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.


Acetamiprid removal was investigated by synthesized Graphene oxide, multiwall nanotube and graphite from an aqueous solution. For this propose, FT-IR, XRD, UV–Vis, SEM and EDS were used to characterize the synthesized nano adsorbents and to determine the removal process. A novel PVC membrane electrode as selective electrode made for determining the concentration of acetamiprid. Batch adsorption studies were conducted to investigate the effect of temperature, initial acetamiprid concentration, adsorbent type and contact time as important adsorption parameters. The maximum equilibrium time was found to be 15 min for graphene oxide. The kinetics studies showed that the adsorption of acetamiprid followed the pseudo-second-order kinetics mechnism. All the adsorption equilibrium data were well fitted to the Langmuir isotherm model and maximum monolayer adsorption capacity 99 percent. Docking data of adsorption have resulted in the same as experimental data in good manner and confirmed the adsorption process. Therefore, there is serious need for unsophisticated, cheap, and valid procedures aimed at monitoring and detecting the pesticide residuals in different food productions5.Insecticides or pesticides have currently led to significant challenges concerning the safety issues and contamination of foods. Careless consumption of such compounds regardless of controlling or optimizing their concentrations may threaten humans’ lives and health due to their toxic nature. Unfortunately, pesticides are used both in agriculture and non-agriculture fields11, clothianidin, and dinotefuran employed for a wide range of pests14. Organophosphates are becoming more and more common as pesticides across the world and have recently replaced organochlorine insecticide15. As chemical pesticides control the pests and different diseases, they play a significant role in increasing agricultural yield while helping in the prevention of diseases brought by insects, such as malaria, dengue, encephalitis, filariasis, and so on in the field of human health16.The design of new members of these compounds has led to promising results in recent years. In fact, the nitro group fixation in a cis configuration for different neonicotinoid types has yielded compounds with certain biologic functions and behaviors, supporting their potential efficiency to elucidate the pharmacology and characteristics of the neonicotinoid binding sites. Neonicotinoids consist of 5-membered ring derivatives, including imidacloprid and thiacloprid, 6-membered ring compounds, including thiamethoxam, and also noncyclic structures such as nitenpyram, acetamiprid18, combined photo-Fenton and biological oxidation19, developed oxidation procedures21, aerobic degradation24, nanofiltration membranes25, ozonation26, coagulation28, fluid extraction30, solid phase extraction31, well as adsorption36. Kyriakopoulos and Doulia37 examined pesticide adsorption on carbonaceous38 and polymeric39 substances from aqueous solutions. In another study, Ahmad et al. and Hameed40 examined pesticide residuals detoxification using activated carbon. Adsorption can be utilized to decontaminate water as it is a popular equilibrium separation procedure and performs effectively in this regard16.Pesticides are mainly regarded as chemical mutagens. Pesticide elimination from the water system has currently become an important environmental issue. This is because, the residuals of these compounds have entered into underground water, resulting in serious concerns due to their significant growth in recent years. Conducting research to find out a unique approach aimed at removing these harmful chemicals worldwide is very challenging due to the extensive scope of pesticides used globally. There are a variety of techniques to remove pesticides, including photocatalytic degradation2 hybridized carbons41. Scientists and researchers have been interested in this material since it was discovered in 2004, because it showed specific characteristics, including considerable quantum Hall impacts, high mobility, great electronic42 and mechanical properties43, specific magnetism and high thermal conductivity44, There have been investigations on the use of graphene-based materials to manage the environmental issues related to pollution45.The current developments in carbon materials have led to graphene as a fundamental component of graphitic materials of all other ranges, representing a single atomic layer of graphite with hexagonally bonded and spIn this study, acetamiprid removal was investigated by three different nano carbon materials. Acetamiprid adsorption with the use of GO, multi-wall nanotubes, and graphite has not been previously examined through comparative studies. However, investigations revealed that time, temperature and acetamiprid concentration variations affected the adsorbents. The study also conducted the theoretical calculations.3, MWNTC, graphite, potassium permanganate, Peroxide hydrogen, sulfuric acid, (provided by Merck), which means that they did not need any additional purifications. Technical materials of acetamiprid, with a purity level higher than 95%, were obtained from Giah Company, Iran.The materials used in this study included reagent grade dioctyl phthalate (DOP), dibutyl phthalate (DBP), dimethyl sebacate (DMS), 2-nitrophenyloctyl ether (NPOE), tetrahydrofuran (THF) and high molecular weight PVC (provided by Merck). The study used highly pure sodium tetraphenyl borate (NaTPB), NaNO47. was utilized to synthesize graphene oxide. The addition of graphite powder (3.2 g) and NaNO3 (3.2 g) into 98% H2SO4 (140 mL) took place in an Erlenmeyer flask (800 mL), in an ice-bath (˂ 10 °C) while constantly stirred. Gradual addition of KMnO4 (18.5 g) to the suspension was done after the compound was stirred for a four-hour period in this temperature, after which the suspension was stirred for another one hour at a temperature of < 15 °C. Then, the gradual addition of 300 mL distilled water to the crude mixture was followed by stirring the mixture for another two hours, raising the temperature to 35 °C with constantly stirring for 2 h. In the next step, addition of 60 mL of a H2O2(30%) aqueous solution led to changes in the color of the mixture from dark brown into yellow. The obtained compound underwent centrifugation at 4400 rpm over a 20-min period, followed by its frequent washing by 5% HCl solution and deionized water to reach a gel-like substance with neutral value. The obtained substance was centrifuged and then vacuum dried at a temperature of 70 °C to produce the powder of graphene oxide.Modified Hummers’ technique with slight changes49 of qe:The present research carried out acetamiprid equilibrium adsorption on graphene oxide, multi-walled nanotubes, and graphite in polyethylene tubes. The study also considered constant concentrations of acetamiprid in different time and temperature conditions with the use of an orbital shaker which operated at 150 rpm agitation speed. It also utilized 10 ppm stock solutions of acetamiprid. Analysis of the solution for concentrations of acetamiprid and its adsorption on the adsorbent at equilibrium time with the use of UV–Visible spectroscopy were conducted following magnetic separation and filtration. The relation below was used to calculate the acetamiprid adsorption on the adsorbent at equilibrium timeIn which, Co, Ce, V, and m represent the primary acetamiprid concentrations (mg/L), acetamiprid concentrations (mg/L) in solution following the adsorption, the solution volume (L), and the mass (g) of graphene oxide, multi-walled nanotubes, and graphite, respectively. the following relation was used to calculate the percentage of acetamiprid removal:In which, Co and Ct indicate the primary acetamiprid solution concentrations (mg/L) and the ultimate concentrations (mg/L) following the process of adsorption, respectively. Relation In which m and V represent the adsorbent dose (mg) and acetamiprid solution volume (L).51. Dissolution of the sensor material, with acetophenone (0.002 g) acting as a plasticizer, and PVC (0.004 g) was done in 2 ml THF. NaTPB (0.001 g) played the role of an additive in several cases and graphite, graphene oxide and multi-wall nanotube (0.002 g) as ionphore. To obtain an oily concentrated compound, slow evaporation of the THF solution was conducted at ambient temperature. Formation of a transparent membrane with a thickness of around 0.3 mm resulted from dipping a Pyrex tube (3–5 mm i.d.) into the obtained compound for an approximately 10-s period. After pulling the tube out of the compound and keeping it at ambient temperature for a 1-h interval, internal filling solution (1.0 × 10−2 M acetamiprid) was used to fill it. Ultimately, electrode conditioning was obtained as it was soaked in a 0.1 M acetamiprid solution for a 36-h period.Preparation of the membrane electrode took place according to previous reports−2 M acetamiprid | sensor membrane | sample solution | reference electrode. A saturated calomel electrode with an Orion digital research pH-mV meter were considered to make the emf observations. The emf of acetamiprid solutions whose concentrations ranged from 10−1 to 10−9 M by serial dilution was measured to investigate the electrode performance. Stirring of the solutions and recording of the reading potentials after reaching stability were performed in the next steps, followed by plotting as a logarithmic function of acetamiprid activities, based on γ as the activity coefficient. Debye–Hückel relation52, which can be also applied to acetamiprid was used for data calculation:The study applied a cell assembly of the type described below: Ag/AgCl | 10In which, µ represents the ionic strength and Z is the valency. The temperature of 25 ± 0.1 °C was considered to carry out all measurements. Fresh preparation of the acetamid solutions took place using precise dilution from their stock standard solution of 0.1 M, with distilled, de-ionized water.The initial step was allocated to full geometric optimization of acetamiprid molecules, carbon nanotubes, graphene oxide, as well as graphite using density functional theory (B3LYP and basis set 6-311G(d)), by drawing 3D structures by guass view 5 software, determining the energy groups and composed molecules of each. The ArgusLab molecular modeling software was used for a quest of conformations, after which the molecular adsorption energies, density of states, ESP, and chemical parameters could be calculated., the functionality of which was supported by Fourier transform infrared spectroscopy. According to Fig. −1 in the area with high frequency due to O–H bond stretching mode, revealing that hydroxyl groups are present in GO. The carboxyl group had a band at 1720 cm−1. The stretching and bending vibrations of OH groups found in water molecules adsorbed on GO resulted in a dramatic or resonance peak at 1615 cm−1. The peaks at 1356, 1225, and 1056 cm−1 denote the C–OH group, C–O–C stretching, and the C–O group vibration mode, respectively.Hummer method was used for synthesis of graphene oxided-spacing. The increasing interlayer distance among the successive carbon basal planes shows associations with the intercalation of oxygen functional groups and water molecules into the structure of carbon layers53.The crystalline characteristics and phase purity of the synthesized graphene oxide are characterized through XRD analyses as shown in Fig. Figure max 620 nm as shown by acetamiprid and acetamiprid-adsorbent UV–Vis spectra in Fig. max of acetamiprid is eliminated following adsorption at the presence of the adsorbent because of perfect omission in the solution6.The highest absorption peaks were found to be at λ−1 because of stretching vibrations of –OH, C=O (carboxyl), C–OH, and C–O. Acetamiprid54 had peaks at 3000 because of –CH stretching, 2240, 1640 and 1500 cm−1 because of CN (nitrile), C=N (Imine) and C=C aromatic, correspondingly as shown in Fig. −1 because of graphene backbone chain skeletal vibrations. The peaks of acetamiprid were at 2976 because of –CH stretching, 2267, 1514 and 533 cm − 1 because of CN (nitrile), C=C aromatic and C–Cl53.IR spectroscopy illustrates acetamiprid adsorption on graphene oxide, multi-walled nanotubes, and graphite. As shown in Figs. −1 because of CN (nitrile), C=C aromatic and C–Cl of Acetamiprid.As shown in Fig. 53.The surface morphologies of the adsorption materials were examined by field emission scanning electron microscopy  as shown in Fig. Figure The study examined the contact time effects related to adsorption characteristics of graphene oxide, multi-walled nanotubes, and graphite, as shown in Fig. Application of Langmuir and Freundlich adsorption isotherms, as shown in Fig. −2 M of the respective solutions). Accordingly, evidence shows that graphene oxide-based PVC membranes could appropriately determine the highest sensitivity of responses in various adsorbent experiments. Besides, EMF responses found for acetamiprid selective were considerably lower compared to Nernst equation predictions, which may be related to the selective behaviors shown in the figure for possible responses of different PVC membrane electrodes with graphite, graphene oxide, and multi-walled nanotubes. The function of graphene oxide in opposition to acetamiprid was compared with different adsorbents while examining the fast exchange kinetics resulted from graphene oxide-acetamiprid complexes. The potential responses with the required stability were produced by the ionophore PVC-based membranes  functioning as selective electrodes for acetamiprid. The solution used in this experiment contained 0.1 M acetamiprid following 36-h conditioning. These treatments led to Nernstian responses in various concentrations of acetamiprid solutions, while the slope was nearly fixed.Graphite, graphene oxide and multi-walled nanotubes are all insufficiently insoluble in aqua while having carbon atoms and heavy electron density, leading to their desirable performance as carriers in the PVC membranes concerning specific pesticides as acetamiprid of appropriate size and charge. Therefore, preliminary studies have employed it as a neutral carrier for the preparation of membrane electrodes based on PVC for acetamiprid. Figure 55, increased in the case of long range interaction, including hydrogen bonding through a Coulomb Attenuating Methodology while restricting electron spin. The Gaussian 0956 software package was used to perform the required calculations, which were carried out in the gas phase as shown in Figs. 24 show their high likelihood to act as the sites for initiating interactions. Nevertheless, constructive reactions can only occur through coordination at the less electron rich C=N moiety. Thus, it can be hypothesized that adsorption initiation in neutral circumstances is carried out through coordination of adsorbent molecules with the nitrogen atoms of the C=N moiety, leading to a bonded complex and allowing close associations of the two reacting species.A moderate, inexpensive, but valid doubly split valence basis set with polarization functions on heavy atoms: 6–311 g (d) was used to optimize and calculate the energies, enabling to accurately map n-electrons and prevent overestimation of excessive energy. Correction of fermion exchanges took place through the 3-parameter hybrid function of Becke (B3), while electron correlations were considered using the model of Lee, Yang and Parr (LYP)The density of states (DOS) necessarily represents the number of various states at a certain level of energy occupied by the electrons, which means how many electron states can be found in each unit volume and unit energy. This function affects different bulk characteristics, including specific heat, paramagnetic susceptibility, and various transport processes associated with solids having conductivity. The overall distribution of states can be determined as an energy function using calculations of density of states, while the energy bands spacing is also determined in semi-conductors. Its mathematical representation shows distribution by a probability density function, typically representing an average across the domains of space and time in different states that the system occupies. DOS has direct associations with the dispersion relations of the system properties. Higher density of states at a certain level of energy reflects availability of a great number of states to be occupied. Continuity is generally a feature of the matter DOS. Yet, the isolated systems, like atoms or molecules in the gas phase, have discrete density distributions similar to a spectral density. Local DOS or LDOS represents local changes which are mostly because of distorted original system.k. It is necessary to know the system-specific energy dispersion relationship between E and k to enable converting between the density of states as an energy or the wave vector function. Overall, the system’s typological features, including the structure of the band, affect the characteristic of DOS significantly. The 3D Euclidean topologies can be observed in the highly popular systems such as neutronium in neutron stars and free electron gases in metals (instances of degenerate matter and a Fermi gas). On the other hand, 2D Euclidean topologies are found in systems with lower popularity, including 2D electron gases of 2DEG in in graphite layers as well as the quantum Hall effect systems in devices of MOSFET type. The 1D typology can be also observed in those systems with the least popularity, including carbon nanotubes, the quantum wire, and Luttinger liquid. Given the achievements in the field of nanotechnologies and nanomaterials, the last two typologies which are less familiar have the potential of becoming more common. As shown in Fig. It is possible to calculate DOS for electrons, photons, or phonons considering the quantum mechanical system, whose results may be provided through a function of energy or the wave vector Table The study examined Acetamiprid/graphene oxide, Acetamiprid/graphene and Acetamiprid/multi-walled nanotubes nanocomposites ability to comparatively remove and adsorb acetamiprid from aqueous solutions. Thorough analysis showed that graphene oxide layers contain a large variety of oxygen functionalities and Based on evidence obtained from experiments, the acetamiprid aqueous solution were independent from pH for the efficiency of their adsorption. Evidence also showed increased absorbent performance in adsorbing acetamiprid, according to which Acetamiprid/graphene, Acetamiprid/graphene oxide and Acetamiprid/multi-walled nanotubes had respective efficiencies of 97.5%, 92.5% and 95.3%. Based on the results obtained from experiments, the adsorption nature was in favor of Langmuir and Freundlich isotherm in the three adsorbents under study. According to data from equilibrium adsorption, the pseudo-second order had the greatest fitness, while the involvement of the chemisorption mechanism could be also observed. The studied adsorbents were increasingly stable and reproducible. Similar results were shown in theoretical and docking as well as empirical analyses.