Graphene is a promising material that may be potentially used in biomedical applications, mainly for drug delivery applications. In our study, we propose an inexpensive 3D graphene preparation method by wet chemical exfoliation. The morphology of the graphene was studied by SEM and HRTEM. Moreover, the volumetric elemental composition  of the materials was analyzed, and Raman spectra of prepared graphene samples were obtained. X-ray photoelectron spectroscopy, relevant isotherms, and specific surface area were measured. Survey spectra and micropore volume calculations were made. In addition, the antioxidant activity and hemolysis rate in contact with blood were determined. Activity against free radicals of graphene samples before and after thermal modification was tested using the DPPH method. The RSA of the material increased after graphene modification, which suggests that antioxidant properties were improved. All tested graphene samples caused hemolysis in the range of 0.28–0.64%. The results showed that all tested 3D graphene samples might be classified as nonhemolytic. Nanomedicine is a new generation of medicine that involves the application of nanoscaled materials. Among them, graphene is an emerging material due to the variety of its properties. In most research, graphene has been studied in electrochemical applications as an electrode material for constructing supercapacitors, photovoltaic cells, and metal–air batteries ,3,4,5,6.By definition, graphene is a paternal 2D material; however, its 3D structuring is possible and may deliver a new sort of material comprising graphene’s unique properties  with ones typically attributed to standard 3D materials (porosity and enhanced surface area) ,13,14.The 2D nature of pristine graphene hinders its applicability to the immobilization of large molecules  and microscopic objects such as cells or bacteria ,16. SuchGraphene has also been studied as an additive to polymeric matrixes. Such hybrid materials have shown improved mechanical properties . GraphenHowever, all modifications should be defined as safe and non-toxic. The aim of the proposed study was to determine graphene properties in terms of its applicability as a material for living-cell-bearing supports of advanced 3D architecture. The scheme of the method used by the authors is shown in In such a case, non-destructive activity towards basic body fluids such as blood and lymph is absolutely essential since such cell-containing structures will stay in permanent contact with fluids. Moreover, the antioxidant properties of graphene-based materials should be tested, since this feature is a part of widely understood blood compatibility. In this work, the so-far-achieved goals in the 3D structuring of graphene flakes will be exploited to receive 3D graphene derivatives, the blood compatibility of which is still unknown. We assume that blood compatibility should be investigated since our 3D structuring protocol involves the addition of a hard template, a cationic surfactant, and other chemical reagents, which have to be used to achieve advanced pore structure. The up-to-date collected knowledge and skill by authors in the area of graphene exfoliation and subsequent 3D structuring has been assumed as a starting point for the design of 3D graphene derivatives that are safe in contact with blood.Graphene and other carbon materials need to be characterized by a set of widely approved experimental methods, as in this study. Such features as surface area, pore structure, and chemical composition are crucial for any carbon-based material apart from biological activity.2CO3 nanocrystals precipitate among exfoliated graphene flakes upon drying the reaction mixture. Thus, the template particles were removed by HCl etching and washing with distilled water. N2 sorption analysis showed that the specific surface area decreased (330–391 m2/g) below the value determined for the used paternal graphene nanoplatelets (750 m2/g). In parallel, changes of the pore structure became visible. The total pore volume decreased in all samples from 0.999 (GF-750) to 0.220 cm3/g (GF_Na_600). Surprisingly, the contribution of the mesopore volume Vme to the total pore volume Vt increased from 36% to 79%. It is notable that for three of four investigated samples the share of mesopores is close to 50% or much higher. The presence of mesopores (average pore diameter from 2 to 50 nm) is significant for applications involving a liquid environment where adsorption/desorption proceeds .The elemental composition and surface parameters for the obtained samples of porous 3D graphene are given in Additionally, the morphology details of representative samples are shown in the HRTEM images . Figure In the case of carbon-based materials, the surface elemental composition may substantially differ from the bulk elemental content. In general, carbon is a reactive element, and surface atoms may come into reaction with gases present in the atmosphere. In particular, oxygen is supposed to yield diversified surface-oxygen-based species. Therefore, two methods need to be applied to determine elemental composition: elemental combustion analysis  and XPS .The results of combustion elemental analysis only allowed the bulk content of three elements to be determined: C, N, and H, while the specific chemical environment of the heteroatoms embedded in the carbon matrix remains unknown. Therefore, obtained samples were additionally supported by X-ray photoelectron spectroscopy (XPS) investigations. A sample with the most promising biological properties was selected for the XPS study based on DPPH and blood compatibility. The XPS spectra of the representative GF_Na_800 sample were determined and are demonstrated in 3 hybridized atoms (band C 1s at binding energy 285.0 eV), which is characteristic of graphene materials. The C 1s spectra of the GF_Na_800 sample are composed of four peaks corresponding to C-C bond (sp3) peak at 285 eV  specimen is the absorbance of solution in contact with tested samples;[OD] negative is the absorbance of blood in physiological saline;[OD] positive is the absorbance of blood in water.For the study of compatibility of the graphene samples with blood, anticoagulated sheep blood (0.2 mL) was added to physiological saline solution (10 mL) which contained 1 g of each sample (n = 3) according to ASTM Hemolysis (Direct Contact and Extract Methods) GLP Report. Two control samples with the addition of fresh blood were prepared: positive (in water) and negative . The mixture was incubated  and the suspension was centrifuged . The absorbance was measured spectrophotometrically at 540 nm by using the Multiscan FC  . FinallyThe applied manufacturing protocol, despite some chemical reagents used as hard templates and cationic surfactants, delivers a blood-compatible porous material potentially applicable for further living-cell-involved manipulations. The antioxidant activity of the material increased after graphene modification. The maximum RSA = 89% was noticed after graphene treatment at 800 °C. Moreover, the increase in the temperature in the preparation process results in an increase in the hemolysis rate. The structural properties indicate a high quality of obtained porous graphene structures with a well-developed surface area and pore structure, which is essential for living cell immobilization. We have proven that the manufacturing protocol is relatively environmentally friendly and low-cost. These aspects of the performed study will be continued in further research.


For EGr/GC, the oxidation peak was higher and appeared at lower potential (+1.12 V) compared with that of bare GC (+1.35 V). The linear range for AZT obtained with the EGr/GC electrode was very wide, 10−8–10−5 M, the sensitivity was 0.68 A/M, and the detection limit was 3.03 × 10−9 M. It is important to mention that the sensitivity of EGr/GC was three times higher than that of bare GC (0.23 A/M), proving the advantages of using graphene-modified electrodes in the electrochemical detection of AZT.An electrochemical cell containing two graphite rods was filled with the appropriate electrolyte (0.2 M ammonia + 0.2 M ammonium sulphate) and connected to the exfoliation system to synthesize graphene (EGr). A bias of 7 V was applied between the anode and cathode for 3 h. After synthesis, the morphology and structure of the sample was characterized by SEM, XRD, and FTIR techniques. The material was deposited onto the surface of a glassy carbon (GC) electrode (EGr/GC) and employed for the electrochemical detection of azithromycin (AZT). The DPV signals recorded in pH 5 acetate containing 6 × 10 For many years, infectious diseases have represented a difficult, persistent, and on-going public health challenge. However, by the mid-1900s, following the introduction of antibiotics, the mortality rate of infectious illnesses had significantly decreased , and theAzithromycin (AZT), an azalide—a subclass of macrolide-type antibiotics, is a second-generation broad-spectrum antibiotic with a long half-life and a high tissue penetration degree , primari2O4 [However, most of the reported methods present serious drawbacks since the microbiological approaches need long incubation periods and are affected by limited detection ability and poor accuracy ,25, whil2O4 ; aniline2O4 ; renewab2O4 ; and ion2O4 . Among t2O4 ,43.With several surprising features in its two-dimensional structure , graphenIn this work, we present a simple approach for modifying a glassy carbon electrode with graphene obtained by a simple electrochemical method, without the use of organic solvents. The modified electrode exhibits improved characteristics both in terms of sensitivity and the detection limit of AZT, in comparison with bare GC. In addition, we used the standard addition method for the quantification of AZT in spiked samples, demonstrating excellent results.High purity (99.995%) graphite rods  were purchased from Sigma-Aldrich  and were employed during electrochemical exfoliation. Ammonium sulphate was bought from Reactivul Bucuresti , while the ammonia solution (25%) was purchased from POCH SA . Azithromycin (AZT) was purchased from HiMedia Laboratories Pvt. Limited , uric acid , dopamine hydrochloride , and dimethylformamide (DMF) were purchased from Alfa-Aesar , while L-tyrosine (Tyr) was purchased from Fluka BioChemika . Glacial acetic acid (99.5%) was purchased from CHEMICAL COMPANY  and sodium acetate was purchased from Reactivul Bucuresti . Sodium dihydrogen phosphate (99.7%) and di-sodium hydrogen phosphate anhydrous (100%) were purchased from VWR Chemicals . Acetate buffer was employed for the preparation of the solutions with pH 3.6, 4.4, and 5. Phosphate buffer was employed for the preparation of the solutions with pH 6 and 7.−2 M AZT solution) was added, so the final concentration of the drug was 2 × 10−7 M AZT. This was denoted as the real concentration  of the drug. Next, three equal volumes (50 µL) from 10−4 M AZT stock solution (in ethanol) were added to the beaker under continuous stirring and the current increase was read after each addition . The current step was plotted versus the concentration, allowing us to determine the AZT (Cx) in the analyzed drug, resulting in Cx = 1.76 × 10−7 M, while Creal = 2 × 10−7 M. The recovery was determined as the Cx/Creal ratio (88.45%). It is important to mention that the recovery data obtained for several drug solutions of different concentrations ranged from 80 to 88.45%, indicating that some of the interfering species may interact with the electrode surface. According to the drug leaflet, the powder also contains sugar, xanthan gum, hydroxypropyl cellulose, anhydrous trisodium phosphate, anhydrous colloidal silicon dioxide, aspartame (E 951), caramel cream flavor, and titanium dioxide (E 171). For azithromycin determination using the standard addition method, the following procedure was applied. In 5 mL of acetate buffer solution (pH 5), a proper amount (0.38 µL) from the commercially available pharmaceutical drug , equipped with a cold field emission gun (CSEG). X-ray powder diffraction (XRD) was employed for the structural investigations of the graphene sample. The sample was dispersed in DMF (0.2 mg/mL) and then deposited onto the surface of an amorphous quartz substrate. A D8 Advance Diffractometer , 40 kV, 0.5 mA, equipped with a LYNXEXE detector (λ = 1.5406 Å) was employed.−1 spectral domain (4 cm−1 resolution). About 0.3 mg of graphene was mixed with KBr powder and pressed in a thin pellet, before recording the spectrum. The FTIR spectrum of graphene was recorded with a JASCO 6100 FTIR spectrometer , within a 4000–400 cmA Christ-Alpha 1–4 LSC freeze dryer equipment  was employed for drying the graphene sample after electrochemical exfoliation of the graphite rods.For the electrochemical measurements  and amperometry), the cell containing three electrodes was coupled with a Potentiostat/Galvanostat Instrument PGSTAT-302N . The GC (3 mm diameter) or the EGr/GC electrode was employed as a working electrode, together with a Ag/AgCl reference (3 M KCl) and a large foil platinum counter electrode. The DPV measurements were generally recorded from 0.5 to +1.5 V vs. Ag/AgCl, at a 10 mV/s scanning rate , while the amperometric measurements were recorded at +1.12 V (for the EGr/GC electrode) and +1.35 V (for the bare GC electrode). An electrochemical cell containing two graphite rods  was filled with the appropriate electrolyte (0.2 M ammonia + 0.2 M ammonium sulphate) and connected to the exfoliation system . The pulse generator allowed us to set the time parameters of the current pulses, such as the duration (0.8 s) and the pause between two pulses (0.2 s) . During N,N-dimethylformamide (DMF) was chosen as the organic solvent for the dispersion of the graphene sample (2 mg/mL). DMF has a higher boiling point (153 °C) in comparison with that of ethanol (78.37 °C) or distilled water (100 °C), which means that it evaporates slower at room temperature. Consequently, the graphene flakes from DMF solution have a longer time to interact by π–π stacking with the electrode surface, leading to the formation of a very stable layer on top of the GC electrode. A total of 10 µL from the graphene dispersion in DMF, previously homogenized with an ultrasonic device (SONICS Vibra-Cell), was deposited by the drop-casting method onto the GC electrode and dried at room temperature for 24 h. After that, the modified electrode (EGr/GC) was used for AZT electrochemical detection and quantification.The structural characteristics of the graphene sample were next investigated by the XRD technique. The pattern of the sample can be seen in −1 can be assigned to the O–H stretching vibrations from the adsorbed water molecules, while the 1383 cm−1 band is assigned to the C–O stretching vibrations of the functional groups attached to graphene. The large shoulder at around 3000 cm−1 may be attributed to the aromatic C–H stretching bands, which are weak-to-moderate bands. The other two important bands are the aromatic ring stretching vibrations centred at around 1638 and 1577 cm−1, which generally appear as a pair of band structures. These bands are characteristic of the vibration modes of the sp2-hybridized carbon atoms (C=C). Next, more information about the vibration characteristics of the functional groups attached to the graphene surface was obtained by the FTIR technique. The spectrum of the sample embedded in the KBr pellet is presented in −1, the graphite (G) band at 1582 cm−1, and the 2D bands at 2708 cm−1, generated by the in-plane vibrations [D/IG ratio [D/IG ratio is inversely correlated with the defect-free domain or the in-plane crystallite size (La):a was determined to be 17.91 nm. This is a large value, so we can assume that the majority of the defects are located at graphene edges. This is in excellent agreement with the SEM/TEM micrographs, which revealed that the basal planes of graphenes are generally smooth and defect-free. Raman analysis is useful for determining the structural properties of graphitic materials. The defect (D) band at 1355 cmbrations , shear mbrations , and thebrations , are thebrations , indicatbrations . HoweverIG ratio , which wIG ratio , the ID/−5 M). The DPV technique was employed, and the selected pH range was 3.6–7.0. Since the pKa of azithromycin is 7.34 [a. As can be seen in p) is well-defined above pH 4.4 and has a maximum value at pH 5. At pH value close to pKa, the peak current starts to decrease .According to the literature , in solup, with the natural logarithm of the scan rate, lnυ, for irreversible reactions (linear sweep voltammetry).a is the charge transfer coefficient, sk, the standard rate constant of the surface reaction, n is the number of electrons involved in the reaction, and E0′ is the formal potential.In order to determine the number of electrons involved in the oxidation reaction, the Laviron Equation (2) was employed . The equ−5 M azithromycin by varying the scan rate from 2 to 100 mV/s. Using the Ep vs. lnυ plot, we determined nαa from the corresponding slope  solution containing 6 × 10ng slope a. The obp and pH was obtained, following the equation: Ep = 1.42 − 0.052 × pH (r2 = 0.972). Since we determined that the number of electrons involved in the redox process is 1, it is reasonable to assume that the number of hydrogen ions taking part in the electrode reaction is also 1.The pH effect on the oxidation peak potential of azithromycin was also investigated b. The pe−5 M AZT revealed significant differences between the two electrodes. For EGr/GC, the oxidation peak is higher and appears at lower potential (+1.12 V) compared with that of the bare GC electrode (+1.35 V). The increased sensitivity of EGr/GC cannot be attributed only to its high electrochemical roughness factor, which was determined to be 1.46 . Other factors are the enhanced electron transfer rate at the edges of graphene sheets and the hydrophobic interaction between AZT and graphene, which may help with the orientation of N3 amine group towards graphene, favouring its oxidation. The electrocatalytic properties of the synthesized material towards AZT oxidation are now discussed in comparison with those of the bare GC electrode . Hence, −6–6 × 10−5 M) in pH 5 acetate buffer, using the DPV technique. The corresponding signals are plotted in −5 M concentration. The linear range for AZT was 10−6–3 × 10−5 M, the sensitivity was 0.043 A/M, and the detection limit was 3.03 × 10−7 M. Further analysis of AZT was investigated by recording the AZT signals at various concentrations , proving the advantages of using graphene-modified electrodes in the electrochemical detection of AZT. Significantly better results were obtained by employing the amperometric technique , as can be seen in −5 M AZT  and for a mixture of analytes, DA + UA + Tyr + AZT (10−5 M each), are presented. All the analytes appear at well-defined oxidation potentials and their mixture led to the slight increase in the AZT oxidation peak and potential, indicating a weak influence of the interfering species.Next, the selectivity of the EGr/GC electrode was evaluated in the presence of various interfering biomolecules such as dopamine (DA), uric acid (UA), and tyrosine (Tyr). In −2 M; 0.38 µL) and the stock solution  was read and plotted versus the concentration, allowing us to determine the concentration of the drug, Cx . It is important to mention that the recovery data obtained for the three drug solutions ranged from 80 to 88.45%, indicating that some of the interfering species may interact with the electrode surface. Since the EGr/GC electrode proved to have a good selectivity, it was additionally tested for AZT quantitative analysis in pharmaceutical drug solution, using the standard addition method a,b. The −8–10−5) for AZT, with excellent sensitivity (0.68 A/M); and the detection limit was 3.03 × 10−9 M  was prepared by the electrochemical exfoliation of graphite rods and characterized by advanced techniques  to obtain information about its morphology and structure. The SEM micrographs revealed that some of the graphene flakes are large  and have a smooth surface, while others are small  and exhibit a wrinkled appearance. The flakes are also thin and randomly oriented, generating a highly porous layer which is beneficial in electrochemical investigations due to the increase in the active area of the modified electrode. The XRD technique evidenced that the sample contains few-layer graphene (95%) and multilayer graphene (5%), while the FTIR technique allowed the identification of the oxygen-containing groups attached to graphene. The graphene-modified electrode (EGr/GC) was next used for azithromycin (AZT) electrochemical detection and quantification. Excellent results were obtained by employing the amperometric technique .


In this work, the adsorption energy and the electronic properties of a graphene/WS2 structure were calculated by first-principles before and after adsorption of NH3. The calculation results indicate that the bandgap of the material was appreciably reduced after NH3 was adsorbed. In addition, a graphene/WS2 gas sensor was prepared. The response of the sensor to NH3 at a concentration of 100 ppm was 2.42% and 1.73% at 30 °C and 60 °C, respectively. Combining simulation with experiment, it is feasible to use graphene composite WS2 to detect NH3, which provides a new idea for applications of graphene and other composite materials in gas sensing.Two-dimensional materials have outstanding application prospects in gas sensing. By constructing composite structures of various gas-sensitive materials, more-efficient and sensitive gas sensors can be further developed. After graphene is compounded with WS In recent years, in-depth research on two-dimensional materials has promoted further development of gas sensor technology, and has enabled gas sensors to move toward high sensitivity, stability, and reliability, as well as a fast response and recovery rate ,2,3. Amo2, MoTe2, and other materials have advantages of, e.g., high sensitivity and function at room temperature [However, because of limitations imparted by the electrical properties of graphene, the performance of gas sensors prepared by using graphene has thus far failed to meet theoretical expectations. Accordingly, researchers have prepared graphene-based composite materials , metal oxides, and functional polymers on the surface of graphene). Rapid transfer of charges at the interface of the composite material can improve the sensing performance ,6,7. In perature ,9.2, a dense layered stacked structure readily forms in the process of forming a charged network inside the material. This stacked structure is not conducive to the contact between the nanosheets and gas molecules [2 with graphene to prepare a graphene/WS2 composite structure. The large specific surface area of graphene can absorb more gas molecules, whereas WS2 can output a higher response. Complementing the advantages of the two materials will facilitate gas detection [As a representative material in TDMs, during electrical testing of WSolecules . Alternaetection ,12.3 based on a graphene/WS2 composite structure. The electronic energy band structures of WS2, graphene/WS2, and graphene/WS2/NH3 were calculated by using first-principles, and the bandgap changes were compared and analyzed. Moreover, a graphene/WS2 composite gas sensor was prepared, and the response sensitivity of the device to NH3 at various temperatures was discussed. A combination of theoretical simulation and experiment helps to better understand detection of NH3 by the graphene/WS2 composite structure.This work discusses adsorption of NH2 composite structure; its properties were studied by using CASTEP based on density functional theory [−5 and 1 × 10−1 eV/nm, respectively. The corresponding maximum stress displacement did not exceed 0.02 GPa and 5 × 10−4 nm, respectively. The BFGS algorithm was used for geometric optimization [The research goal is adsorption of ammonia gas by using a graphene/WSl theory ,14. To el theory . The Brimization . Figure 2 substrate by using wet transfer technology. A comb-shaped electrode was deposited onto the substrate. The space of the electrode was 200 μm. WS2 nanosheets were prepared by a one-step hydrothermal reaction. Tungsten chloride  and 25 mmol of thioacetamide (CH3CSNH2) were dissolved in 35 mL of deionized water and stirred for 40 min; then, the solution was transferred to a Teflon-lined stainless steel autoclave, held at a temperature of up to 220 °C, and kept at this temperature for 24 h. After cooling, the reaction products were washed and freeze-dried. Then, WS2 was ultrasonically dispersed in deionized water. The concentration of the WS2 suspension was 10 mg/mL. A 1.5-µL pipette was used to apply one to two drops of WS2 suspension onto the graphene above the substrate. After drying at room temperature in air, it was aged at 60 °C for 2 h to achieve stability. After the device was prepared, a WS-30B system was used to test the response characteristics of the graphene/WS2 composite gas sensor in a 100 ppm NH3 atmosphere. The temperature, humidity and gas concentration are automatically controlled by the gas sensing test bench, and the atmosphere of the test chamber is controlled by software. To reduce the influence of humidity in the experiment, the humidity of the test system was controlled to 25%, and the test temperatures were 30 °C and 60 °C. Before the test, background gas  was introduced, and then NH3 was introduced for testing and signal acquisition.The graphene grown by CVD was transferred onto an Si/SiO2 directly affect the electron transport properties inside the material. Therefore, the energy band structure of the composite material before and after the adsorption of gas was calculated. The bandgap of graphene is zero, and thus in this work only the energy band of WS2 and graphene/WS2 was calculated. 2. 2 was 1.945 eV. Both the bottom of the conduction band and the top of the valence band are located at point G, indicating that WS2 is a direct bandgap. This value is similar to previous results [The interfacial interactions of graphene/WS results ,18, whic2 before and after adsorption of NH3. When graphene was compounded with WS2, the bandgap of the composite structure changed significantly. The top of the valence band was at the high symmetry point G, and the bottom of the conduction band was at point G. The composite structure transformed into an indirect bandgap; the bandgap value was 1.187 eV. Concomitantly, the independent energy band structure of WS2 was largely retained when the two materials were composited. The conduction band shifted to the Fermi level with a low bias, such as in n-type doping. This is attributable to the electrons provided by the graphene transition to the conduction band. The energy height of the electrons that left the bottom of the conduction band was reduced. When the composite structure adsorbed NH3 molecules, its energy band structure did not change significantly compared with the structure of not-yet-adsorbed gas; but the bandgap value decreased to 1.246 eV, and the conduction band continued to move toward the Fermi level. Thus, after NH3 was adsorbed, electrons more readily migrated from the top of the valence band to the low conduction band, and the conductivity of the composite material was significantly improved [improved .2 before and after adsorption of NH3. After adsorption of NH3, the total density of states shifted slightly to the left, which indicates that charge transfer was facilitated [3 changed the strong hybridization and bonding between the atomic orbitals, which changed the charge transfer. In addition, under this simulation system, the adsorption energy Ead of graphene/WS2 for NH3 can be calculated as follows [ilitated . Concomi follows ,22.Ead 3 adsorption was −94,137.75 eV, whereas the total energy of the composite material without adsorbed gas ad of graphene/WS2 to NH3 was calculated as −1.25 eV. This value indicates that the composite material can readily achieve NH3 adsorption.In this simulation system, the total energy 2, the electron transport inside the material changed. Moreover, the composite material readily adsorbed NH3 and the bandgap of the adsorbed material was reduced. This indicates that the composite of graphene and WS2 will facilitate construction of a sensor for detecting NH3.Through analysis of the simulation results, it was evident that after graphene was compounded with WS2 prepared by the hydrothermal method. 2 was composed of agglomerated flocculent particles. After partial magnification in 2 nanosheets were found at the edge of the WS2 block and presented a loose structure, indicating the high specific surface area, which was beneficial to NH3 adsorption and improving the gas sensitivity. 2, and the XRD pattern is consistent with pure hexagonal WS2 (JPCDS No. 084-1398). The reflections at 14.22°, 34.04°, and 56.78° (corresponding to the (002), (100), and (110) planes, respectively) confirm the presence of the hexagonal phase. The sharp peaks indicate the crystallinity of the as-prepared sample. Some peaks were evident, along with slight rightward shifts of the peak positions and broadening, which indicates that there was some residual compressive stress on the surface of the WS2 nanosheets after freeze-drying.2 gas sensor when exposed to 100-ppm NH3 at 30 °C with 25% humidity. In accordance with the NH3 introduction time, the resistance of the gas sensor first increased and then decreased. This trend indicates that the device adsorbed gas molecules in an NH3 atmosphere, and detected NH3. As the concentration of gas absorbed onto the surface of the graphene and WS2 increased, charge transfer occurred between the gas molecules and the composite material. The type and concentration of carriers inside the composite material changed, which eventually led to the change in sensor resistance. This change corresponds to the gas response of the device [e device .The response time (recovery time) was defined as the time required to reach 90% of the change in sensor resistance upon exposure to the target gas (air). 2 sheet failed to desorb, which reduced the resistance of the WS2, improved the barrier between graphene and WS2, accelerated the transfer between electrons, and changed the overall electrical performance of the devices [A single response/recovery curve can indicate the response and recovery time of the device, but the overall performance of the device must be measured over a long period of time. During the entire experiment, the humidity was set to be 25% and the temperature was 30 °C, which eliminated the interference that would have been caused by temperature and humidity changes.  devices . Temperature corresponds to the diffusion rate of gas molecules. The detection performance of the sensor differed at different temperatures. Therefore, after adjusting the temperature to 60 °C, one can test the sensor again. Other experimental conditions are the same as those at 30 °C.2 gas sensor when the NH3 concentration was 100 ppm and temperature was 60 °C. Similarly to the test result at 30 °C, the sensor still detected NH3 molecules at 60 °C. At 60 °C, the response time of the gas sensor was 300 s and the recovery time was 600 s. The response time was the same as that at 30 °C, but the recovery time was significantly shortened. This is because the increase in temperature aggravated the movement of NH3 molecules. Molecules that might be attached to the gas-sensitive material at 30 °C will also accelerate desorption as the temperature increases. In accordance with the various means by which gas molecules are adsorbed on the surface of the material, the means of gas adsorption can be divided into physical adsorption and chemical adsorption. Physical adsorption mainly depends on the interaction forces between molecules, whereas chemical adsorption depends on the formation of chemical covalent bonds between gas molecules and the surface of the composite material. In general, physical adsorption precedes chemical adsorption [3 molecules mostly remain on the surface of composite materials by physical adsorption.sorption . The tem3 concentration was 100 ppm and the temperature was 60 °C. The device responded and recovered many times within more than 1 h of test time.3 such that NH3 underwent partial chemical adsorption concomitantly with physical adsorption. Over the course of desorption, a temperature of 60 °C is insufficient to destroy the stable chemical covalent bond. Chemical adsorption of NH3 on the surface of the material prevented the device from responding in the next cycle. As a result, there was a smooth region below the curve. 3 molecules that were not desorbed gathered on the surface of the composite materials. The aggregated molecules hindered the desorption and slowed the recovery rate. Therefore, the recovery time exhibited a gradually increasing trend [The device can maintain the excellent response after passing many cycle tests. Due to multiple recovery processes, many NHng trend . 0 − Rt|)/R0 × 100%. In the aforementioned formula, R0 is the initial resistance value of the gas sensor in air and Rt is the resistance value of the gas sensor after adsorbing NH3 molecules. At temperatures of 30 °C and 60 °C, the response of the sensor was 2.42% and 1.73%, respectively. The response of this type of device was lower than that in previous studies, but exhibited a faster response time (The response can intuitively reflect the performance of the device. The response (S) of the sensor was defined as (|Rnse time .2 is not obvious and there is stacking, which cannot provide sites for more gas molecules. The electron migration ability is reduced, and the composite material fails to assume the role of a bridge for electron transport.The low response mainly corresponds to the method used to prepare the device. Especially after one transfers the graphene to the electrode, the graphene will break, curl, and leave contaminants. In addition, the layered structure of WS3 by graphene/WS2 composites through simulation. After adsorption of NH3, the bandgap value of the material was significantly reduced, and the adsorption energy was calculated: 1.25 eV. The simulation results indicate that the use of graphene composite WS2 to detect NH3 is feasible under certain conditions. A graphene/WS2 gas sensor was also prepared, which realized detection of NH3 and verified the assumptions of the simulation results. At 30 °C and 60 °C, the response to 100-ppm NH3 was 2.42% and 1.73%, respectively. This research provides new ideas for applications of graphene and other composite materials in gas sensing.This work analyzed adsorption of NH


Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in  ATAC-seq is also robust to small sample sizes and has been adapted for single-cell applications5. Thus, ATAC-seq is quickly becoming the assay of choice for chromatin studies.Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is a relatively new technique that has advanced our understanding of chromatin-mediated gene regulation. The technique offers several advantages over other sequencing techniques that examine chromatin, such as DNase-seq, micrococcal nuclease (MNase-seq), or formaldehyde-assisted isolation of regulatory elements (FAIRE-seq), including low input requirements 6, and many studies using ATAC-seq focus on mammalian or human-derived tissues9. Recent studies used ATAC-seq to investigate other tissue types, including tissues from plants10, zebrafish11, and livestock12. Some ATAC-seq studies using Drosophila melanogaster have been published, though many of these studies focus on embryogenesis and larvae15. One recent report performed ATAC-seq from central brains after fluorescent sorting of GFP-positive cell bodies16. However, the physical dissection and dissociation of many brains can be labor-intensive and yield relatively few cells, in that case 6000–10,000 cells16. We were interested in optimizing the standard ATAC-seq protocol to allow efficient library preparation while taking advantage of the many genetic tools available for labeling specific Drosophila neurons17.ATAC-seq was developed using peripheral blood samples6. Additional studies optimized ATAC-seq library preparation from various tissue types in several storage conditions21. Most studies in Drosophila larvae treat the tissue similarly to mammalian tissues, only adding an additional washing step to clean the embryos or larvae before homogenization22. ATAC-seq library preparation using adult Drosophila poses challenges that are not present in other tissues. For example, the insect cuticle is a chitin structure that is difficult to process by simple homogenization. Another challenge is eye color, which is commonly used to identify the presence of transgenic insertions, and many transgenic fly lines contain the mini-white (wmC+) gene. In a w- background, mini-white confers a yellow to red eye color, so eye color can range from white (w−) to red, depending on the genotype. Some Drosophila retinal pigments emit fluorescence24 that can complicate fluorescent-activated nuclei sorting (FANS). Finally, the Drosophila genome is considerably smaller than mammalian genomes26, so established ATAC-seq parameters may use higher-than-optimal enzyme concentrations and long incubation times that may lead to low-quality ATAC-seq libraries when prepared from fly tissues. Thus, optimizing each parameter for ATAC-seq library preparation using adult flies should be performed prior to sequencing. However, these experiments add considerable sequencing costs, putting the approach out-of-reach for many labs.ATAC-seq library preparation has several parameters that can be optimized based on the input tissue. Many of these parameters are addressed in the original ATAC-seq protocolDrosophila neurons. Our goal was to optimize each variable in the library preparation process, with particular attention to challenges specific to Drosophila model systems. Our protocol provides guidelines for ATAC-seq library preparation using Drosophila and outlines the optimal parameters for each library preparation step. This protocol will accelerate studies of chromatin-mediated gene regulation in flies by coupling the powerful genetic tools in Drosophila with high-throughput, in-depth examination of chromatin structure.Identifying cell- and tissue-specific roles in organism function is a major goal of many current studies, which require specific and sensitive detection of target cells, especially for rare cell types. To facilitate these studies, we developed a protocol to generate ATAC-seq libraries from adult Canton S (BL64349), tub-Gal4 (BL5138), elav-Gal4 (BL458), nSyb-Gal4 (BL51635) UAS-GFP-nls , UAS-Stinger , TH-Gal4 (BL8848), Cha-Gal4.19B (BL6798), and vGAT-Gal4 (BL58980). w* Berlin flies have been in the lab for many generations27 and were originally a gift from Martin Heisenberg. Flies were reared in bottles containing standard cornmeal agar and grown at 25 °C with 70% relative humidity and a 12-h light/dark cycle.The following fly lines were obtained from the Bloomington Drosophila Stock Center  and used in this study: 2, 0.1% Tween-20, 0.1% Nonidet P40 substitute, 0.01% digitonin , and 1% bovine serum albumin] in a Dounce homogenizer. The tissue was homogenized with the A pestle until the resistance disappeared. The crude homogenate was passed through a 40 μm filter and homogenized with 15 slow strokes with the B pestle. The crude nuclei were washed with wash buffer  and centrifuged at 500g for 10 min per wash. The washed nuclei were resuspended in 1 mL wash buffer with 3 mM DAPI for evaluation and sorting.All equipment and buffers were pre-chilled to be ice-cold. All plastic and glassware used for nuclei isolation was pre-treated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Flies were collected into empty bottles and frozen at − 80 °C. Then, the flies were placed in pre-chilled (in a − 80 °C freezer) sieves and agitated for 1 min to separate the heads from the bodies. The fly heads were added to lysis buffer , 16.5 μL 1× PBS, 0.5 μL Tween-20, 5.5 μL water, and Tn5 enzyme . The mixture was incubated at 37 °C for various times and the DNA was purified using a MinElute PCR purification kit . Then, the purified DNA was mixed with CD index primers (Illumina) and Phusion High Fidelity PCR MasterMix  and was amplified by 1 cycle of 72 °C for 5 min and 98 °C for 30 s, followed by 5 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min in a C100 thermocycler . 5 μL of this reaction was removed and used for a qPCR side reaction. The aliquot was mixed with the same CD index primers and SsoFast EvaGreen Supermix (BioRad) and amplified for 1 cycle of 98 °C for 30 s and 40 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min in an Applied Biosystems 7900HT qPCR instrument (ThermoFisher Scientific). Total fluorescence was calculated from the background-subtracted maximum fluorescence of each sample. The cycle number corresponding to a portion of the total fluorescence was determined (~ 8–10 cycles) and the DNA was amplified for this number of additional cycles. After amplification, the DNA was purified with 0.5× and 1.1× AMPure XP beads  to remove primer-dimers and high-molecular weight DNA (> 5000 bp). The purified DNA was stored at − 20 °C until further analysis.Purified nuclei were centrifuged for 10 min at 500ATAC-seq library quality was assessed using an Agilent 2200 TapeStation and High Sensitivity D5000 ScreenTape assays  at the Huntsman Cancer Institute High Throughput Genomics core facility or the Genome Technology Access Center of the McDonnell Genome Institute at the Washington University School of Medicine .28. Briefly, Fastq files were quality checked using FastQC (v 0.11.9). Adapter sequences were removed with CutAdapt (v3.4). The sequenced reads were aligned to the dmel_r6.26 genome assembly using Bowtie2 . Aligned reads were sorted with Samtools  and deduplicated with Picard (v2.23) using the MarkDuplicates command. Aligned, filtered, and deduplicated reads were used to call peaks using MACS2 software . Then, featureCounts  was used to generate the count matrix. Differential accessibility analysis was performed using DESeq2 . Insert size distributions were calculated using Picard. Peaks were visualized using the IGV browser (v2.7.2).ATAC-seq libraries constructed using nuclei isolated from dopaminergic and GABAergic neurons were sequenced on a Novaseq 6000 instrument (Illumina) using 50-bp paired-end reads. The sequencing data was analyzed as previously describedThe data are represented as means ± standard deviation. Comparisons between homogenizer size and fly volume were analyzed by two-way ANOVA with Bonferroni’s multiple comparison tests. Freezing time was analyzed by one-way ANOVA using Tukey's multiple comparison tests. Eye color experiments, % read alignment, and fractions of reads in peaks (FRiP) were analyzed using Student's t-tests. Gal4-UAS and washing experiments were analyzed by simple linear regression, and analysis of covariance (ANCOVA). All statistical analyses were performed using GraphPad Prism 9 software .Drosophila. We first asked if the number of flies used for head isolation and the size of the Dounce homogenizer play a role in the number of extracted nuclei that are labeled with DAPI. Using a larger volume of flies increased the number of extracted nuclei  = 15.6, p = 0.004). The size of the Dounce homogenizer used for extraction did not significantly affect the total extracted nuclei if analyzed by 2-way ANOVA  = 2.0, p = 0.20; Fig. Our goal was to identify the optimal conditions for nuclei isolation, purification, and ATAC-seq library preparation using adult  ANOVA, F,8 = 15.6Drosophila is the availability of sophisticated genetic tools that allows the easy identification of target cells, which can then be isolated by flow cytometry. In our nuclei isolation procedure, live neuronal synaptobrevin (nSyb)-Gal4 flies expressing pan-neuronal GFP-nls are frozen at − 80 °C for 5 min, which is similar to slow freezing, since the flies are not immediately frozen when placed in the freezer, as would occur when snap-freezing in liquid nitrogen. Snap-freezing can decrease GFP fluorescence34 and damage neurons35, thus decreasing the number of viable GFP-positive nuclei collected by flow cytometry. We hypothesized that extended freezing at − 80 °C could reduce GFP fluorescence or nuclear viability, so we performed a time-course experiment to examine whether freezing time influences the detection of GFP-positive nuclei by flow cytometry. Five minutes is about the minimum time per sample in which flies can be frozen at − 80 °C and homogenized when handling multiple samples. We observed that GFP fluorescence decreased quickly with additional time on dry ice but reached a plateau at ~ 60% fluorescence in the time analyzed . Therefore, we suggest that flies should only be frozen briefly (5 min or less) at − 80 °C when used for nuclei isolation and ATAC-seq library preparation.One advantage to using 18, which reduces the number of reads aligning to the nuclear genome. Since Drosophila can be used to easily label target nuclei, fluorescence-activated nuclei sorting is a convenient way to isolate nuclei of interest while simultaneously reducing mitochondrial contamination. Sorting GFP+ nuclei using a flow cytometer is dependent on the gating scheme for the detection of green fluorescent signal. Detection schemes for stains usually come at a low gate  and a high gate . To determine the sorting sensitivity at these gates, we ubiquitously expressed a brighter version of nuclear GFP, UAS-Stinger, using a strong Tubulin-Gal4 driver. At the low gate, we recovered ~ 80% of nuclei from ~ 100,000 DAPI+ nuclei . These numbers would significantly contaminate the nuclei sorted from a genotype where Gal4 is expressed in a subset of only hundreds of neurons. The high gate will produce fewer false positives, thus increasing the specificity for the Gal4-expression pattern, but the high gate will also reduce the sensitivity and recovery of those Gal4 + neurons. Therefore, we asked whether we could increase the recovery via genetic means by increasing the number of Gal4 or UAS-GFP-nls transgenic constructs to boost GFP expression. We used ics-Gal4 UAS-GFP-nls flies and crossed them to themselves, ics-Gal4, UAS-GFP-nls, or w* Berlin to generate flies with 4, 3, 3, or 2 combined copies of Gal4/UAS insertions, respectively. ics-Gal4 expression includes many neurons and the expression pattern is not different between ics-Gal4/ + hetero- and ics-Gal4 homozygotes36. Expanding the copies of Gal4/UAS insertions increased the number of detected GFP+ nuclei , or to red-eyed (w+) virgins and then collected the male progeny. Both of these male genotypes contain 2 wmC+ alleles from the transgenes, but one group is wild-type red-eyed (w+) and the other is orange-eyed (in a w- background). At the low gate, we recovered almost twice as many ‘GFP+ nuclei’  in mammalian cells may not be appropriate for fly nuclei. To determine the optimal Tn5 reaction conditions, we sorted GFP+ nuclei from fly heads and constructed ATAC-seq libraries. We evaluated the library quality using the guidelines recommended by Buenrostro et al.37. We first examined how reaction time affects the fragment distribution by sorting pan-neuronal GFP+ nuclei from nSyb-Gal4 UAS-GFP-nls fly heads. Then, we prepared ATAC-seq libraries using 1× Tn5 enzyme and sample incubation for 5, 23, and 60 min  to determine the number of PCR cycles needed for library amplification before sequencing  and 5.01e−53 (2); DAT: p = 1.03e−05 (3) and 1.04e−05 (4); hth: p = 0.009 (5), 1.69e−36 (6), and 1.14e−21 (7)]. Peaks associated with dopamine-neuron specific genes were much smaller or absent in GABA neurons , 4.42e−08 (2), 0.04 (3), 0.01 (4), and 6.6e−08 (5); Lim3: p = 1.39e−05 (6) and 0.001 (7); CG14989; p = 0.1]. These peaks were much smaller or absent in dopaminergic neurons. These results confirm that our nuclei isolation and library generation protocol yields high quality data, including differential chromatin accessibility in known cell-type markers41.Finally, we combined our optimized parameters 51. We recommend that specific Gal4 and UAS-GFP-nls combinations be tested to determine GFP+ nuclei recovery prior to performing actual ATAC-seq experiments, particularly for sparsely expressing target neurons.An advantage of using Drosophila genome is ~ tenfold smaller than mammalian genomes, so enzyme-mediated steps during ATAC-seq library preparation may be affected by the amount of available DNA. Tn5 tagmentation in mammalian samples is generally performed using undiluted Tn5 enzyme included in a library preparation kit (1×) for 30 min at 37 °C37. Because the amount of accessible chromatin in Drosophila nuclei may be lower than in mammalian nuclei, we investigated the effects of changing the parameters of the enzymatic reactions (Tn5 tagmentation and PCR amplification) on fragment distribution and library quality. Previous studies showed that decreasing the Tn5 concentration in mouse embryonic stem cells only decreases tagmentation efficiency when diluted to 10 nM or lower18. Notably, this experiment used homemade Tn552, which may have different kinetics or concentrations than commercially-available Tn5 enzyme. Increasing the tagmentation time decreased library complexity, while decreasing the tagmentation time increased the number of mid-length fragments but did not improve nucleosomal periodicity. We also examined the number of PCR cycles needed to amplify ATAC-seq libraries before sequencing. Protocols for generating ATAC-seq libraries from mammalian tissues call for amplification to one-third the total fluorescence in a qPCR side reaction37. Because of the smaller genome size in Drosophila, amplifying ATAC-seq libraries to one-third of the total qPCR fluorescence may introduce GC and size bias. Indeed, amplifying our libraries to one-third qPCR fluorescence introduced a PCR bubble, indicating that the available primers were depleted during amplification38, causing amplification to occur using annealed adaptor sequences, which increased the mid-size fragments in the library. While these libraries may be sequenceable after one cycle of reconditioning PCR39, libraries with PCR bubbles should be quality-checked to verify that the PCR bubble was resolved before sequencing. Based on these results, we recommend transposition with 1× Tn5 for 23 min, followed by amplification of the ATAC-seq libraries to 25% qPCR fluorescence, which preserves library complexity and does not introduce GC or size bias. One difference between our optimized protocol and the original ATAC-seq protocol is the enzyme used for PCR amplification. While the Phusion enzyme we used has been previously used for ATAC-seq library preparation53, the Q5 enzyme listed in the original protocol may be more appropriate for transcription factor footprinting or single nucleotide polymorphism profiling due to its higher fidelity.The Drosophila dopaminergic and GABAergic neurons. These libraries were of high quality, with the expected nucleosomal banding patterns and fragment size distributions. Additionally, these libraries had high alignment rates, further confirming their quality. Therefore, we propose that ATAC-seq libraries from Drosophila tissues should use the following parameters (Fig. 37, a qPCR-based method55, or both. This will ensure that the sequenced libraries are high-quality, which will ultimately reduce experimental cost. Our protocol harnesses the powerful genetic tools available in Drosophila to achieve excellent cell-type specificity followed by ATAC-seq, which is used to interrogate the chromatin landscape with unprecedented resolution. Thus, our protocol will enable detailed investigations of the role of chromatin-mediated gene regulation in normal and pathological states.Using these optimized conditions, we generated ATAC-seq libraries from 65,000 nuclei isolated from ers Fig. : freezinSupplementary Table 1.Supplementary Information.


Understanding and ameliorating neurodegenerative diseases represents a key challenge for supporting the health span of the aging population. Diverse protein aggregates have been implicated in such neurodegenerative disorders, including amyloid-β, α-synuclein, tau, fused in sarcoma (FUS), and transactivation response element (TAR) DNA-binding protein 43 (TDP-43). Recent years have seen significant growth in our mechanistic knowledge of relationships between these proteins and some of the membrane-less nuclear structures that fulfill key roles in the cell function. These include the nucleolus, nuclear speckles, and paraspeckles. The ability of macromolecular protein:RNA complexes to partition these nuclear condensates through biophysical processes that involve liquid–liquid phase separation (LLPS) has also gained attention recently. The paraspeckle, which is scaffolded by the architectural long-non-coding RNA nuclear enriched abundant transcript 1 (NEAT1) plays central roles in RNA processing and metabolism and has been linked dynamically to TDP-43. In this mini-review, we outline essential early and recent insights in relation to TDP-43 proteinopathies. We then appraise the relationships between TDP-43 and NEAT1 in the context of neuronal paraspeckles and neuronal stress. We highlight key areas for investigation based on recent advances in our understanding of how TDP-43 affects neuronal function, especially in relation to messenger ribonucleic acid (mRNA) splicing. Finally, we offer perspectives that should be considered for translational pipelines in order to improve health outcomes for the management of neurodegenerative diseases. Neurodegenerative diseases are a diverse set of conditions characterized by the progressive degradation of the structure and function of the central or peripheral nervous systems . Major nPathological deposition of intracellular or extracellular protein aggregates is associated with several neurodegenerative disorders. Depending on the specific protein, these conditions can be grouped into amyloidosis (amyloid-β), α-synucleinopathies (α-synuclein), tauopathies (tau), FUSopathies [Fused in sarcoma (FUS)], and TDP-43 proteinopathies [transactivation response element (TAR) DNA-binding protein 43 (TDP-43)] . Such prC-terminal fragments (SOD1), FUS, optineurin (OPTN), TANK binding kinase 1 (TBK1), and C9ORF72  DNA-binding protein 43 is a highly conserved heterogeneous ribonucleoprotein (hnRNP) multi-domain protein first identified as a 43-kDa protein that bound the TAR in human immunodeficiency virus . Under nThere is growing recognition that TDP-43 undergoes liquid–liquid phase separation, (LLPS) a biophysical process that gives rise to biomolecular condensates reminiscent of an oil-in-vinegar mixture . Driven The nucleolus was established as the site for ribosome biogenesis in the 1960s and through a variety of technological innovations, we now recognize that key nucleolar proteins involved in rRNA transcription shuttle rapidly between the nucleolus and nucleoplasm . FurtherNuclear speckles (interchromatin granule clusters) are highly dynamic structures enriched for transcription factors, chromatin remodeling factors and other proteins that enable nuclear speckles to function as a hub for coordinated regulation of the various steps associated with gene expression . These iO-methylation of U1 and U2 small nuclear RNAs  are multifunctional biomolecular condensates that support the maturation of small nuclear ribonucleoproteins (snRNPs). The snRNPs are multimeric protein-RNA complexes that form the spliceosome and it is thought that snRNPs are concentrated in CB in order to promote the assembly of the individual monomers into higher order complexes . Cajal bear RNAs .Interestingly, early work showed CB interactions with replication-dependent histone (RDH) loci . These hnuclear enriched abundant transcript 1 isoform 2 (NEAT1_2) lncRNA in the paraspeckle, (b) ribosomal intergenic spacer (IGS) lncRNA that drives amyloid body assembly for local nuclear protein synthesis (Drosophila heat shock RNA (Hsr) omega in the omega speckle (Three long non-coding RNAs (lncRNAs) have been also been categorized as scaffolding or architectural RNAs associated with nuclear bodies in human cells . These aynthesis , and (c)ynthesis , 2008. I speckle  and the  speckle  also mee speckle .In healthy cells, TDP-43 resides in the nucleus and regulates RNA processing and translation, functions that are lost following of mis-localization of TDP-43 as cytoplasmic inclusions . Both thLandmark proteomics studies on post-mortem brain extracts from FTLD and ALS patients in 2006 uncovered TDP-43 as a key protein associated with FTLD, specifically ubiquitin-positive but tau- and alpha-synuclein negative FTLD . This prIn an early study of protein:RNA interactions in the post-mortem brain tissue by In addition to ALS and FTLD, roles for TDP-43 in the pathology of AD have also been established. Several studies have observed TDP-43 immunoreactivity in a subset of AD cases with neuroanatomical distribution pattens that were broadly distinct from those observed in FTLD . The finEarly work revealed that the amygdala was particularly vulnerable to TDP-43 in advanced AD region . FurtherSTMN2), a regulator of the microtubule cytoskeleton  non-POU domain-containing octamer-binding protein (NONO) and SFPQ (splicing factor proline and glutamine rich), with NONO forming dimers that support oligomerization with other PSPs . From a NEAT1-deficient mice, beyond the loss of paraspeckles although subsequent work revealed a high expression of NEAT1 in the corpus luteum of mouse ovaries, impaired formation of luteal tissue, low serum progesterone, and a stochastic inability to become pregnant  cells as well as Drosophila neurons  for spinal muscular atrophy  shown by Interestingly, inopathy . Exposurinopathy . These p neurons . Nonethe neurons . Whether atrophy , casimer atrophy  and vola atrophy , the pro atrophy . It willvia machine learning) to uncover similar PS-ASOs that condense NEAT1 and TDP-43? In this regard, it is noteworthy that an attempt to deploy AlphaFold and other computational approaches in expanding the targetable chemical space of TDP-43 has already been reported (It will also be important to determine whether ASOs or bait oligonucleotides ASOs can support the generation of the neuroprotective TDP-43 nuclear bodies that reported . Howeverreported , is a reSTMN2 or UNC13A they do nothing to address any pathology that may be due to cytoplasmic TDP-43 aggregates. Hence such strategies will need to demonstrate that therapeutic restoration of STMN2 or UNC13A protein expression enhances neuronal survival even in the presence of cytoplasmic TDP-43 inclusions. More pragmatically, small molecules that have shown potential as disruptors of TDP-43 aggregates, such as an acridine derivative generated by One obvious limitation of the above approaches, however, is that while they may restore expression of functional The neuroprotective abilities of NEAT1_1 in TDP-43 proteinopathies reported by Escherichia coli, a whole new field of “swimming microrobots,” “microswimmers,” or “miniature medical robots” has emerged in recent years for a wide range of biomedical applications (Taking inspiration from the motility of single-cell biological entities such as sperm and ications . The preications . Magnetiications . Howeverications . Interesications . We are Much progress has been made in deciphering how TDP-43 mislocalization and dysfunction contributes to neurodegeneration. As argued above, it seems that the induction of paraspeckles in the neuronal nucleus or the retention of TDP-43 as nuclear bodies can support neural viability. However, further studies are needed to clarify the relationships between induced paraspeckles and TDP-43 localization and function, especially in relation to mRNA splicing.The emergence of NATs spanning ASO and other modalities such as short-interfering RNA  offer a KR, DT, and DS conceptualized the idea. KR wrote the first draft. All authors read the manuscript and approved the submitted version.


Issues of medication adherence, multimorbidity, increased hospitalisation risk and negative impact upon quality of life have led to the management of polypharmacy becoming a national priority. Clinical guidelines advise a patient-centred approach, involving shared decision-making and multidisciplinary team working. However, there have been limited educational initiatives to improve healthcare practitioners’ management of polypharmacy and stopping inappropriate medicines. This study aimed to evaluate the impact of a polypharmacy Action Learning Sets (ALS) tool across five areas: i. healthcare practitioners’ confidence and perceptions of stopping medicines; ii. knowledge and information sources around stopping medicines; iii. perception of patients and stopping medicines; iv. perception of colleagues and stopping medicines and v. perception of the role of institutional factors in stopping medicines.n = 24] and pharmacy professionals [n = 9]. A pre-post survey with 28 closed statements across five domains relating to the study aims [n = 32] and a post evaluation feedback survey with 4 open-ended questions [n = 33] were completed. Paired pre-post ALS responses [n = 32] were analysed using the Wilcoxon signed-rank test. Qualitative responses were analysed using a simplified version of the constant comparative method.The ALS tool was delivered to a multi-disciplinary group of healthcare practitioners: GPs [The ALS tool showed significant improvement in 14 of 28 statements in the pre-post survey across the five domains. Qualitative themes (QT) from the post evaluation feedback survey include: i. awareness and management of polypharmacy; ii. opportunity to share experiences; iii. usefulness of ALS as a learning tool and iv. equipping with tools and information. Synthesised themes (ST) from analysis of pre-post survey data and post evaluation feedback survey data include: i. awareness, confidence and management of inappropriate polypharmacy, ii. equipping with knowledge, information, tools and resources and iii. decision-making and discussion about stopping medicines with colleagues in different settings.This evaluation contributes to developing understanding of the role of educational initiatives in improving inappropriate polypharmacy, demonstrating the effectiveness of the ALS tool in improving healthcare practitioners’ awareness, confidence and perceptions in stopping inappropriate medicines. Further evaluation is required to examine impact of the ALS tool in different localities as well as longer-term impact. Polypharmacy refers to the routine use of at least five or more medicines –3. It isTo address issues relating to polypharmacy, a distinction can be made between ‘appropriate’ and ‘problematic’ polypharmacy. Appropriate polypharmacy refers to the optimisation of medicine using high quality-evidence to prescribe for a person with complex and/or multiple conditions, which is contributing to increased life expectancy and better quality of life. Problematic polypharmacy refers to inappropriate prescribing of multiple medications and is associated with greater risk of drug interactions and problematic reactions , 10.A review of barriers and enablers to stopping inappropriate medicines by the University of York et al. , identifClinical guidelines by NICE , 15, theOther recommendations include the adoption of a multidisciplinary team approach, organised medication reviews, and the use of a screening tool (e.g. STOPP/START) and individually tailored management plans for patients, to accommodate their aspirations, preferences and needs , 15. HowIssues of medication adherence, multimorbidity, increased hospitalisation risk and negative impact upon quality of life have led to the management of polypharmacy becoming a national priority. However, there have been a limited number of educational initiatives to improve healthcare practitioners’ management of inappropriate polypharmacy and in stopping inappropriate medicines.A range of interventions have been advanced to address the issues associated with inappropriate polypharmacy , 20. TheInterventions to improve appropriate polypharmacy include professional , organisational  or multidimensional pharmaceutical focused approaches , 19, 20.screening tool of older people’s prescriptions), START , more defined approaches to deprescribing (The 7 Steps), and/or the Drug burden index  and a post evaluation feedback survey collecting both qualitative and quantitative data [n = 33].The evaluation consisted of a pre-post polypharmacy ALS survey collecting quantitative data .There was a statistically significant pre-post improvement in participants’ confidence about stopping medicines safely for patients with a medium effect size  [Q5] and agreed to a greater extent that they had enough information to help make decisions about stopping medicines  [Q6].Participants were more aware of current evidence about stopping medicines  [Q7], would use online information to help make decisions about stopping medicines  [Q8] and were more confident about deciding when it was appropriate to stop a medicine(s)  [Q10].Participants agreed to a greater extent that they would use e-learning to help make decisions about stopping medicines  [Q9].Participants’ willingness to increase their knowledge about stopping medicines safely was higher before the three ALS  did not change [Q11].There were statistically significant pre-post improvements in relation to perception of patients and stopping medicines with medium effect sizes.p = 0.001, r = 0.41) [Q12], negotiating with patients about when to stop a medicine  [Q13] and on negotiating with patient’s family members (where appropriate) about when to stop medicines  [Q14].Participants were more confident about deciding when it was appropriate to stop medicines  [Q15], their confidence in negotiating decisions about stopping medicines with colleagues in general  [Q16], with colleagues within their organisation  [Q20], with colleagues outside their organisation  [Q21] and with pharmacists  [Q17].Non-significant pre-post differences with low effect sizes were recorded for the practitioners’ ability to negotiate decisions about stopping medicines with other practitioners/health care professionals where necessary  [Q22] or their willingness to use e-learning to help in making decisions about stopping medicines with colleagues  [Q23].Likewise, the ALS tool did not significantly influence participants’ perceptions with respect to having enough time to negotiate decisions about stopping medicines with colleagues  [Q18] and with hospital colleagues  [Q19] with medium and low (nearly medium) effect sizes, respectively.However, there were significant positive pre-post improvements in confidence in negotiating decisions about stopping medicines with nurses  [Q24], their belief that other priorities will not get in the way of them being able to stop medicines  [Q26] and their confidence in making decisions about stopping medicines in different settings   [Q28].Significant pre-post improvements with both low and medium effect sizes were found for the professionals’ feelings of being more supported making decisions about stopping medicines in their place of work  [Q25] and the availability of the necessary resources (e.g. equipment/staff) to support the process of stopping medicines for patients  [Q27].Conversely, non-significant changes with low and negligible effect sizes respectively, were recorded for having enough time to make decisions about stopping medicines 1). ThemeMany participants reported how the ALS tool had increased their awareness and confidence in managing polypharmacy (for e.g. stopping inappropriate drugs), both in terms of their experiences over the duration of the ALS, but also prospectively, in relation to how they would apply learning from the ALS to their future practice.‘It has inspired me to be more proactive in the management of polypharmacy’ (GP)“Looked at the different facets affecting the decisions surrounding polypharmacy e.g. when to stop, when to start and the personnel involved in these processes” ‘I will be more confident with reviewing and stopping medications for my elderly patients’ (GP)Participants stated how the ALS tool provided an opportunity to share experiences of managing polypharmacy with other attendees. This was perceived as valuable in gaining different professional perspectives and settings other than their own as well as providing time and a ‘safe’ space for reflection and discussion:‘Just having the time and space to discuss the issues especially in a multidisciplinary setting is useful’ (GP)‘Good skills mix and interesting to see what problems different people may be facing when looking at the various aspects of deprescribing’ Very useful. Non-judgemental and non-threatening environment. Good to share views (GP)Sharing learning from the ALS also featured as a future intention for some participants, in terms of disseminating learning with their colleagues from different professions and settings:‘Share information with GP colleague i.e. information for review/tools’ ‘Attend multidisciplinary team meetings/network with specialists in the areas I lack experience and confidence.’ The usefulness of the polypharmacy ALS as a learning tool also featured as a core theme. Key elements included the style and presentation of the sessions:‘All three sessions were different; useful recapping at the beginning what had been covered. All the speakers were good and added to the action sets’ (GP)‘It was very inspiring and well presented’ (GP)‘Refreshing course, has helped me to learn an important and neglected area of general practice’ (GP)Many respondents identified how the ALS tool equipped them with tools and information in managing polypharmacy both over the course of the ALS but also in terms of their future application of learning to practice:‘Has given me tools to help with deprescribing’ (GP)‘They were very informative and provided insight into many aspects of the challenge of dealing with polypharmacy in patients’ ‘Gave me more insight, tools and confidence to discuss what patients want’ (GP)‘Will use ‘Numbers needed to treat’ [concept] much more frequently’ (GP)‘Explore patient tools to visually explain the rationale behind stopping meds.’ In addition to the qualitative thematic findings, the majority of respondents (85%), reported that the polypharmacy ALS tool met their expectations, whilst 15% either reported that it had either not met or had not stated whether it had met their expectations. There was a high level of comprehension of the ALS; with 73% of respondents stating that there was not anything they did not understand during the ALS. Just over half or 55% of respondents stated that there were not any parts of the ALS which were unnecessary or not worthwhile, with the remaining respondents either not stating if there were any parts of the ALS they found unnecessary (24%) or (21%) indicating that they had found parts of the ALS unnecessary. These comments related to some parts being repetitive or suggestions to condense some sessions. Most respondents (97%) reported that they would recommend the polypharmacy ALS tool to colleagues.Synthesised themes (ST) from analysis of the pre-post survey and the top four qualitative themes (QT) in the post evaluation feedback survey are provided in Table Building on the limited research and evaluation surrounding the role of initiatives in addressing inappropriate polypharmacy , 17, thiAnalytical synthesis of findings from the pre-post ALS survey and post evaluation feedback survey, revealed the effectiveness of the ALS tool in accordance to three synthesised themes; Awareness, confidence and management of inappropriate polypharmacy (ST1); Equipping with knowledge, information, tools and resources (ST2) and Decision-making and discussion about stopping medicines with colleagues in different settings (ST3).Due to the substantial medical, social and economic issues associated with inappropriate polypharmacy , 2, 5–10Pre-post survey results indicate significant improvements in relation to participants: being aware of current evidence; having enough information to help make decisions; use of e-learning and online information; desire to increase knowledge about stopping medicines and confidence about deciding when it is appropriate to stop medicines. The fourth theme of equipping with tools and information from analysis of post evaluation feedback survey findings, supports these findings, demonstrating the value of the ALS in providing knowledge through information, tools and resources to manage polypharmacy over the course of the ALS but also through future application of learning to practice. The third qualitative theme of usefulness of the ALS as a learning tool, also recognises the overall value of the ALS tool as a learning instrument, with many participants positively reporting upon its style and presentation. In addition, most participants reported that the ALS tool met their expectations, that it was comprehensive, and they would recommend it to colleagues.Significant improvements in making decisions with nurses and hospital colleagues as well as about making decisions about stopping medicines in different settings  were evident in analysis of pre-post survey data. Though making decisions with colleagues more generally, or with pharmacists or colleagues within or outside their organisation did not constitute a significant area of improvement. The opportunity to share experiences with colleagues from other professional backgrounds and settings, featured as a qualitative theme in analysis of the post evaluation feedback survey, both in terms of participants perceptions and experiences during the ALS, but also in terms of future intentions in applying learning from the ALS, for example, through discussions with colleagues from other professions in multidisciplinary meetings. This improvement supports Mecca’s et al.  finding,In addition to ST1-ST3, other significant areas of improvement identified in analysis of the pre-post survey related to the domain of perception of patients and stopping medicines [D3], including confidence; in deciding when it is appropriate to stop medicines, negotiating with patients and negotiating with patients’ family members (where appropriate) about when to stop a medicine. These findings demonstrate the effectiveness of the ALS for respondents in supporting the patient-centred approach advocated by NICE recommendations , 15, thePre-post survey analysis also indicated substantial improvements in participants feeling supported in making decisions about stopping medicines where they work as well as their perception of other priorities not getting in the way of them being able to stop medicines. Interestingly the concepts of support and other priorities, represent factors which are arguably extrinsic to an individual’s behaviour to influence, or contextual in nature , suggestThe results of this study must be interpreted with caution as they are subject to limitations arising from i. a small sample size, ii. participants’ limited occupation range, iii. the locality of the study, iv. unevidenced clinical benefit for patients, v. unevidenced longer term impact and vi. participants being an ‘engaged sample’. Due to the small size of the study and the greater representation of GPs in the sample compared to pharmacy professionals (75%/25%), further evaluation is required to examine the impact of the ALS upon the confidence, perceptions and experiences of healthcare practitioners from different professions within and across localities to enable further corroboration and synthesis of statistical and thematic findings. Limitations regarding sample size have been mitigated by reporting effect sizes. Moreover, further research is needed to evaluate the clinical benefit of the ALS tool for patients when applied in daily practice.As the duration between the first and final ALS session was 1 month the study did not evaluate the longer-term impact of the polypharmacy ALS upon healthcare practitioners’ confidence, perceptions and experiences of stopping inappropriate medicines, which would provide insight into the extent to which evidence relating to the effectiveness of the ALS tool was applied and sustained.It is recognised that by attending the ALS sessions, participating healthcare practitioners represent an ‘engaged sample’, who may already be aware and/or interested in improving their learning and practice of stopping inappropriate medicines.By demonstrating the effectiveness of the ALS tool in improving important areas such as healthcare practitioners’ awareness, confidence and perceptions in stopping inappropriate medicines, this paper contributes to developing an understanding of the role of educational initiatives in addressing inappropriate polypharmacy. Key areas of improvement include awareness, confidence and management of inappropriate polypharmacy, equipping with relevant knowledge, information, tools and resources and decision-making and discussion about stopping medicines with colleagues in different settings. In addition, the ALS tool was well received as an educational learning tool in terms of being comprehensive, meeting expectations and strongly recommended. The style and presentation of the ALS tool was also well received. Further research evaluating the impact of the ALS tool upon the perceptions and experiences of a variety of healthcare practitioners (e.g. nurse practitioners) as well as the longer-term impact are required to corroborate the findings.


By performing first-principles calculations, we studied hexagonal-boron-nitride (hBN)-supported graphene, in which moiré structures are formed due to lattice mismatch or interlayer rotation. A series of graphene/hBN systems has been studied to reveal the evolution of properties with respect to different twisting angles . Although AA- and AB-stacked graphene/hBN are gapped at the Dirac point by about 50 meV, the energy gap of the moiré graphene/hBN, which is much more asymmetric, is only about several meV. Although the Dirac cone of graphene residing in the wide gap of hBN is not much affected, the calculated Fermi velocity is found to decrease with the increase in the moiré super lattice constant due to charge transfer. The periodic potential imposed by hBN modulated charge distributions in graphene, leading to the shift of graphene bands. In agreement with experiments, there are dips in the calculated density of states, which get closer and closer to the Fermi energy as the moiré lattice grows larger. For praD semimet in SiO2 ,4. Carri in SiO2 ,6,7. In  in SiO2 . Being f SiO2 [2 . It was  SiO2 [2 ,11,12,13Not only have qualities of graphene been much improved by growing on hBN flakes, but also many fundamental physical phenomena have been discovered in graphene/hBN heterostructures ,28,29,30In particular, great efforts have been devoted to the clarification of the band structure modulation around the Dirac cone induced by the hBN superlattice potential ,44,45,46In this paper, we will study graphene/hBN systems by first-principles calculations to address effects of the periodic potential induced by moiré patterns. The joint action of global symmetry breaking and the local stacking order in the moiré pattern will be discussed. The weak interlayer van der Waals interaction was usually believed to have little influence on graphene and was deemed as no more than a mechanical supporting substrate. In our study, it is found that, although the basic properties of graphene are preserved, the BN sublayer has salient effects on graphene, such as charge transfer, charge redistribution, band shift, and energy gap modification, which will be detailed in the following section. As it is known, the size of the observed graphene/hBN moiré structure is too large for normal density functional theory (DFT) calculations. Therefore, the aim of our DFT calculations is to study the evolution trends of the gap, Fermi velocity, and density of states (DOS) with respect to the twisting angle, rather than to investigate properties that are only manifested in very-large-sized Gr/BN systems. Simultaneously, we also attempt to give a more unified understanding of the change of the Fermi velocity, bands, and DOS based on charge redistribution and transfer induced by interlayer interactions.In the following section, BN always refers to hexagonal BN. The graphene/hBN system is briefed as Gr/BN. Gr/BN-θ denotes that graphene and BN have a relative twisting angle of θ. In Gr/BN, graphene is unstrained, whereas the lattice of BN is adapted to match with graphene. The experimental lattice constants of graphene and boron nitride are 2.460 and 2.504 Å, respectively. In reality, the moiré pattern formed in Gr/BN is owing to either the primitive lattice mismatch or the twisting angle or both. In the following section, these two factors are first discussed separately and then considered simultaneously. In our study of the Gr/BN moiré super lattice, the graphene super lattice is kept unstrained.We first consider the untwisted Gr/BN system without lattice mismatch. For Gr(1 × 1)/BN(1 × 1) systems, the energy gap is found to be 62.5 meV for AA-stacked Gr/BN, whereas it is reduced to 57.6 (50.5) meV for AB1 (AB2) stacking with half of the C atoms facing the B (N) atoms, as shown in In untwisted Gr/BN systems, there is interlayer in-commensuration due to the lattice mismatch. The number of C atoms per unit area is not equal to that of B and N atoms, and therefore the interlayer atomic registry becomes disordered in comparison with that of the perfect AA- or AB-stacked Gr/BN. The realistic untwisted system is Gr(56 × 56)/BN(55 × 55) with a periodicity of about 14 nm. It contains 12,322 atoms, which is well beyond the reach of the normal density functional theory method. Close examination found that Gr(56 × 56)/BN(55 × 55) features local quasi-AA and -AB stackings, as shown in In In Gr(13 × 13)/BN(12 × 12), although BN is stretched by about 6.4%, which seems to be fairly large, it can be served as a model system to represent a system with local quasi-AA and -AB stacking. To be more realistic, we also calculated the untwisted Gr(28 × 28)/BN(27 × 27) system with up to 3026 atoms using DFTB+ , in whicSince the lattice mismatch between the primitive lattices of graphene and BN is smaller than 1.8%, it is usually assumed that the two layers take the same primitive lattice constant (2.460 Å) in the twisted Gr/BN-θ systems , which wFollowing Ref. , we consIn the twisted Gr/BN-θ, graphene basically maintains its intrinsic lattice periodicity, and, at the same time, it is modulated by an external periodic potential imposed by BN. Below, we will focus on the Gr/BN-7.34° system, as depicted in BN has a large energy gap, and its band edge is far from the Dirac point of graphene. Therefore, the Dirac cones of graphene falling within the gap of BN are almost not affected by BN b. The caz states of C atoms. The strength of the C_pz state interaction determines the slope of the Dirac cone. The larger the interaction strength, the larger is the slope, which is proportional to the Fermi velocity z states of the C atoms lost electrons . To illustrate if this little strain will appreciably change the above-discussed evolution of the electronic structures of the twisting Gr/BN systems, we searched possible Gr/BN moiré systems via taking account of the primitive lattice mismatch and the rotation between the Gr and BN layers simultaneously. It is found that a Gr/BN-13.90° system has an almost-perfect superlattice match, in which the BN layer has a tiny strain of 0.09%, which is much smaller than 1.8%. There are 242 C atoms and 234 B and N atoms in Gr/BN-13.90°, and the superlattice constant is 27.06 Å, as shown in We also found a nearly perfectly matched system, Gr/BN-1.95°, in which the BN layer has a tiny strain of 0.11%. The super lattice constant is 63.96 Å, containing 2654 atoms in total, with 1352 C and 1302 B and N atoms. Its electronic properties are calculated by DFTB+ . The banThe first-principles calculation is performed within the framework of density functional theory using local density approximation, as implemented in the Vienna Ab-initio Simulation Package ,55,56,57z states and inducing an in-plane electric field in graphene, which will give rise to an appreciable reduction of the Fermi velocity. The periodic potential imposed by BN in the moiré Gr/BN modulates the charge distribution, resulting in the upshift of the valence bands of graphene. In comparison to the smooth DOS of perfect graphene, there are dips in the DOS of the twisted moiré Gr/BNs, which approaches the Fermi energy as the size of the moiré super lattice increases; this is in good agreement with the experiments. Depending on the high electronic qualities of semiconducting graphene, promising applications are expected in next-generation digital logic devices by enhancing the energy gap in twisted Gr/BN systems.In conclusion, we have made a computational study on the untwisted and twisted Gr/BN systems. The moiré pattern is formed as a result of a small lattice difference between graphene and BN, and the relative interlayer twisting. It is found that the Dirac cones of graphene are well preserved in the heterostructure, and the graphene overlayer remains almost flat. In the perfectly matched AA- and AB-stacked Gr/BN, there is a sizable energy gap of several tens of meV, but the gaps have a staggered alignment. Therefore, in the moiré Gr/BN with both quasi-AA and -AB stacked regions, the overall gap  is almost closed. There is an inhomogeneous electron transfer from graphene to BN, reducing the interaction of the C_p


He investigated instrument instability induced by environmental parameters, yet did not find a clear coincidence with local temperature, atmospheric pressure, and relative humidity. Parkhomov hypothesised that gravitationally-focussed ‘slow’ cosmic neutrinos influenced beta decay. In the current work, environmental conditions in the Moscow area at the time of the experiment are presented. There appears to be a resemblance of the shape of the annual 90Sr/90Y decay rate anomalies with the inverse of the absolute air humidity, albeit with an apparent time shift of 0.05–0.15 year. Humidity may have influenced the range of beta particles in air, as well as geometric and electronic properties of the detection set-up, however causality could not be unambiguously demonstrated. The instabilities in the 60Co data were more difficult to correlate with environmental data, except for some similarities with temperature and external dew point.Parkhomov published decay rate measurements of  The most notable cases in which the effect occurred pertained to counting of beta emissions with Geiger–Müller (G–M) counters6 or set-ups with unstable conditions8, whereas it is mostly negligible in measurements with the accurate techniques13 of primary standardisation laboratories23 and other laboratories working under stable measurements conditions29. Parkhomov5 published decay rate measurements of 90Sr/90Y and 60Co beta decay sources with G–M counters and observed annual cyclic perturbations with less than 0.2% amplitude. There is no reason to associate these fluctuations with anomalies in the decay process itself; Accurate activity measurements of 90Sr/90Y with the TDCR technique (Triple-to-Double Coincidence Rate counting12) are free of any annual oscillation exceeding 0.004% amplitude18. Ionisation chamber measurements11 of 60Co sources are free of annual oscillations at an accuracy level of 0.007%18.In recent years, there has been a lively discussion on the origin of observed anomalies in some time series of measured decay rates32S beta decay at Brookhaven30, 32P beta decay at Ohio State University30, radon decay in a closed canister at the Geological Survey of Israel33, various beta emitters at the Technical University of Valencia35, and γ-ray spectrometry measurements of 145Sm and 171Tm at the Paul Scherrer Institute37. The perturbations from a smooth exponential decay curve revealed strong correlations with humidity represented by the dew point, a humidity accumulation model, or absolute air humidity derived from historic data of nearby weather stations.The most likely explanation is found in environmental conditions that influence the electronic operation and geometric conditions of the experiment. Recent studies have revealed that ambient air humidity can significantly influence decay rate measurements, even inside temperature-controlled laboratories. Notable examples are time series of 1. Metrologists have often argued that there is no metrological ground for these assertions, since the best measurements point to the validity of the exponential-decay law39. Parkhomov hypothesised that gravitationally-focussed ‘slow’ cosmic neutrinos influenced beta decay. In this work, environmental conditions in the Moscow area at the time of the experiment are presented. Particular attention is paid to the annual cycles of absolute air humidity, which may have influenced the experiment.In spite of this straightforward explanation of instrumental instability, some authors have questioned the invariability of the decay constants and made assertions that a significant fraction of radioactive decay is induced by solar and cosmic neutrinos90Sr/90Y beta decay rates with the SBM-12 and STS-5 Geiger-Müller counters presented in Fig. 90Sr/90Y parent-progeny pair is in equilibrium, with 90S emitting a soft beta spectrum with 546 keV endpoint energy and 90Y a harder beta spectrum up to 2.3 MeV. Both beta spectra are detected in the first counter, type SBM-12, located in the air cavity at a distance of 2 cm from the source. The second counter, type STS-5; is separated from the source by additional layers of aluminium and polyvinylchloride, thus recording mainly the high-energy fraction of the 90Y beta spectrum. The temperature in the installation is stabilised at 31 °C ± 0.1 °C. The container with source and detectors is filled with quartz sand to absorb externally emitted beta particles.At the Russian Academy of Natural Sciences in Moscow (Russia), Parkhomov built a set-up to investigate rhythms and fluctuations in radioactivity and various processes , and simultaneously recorded temperature and radiation background. Of particular interest are the measurements of An extensive set of intermediately processed data that were the basis of the final data prior to further filtering and correcting.A set of processed data, corrected for decay and change in response of the detector with time, averaged over 31–38 h (SBM-12) or 38–48 h (STS-5), and excluding data for which the thermostat indicated less than 20 °C or more than 31 °C.5. These graphs are similar but not identical to data set #2.Visually matching analogues of the original graphs in Parkhomov’s publicationsIn private communication, Parkhomov has kindly provided measurement data in an effort to reproduce the departures from a smooth exponential decay curve, resulting in three data sets:The three versions of the SBM-12 and STS-5 data sets are compared in Fig. 40. Even though the laboratory is at the other side of Moscow, it can be assumed that the general trends of the weather conditions run largely in parallel. The graph shows the dew point, the absolute humidity , temperature in reverse scale, as well as the ambient air pressure in normal scale. It appears that the shape of the oscillations in the air humidity shows some resemblance to the instabilities in the 90Sr/90Y decay rate measurement results.The data sets #3 are shown in Fig. In Fig. Humidity may have influenced the range of beta particles in air, as well as geometric and electronic properties of the detection set-up. Since the beta particles enter the detector with a continuous energy spectrum, the threshold level separates the detected from the non-detected fraction. Any change in the threshold energy, the amplification of the gas detector, or the energy spectrum of the electrons (after straggling in air with a variable density) will result in a different effective detection efficiency. It is interesting to note that Parkhomov does not consider any variability in the threshold region and assumes that variations merely occur at the high-energy side resulting from inverse beta decay.It is tempting to ascribe the variations in the decay rates to the influence of humidity, however the consideration that the effect of the humidity appears to be delayed by about 55 days puts this hypothesis under pressure. It is conceivable that two environmental conditions have executed a combined effect on the set-up. A weighted combination of temperature and humidity can result in waves that are delayed in time.60Co decay rate measurements were performed with a miniature  halogen SBM-12 G–M counter. A 0.1-mm-thick kovar alloy  tube was tightly clad to the counter. The 60Co was formed by irradiation of this alloy in a nuclear reactor. The 60Co decay counting rate during the measurement campaign decreased from 16 to 5 s−1, whereas the background counting rate was about 0.01 s−1. For statistical precision, the data were averaged over long periods, from 2.5 days at the beginning to 8 days at the end. The SBM-12 detector was not thermally stabilised in view of the assumed weak dependence of the counting rate on temperature (0.0006% per °C).The 60Co decay rates show annual oscillations that look more acute than a sinusoidal function, as shown in Fig. 90Sr/90Y. However, the 60Co source was not surrounded by sand, therefore humidity was not buffered by a surrounding medium. The shape of the oscillations resembles the time dependency of the dew point, albeit with an unrealistic time shift of 0.3 year. In spite of the similarity of both time series, the large time gap suggests that the similitude may be merely fortuitous.The 60Co count rates are compared with the temperature inside the laboratory shifted by 0.1 year to left. There is a vague resemblance in the time region 1999–2002, however there is no satisfactory evidence for causality.The reality may be more complex and it is difficult to disentangle the effects of environmental factors retrospectively. The decay rates were not exclusively influenced by outside weather conditions, since the laboratory was heated in the cold season (from September to May), electric heaters were used during cold snaps, and air conditioners during hot weather. The difference between outside temperature from the weather station and the temperature measured inside the laboratory by Parkhomov is shown in Fig. 90Sr/90Y beta decay rates show a marked resemblance with the annual variations in the absolute air humidity in the Moscow region, except that the humidity cycle precedes the decay rate cycle by about 0.05–0.15 year. The 90Sr/90Y source and counters were surrounded by sand, which may have acted as a buffer for humidity, which in turn may have influenced the range of beta particles in air and capacitance in electronics. The 60Co decay rates were measured in other conditions and were difficult to interpret. The variations may have been caused by a combination of environmental parameters, e.g. humidity and temperature, as well as artificial heating and air conditioning inside the laboratory.The annual variations in the measured


Invasive fusariosis (IF) is an uncommon opportunistic mold infection primarily affecting patients (pts) with leukemia and allogeneic hematopoietic cell transplant (HSCT). Historically, IF had poor outcome when there is lack of recovery from immunosuppression. However, IF incidence and outcome in the in the era of new mold-active triazoles and leukemia regimens that incorporate molecularly targeted drugs is unknown.We retrospectively studied the incidence, risk factors, clinical features, and outcome of microbiologically documented IF (revised EORTC/MSG criteria) in pts with leukemia at MD Anderson Cancer Center in the last 12 years (June 2009-October 2021). The independent risk factors for 42-day (d) mortality from IF diagnosis were assessed by a binary multivariable logistic regression model. Annual incidence density (1998-2021) was estimated using Poisson regression analysis.Figure 1).Among 140 IF pts , 100 (71%) had pneumonia, 88 (63%) disseminated infection, and 32 (23%) sinusitis, with no apparent changes in relative frequency over the years. 124 pts (89%) had neutropenia (ANC < 500) at IF diagnosis, 118 (84%) had relapsed/refractory (R/R) leukemia, and 43 (31%) had prior HSCT. 19% (18/97 pts) of IF pts had positive serum galactomannan and 55% had coinfections. In the last 5 years of the study, 45/58 IF cases (78%) were breakthrough infections to mold active triazoles . 74 (53%) out of the 140 IF pts died by d42. Only 6 pts (9%) had neutrophil recovery at time of death compared to 47 (64%) who survived until day 42 (P < 0.001). Neutrophil recovery , SOFA score , and pneumonia  were independent predictors of 42-d mortality. Since 1998, the incidence density of IF continuously increased (P = 0.006) at an annual ratio of 1.05 , AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Merck: Advisor/Consultant.