the diagnosis of SFN. The presence of a small nerve fiber dysfunction in a patient is mainly based on clinical criteria, such as a neurological examination and validated scales  ,21,22,23A prospective comparative study was performed in 2018–2019 yy at the St. Petersburg Research Institute of Phthisiopulmonology and at the St. Petersburg City Public Health Institution “City Multi-disciplinary Hospital No. 2”. The study was approved by the independent ethics committee of the St. Petersburg Research Institute of Phthisiopulmonology (extracted from protocol No. 46.1 of 04/20/2018). All of the study participants signed an informed consent.n = 25, average age 33.4 ± 8.5 y.o.) and healthy subjects . There were no statistically significant differences in gender and age in the patient groups. The inclusion criteria were age between 18 and 65 y.o. and signing the informed consent to participate in the study. The patients from group 1 were diagnosed with stage I-II sarcoidosis. The diagnosis of pulmonary sarcoidosis was performed according to the criteria of the American Thoracic Society (ATS), the European Respiratory Society (ERS), and the World Association of Sarcoidosis and Other Granulomatous Diseases (WASOG).The study included 50 patients with lung sarcoidosis and healthy subjects (average age was 38.4 ± 7.2 y.o.). The first group consisted of patients with lung sarcoidosis , the presence of other infectious diseases , a history of cancer that was treated with chemotherapy, the presence of other diseases , the use of medications  as well as a medical history of autoimmune diseases.According to the design of the study, the patients with sarcoidosis underwent a routine examination using X-ray, morphological, bacteriological, and molecular genetic methods. A neurological examination assessing superficial and deep pain sensitivity, muscle strength, and muscle-tonic reflexes was performed. For the clinical verification of the SFN, the validated questionnaire for the detection of small fiber neuropathy was used . The qA total of 23 patients with lung sarcoidosis agreed to undergo a skin punch biopsy , which was then fixed in n Zamboni solution, and the performance of the enzyme immunoassay with the primary PGP 9.5 antibodies (Abcam) and the secondary AlexaFluor goat-anti rabbit antibodies 488 (Abcam) was evaluated. The results were compared to the normal values that were obtained in the worldwide normative reference study [Statistical analysis was performed using the Statistica 8.0 software package . The distribution of patients into groups was carried out based on the presence of a verified diagnoses of pulmonary sarcoidosis or based on the absence of verified somatic diseases .All of the data that were collected during the study were analyzed using descriptive statistical methods. Normally distributed data are presented in the form of the “mean ± standard deviation” formula. Data that were not normally distributed are presented in the form of the “median (interquartile range 25—75 quartiles)”, where the confidence interval was also calculated. Normality testing was performed using the Shapiro–Wilk test.p < 0.05.All of the data were analyzed using parametric and nonparametric statistical methods and the Chi-square test, and statistical comparison methods for two (Mann–Whitney U-test) and three  groups were performed; correlations were determined using the Spearman coefficient. Differences or association rates were considered to be statistically significant at a The average score of the SFN screening list scale (SFN-SL) in patients with sarcoidosis was 2.0 , and in healthy volunteers, it was 0  points .p < 0.0001). Thus, in the first group, a higher average SFN-SL score was observed than it was in the second group, while the results of Groups 1 and 2 exceeded those of healthy individuals.Statistically significant differences were found in the results of patients with sarcoidosis and in the results of healthy subjects , pain in the chest or in the extremities, and blurred vision. It is important to note that in 76% of patients with sarcoidosis, the clinical symptoms had a severity of no more than 1 point on the SFN-SL scale and, in most cases, these symptoms did not bother patients or lead to a decrease in quality of life .A total of 13 biopsy specimens were obtained from the patients with sarcoidosis, where the intraepidermal density (IEFD) of the small nerve fibers was calculated .A total of 13 of the biopsies were obtained from patients with sarcoidosis . The calp = 0.0102). Additionally, a negative, statistically significant correlation between the IEND and SFN-SL score was described .Thus, in all of the examined patients, the number of small nerve fibers was within normal limits but below average values. A negative, statistically significant correlation was revealed between the IEND results and in the SFN-SL scores of patients with sarcoidosis , e.g., the development of palpitations and dizziness. In 32% of cases, patients noted pain in the chest or limbs, which was often accompanied by allodynia, a subjective perception of tactile touch as pain. In some cases, this can result in sleep disturbances due to the pain sensations that are caused by bed linen touching the skin. Another symptom that is often noted by patients with sarcoidosis is blurred vision, which is described in 20% of cases. A physician needs to clarify whether these visual impairments are transient or permanent in nature in order to determine a differential diagnosis of ophthalmic pathology. In the neuropathy of small fibers, blurred vision is transient, arising while overworking or during physical exertion. Gastrointestinal tract disorders are less commonly reported with patients with sarcoidosis. In 12% of the cases, the patients complained of impaired intestinal motility, with the development of both diarrhea and constipation, which occurred simultaneously with the onset of sarcoidosis. This also includes subjective complaints of swallowing dysfunction, which is associated with both impaired muscle innervation and with the progression of mouth dryness.p = 0.0102, and r = −0.7382, p = 0.0064), a decrease in the density of the small nerve fibers in patients with pulmonary sarcoidosis was more prominent.While a negative, statistically significant correlation between the IEND and SFN-SL scores was described in both groups (Spearman coefficient, r = −0.3508, A major limitation of this study was that conventional nerve conduction studies and autonomic function tests were not performed. The small sample size also could also be considered as a limitation.Thus, the neuropathy of small fibers seems to be a widespread pathology that results in the development of multiple organ dysfunction. Disorders in small fiber neuropathy, along with the typical complaints of patients with lung sarcoidosis, such as cough and shortness of breath, can significantly contribute to a decrease in the quality of life. At the same time, when focusing on the main complaints as well as on instrumental and laboratory deviations, clinicians often do not attach the necessary importance to the manifestations of SFN. It is necessary to identify the main causes that are responsible for a patient’s quality of life deteriorating in order to determine a treatment strategy and to improve the prognosis of the patient’s disease ,32.Given the low awareness of both medical specialists and patients about the development of this complication and the difficulties that are involved in its diagnosis, further study into this issue is required.In patients with pulmonary sarcoidosis, small fiber neuropathy may develop as a result of systemic immune-mediated inflammation. The validated questionnaires and histologic verification of the diagnosis help to establish the severity of the neuropathy of small fibers to determine the prognosis and to plan the treatment strategy.


We found the resulting graphene sheets to have high crystalline basal planes, lateral sizes of several μm, and a thickness of <5 nm. Furthermore, the high aqueous dispersion stability of as-prepared graphene could be demonstrated using a multi-light scattering technique, showing very little change in the optical transmittance and the terbiscan stability index over time.The electrochemical exfoliation of graphite has been considered to be an effective approach for the mass production of high-quality graphene due to its easy, simple, and eco-friendly synthetic features. However, water dispersion of graphene produced in the electrochemical exfoliation method has also been a challenging issue because of the hydrophobic properties of the resulting graphene. In this study, we report the electrochemical exfoliation method of producing water-dispersible graphene that importantly contains the relatively low oxygen content of <10% without any assistant dispersing agents. Through the mild  In order to obtain a stable water dispersion of graphene, electrostatic repulsion should dominate over van der Waals attraction . With thin situ sulfate functionalization of graphite manipulated by controlling the reaction of persulfate (S2O82−) ions to graft sulfate (R–OSO3−) groups onto the graphene sheets during the electrochemical reactions. The as-functionalized sulfate groups on graphene are believed to induce the stable water dispersion of graphene rather than the conventional oxygen functional groups such as carboxyl and hydroxyl groups; the strongly acidic sulfate groups are expected to be fully dissociated and induce strong negative charges under all relevant solution conditions, but the carboxyl or hydroxyl groups offer negative charges at a relatively higher pH value , potassium hydroxide (>97%), and ammonium acetate (>97%) were purchased from Sigma-Aldrich. Graphite foil (99.8%) was purchased from Alfa-Aesar. All chemicals were used as received without any further purification.In a typical procedure, graphite foil was used as the anode for the electrochemical exfoliation of graphite, with Pt foil being used as the cathode. As the electrolytes for the electrochemical reaction, ammonium persulfate (as an oxidant) and ammonium acetate (as pH buffer) were dissolved in deionized water using a mechanical stirrer to obtain 0.35 and 0.5 M solutions, respectively. The pH of the solution was adjusted by adding potassium hydroxide (KOH) to prevent the solution from becoming acidic (below pH 7) during the electrochemical reaction. The electrochemical exfoliation reaction was carried out by applying a constant voltage (10 V) to the graphite-foil electrode at 25°C for 1 h. The reaction product was collected by vacuum filtration and washed several times with deionized water. The resulting wet black powder was ultrasonicated in deionized water for 30 min, and a stable graphene nanosheet dispersion was obtained without any additional sedimentation and/or centrifugation processes. The dispersion stability of the graphene nanosheet could be controlled by changing the initial pH of the electrolyte.−9 Torr). The Raman spectrum of the graphene sample was measured using a UniRAM Raman microscope (Uninanotech) at room temperature and an excitation wavelength of 532 nm. The attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrum of the graphene was collected using a Nicolet iS10 FT-IR spectrometer (Thermo Scientific). The sample dispersion stability was characterized using a Leanontech Turbiscan Lab Expert instrument with a near-infrared (NIR) light source (880 nm). The zeta potential was obtained by using an ELSZ-1000 (Otsuka Electronics).Electrochemical exfoliation was conducted by using a ZIVE MP1 electrochemical workstation (WonATech). Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of the graphene samples were acquired using a JEOL JEM-2010 microscope at an accelerating voltage of 200 kV. Atomic force microscopy (AFM) images were obtained using an XE-100 (Park Systems) instrument. X-ray photoelectron spectroscopy (XPS) results were obtained using a Kratos AXIS Ultra DLD with an Al Kα radiation source  in an ultrahigh vacuum chamber (7 × 102O82−), a strong oxidant (E0= 2.01 V) (4˙−), which can be readily converted to hydroxyl free radicals (˙OH) in aqueous solution via a radical interconversion reaction. In particular, under acidic conditions, the breakdown of S2O82− can be further acid-catalyzed; as a result, most of the S2O82− converts into ˙OH (The persulfate anion (S 2.01 V) , undergointo ˙OH . For thiinto ˙OH .In the specific two-electrode system with the use of graphite foil as the anode and Pt foil as the cathode, the electrolyte solutions were first prepared by dissolving ammonium persulfate in deionized water to obtain a 0.35 M aqueous solution, and the pH value was adjusted using potassium hydroxide with ammonium acetate buffer solution (pH 11). Subsequently, a constant voltage of 10 V was applied to the graphite-foil electrode at a temperature of 25°C to prevent the thermal decomposition of persulfate ions . For 10 2-type carbon as per the peak at 284.0 eV with a small amount of sp3-type, hydroxyl-type, and carboxyl-type carbon, corresponding to peaks at 284.8, 286.3, and 288.8 eV, respectively (3−) has not been grafted on the surface of graphene. Instead, we could observe the chemical bonds between carbon and oxygen (533.3 eV for C–O and 531.5 eV for C=O) and between sulfur and oxygen   is found in the S 2p spectra, without other peaks corresponding to the sulfonate  bond (3−) instead of sulfonate (R–SO3−). Furthermore, the FT-IR spectrum of graphene collected in the ATR mode also revealed that water-dispersible graphene has various types of functional groups in the structure , which depicts the detailed chemical composition in the form of the peak binding energies of C 1s, O 1s, and S 2p. The sulfate-functionalized graphene could be identified as mostly consisting of spectively  (Parvez ectively . As the ectively  could no32.2 eV)  (Kwan et32.2 eV)  from theeV) bond  (MassonneV) bond . Based otructure . The diste group , while tectively .in situ sulfate functionalization of graphene employed in our study for the production of water-dispersible graphene could be confirmed in comparison to the hydrophobic graphene produced without maintaining the basic pH of the electrolyte during the electrochemical reaction. Different from the specific electrochemical reaction of water-dispersible graphene, we conducted the same electrochemical reaction without using the buffer reagent. In detail, we carried out the electrochemical reaction in the solution with the concentration of 0.35 M, adjusting the pH of the electrolyte to 11, applying the constant voltage of 10 V at a temperature of 25°C, continuing the reaction for 1 h, and only without using the buffer reagent of ammonium acetate. Without using the buffer reagent, the pH value of the electrolyte changes from 6.5–7 to 2 during the electrochemical reaction because the persulfate ions dissociate into sulfuric acid as the reaction proceeds  in neutral deionized water . It is nThe microstructures of water-dispersible graphene were observed by high-resolution transmission electron microscopy (HRTEM), as shown in −1, the G band at 1,570 cm−1, and the weak 2D band at 2,680 cm−1 (D/IG) is used to investigate the degree of disorders in graphene. Contrary to the TEM analysis result, it shows a high ID/IG value of 1.03, similar to rGO (a = 4.35(ID/IG)−1, the size of the sp2 carbon domains (La) in the water-dispersible graphene was found to be 4.23 nm  over 3 days was quantitatively analyzed using a multi-light scattering technique , in whicvia mild in situ sulfate functionalization by controlling the reaction of S2O82− ions. Under the basic pH conditions (>pH 7) during the electrochemical exfoliation of graphite, the sulfate functionalization of graphene could be effectively induced, and as a result, the water-dispersible graphene could be prepared with high crystallinity and a relatively low oxygen content of <10% compared with graphene obtained from conventional electrochemical exfoliation. The aqueous dispersion of sulfated graphene was highly stable over a long time without any additional separation processes of sedimentation and/or centrifugation, exhibiting constant transmittance regardless of the solution height and very little change in the transmittance and the TSI. This result indicates that the sulfate functional group attached to the graphene sheet effectively stabilizes the dispersion from electrostatic repulsive forces by increasing the surface charge even when only a small amount of sulfate functional groups is present. More importantly, our study shows that a comprehensive understanding of the persulfate reaction can provide a tool to introduce sulfate functional groups in various materials and increase dispersion stability without inhibiting their original characteristics.In summary, we report the preparation of highly water-dispersible graphene using electrochemical exfoliation of graphite


After this treatment, the synthesized samples were then characterized by using FTIR, SEM, and DTA/TGA techniques for analyzing humidity sensing properties. The polypyrrole samples with the best morphological structure and properties were then incorporated on interdigitated electrodes. For the fabrication purposes of this thin film structure, at first a few drops of polyvinyl alcohol (PVA) were placed over interdigitated electrodes (IDE) and then the synthesized polypyrrole composite was uniformly deposited in the form of a thin film over it. The plots show that this is a good resistive-type humidity detection device for the relative humidity range of 30% to 90%. The response and recovery times of this newly fabricated humidity sensor were reported to be the same as 128 s at room temperature. Additionally, the stability and the repeatability response behavior of this Ppy sensor were verified up to five cycles of multiple repetitions. This presents an excellent stability and repeatability performance of the sensor. Furthermore, the capacitances versus humidity response and recovery properties of the designed sensor were studied too. This illustrates an excellent capacitive verses humidity response and shows a linear and an active behavior. Lastly, the experimental result proves that polypyrrole composite thin film shows a reasonable best performance up to a temperature of 100 °C.In this research article, an organic polymer based polypyrrole (Ppy) composite material has been synthesized and analyzed for the design and fabrication purposes of a fast-responsive, highly sensitive, and an economical resistive-type novel humidity detection sensor. This humidity sensor most suitably serves the purpose for industrial humidity  detection applications. First, a polypyrrole composite material (a mixture of polypyrrole, polypyrrole-NiO, polypyrrole-CeO Being a famous conductive polymer, it shows a promising behavior for the stability of a product and or device along with the conversion of the corresponding cation to anion radicals. Several methods exist for the preparation of polymers, but the most widely used methods are the electrochemical and chemically loss of electrons . The synthesis of polymer composites from any one of these two methods depends on its type of application in practical devices. The electrochemical synthesis technique shows good results in the formation of polymers with good order. Therefore, it is the most commonly used technique to synthesize electrically conducting polymers. In 1979, Diaz et al. performed electrochemical polymerization of pyrrole, and obtained a free-standing polypyrrole film with a high conductivity of σ = 100 Scm−1 , as the −1 . Finally−1 . The thi4H2NH)nH) composite, the pyrrole (C4H4NH) monomer is polymerized through the chemical oxidation polymerization method and by using oxidants such as ammonium per-sulfate ((NH4)2S2O8) within a host polymer solution. In this method, the distribution between polypyrrole and host polymer is good, and due to this, the mechanical properties and electrical conductivity of the newly prepared material considerably increases. The non-conducting state of polypyrrole is considered of having a benzenoid structure and such state is known as natural state of polypyrrole. The natural state in the benzenoid structure of polypyrrole is the same as shown in Nowadays, polymeric conductive composites can be prepared by an easy and effective method which is called chemical oxidation polymerization. In the preparation of polypyrrole , etc. Humidity measurements also play a vital role in human breath monitoring system technologies ,12,13,14Thus, different types of humidity sensors have been designed for this purpose from the applications of diverse hygroscope composite materials ,17. The The role of miniaturization technologies in humidity sensors offers a variety of advantages, e.g., compactness, low cost, and low density ,27, batcHumidity sensors calculate the volume of aqueous vapors present in gases. It could be a mixture of a pure gas or of an air, such as nitrogen or argon. A detailed discussion on types of humidity measuring techniques and units has been added in Relative humidity sensors are the most commonly available humidity sensors in the market. RH sensors can be sub-divided based on their operating principles. According to Yamazoe’s and Shimizu’s classification which appeared in the 1980s, various sensing elements were placed in the main groups of electrolytes, ceramics (porous), and polymers (organic) . In 1995All of the abovementioned kinds of humidity sensors utilize the basic principle of change in the electrical  and physical properties of the sensitive elements, when exposed to different atmospheric humidity conditions. This provides a measure of humidity in the form of some amount of adsorption and desorption of water vapor molecules. It has been analyzed and reported that the sensitivity of humidity in porous film is higher than non-porous counterparts ,42. AmonCeramic humidity sensors are metal oxide-based sensors that have advantages over polymer film type sensors from the perspective of their thermal capability, mechanical strength, resistance to chemical reactions, and physical stability. Ceramic humidity sensors are divided into impedance type sensors and the capacitive type, the impedance type sensor is then further divided into electronic conduction and ionic conduction types ,50. The Nowadays there is an increase in the usage of different type of composite materials for humidity sensing applications; these materials are prepared by mixing several organic and inorganic substances. An important property of the ceramic system is the chemical and physical adsorption of water vapor molecules to its surface. That is why in ceramic sensors the sensitivity and response time depends upon the surface type with certain characteristic properties ,51. In cMacromolecules are organic polymers and represent the structure of a unit. A polymer is a combination of a carbon-hydride compound and its derivatives. The synthetic polymers are classified by monomers . Polymers are classified by two or more various kinds of monomers which are known as co-polymers. Most of the polymeric sensors are based on porous polymer thin film (even thinner than millimeters) and having the same sensing principle as of ceramic sensors. The water vapors are coated in a polymer film with micro pores for condensation, and some measurable physical properties change due to water absorption phenomenon. The polymeric humidity sensors are categorized into two main types, the capacitive-type humidity sensor and the resistive-type humidity sensor.Capacitive type humidity sensors are based on principle of change in dielectric constant in the thin film due to the absorption of water molecules. This change can be caused by the capillary condensation in porous structures or by the swelling of the polymer film. The electrode geometry and hygroscopic material determine the properties of such sensors. Because the polymer is highly sensitive to high temperatures, so nearly all the polymer-based humidity sensors operate at room temperature. Korvink et al. considered four electrode geometries for capacitive humidity sensors in . The eleResistive-type humidity sensors are the sensors whose resistance changes with humidity variations. The humidity-dependent resistance changing of such sensors can be approximated by applying the following Equation (1),In these sensor materials the variations in resistance are caused by a chemical reaction occurred in water molecules and surface atoms, and it could be either electronic or ionic type. A good conductive material, such as a polymer composite, produces big changes in the resistance  in the presence of water molecules, thus exhibit suitable humidity sensing properties. In this work, an organic polymer based polypyrrole (Ppy) composite material has been synthesized and analyzed for the design and fabrication purposes of a fast-responsive, highly sensitive, and an economical resistive-type novel humidity detection sensor. The novelty of this work can be seen in a number of ways, e.g., the composition of different materials used during the fabrication of this humidity sensor has not yet been reported in the entire existing literature. Furthermore, the selection of these materials has been made on the bases of their structural morphology, conductance properties, cheaper prices and easily availability. These are such attributes which can be taken care of during the fabrication of humidity sensors. Moreover, capacitive-type humidity sensors have been reported in the existing literature and are available in the market till now. However, this is a new addition in the resistive-type humidity sensors family. Properties of any sensor such as response time, recovery time, sensitivity and repeatability play an important role in the performance and suitability of the sensor. The newly fabricated sensor showed better properties in this respect too than that of existing works in this field. For example, the response and recovery times were calculated to be around 168, 1500 , 45, and4H4NH), niobium pentoxide (Nb2O5), cerium oxide (CeO2), nickel oxide (NiO), and anhydrous iron (III) chloride (AR-grade) were all purchased from Sigma Aldrich, Korea. Pyrrole monomers were purified under low pressure through a distillation process and then were stored in a dark place at 4 °C.Pyrrole  chloride hexahydrate” was added drop wise and with continuous stirring in this solution. The solution was left for polymerization for 24 h. The suspension was filtered out and was washed with acetone and distilled water to get ferric chloride. Finally, a black precipitate of polypyrrole was obtained. The obtained black precipitate was dried in a vacuum oven for 3 h and from 60 to 70 °C.For polymerization purposes of 3.4 mL pyrrole, FeC3 was dissolved in 25 mL of ethanol and 3.4 mL of polypyrrole and stirring was performed for nearly 15 min. Then 25 mL of FeCl4.6H2O was added in a drop wise manner in an ethanol solution of pyrrole. Fine graded powder of nickel oxide (NiO) was adjusted in percentages of weights and was added to the polypyrrole solution. Then the entire mixture reaction was starved for 3 h at 0–5 °C with the help of a magnetic mixer to disperse NiO in this polymeric solution. The acquired materials were purified and properly rinsed with distilled water and acetone to eliminate the unreacted Pyrrole and the excessive amount of ferric chloride. Finally, the sample was dried in a vacuum oven for 2 h at 60 to 70 °C.FeC3.6H2O was added in drop wise fashion in an ethanol solution of Pyrrole. Fine graded Niobium Pentoxide powder (Nb2O5) was taken in different volumes and then was added to the polypyrrole solution. Again, the sample was dried in a vacuum oven for 2 h at 60–70 °C.For this step, 3.4 mL of pyrrole was put in 25 mL ethanol and the mixture was properly allowed to dissolve, after that stirring was done on this mixture for 10 min. Then, 25 mL of FeCl3·6H2O was added drop wise in the ethanol solution of Pyrrole. Here, fine graded Cerium Oxide powder (CeO2) was taken in volumes of 10, 20, 30, 40, and 50, and was added to the polypyrrole solution. The mixture was starved for 3–4 h at 0–5 °C with a magnetic stirrer to disperse CeO2 from the polymer solution. Lastly, the sample was dried down in a vacuum oven for one hour and 30 min at 60–70 °C.In this step, 3.4 mL of pyrrole was added in 25 mL ethanol and then starved for almost 10 min. Then, 25 mL of FeCl2, and polypyrrole-Nb2O5 composites obtained from above synthesis techniques were mixed  is the study of changes in mass of samples occurring in variations of heat . The PpySEM was developed for scanning the surface of a sample of a substance. SEM can be used to examine and obtain certain information about chemical compositions and surface topography that compose the crystalline structure, giving a magnified image of the sample of a substance . The spe2O3), and the top deposited layer is of Ppy composite material. For fabrication purposes of the Ppy composite based thin film sensor, at first a few drops of polyvinyl alcohol (PVA) were placed over an interdigitated electrodes (IDE) and then the synthesized polypyrrole composite was uniformly deposited in form of a thin film over it. Note that the composition of PVA and Ppy is different: the amount of Ppy is five times the amount of PVA. The plot comprising the capacitance verses % RH of the newly fabricated device was also obtained and is shown in It is further revealed from the Subsequently, the newly fabricated device was placed in the humidity test chamber again and the resistance verses % RH values of the device were obtained for the same operating frequencies  and at a constant 1 volt. It can be carefully observed that when the value of % RH rises then the value of resistance reduces in an exponential form (as discussed in ) for lowA hysteresis plot is another important feature among several others of humidity detection sensors which highlights their performance and various other properties. It shows a lag between humidification and dehumidification of the Ppy composite thin film sensor in This plot shows that during the humidification curve  the value of resistance increases with an increase in time. In the same way, during the dehumidification curve  the value of resistance decreases with a decrease in time. In such types of ideal cases, it can be stated that the forward process and reverse process are almost identical to each other with a minimum hysteresis loss value, this in turn also demonstrates that the stability performance of the sensor is reliable and can be used for application purposes.Real-time visualization of humidity values of the sensor plays an important role in the final verification process. air and Rair denote the capacitance and resistance due to the dielectric of air occupied in the pore area. The CPPy and RPPy give the capacitance and resistance due to the dielectric of Ppy composite itself when exposed to the surrounding environmental parameters such as relative humidity, light, and temperature, respectively. The capacitance, Csilver, and resistance, Rsilver, are due to the dielectric of the silver substrate. As, the electrical properties of Ppy thin film are dominant over other materials properties, that is why the capacitance and resistance properties of the PPy are only taken into account, and an equivalent circuit has been drawn in Schematic diagrams of Ppy composite polymer resistive-type thin film sensors  in a detailed and equivalent form are drawn in 2, and polypyrrole-Nb2O5) has been synthesized and analyzed for the design and fabrication purposes of a novel resistive-type humidity detection sensor. The polypyrrole samples with best morphological structure and properties were incorporated on alumina interdigitated electrodes. The synthesized polypyrrole composite has been treated at various temperatures, i.e., 100, 150, and 200 °C, respectively. It was observed from the resistance verses % RH plot that if the % RH rise’s then the resistance reduces exponentially. Additionally, a linear behavior was found between % RH and capacitance values for lower range of operating frequencies. The FTIR spectrum of Ppy composite is predicting the presence of hydroxyl (OH) by showing a very strong and broad peak at 3299 cm−1.An organic polymer based polypyrrole composite material  of the synthesized Ppy, performance of IDE sensors can considerably be improved.


Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations.Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities.We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa.The Markov model we present here indicates that Video AbstractThe online version contains supplementary material available at 10.1186/s40168-021-01096-9. Lactobacillus species that can be classified into one of two CTs—CT3, which is dominated by Gardnerella vaginalis, and CT4, which features diverse taxa but is generally Prevotella-rich. The remainder of women in this cohort has low-diversity, Lactobacillus-dominant microbial communities dominated by either Lactobacillus crispatus  or Lactobacillus iners . The low population frequency of L. crispatus, which has been replicated in other sub-Saharan African cohorts [Lactobacillus species with a higher prevalence of L. crispatus-dominated communities than of L. iners [Lactobacillus-dominated communities. In addition, CT4 is associated with higher numbers of activated cervical CD4+ T cells  and an over four-fold higher risk of HIV acquisition [Lactobacillus abundance are a characteristic feature of clinical bacterial vaginosis (BV), a common condition associated with vaginal symptoms such as discharge, malodor, and discomfort [Lactobacillus-dominated state [The cervicovaginal microbiome impacts several important reproductive outcomes, including preterm birth , 2, fert cohorts , 12, 13,L. iners –16. Impouisition . Notablyscomfort . BV can ed state , 19 thatWhile cross-sectional surveys provide valuable insight into FGT bacterial population structure and disease associations, they fail to capture important information about temporal changes in bacterial composition. Longitudinal studies performed in high-resource countries have shown that cervicovaginal communities can be dynamic, with composition shifting between community state types over the span of days to weeks –24. No sL. iners-dominant) acted as a gateway to more diverse and stable CT3 and CT4 communities, which were previously associated with elevated genital inflammation [L. crispatus-dominant communities (CT1), which are associated with lower levels of mucosal inflammation and more favorable reproductive health.In order to elucidate the longitudinal dynamics of FGT bacterial composition, we performed culture-independent bacterial 16S rRNA gene sequencing and bacterial community profiling on cervical swabs collected at 3-month intervals from a cohort of healthy, HIV-uninfected, Black South African women, and classified them into CTs. We constructed a Markov-based model of CT transitions and performed Monte Carlo simulation and parameter sensitivity analyses to predict interventions that could effectively shift CT stationary distributions from high-inflammatory CT3 and CT4 to low-inflammatory CT1. We found that CT2  cohort . ParticiLongitudinal sampling covered a median follow-up duration of 9 months per participant range 3–21 months) with a median of 3 samples per participant (range 2–7 samples) for a total of 316 samples  on Bray-Curtis dissimilarity of species-level relative abundances Fig. B. Using We next implemented a discrete-time stochastic model for probabilistic forecasting over the CT state space in order to better understand the longitudinal transitions between communities. To force tractability of the model, we assumed memorylessness—namely, the model was conditional on the present CT where the future and past states were independent, an approach that has previously been used to model dynamics of vaginal bacteria in Western populations , 24. TheTo further explore the adherence to each CT, we calculated the mean number of timesteps required to transition between each pair of CTs Fig. B. We fouL. iners-dominant CT2 acts as an intermediary or “gateway” state between L. crispatus-dominant CT1 communities, which are most strongly associated with favorable reproductive outcomes, and the more diverse and higher-risk communities, CT3 and CT4  from the same cohort   and those that transitioned to a different CT  using t states , 27, 28,t states , 29, 30.After demonstrating the transient effects of time-discrete interventions on population-level CT prevalence, we next investigated the impact of changes on the underlying transition probabilities of the chain. Specifically, we modeled the effects of two types of simulated interventions on predicted population equilibrium distributions of CTs: “boosting,” which increases the probability of a directional transition between two CTs, and “blocking,” which reduces the probability of a directional transition.L. iners dominance, blocking or boosting transitions between CT2-CT3 and CT2-CT4 had a negligible effect on the proportion of CT1 communities at equilibrium  and low (CT1 and CT2) diversity communities, calculating new CT stationary distributions based on the modified transition probability matrices Fig. . We focuium Fig. E, D. Theium Fig. A. For exL. iners dominated communities (CT2) act as a gateway between L. crispatus dominated communities (CT1), previously associated with favorable reproductive outcomes, and stable, high-diversity communities (CT3 and CT4), which have been associated with adverse reproductive outcomes [L. iners is found in high abundance in two CSTs, CST3 and CST9. Under the CT definition described in Anahtar et al. 2015 for our South African population, there is only one community with high abundance of L. iners, CT2 [L. iners-dominant state allowing us to compare transition probabilities across populations. This reveals that in North American women the most likely transition from L. crispatus-dominated CST1 was to a community dominated by Lactobacillus jensenii , a species which is rarely found in women in sub-Saharan Africa. The largest transition probability from CST5 was back to CST1 (mean weekly transition probability of 0.13). The summed transition probability of moving from CST1 or CST5 to L. iners dominated communities CST3 and CST9 was lower than in our study at 0.07 and 0.1, respectively. Both CST1 and CST5 had a high probability of remaining . These differences in transition probabilities of community states within North American and South African populations may explain the observed higher degree of diverse communities in South African compared to North American women.The FGT microbiome is associated with adverse reproductive outcomes in sub-Saharan Africa . Althougoutcomes . A similoutcomes , 24, 31.ers, CT2 . These dLongitudinal studies have performed sampling over varying intervals, including daily , weekly L. crispatus-dominant) are relevant because standard BV treatment tends to promote dominance by L. iners rather than L. crispatus [L. crispatus-dominant communities would be to simultaneously boost transition from L. iners- to L. crispatus-dominant communities, while blocking the reverse transition from L. crispatus- to L. iners-dominant communities. Possible interventions to shift CT2-CT1 transitions may include introduction of a L. crispatus probiotic along with approaches to selectively kill or inhibit L. iners growth.We simulated the effects of various interventions on shifting population prevalence of different microbial community states. Our modeling predicted that time-discrete interventions  had relatively transient effects at the population level, with the population returning to its equilibrium distribution within 9 months if shifted to CT2, CT3, or CT4. Shifting to CT1 provided moderately more durable effects, with a return to equilibrium distribution estimated to require 15 months. The longer-lasting effects of shifts to CT1  prospective observational study in Umlazi, South Africa . Exclusihttps://benjjneb.github.io/dada2). Taxonomic assignments were further curated using an internally developed taxonomic database . The average steps required to return to a CT were calculated with the “mean first passage time” (mfpt_to) function in Pykov.Markov chain stationary distributions were calculated using the inverse iterative method in Pykov . Multivariate Gaussian noise was applied to the PCoA coordinates before animation.The boost interventions shifts α probability density in the transition matrix from returning to CTb instead of transitioning to CTa as can be seen in the pseudocode below:The block intervention shifts Β probability density in the transition matrix from transitioning to CTb to instead returning to CTa as can be again seen in the pseudocode below:Differences in CT Shannon alpha diversity were calculated using a non-parametric two-sample Monte Carlo t tests. P values are two-sided and are not adjusted for multiple hypothesis testing. Statistical analyses were performed in Python with SciPy  and NumPAdditional file 1. Supplementary fig. 1 PCoA of Bray-Curtis dissimilarity of species-level relative abundances of 316 samples from 88 women, split by CT of origin, with arrows depicting the movement of a community between sequential time points.Additional file 2. Supplementary fig. 2 Table showing the number of transitions between CTs observed in the study.Additional file 3. Supplementary fig. 3 Statistical power calculation for identifying a given difference between empiric and equilibrium distributions.Additional file 4. Supplementary fig. 4 The number of iterations taken for the models to pass a p-value significance threshold of 0.05 (chi-sq between perturbed and equilibrium distributions).Additional file 5. Supplementary file 1 Taxonomic assignment to ASVs.Additional file 6. Supplementary movie 1 Monte Carlo simulation showing the possible movement, at a 3-month time interval, of an individual through the PCoA-space displayed in Fig Additional file 7. Supplementary table 1 Cohort demographics.


In this review, we will explore the complex interplay between TDP-43 pathology, ER stress, and the UPR by breaking down the evidence available in the literature and addressing the reasons behind these discrepancies. We also highlight underexplored areas and key unanswered questions in the field. A better synchronization and integration of methodologies, models, and mechanistic pathways will be crucial to discover the true nature of the TDP-43 and ER stress relationship and, ultimately, to uncover the full therapeutic potential of the UPR.Nuclear depletion, abnormal modification, and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) are linked to a group of fatal neurodegenerative diseases called TDP-43 proteinopathies, which include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although our understanding of the physiological function of TDP-43 is rapidly advancing, the molecular mechanisms associated with its pathogenesis remain poorly understood. Accumulating evidence suggests that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are important players in TDP-43 pathology. However, while neurons derived from autopsied ALS and FTLD patients revealed TDP-43 deposits in the ER and displayed UPR activation, data originated from  The abnormal distribution, modification, and aggregation of TAR DNA-binding protein 43 (TDP-43) are the pathological hallmarks of a group of neurodegenerative diseases that are collectively known as TDP-43 proteinopathies . Accumulation of excess or abnormally localized TDP-43 contributes to an increase in cytoplasmic aggregation that leads to the sequestration of nuclear TDP-43, other off-target proteins, and mRNAs from the environment resulting in a toxic gain of function  generated by caspases and calpain proteases  is a membranous organelle largely responsible for protein maturation within the cell. Proteins that pass through the ER undergo post-translational modifications including glycosylation, oligomerization, disulfide bond formation, pro-isomerization, and subsequent folding with the help of resident ER chaperones, glycosylating enzymes, and oxidoreductases  to restore proteostasis. There are three UPR pathways named after their respective signalers: Inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), Protein kinase RNA-like endoplasmic reticulum kinase (PERK), and Activating transcription factor 6 (ATF6). These pathways operate in parallel and act to coordinate a series of proadaptive cascades in an effort to restore proper protein folding capacity . HoweverThe first branch of the UPR is mediated by the kinase and endoribonuclease membrane-bound protein sensor, IRE1 , and reduces the burden of protein inputs to mitigate luminal protein accumulation and misfolding. However, when ER stress is unmanageable, the UPR alters its function to promote apoptotic programs. This dynamic function of the UPR in determining cell fate is especially relevant in the context of neurodegenerative diseases characterized by severe proteostatic imbalances.As described above, abnormal accumulation of TDP-43 in the cytoplasm, as well as TDP-43 protein modifications, have largely been associated with the development of several neurodegenerative disorders including ALS and FTLD , a protein closely associated with ER-mediated apoptosis whose regulation is mainly mediated through the PERK/eIF2α/ATF4 pathway  signaling being responsible for the increase in TDP-43-associated apoptosis since immortalized cells and primary neurons carrying TDP-43-M337V and TDP-43-A382T mutations presented a 50% reduction of luminal ER Ca2 + levels, and delayed Ca2 + flux when compared to TDP-43-WT  exhibited significantly increased levels of the ER markers XBP1 and ATF6 when compared to control cells  formation, refolding and degrading misfolded proteins; or, if instead, ER stress promotes TDP-43 mislocalization, post-translational modifications, and aggregation that may accelerate cell death. To address this, several groups investigated the interplay between TDP-43 and ER stress through overexpression paradigms, although experimental procedures relying on pharmacological regulation of ER stress have been the preferred approach.In 2013, Walker and collaborators used the ER stressor thapsigargin on Neuro2a cells expressing seven different forms of TDP-43 associated with ALS and FTLD . The authors observed that while in normal conditions all studied isoforms localized mostly in the nucleus with only moderate cytoplasmic localization after thapsigargin treatment TDP-43 mislocalization to the cytoplasm significantly increased for all TDP-43 variants when compared to untreated cell cultures  caused a decrease in the levels of p-eIF2α leading to a significant improvement of the locomotor deficit caused by TDP-43 expression. Conversely, knocking-down of Gadd34 resulted in increased eIF2α-phosphorylation that translated into exacerbated motility impairment in the fly , guanabenz (an α2-adrenergic receptor agonist that interferes with stress-induced dephosphorylation of eIF2α), and phenazine , resulted in improved motor symptoms and reduced neurodegeneration in both animal models. In correlation with this phenotypical improvement, the authors also detected a significant decrease in the amount of insoluble TDP-43 upon treatment, likely due to an enhancement of protein clearance  pathways mediated by PERK, IRE1α and ATF6 play a fundamental role in the maintenance of protein homeostasis. This role is even more crucial in the context of neurodegenerative disorders where protein synthesis, refolding and degradation might dictate the fatal outcome of neuronal cells and, therefore, the organism. The accumulation of pathological inclusions composed of TDP-43 aggregates is a common feature of many proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Therefore, understanding the specific contribution of TDP-43 to ER stress and how ER stress harnessing could modulate or even alleviate TDP-43 aggregation and toxicity, are two of the most important puzzles in the field. In this regard, we have identified key questions that deserve serious consideration. For instance:What is the mechanism by which TDP-43 upregulates CHOP independently of UPR signaling?2+ dyshomeostasis, TDP-43, and ER stress  to understand the complex relationship between TDP-43 and ER stress over the last decade. Despite initial reports, where the specificity and sensitivity of the assays might have masked or altered the results, it seems that overexpression of mutant forms of TDP-43 and, to a lesser extent, TDP-43-WT might activate the UPR as a protective response mechanism by the cell. In turn, potentiation or pharmacological activation of ER stress could serve as a mechanism to modulate TDP-43-mediated toxicity. However, the inconsistent results between studies might cloud this approach or call for caution. The differences between cell and animal models, TDP-43 transgenes, ER stressors, and even experimental methodology could easily account for many of the observed discrepancies. Moreover, members of the UPR branches are differentially expressed in different cell types (Walter and Ron, Several groups have used different approaches and models (both LM, JL-S, and DER-L conceived the original idea and revised the manuscript. LM and JL-S designed the outlines of the study, performed literature review, wrote the first draft, and prepared the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, high-mortality lung disease, but its pathogenesis is still unclear. Our purpose was to explore potential genes and molecular mechanisms underlying IPF.  IPF-related data were obtained from the GSE99621 dataset. Differentially expressed genes (DEGs) were identified between IPF and controls. Their biological functions were analyzed. The relationships between DEGs and microRNAs (miRNAs) were predicted. DEGs and pathways were validated in a microarray dataset. A protein-protein interaction (PPI) network was constructed based on these common DEGs. Western blot was used to validate hub genes in IPF cell models by western blot.  DEGs were identified for IPF than controls in the RNA-seq dataset. Functional enrichment analysis showed that these DEGs were mainly enriched in immune and inflammatory response, chemokine-mediated signaling pathway, cell adhesion, and other biological processes. In the miRNA-target network based on RNA-seq dataset, we found several miRNA targets among all DEGs, like RAB11FIP1, TGFBR3, and SPP1. We identified 304 upregulated genes and 282 downregulated genes in IPF compared to controls both in the microarray and RNA-seq datasets. These common DEGs were mainly involved in cell adhesion, extracellular matrix organization, oxidation-reduction process, and lung vasculature development. In the PPI network, 3 upregulated and 4 downregulated genes could be considered hub genes, which were confirmed in the IPF cell models.  Our study identified several IPF-related DEGs that could become potential biomarkers for IPF. Large-scale multicentric studies are eagerly needed to confirm the utility of these biomarkers. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, irreversible lung disease, characterized by an irreversible decline in lung function, progressive pulmonary scarring, and common interstitial pneumonia –3. It afLong-term clinical work has shown that clarifying the pathogenesis of IPF helps to diagnose early disease, which is of great significance for the treatment of this disease, and has long-term clinical results to improve this fatal disease , 10. HowmiRNAs, a class of noncoding small RNAs, are involved in RNA silencing, posttranscriptional regulation, and other biological processes . It has http://www.ncbi.nlm.nih.gov/geo) is a public functional genomics data repository. Based on the filter criteria of keywords: IPF, organism: homo sapiens, and experiment type: expression profiling by high-throughput sequencing or expression profiling by array, two datasets GSE99621 and GSE110147 were included for this study. RNA-seq data of IPF were obtained from the GSE99621 dataset. This dataset contained 8 affected areas of the lung (IPF_S), 10 unaffected areas of the lung (IPF_N), and 8 healthy controls (N_CTL) on the GPL16791 platform [Q < 20), retaining sequences with Q > 20 bases above 90% and total length > 35 bp. The clean reads were mapped to the protein-coding gene sequence of Homo sapiens (assembly GRCh38.p12) using the HISAT2. Then, bedtools was utilized to calculate the number of reads and the RPKM expression value by a localized Perl script. After that, the GeneCluster3.0 was used for the systematic hierarchical clustering of samples. The principal component analysis (PCA) was then conducted. Microarray data of IPF were also retrieved from the GSE110147 dataset on the GPL6244 platform, including 22 IPF tissues and 11 normal lung tissues [The Gene Expression Omnibus (GEO) (platform . The ada tissues . The flohttp://bioconductor.org/packages/release/bioc/html/limma.html) [p value ≤ 0.05 and fold change ≥ 1.5 were screened as upregulated genes. Meanwhile, those with adjusted p value ≤ 0.05 and fold change ≤ 1.5 were identified as downregulated genes.Differential expression analysis between the IPF_S, IPF_N, and N_CTL samples was performed using the limma package in R (ma.html) . Genes whttp://david.abcc.ncifcrf.gov/) [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were carried out by a functional annotation analysis tool online, Database for Annotation, Visualization and Integrated Discovery  . In the http://mirwalk.uni-hd.de/) is a publicly available comprehensive resource that hosts predictive and experimentally validated miRNA-target interaction pairs. This database allows for possible miRNA binding site predictions within the complete sequence of all known genes in the three genomes , including ten different prediction datasets [The miRWalk 2.0 database was used to predict the relationships between DEGs and miRNAs. The miRWalk  [http://www.ebi.ac.uk/intact) [It is helpful to clarify the key mechanisms of disease development and reveal key cellular functions and biological processes by studying the interactions between transcripts or proteins. The data of BioGRID (rid.org)  and IntA/intact) , two pro2. BEAS-2B cells were treated with 0.5 ng/ml TGF-β1 for 48 h to construct an IPF cell model. The IPF model was validated by examining α-SMA, fibronectin, and Col I expression levels.Human normal lung and bronchus epithelial cell line BEAS-2B  were grown in BEGM kit from Lonza  at 37°C in 5% COβ1-induced BEAS-2B cells and controls were lysed by RIPA lysates . After the lysates were centrifuged at 12,000 × g for 20 min, the supernatant was harvested. The extracted protein was resolved by 10% SDS-PAGE and transferred onto PVDF membranes. Following blocking, the membranes were incubated with α-SMA , Fibronectin , Col I , GABARAPL1 , GPX8 , SGTA , VCAM1 , ARRB1 , and GAPDH , followed by secondary antibodies. The optical density of the bands was quantified using ImageJ software.TGF-t-test. p value < 0.05 indicated statistical significance.All statistical analyses were carried out by R packages and GraphPad prism software. Each experiment was independently repeated at least three times. Data were presented as the mean ± standard deviation. Comparisons between two groups were presented by Student's p < 0.05 and fold change ≥ 1.5, upregulated genes were screened between the IPF_S, IPF_N, and N_CTL samples  and analyzed potential pathways (such as immune and inflammatory pathways) by comprehensively analyzing IPF-related RNA-seq and microarray datasets. Combining the PPI network, miRNA-target network, and functional enrichment analysis, we screened out potential biomarkers and their related regulatory mechanisms in IPF. These biomarkers might provide novel ideas and clues for further experimental research.In this study, we analyzed DEGs between IPF_S and N_CTL. Functional enrichment analysis showed that upregulated genes in IPF_S were mainly enriched in immune response, chemokine-mediated signaling pathway, and cell adhesion and the like. Numerous molecules involved in immune response have been proposed as potential biomarkers for IPF . For exaβ1 expression upregulation and increase fibroblast expression in IPF [It has been well recognized that small sample sizes, different platforms, and different statistical methods could lead to inconsistent results , 42. Then in IPF . Furthern in IPF . Methylan in IPF . Based oIt has been confirmed that miRNA-mRNA interactions play a critical role in the development of IPF –50. HereCollectively, this study provided several novel draggable-target molecules for IPF by bioinformatics. The reliability of results for biological investigations was verified in IPF cell models. The consistent results between bioinformatics and biological investigation suggested convincing evidence that hub genes including GABARAPL1, SGTA, ARRB1, GPX8, and VCAM1 were abnormally expressed in IPF and could be utilized as a promising novel target for IPF treatment. However, there are several limitations in our study. First, although we validated hub genes in IPF cell models by western blot, their functions in IPF should be clarified. Second, although several pathways were identified for IPF, molecular experiments should be presented to prove more reliable evidence for the phenotypes and pathways underlying IPF. In conclusion, our study identified several IPF-related DEGs that could become potential biomarkers for IPF. Large-scale multicentric studies are eagerly needed to confirm the utility of these biomarkers in our future studies.In this study, we comprehensively analyzed IPF-related DEGs and potential signaling pathways using the RNA-seq and microarray datasets. By combining the PPI network, miRNA-target network, and functional enrichment analysis, we identified potential biomarkers including GABARAPL1, SGTA, ARRB1, GPX8, and VCAM1 for IPF. Among them, GABARAPL1, SGTA, and ARRB1 exhibited lower expression levels in IPF while GPX8 and VCAM1 were both downregulated in IPF. These biomarkers might provide novel insights for further experimental research.


Graphene as a material for optoelectronic design applications has been significantly restricted owing to zero bandgap and non-compatible handling procedures compared with regular microelectronic ones. In this work, nitrogen-doped reduced graphene oxide (N-rGO) with tunable optical bandgap and enhanced electrical conductivity was synthesized via a microwave-assisted hydrothermal method. The properties of the synthesized N-rGO were determined using XPS, FTIR and Raman spectroscopy, UV/vis, as well as FESEM techniques. The UV/vis spectroscopic analysis confirmed the narrowness of the optical bandgap from 3.4 to 3.1, 2.5, and 2.2 eV in N-rGO samples, where N-rGO samples were synthesized with a nitrogen doping concentration of 2.80, 4.53, and 5.51 at.%. Besides, an enhanced n-type electrical conductivity in N-rGO was observed in Hall effect measurement. The observed tunable optoelectrical characteristics of N-rGO make it a suitable material for developing future optoelectronic devices at the nanoscale. The heteroatom atom doping of carbon-based nanomaterial, i.e., graphene, graphene oxide, and carbon nanotube, among others, has gained great attention in material science and research . The uniIn its intrinsic form, graphene is nearly transparent, with a lower light absorption coefficient and extreme thickness. When graphene is utilized as light active material in solar cell applications, it appears to have low-efficiency energy conversion compared with conventional solar cells . Therefop-type or n-type semiconducting behavior, depending on the bonding configuration. Particularly, lower charge carrier mobility and electrical conductivity were reported in nitrogen-doped graphene compared with intrinsic graphene, which is attributed to the existence of nitrogen contents and the induced defect in pristine graphene. However, nitrogen doping shows the capability to function at the scattering center, which hinders the hole and electron transportation [Nitrogen as a dopant is the most widely investigated element for modifying the optical and electronic properties of graphene or reduced graphene. Nitrogen-related defect sites induced in graphene enhance electrochemical activity in graphene and reduce graphene oxide . Besidesortation .Nitrogen-doped reduced graphene oxide (N-rGO) is produced by chemical substitutional doping of graphene oxide with nitrogen as a dopant element. N-rGO shows ideal physicochemical properties for optoelectronic design applications such as higher chemical stability and large surface area. Optical and electronic properties can also be potentially optimized by bonding configuration . SeveralThe N-rGO synthesis is still fascinating and challenging, where the whole process has not been well understood and needs further exploration. The optimized synthesis of N-rGO with a certain level of control over doping concertation and the formation of bonding configuration is still lacking. Previously, different synthesis techniques have been employed for N-rGO syntheses, such as thermal annealing , plasma Microwave-assisted and hydrothermal reduction synthesis of nitrogen-doped graphene has been demonstrated in several applications, such as electrode design, supercapacitor, lithium-ion batteries, and field-effect transistors . In this2 selected area. FESEM analysis and EDX mapping revealed that nitrogen doping significantly affects the morphology and the homogeneous distribution of the elements, as shown in The field emission scanning electron microscopic (FESEM) images of GO and N-rGO and elemental mapping of N-rGO are shown in −1 and 1600 cm−1, which can be assigned to the D and G band, respectively. The D band intensity peak at ~1348 cm−1 found in the GO spectra was ascribed to the disorder and defects in the graphite lattice owing to the presence of oxygen-related functional groups [−1, ascribed to the phase of the two second-order phonon vibrations [−1, respectively, corresponding to the vibrational scattering of E2g symmetry in the graphitic lattice.Raman spectroscopic analysis is an effective and valuable technique for evaluating the degree of disorder in nanomaterials . Figure l groups . Most imD/IG) describes the nanomaterial quality and a corresponding concentration of SP3 defects in the SP2 hybridized graphene layer [D/IG ratio of N-rGO samples was examined, i.e., GO (0.97), N-rGO1 (1.27), N-rGO2 (1.31), and N-rGO3 (1.38). Moreover, an increment in the (ID/IG) ratio indicates the reduction of GO and the existence of nitrogen content owing to the extra scattering effect induced by electron doping [−1 in N-rGO1, N-rGO2, and N-RGO3 samples indicates the formation of few-layered graphene [Furthermore, an increase in the D band intensity peak compared with the G band was examined in the Raman spectra of N-rGO. The intensity peak ratio (Ine layer . The incn doping . The redgraphene . −1), D peak (~1358 cm−1), D″ peak (~1555 cm−1), and G peak (~1598 cm−1). The Raman spectra of N-rGO was deconvoluted into four peaks, i.e., I peak , carboxyl (COOH), and the epoxide (C–O–C) groups, respectively [−1 can be assigned to the C–O groups stretching vibrations [−1. Particularly, in N-rGO synthesis process, the broad spectrum (−OH) stretch was reduced to a great extent, and N-C-related peaks were discovered at 1605 and 1265 cm−1, which are in good agreement with the reported values [The other peaks at 1715, 1415, and 1227 cmectively ,45. Morebrations . In the d values .2 bonding of carbon atoms, which indicates the existence of the majority of carbon atoms in the honeycomb structure [tructure . In O 1structure . Moreovetructure .d, θ, n, and λ are the interlayer distance, scattering angle, order of reflection, and wavelength of the incident X-ray source (1.54 Å), respectively. The interlayer distance for GO was determined to be about ~8.05 Å, which shows an agreement with [The interlayer distance of the GO and N-rGO crystalline plane was obtained using Bragg′s law, as given in Equation (1) .(1)λ=2dent with ,52. Furtent with . c) (The XRD peak intensities (002 planes) were increased with an increase in the doping concentration of nitrogen. The difference in peak shape reflects overlapped N-rGO sheets along the c-axis  and highc) , which w2 bonding configuration owing to the SP3 bonding formations, i.e., COOH, C–O–C, and C–OH, which cause the random dispersions [2 hybridized ring from the SP3 hybridized ring in the GO structure also prompts the insulation behavior. The enhanced electrical conductivity in N-rGO1, N-rGO2, and N-rGO3 samples was recorded to be ~0.625, 0.757, and 0.781 S/cm, respectively, where p-type electrical conductivity of GO was measured to be about 1.567 × 10−6 S/cm. Therefore, enhanced electrical conductivity in N-rGO samples was ascribed to the further reduction of graphene oxide, increment in SP2 related domains, and existence of nitrogen doping concentration. In addition, the pyrrolic-N bonding configuration in N-rGO material samples was more dominant in XPS analysis, which enhances the π-bond in carbon atoms by reducing the Stone–Wales effect and forming percolation ways for electrons, which ultimately results in quitting a conduction gap and enhancing n-type electrical conductivity.The Hall effect measurements of GO and N-rGO samples were carried out at 300 K, in order to examine electrical conductivity, charge carrier mobility, and carrier type, as summarized in persions . The iso2 domains in the graphene sheet. In contrast, an additional absorption shoulder in the absorption spectrum of N-rGO samples (from 500 nm) can be ascribed to the n to π* related transitions. The n–π* related transitions also reveal the existence of pi* states and the homogeneous distribution of nitrogen atoms in N-rGO material samples.The optical bandgap of GO and N-rGO samples was determined through ultraviolet−visible (UV/vis) spectroscopy analysis. The UV/vis absorption spectra of GO and N-rGO-based thin film (deposited on a glass substrate) are illustrated in The Tauc plot calculation for GO and N-rGO samples is presented in 2SO4, 97%), potassium permanganate , phosphoric acid , hydrochloric acid , and hydrogen peroxide , with known concentrations, were ordered from R&M, Malaysia, Selangor. Ammonia solution  was obtained from Sigma Aldrich, Malaysia, Selangor. GO was synthesized via an improved Hummer′s method, as described in [Graphite flakes (about 50 μm), ethanol (95.0%), sulfuric acid  solution, which produced a dark green solution. The entire reaction was performed in an ice bath to avoid excessive heating and maintain the reaction temperature. The resulting solution was stirred for 2 h to exfoliate and oxidize the graphite flacks to few-layered graphene oxide. On completion of the oxidation reaction, hydrogen peroxide (H2O2) was added to avoid additional reactions. At this same point, the color of the resultant solution changed to dark yellow-brown. Afterwards, 1 L of deionized (DI) water was added to the solution to dilute it and adjust the pH level to ~1. The resultant mixture solution was then centrifuged at 7000 rpm to collect oxidized graphite flakes and to remove the acidic contents. The collected GO flake was further treated with 1 M HCl solution to eliminate impurities such as manganese and potassium ions, usually found in solids. Afterwards, the residual mixture was washed several times using DI water until the pH level exceeded ~1. Powder-like GO flakes were then extracted by centrifugation at 10,000 rpm. The GO particles were dry-frozen and finely ground into powder, referred to as GO flakes.Briefly, 360 mL of HInitially, GO dispersion solution (75 mg/150 mL) was prepared in deionized water accompanied by 2 h constant stirring, followed by sonication of ~30 min. Subsequently, 5 mL ammonia solution was added to the resultant solution and continuously stirred for 12 h at 60 °C. The resulted solution was then poured into a 200 mL Teflon tube and heat-treated at 200 °C for 10 h in an autoclave machine using the oven. The collected N-rGO flakes were then exposed to microwave radiation using a microwave oven . The microwave intensity was adjusted to an optimum level to improve the reduction of oxygen-related functional groups and absorption of unreacted nitrogen contents. The N-rGO material samples were obtained at optimized parameters (time = 40 s and power = 800 W).The resulting N-rGO sheets were washed several times with DI to extract the untreated nitrogen source. The dry N-rGO powder was eventually obtained through the dry freeze process. Subsequently, three samples of N-rGO, namely, N-rGO1, N-rGO2, and N-rGO3, were synthesized with three different concentrations of ammonia solution, 2, 5, and 10 mL, respectively.−8 mbar) using a 1486.6 eV Al K-alpha radiation source from the range of 0 to 1200 eV. Crystallinity and interlayer distance: the X-ray diffraction (XRD) patterns were recorded for the GO and N-rGO powder samples to conform to the material’s crystallinity using X’Pert3-Powder, U.S.A., Westborough, wherein XRD patterns were recorded from 5 to 80° with a step size of 0.01 °/s. Imperfections and defects: the Raman spectra of GO and N-rGO samples were also recorded using the Raman spectrometer  to examine the defects and imperfections. GO and N-rGO sample material morphology was determined using FESEM . Functional group analyses of GO and N-rGO powder samples were executed using KBr-FTIR . Functional groups in GO and N-rGO samples were also conformed through XPS spectral analysis using Origin software tools , Gaussian deconvolution and curve fitting of XPS multiple peaks were performed, wherein the baseline line estimations were also performed with the help of the adjacent averaging technique. The XPS spectra of GO and N-rGO samples were recorded using the XPS spectrometer , where the XPS spectrum for each sample was recorded , at ambient conditions.The optical bandgap of synthesized material samples was obtained through UV/vis spectroscopy. NMP (N-Methyl-2-pyrrolidone) was used as a solvent to make an N-rGO-based dispersion solution, where an N-rGO dispersion solution with a concentration of 0.20 mg/mL was used to deposit thin films. Before a thin film deposition, a homogenous dispersion solution was obtained via ultrasonication of each material sample for 40 min, followed by magnetic stirring (1 h). Before thin film deposition, N-rGO-based dispersion solutions were centrifuged at 7000 rpm to separate the unexfoliated graphite structures from the dispersion solution to deposit uniform thin film. The POLOSTM spin coater was utilized to deposit thin film, where a uniform thin film of GO- and N-rGO-based samples was deposited on the glass substrate at 4000 rpm.From a theoretical point of view, the correlation between parametric values was attained via UV/vis spectra, i.e., transmission (T), absorption (A), and reflectance (R), which may be established through Equation (2) .(2)R+A+a is the absorption coefficient, I(d) is the intensity at a depth of thickness d, and oI is the intensity at zero thickness. Moreover, the energy bandgap can also be deduced from absorption coefficients [gE) can be calculated from Equation (6).gE is optical bandgap energy, hv is the photon energy, a is defined as the absorption coefficient, and β is the disorder parameter constant. Significantly, the nature of transitions is defined by j parametric values. Specifically, J parametric values, 1/2 describes the directly allowed transitions, 3/2 defines the directly forbidden transitions, 2 defines the indirectly allowed transitions, and 3 describes the indirect forbidden bandgap-related transitions [ahv2 versus bandgap hv.The Beer–Lambert law, defined in Equation (3), defines a correlation between the light attenuation and certain material properties, at which the relationship between light absorbance and transmittance may be defined . The absficients . The optnsitions . ConsequIn this study, the facile synthesis of nitrogen-doped reduced graphene oxide was demonstrated using the microwave-assisted hydrothermal method. The successful synthesis of GO and N-rGO was confirmed by Raman, FTIR, XRD, XPS, FESEM, and EDX mapping and spectroscopic techniques. In XPS analysis of N-rGO, the maximum nitrogen doping concentration of ~5.51 at.% was conformed. In UV/vis spectroscopic analyses of GO and N-rGO, the tunable optical bandgaps were determined at about 3.4, 3.1, 2.5, and 2.2 eV, respectively. An enhanced electrical conductivity was observed in N-rGO samples, where an increment in electrical conductivity was observed with an increase in nitrogen doping concentration. More specifically, pyrrolic-N bonding configuration was found to be more dominant in N-rGO material samples, which enhance the π-bond in carbon atoms by reducing the Stone–Wales defect and forming percolation ways for electrons, which ultimately results in quitting a conduction gap and enhancing n-type electrical conductivity. The findings suggest that the synthesis approach adopted in the current study is suitable to produce N-rGO. The tunable optical bandgap in the visible region and enhanced n-type electrical conductivity (~0.781 S/cm) observed make it a promising material for developing future optical and electronic applications at the nanoscale.


The adsorption—for separation, storage and transportation—of methane, hydrogen and their mixture is important for a sustainable energy consumption in present-day society. Graphene derivatives have proven to be very promising for such an application, yet for a good design a better understanding of the optimal pore size is needed. In this work, grand canonical Monte Carlo simulations, employing Improved Lennard–Jones potentials, are performed to determine the ideal interlayer distance for a slit-shaped graphene pore in a large pressure range. A detailed study of the adsorption behavior of methane, hydrogen and their equimolar mixture in different sizes of graphene pores is obtained through calculation of absolute and excess adsorption isotherms, isosteric heats and the selectivity. Moreover, a molecular picture is provided through z-density profiles at low and high pressure. It is found that an interlayer distance of about twice the van der Waals distance of the adsorbate is recommended to enhance the adsorbing ability. Furthermore, the graphene structures with slit-shaped pores were found to be very capable of adsorbing methane and separating methane from hydrogen in a mixture at reasonable working conditions (300 K and well below 15 atm). Methane and hydrogen gases have raised considerable attention because of the advantages they have over more traditional fossil fuels in the context of energy storage and delivery . While tnditions ,8. Asidenditions . Anotherural gas ,11.Clearly adsorbent materials are needed that can store (for transportation), capture or separate gases ranging from pure hydrogen to pure methane and mixtures of interest in between. Multiple materials have been suggested for this purpose such as metal organic frameworks ,13, actiAs in all separation processes, the operating conditions are of utter importance for its applicability. In order to improve the current storage possibilities for hydrogen and to facilitate the processes described above, adsorption preferentially takes place at low pressures and around room temperature. This is essential because large pores can accommodate many molecules under high pressure, but will most likely be less selective . SmallerIn many molecular simulation studies, such systems are modeled using the Lennard–Jones potential to represent the dispersion interactions, despite the known limitations-at both long and short distance range-of this potential ,27. In sThe ILJ potential has been extensively tested in molecular dynamics (MD) simulations with success in the past years, but has never been used in a Grand Canonical Monte Carlo (GCMC) setting to the best of our knowledge ,38,39. WThe paper is oulined as follows: In GCMC simulations were performed by means of the LAMMPS package ,46 with i. The absolute amount adsorbed of a given simulation is the number of molecules that are present in the simulation box after convergence. i and For the adsorption isotherms, both the absolute and excess amounts of molecules were calculated whereby the latter was obtained by subtracting the amount that would be present in a bulk gas with the same volume, from the absolute amount adsorbedof state .P is the pressure, i and t is a fitting parameter. The value of t as extrapolated from the data in ref. [It has been experimentally verified that methane adsorption obeys the Toth equation (2)nia=q in ref.  is 0.91  in ref. ,37. In f in ref. ,51,52,53This choice is further validated by the quality of the Langmuir fittings with x is the mole fraction and a and b represent different molecules Qm=〈UThe force field used to model the gas-gas and the graphene-gas interactions assumes that the intermolecular interactions are pairwise additive and both the gas molecules and graphene sheets are rigid . The only considered contribution to the intermolecular interaction is a van der Waals term, namely the ILJ potential ,31(6)V, it is seen that, as for methane, the smallest pore adsorbs only a very limited amount of pure hydrogen, 0.08 mmol cmLooking at the hydrogen uptake from the methane/hydrogen mixture, it is seen that there is a strong influence from methane on the adsorption behavior of hydrogen for the three largest pores. Indeed, the presence of methane seriously hinders the adsorption of hydrogen, lowering the molecular uptake substantially to 0.05 mmol cmk, decreases. Since a larger k constant indicates a strong interaction with the solid material, this suggests that better adsorption will be possible in smaller pores. The same trends are observed for hydrogen, but the equilibrium constants are substantially smaller as could be inferred from the adsorption isotherms themselves. For the mixture, it is seen that the presence of the other gas, clearly influences the fitting parameters of the isotherm. For the methane gas, Langmuir models were fitted to the adsorption isotherms for the three larger pores  for which the parameters are given in A comparison between the uptake in the pore and the possible uptake of bulk gas in an empty container, is given as excess adsorption isotherms in For pure methane, it is seen that the 5 Å pore, which showed close to no adsorption, has an excess amount of molecules adsorbed of 0.04 mmol cmAs already indicated by the absolute adsorption isotherms, the adsorption efficiency slightly lowers when going from pure methane to methane from the mixture. Indeed, these numbers drop slightly to −0.04 mmol cmFor pure hydrogen in the 5 Å pore, the excess adsorption is mostly strongly negative  instead of forming bulk gas as was the case for hydrogen. In the 5 Å pore, there is still no considerable adsorption found, even at this high pressure, due to steric effects as already mentioned.It was shown in the previous sections that the higher the pressure, the lower the advantage of the graphene pore becomes compared to an empty container at the same pressure. Again, this behavior can be understood by looking at the z-density profile and considering the equilibrium distance for the graphene-methane interaction, the pore of 8 Å allows methane molecules to enter the pore and sit in a position where the centre of mass is located more or less at the ideal distance from both graphene sheet feeling optimal attraction from both. There is, however, only a small region where this is possible and once this layer is saturated, the amount of molecules that further enter the system lowers. Newly entering molecules will now be forced to be placed closer to the graphene sheets, costing more energy. For the larger pores of 14 Å and 20 Å, the saturation of the pore requires larger pressures. There are now two ideal positions for the methane molecules, at either graphene sheet. As these ideal positions gets filled up, there is still room left in the middle of the pore to take up less ideal positions.For hydrogen, it is interesting that, whereas at 1 atm, the 5 Å pore showed the highest adsorption peak, at 70 atm this is replaced by the 8 Å pore, where an overlapping double layer is found. In general, as expected, much larger quantities of molecules are adsorbed under these high pressures. It is also notable that no third or fourth adsorption layer is observed in the larger pore, but rather a large bulk gas is present in the middle of the pore reinforcing the results that, at these conditions of temperature and pressure, hydrogen is not attracted by graphene.Interestingly, the effect of the high pressure is rather limited on the behavior of the methane gas within the mixture. Whereas it could be expected that large pressures make any molecule adsorb, rendering the pore less selective, this effect is quite limited. Indeed, the z-density profile for methane in the mixture is rather similar to the profile of the pure methane gas. Conversely, the hydrogen is significantly prohibited to adsorb in the pore in the presence of methane, leaving only small amounts of adsorption. The exception being of course the 5 Å pore, where methane molecules do not enter and thus not hinder the hydrogen adsorption.In this work, the adsorption of methane, hydrogen and their equimolar mixture in graphene structires with slit-shaped pores of different sizes was investigated at a large pressure range and a temperature of 300 K using GCMC simulations and a tailor-made ILJ force field. Specific attention was dedicated to the role of the pore size, by means of varying the interlayer distance (between 5 Å and 20 Å) between the graphene pore walls. Absolute and excess adsorption isotherms, isosteric heats, selectivities and z-density profiles were calculated.The obtained adsorption isotherms showed that the 8 Å, 14 Å and 20 Å pores are capable of adsorbing a large amount of methane, substantially outperforming an empty container of the same size. Thereby, the 8 Å pore was the most efficient one at pressures below 40 atm and the 14 Å was most efficient at higher pressures leading to an isosteric heat of about twice the value compared to that obtained for larger pores. For pure hydrogen systems at these conditions, only the 8 Å pore showed some benefit compared to an empty container, while the 14 Å and 20 Å pores barely influenced the presence of hydrogen in the simulation box. The smallest considered pore of 5 Å pore proved too be to narrow to adsorb substantial amounts of methane and/or hydrogen. For the mixture, it was shown that methane is strongly favored over hydrogen since almost no hydrogen was adsorbed in all considered pores and the methane adsorption was only hindered to a small extent by the present hydrogen. This is very good, for instance, to remove CHThe isosteric heats of adsorption showed a decreasing trend with increasing pore size due to the diminishing interaction of the adsorbate with the opposing graphene sheets. Indeed, in the 8 Å pore, the methane molecules are capable of positioning themselves at the ideal distance of either graphene sheet. For larger pores, the adsorbed molecule is forced to ’choose’ between either graphene sheet and the interaction with the other sheet diminshes with increasing interlayer distance. This is also reflected in the z-density profiles where it is seen that the adsorption layers on a given graphene sheet are located closer to that graphene sheet when the interlayer distance increases and the attraction of the opposing sheet dwindles. In accordance with the adsorption isotherms, the isosteric heat for hydrogen is about 4 to 5 times lower than for methane.Given the clear preference of the pore walls for methane over hydrogen, a very high selectivity was found for the 8 Å, 14 Å and 20 Å pores. Especially the 8 Å pore showed a selectivity of over 80 at relatively low pressures, but also the larger pores show selectivities above 10 over the entire pressure range. Overall, the use of fine-tuned graphene pores seems very promising for the separation of methane/hydrogen mixtures at reasonable operating conditions. Given the use of more advanced pore materials at optimized interlayer distances, such as graphyne, gyroid materials, pillared materials or square pores, the adsorption and separation capacities may be further increased.