These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the  RNA recognition motifs (RRMs) play diverse roles in post-transcriptional gene expression events such as RNA transport, localization, stability, and mRNA and rRNA processing. RRM is also known as the ribonucleoproteins (RNP) domain, as it contains the short and conserved elements RNP1 and RNP2, or RNA binding domain (RBD), that are abundantly distributed in higher vertebrates . RRMs arThe transactive response DNA-binding Protein 43kDa (TDP-43) is a RRM-containing protein, which plays important functions in mRNA metabolism regulation, including transcription repression, exon skipping, and RNA splicing ,6. TDP-4The dysregulation of TDP-43 is hence associated with a variety of human diseases, especially neurodegenerative diseases, e.g., frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), brain ischemia, aging, and Alzheimer’s disease ,12,13. FApart from the TDP-43 involvement in neurodegenerative diseases, an accumulating amount of evidence suggests that TDP-43 is a cancer responsive factor. TDP-43 positively contributes to the anticancer activity for curcumin in MCF-7 cells  and as a®, technology was used to characterize DNA and RNA oligonucleotides  binding to TDP-43 and their interaction inhibition was assessed [Although more is known about the TDP-43 biology and its association with neurodegenerative and cancer diseases, the development of treatments toward TDP-43 is mostly lagging behind those targeting other proteins involved in such diseases . RRM andassessed . Later, assessed , but infassessed . HoweverNMR spectroscopy is a powerful approach which has been extensively used by the pioneers in fragment-based drug discovery for detecting molecular interactions between the target and the fragment libraries ,38,39 an15N labeled TDP-43 tandem RRMs demonstrate that these hits bind to the same site, mainly on the RRM2 domain. It has also been validated by the chemical shift perturbation experiments for TDP-43 RRM2 alone. The CSP-driven HADDOCK was used to generate the protein-hits binding mode. Collectively, our work provides a class of compounds for further hit-to-lead evolution of the TDP-43 RRM domain and paves the path for targeting protein-RNA interactions using the fragment-based approach.Here, we carried out automated NMR fragment-based screening  to identStructurally, TDP-43 tandem RRMs are approximately 160 amino acids long and display a β1α1β2β3α2β4 arrangement of secondary structure, with an additional β-hairpin named β3’β3’’  or β5 4,49 whichNMR ligand-observed methods detect the weak protein-ligand binding by detecting changes in the characteristics of the ligand spectrum that occur upon binding to the protein. Using the ligand-based experiments, i.e., saturation transfer difference (STD) , water l15N-labeled tandem RRMs of TDP-43 and 3 of them induced significant chemical shift changes of the tandem RRM  of the ndem RRM . This apndem RRM , which sndem RRM . Howeverndem RRM c.Consistently, hits 2 and 3 titrations also point to the same binding topology in the tandem RRM of TDP-43 . For exa15N-labeled RRM2 domain of TDP-43  nucleotides are accommodated on the RRM1 β-sheet and the following two nucleotides (A6A7) act as a connector between two RRMs, while the next three nucleotides (U8G9A10) lie on the RRM2. The U8 nucleotide of RNA is recognized on S258 (β4) through hydrogen bonds, on the backbone carbonyl oxygen of N259 (β4), and the backbone amide of E261 from the C-terminus [8 nucleotide in tandem RRM  interact with RRM1 and RRM2 (PDB code: 4BS2) . Among tterminus . Comparandem RRM a. Furthendem RRM c,d.Accordingly, the crystal structure of TDP-43 RRM2 in complex with ss-DNA 5′-GTTGAGCGTT-3′ (PDB entry: 3D2W) reveals that only three 5′ end nucleotides  make extensive contacts with β-sheet residues of RRM2, whereby T3 particularly contacts with S258, Asn259, and Glu261 through hydrogen bonds , while iTo further characterize the binding mode, a data-driven approach, HADDOCK , was useProteins containing RRM domains function in important aspects of the posttranscriptional regulation of gene expression, mRNA maturation, and other RNA processing machinery. These proteins perform their diverse roles depending on the dual ability to recognize RNA and to interact with other proteins by using their RRM domain . As TDP-We uncovered three small molecules binding to the tandem RRM domain of TDP-43 by using NMR fragment-based screening techniques. The NMR spectroscopy, one of a plethora of biophysical methods, is particularly powerful to detect even ultra-weak protein-ligand interactions. Accordingly, chemical shift perturbations observed in the heteronuclear single-quantum coherence (HSQC) spectra or the linewidth analysis of the small molecules allow the determination of binding affinity ,59. ThisNMR is extensively applied in fragment-based lead discovery . The cenAlthough the 4-aminoquinolines molecules have been discovered through high throughput screening against the full-length TDP-43 , the enl6 tag. The RRM2 domain was amplified from the tandem RRM construct and then sub-cloned into the pET22b vector  with the His6 tag. The constructs were transformed into Escherichia coli BL21 and cultivated in 1 L LB media, incubated at 37 °C. The proteins were expressed at 16 °C after induction by 0.5 mM isopropyl β-D-thiogalactosidase (IPTG) for 20 h. The bacteria were harvested by centrifugation , resuspended in lysis buffer , and then lysed by sonication. The cell lysates were centrifuged . The collected supernatant was purified on a column filled with Nickel-chelated resin . The impurities were washed out using a buffer  containing a linear gradient of 20–40 mM imidazole, then the same buffer containing 500 mM imidazole was used to elute out the target proteins. All proteins were further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 75 column . The target proteins were confirmed by SDS-PAGE.The tandem RRM domain of TDP-43 (residues 101–269) was synthesized by GENEWIZ  and sub-cloned into the pET22b vector  with the His15N-labeled proteins, the cells were first cultured in 1 L LB media, harvested when A600 reached 1.0 and then transferred to 1 L M9 media containing 15NH4Cl. The cells were induced by 0.4 mM IPTG to express the proteins (tandem RRMs and RRM2 domains). The purified proteins were concentrated in PBS buffer plus the 5 mM DTT at pH 7.5.For 1H) were acquired against the 890 fragments library  as described previously in detail [2O (50%). To further confirm the identified primary hits, secondary screening was individually carried out for single hits using the same buffer and NMR experimental settings. We then automatically processed and visualized the primary and secondary data with our ACD/Labs scripts, as previously described [All NMR fragment screening experiments were carried out at 25 °C using an Agilent 700 MHZ spectrometer equipped with a 96 well auto-sampler and a 5 mm cryoprobe. During the primary screening, the ligand-based NMR spectra , in PBS buffer , containing NaCl (200 mM), dithiothreitol (5 mM), and D2O (10%) were titrated by small molecules stocked in DMSO at a concentration of 200 mM, using a series of hit/protein molar ratios of 0.0, 0.5, 1.0, 2.0, 4.0, and 8.0 for TDP-43 tandem RRMs and 0.0, 0.5, 1.0, 2.0, and 4.0 for RRM2, respectively. Spectra were processed in NMRpipe and analyzed with Sparky. The chemical shift changes (∆δ) relative to the free form of protein were defined as follows:δ1H and δ15N are the chemical shift differences of the 1H and 15N dimensions, respectively. We referred to the following chemical shift assignments previously deposited in the Biological Magnetic Resonance Data Bank: RRM1 (BMRB Entry 18765), RRM2 (BMRB Entry 19922), and tandem RRM (BMRB Entry 19290). All structures figures were prepared by Pymol .NMR HSQC spectra were acquired at 25 °C on either an Agilent 700MHZ spectrometer equipped with a cryoprobe or an Agilent 500MHz spectrometer equipped with a room temperature probe. The 1 PDB file was generated by the PRODRG [HADDOCK is an information-driven docking technique used for modeling biomolecule structures by using experimental or predictive restraints ,62. The e PRODRG . The docThe NMR HSQC spectra at molar ratios of 0:1 and 8:1 (hit/protein) were processed using the same NMRpipe script, e.g., 2-fold zero-filling, Fourier transformation, and phase corrections. The spectra were then analyzed, with randomly selected peaks, using Sparky. After peak integration, the linewidth, i.e., the full width at half the peak height, was automatically estimated by Sparky.


Astaxanthin (Asta), red pigment of the carotenoid family, is known for its anti-oxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties. In this study, we evaluated the effects of Asta on isolated human sperm in the presence of human papillomavirus (HPV) 16 capsid protein, L1. Sperm, purified by gradient separation, were treated with HPV16-L1 in both a dose and time-dependent manner in the absence or presence of 30 min-Asta pre-incubation. Effects of HPV16-L1 alone after Asta pre-incubation were evaluated by rafts (CTB) and Lyn dislocation, Tyr-phosphorylation (Tyr-P) of the head, percentages of acrosome-reacted cells (ARC) and endogenous reactive oxygen species (ROS) generation. Sperm membranes were also analyzed for the HPV16-L1 content. Results show that HPV16-L1 drastically reduced membrane rearrangement with percentage of sperm showing head CTB and Lyn displacement decreasing from 72% to 15.8%, and from 63.1% to 13.9%, respectively. Accordingly, both Tyr-P of the head and ARC decreased from 68.4% to 10.2%, and from 65.7% to 14.6%, respectively. Asta pre-incubation prevented this drop and restored values of the percentage of ARC up to 40.8%. No alteration was found in either the ROS generation curve or sperm motility. In conclusion, Asta is able to preserve sperm by reducing the amount of HPV16-L1 bound onto membranes. HPV (Human Papilloma Virus) is responsible for the 5.2% (3% in women and 2% in men) of cancers in the world, with prevalence in cervical, ano-genital, head, and neck cancers . OncogenBesides sexual inter-infections between sexual partners leading to the wider virus propagation, it has been recently shown that HPV DNA can cause a detrimental effect on early embryo development and clinical reproductive outcomes, since HPV DNA can be harbored inside the blastocyst stage by spermatozoa carrying HPV virions, which represent the viral DNA included inside the L1–L2 capsid ,6,7. HPV infection is considered a cause of male infertility or subfertility , even ifL1, the main constituent of the capsid envelope, spontaneously self-assembles into virus-like particles (VLPs), that are an icosahedral structure composed of 72 pentamers of L1 and an unknown number of the L2 minor coat proteins. Due to its ability of self-assembling in VLPs also in the absence of the L2 protein, L1 has been considered for vaccine development against HPV infections, since the L1-based VLPs share the same immunogen properties of native HPV, but lack genome and other proteins ,11.Haematococcus pluvialis in the presence of stressing conditions including, deficiency of nitrogen, high salinity, and high temperature [Astaxanthin (Asta) is mainly produced by the microalgae perature .H. pluvialis extracts prevents the development of human colon cancer cells by blunting the progression of the cell cycle, ameliorating apoptosis, and suppressing the expression of inflammatory cytokines  [For its molecular structure, Asta belongs to the carotenoid family, with an extended nonpolar zone in the middle, which is made up of a series of carbon–carbon double bond termed “conjugated atoms” and two polar regions at either ends. This nonpolar–polar structure allows Asta to fit precisely into the polar–nonpolar-polar area of the cell membrane ,14 with d IL-1β) ,16,17,18d IL-1β) . Recent evidence has demonstrated that Asta treatment improves human sperm capacitation by inducing the membrane rafts relocation ,21. DuriThe aim of this study was to evaluate the effect of HPV capsid protein L1 on human sperm capacitation, and the effect of Asta in L1 binding to plasma sperm membranes.0), or after incubation in capacitating conditions, in the absence (C), or presence of 1 (L1 1), 10 (L1 10) or 13 (L1 13) µg/mL of L1 protein   b. Due top < 0.0001 compared to control), which was progressively more marked by prolonging incubation time up to 3 h.Experiments with L1 10 µg/mL were carried out for 30, 60, 90, 180 min of incubation and sperm fluorescence evaluated. Interestingly, already after 30 min of L1 incubation, 91.8% ± 3.7% of sperm presented L1 related fluorescence . In the dose-dependent experiments, almost 100% of the marked cells showed fluorescence at the mid-piece, eligible as the principal binding point right after the first 30 min of incubation with 10 μg/mL, HPV L1 c. Intere0, meaning that capacitation had occurred leading to hyper-motility. However, when values from treated cells were compared to C (capacitated without Asta and/or L1), there was no significant difference, also for samples pre-incubated alone or with Asta and L1.Aliquots were then assessed for motility by computer-assisted sperm analysis (CASA) . SamplesThese results suggest that capacitated-related hyper-motility was not affected by Asta, by L1, or by both Asta and L1.We previously showed that capacitation of sperm is linked to membrane rearrangement with lipid rafts relocation to the apical part of the head . This rep < 0.001). L1 incubation affected rafts relocation , CTB, wip < 0.001), followed by the net reduction of the percentage of cells showing Tyr-P of the head  (In these conditions only a reduced percentage of cells underwent the acrosome reaction compared to the C sample (19.4% ± 2.8% of acrosome-reacted cells (ARC) in the L1 10 µg/mL sample compared to 65.7% ± 4.2% in C sample, < 0.001) .p < 0.001, p < 0.001), Lyn relocation from 16.3% ± 3.5% to 43.2% ± 3.0% (p < 0.001) and Tyr-P of the head from 11.3% ± 1.7% to 43.6% ± 3.9% (p < 0.001) (p < 0.001), which, also if still far from the 65.7% ± 4.2% of the C sample, accounts for twice the value of the L1-treated sample (L1 10 μg/mL: 19.4% ± 2.8%).When Asta was added to the capacitating medium, the values of capacitation only slightly improved, as previously shown , since s< 0.001) , shiftinSamples were also analyzed for the L1 protein binding to the subcellular fractions. After incubation in the absence or presence of Asta, L1, or both, aliquots from each sample were sonicated, sub-cellular fractions were separated on gradient and further centrifuged, as described in Methods. Membranes, cytosol, heads, and flagella were subjected to Western blotting and revealed with ant-L1 antibodies.0, C and Asta samples did not show any response, as expected. The sample treated with Asta+L1 showed a reduction in the L1 content, with Asta reducing the amount of L1 by about 30%, compared to L1 sample (206 ± 17 vs. 145 ± 14 ng of L1 protein/30 × 106 cells p < 0.001). L1 was detected on membranes from L1-treated sperm,  (Compared to data in < 0.001) , data frWhen the effects of the different treatments were assayed on the ROS generation curve , samples2O2 formation in sperm. All samples showed similar curves of ROS generation compared to the control (C), thus suggesting that none of the treatments affected HTo initiate a successful infection, HPV must bind to heparan sulfate proteoglycan (HSPG) by exposing L1 from its capside structure and through L1 form an endocytic complex , that isIt has been recently confirmed that also in human sperm, GAGs mediate the viral binding to the cell surface, in particular the GAG syndecan-1 (Synd-1) seems to interact with the viral capsid protein L1 of HPV16 ,28. SyndIn the present study, the L1 binding would act as an anchor complex, preventing further rafts translocation and relocation to the sperm head. This hypothesis is consistent with the results of Chen et al.  who demoRecombinant HPV16 L1 protein was purchased from Abcam . Anti- HPV16 L1 mouse monoclonal, goat anti-mouse and anti-rabbit IgG- fluorochrome fluorescein isothiocyanate (FITC) conjugate antibodies were purchased from Santa Cruz Biotechnology . Anti-P-Tyr mouse monoclonal and anti-Lyn rabbit polyclonal antibody were obtained by Upstate  and Millipore , respectively. Density gradient (Pure Sperm 40/80) and pure sperm wash buffer (PSW) were obtained from Nidacon International AB . Asta was supplied by FERpharma s.r.l. . 12-myristate-13-acetate phorbol ester (PMA) was purchased from Calbiochem  and all other reagents from Sigma-Aldrich .Forty healthy male donors  were enrolled at the Centre of Assisted Reproduction-U.O.C. Obstetrics and Gynecology Clinic–Padua, Italy. After 3 days of abstinence, semen samples were collected by masturbation in a sterile container and then assessed for sperm parameters. All sperm samples used in this study were normal in terms of sperm count, motility, morphology, volume, and pH, according to the World Health Organization criteria . All samg for 30 min at room temperature. The seminal plasma and sperm from the 40% gradient interface were discarded, and the sperm cells from the bottom pellet (80% gradient) were gathered. After gradient separation, sperm samples were washed with PSW and collected three by three in a single pool (stock sample) to obtain a sufficient number of cells to perform all tests. Stock samples (concentration adjusted to 80 × 106 sperm cells/mL in PSW) were divided in aliquots, analyzed immediately (T0), or incubated for up to 180 min in capacitating conditions, in the absence (C) or presence of Asta 2 µM (Asta) from stock solutions of 100 mM dissolved in dimethyl sulphoxide (DMSO), L1 1, 10 or 13 µg/mL (L1), or together with 30 min-Asta pre-incubation (Asta+L1 10).After semen analysis, samples were laid on a discontinuous gradient (Pure Sperm 40%/80%) and centrifuged at 500× Sperm motility and hyperactivation were analyzed using a computer-assisted sperm analyzer . For each sample, the following parameters were evaluated: the percentage of motile spermatozoa and VAP (average path velocity), VSL (straight-line velocity), and ALH  to determine the percentage of hyper-activated (HA) cells . All mea6 cells) from each sample were accurately washed with phosphate buffer saline (PBS) containing vanadate 1 mM and protease inhibitor cocktail, fixed with 2% (w/v) paraformaldehyde and incubated overnight at 4 °C on slides pre-coated with poly-L-lysine [v/v) Triton X-100 for 15 min at 4 °C and then incubated with anti-L1, anti-P-Tyr, or anti-Lyn antibodies for 1 h at 37 °C in a humid chamber. Slides were washed with PBS, stained with anti-mouse or anti-rabbit IgG-FITC conjugate for 1 h at 37 °C in a humid chamber and then rinsed with PBS and mounted. Staining without primary antibody was used as negative control. Fluorescence was detected with the UltraView LCI confocal system .Aliquots of sperm  from each sample were mixed with an equal volume of CTB (50 µg/mL) and incubated for 15 min at 37 °C. The sperm cells were then washed twice in PBS before being fixed in 2% paraformaldehyde for 30 min, mounted on poly-L-lysine coated glass microscope slides, and viewed using the confocal microscope as described above. For each treatment, at least 200 cells were counted. GM1 membrane raft marker was visualized in live human spermatozoa by staining with the cholera toxin subunit B (CTB)-FITC ,34. For Arachis hypogaea) agglutinin (FITC-PNA) in conjunction with DNA-specific fluorochrome propidium iodide (PI) as a viability test [6 cells) of each sample were incubated for 30 min at 37 °C, in the presence of 10 µM Ca2+ ionophore A23187. Samples containing DMSO, but not ionophore, were used as control. After incubation sperm cells were centrifuged, resuspended in PBS, and treated for 10 min at room temperature with 12 μM PI. Sperm was washed with PBS, fixed with 2% (w/v) paraformaldehyde and incubated overnight at 4 °C on poly-L-lysine-treated slides. Permeabilized sperm cells, as described above, were stained with 1 mg FITC-PNA/mL for 15 min at 37 °C in the dark, washed and mounted. At least 200 cells were evaluated for each sample, and fluorescence was detected as described above. Only sperm cells showing evenly distributed fluorescence over the acrosomal region were considered acrosome-intact.Acrosome status was monitored with acrosome-specific FITC-labeled peanut  from each treatment were accurately washed in PBS-VI (containing vanadate 1 mM and protease inhibitor cocktail), resuspended in 200 μL of the same PBS-VI, and sonicated 3 times (30 s followed by a 10-s rest period each) on ice. Heads and flagellar fragments were then separated by a 15-min centrifugation (700× g) at 4 °C through a 75% Percoll layer in PSW. Flagellar fragments were recovered at the surface of the Percoll layer while the heads were found in the pellet. The purity of each fraction was assessed by microscopy prior to proceeding to analysis. The supernatant was centrifuged for 10 min  and the resulting supernatant was further centrifuged  to separate the membrane from the cytosol [To determine whether and how much L1 was present in the plasma membrane, head or flagellum, intact spermatozoa  or corresponding cytosol  were solDensitometric analysis. Bands corresponding to L1 protein bands were counted by ImageJ software 1.48v.6 cells/mL. ROS levels were determined by a luminometer  in the integrated mode for 180 min at 37 °C. Results are expressed as Relative Luminescence Units (RLU) per 2 × 106 sperm cells. Lastly, 2 µL of a 10 mM N-formylmethionyl-leucyl-phenylalanine (FMLP) stock was added and, after a further 10 min of incubation, 4 µL of a 1 nM stock solution of PMA was added, to exclude leukocyte contamination. Only samples with negative response to FLMP and PMA were processed.Production of ROS was measured by the chemiluminescence assay method with luminol  as probe ,24. Briep < 0.05 (two-tailed). Statistical analysis were performed with JMP® 13 software .Results are expressed as means ± SD. Comparisons among multiple groups were obtained with ANOVA followed by Dunnett’s test. Statistical significance was set at Recent evidence substantially strengthens the possibility that HPV is responsible not only for HPV-associated cancers, but also for idiopathic infertility due to its detrimental effect on sperm parameters ,37,38 an


Apis cerana and Apis mellifera). We also evaluated the changes in the activity of antioxidant and detoxification enzymes in their body. We observed that under higher temperature and humidity conditions, the survival rate of A. mellifera was higher than that of A. cerana. On the other hand, a comparison of water loss between the two species revealed that A. mellifera lost more water. However, under extremely high temperature conditions, A. cerana was more tolerant than A. mellifera. Moreover, under higher temperature and humidity conditions, the activity of antioxidant and detoxification enzymes in bees was significantly increased. Overall, these results suggest that high temperatures can adversely affect bees. They not only affect the survival and water loss, but also stimulate oxidative stress in bees. However, unlike our previous understanding, high humidity can also adversely affect bees, although its effects are lower than that of temperature.The ambient temperature and relative humidity affect the metabolic and physiological responses of bees, thus affecting their life activities. However, the physiological changes in bee due to high temperature and high humidity remain poorly understood. In this study, we explored the effects of higher temperature and humidity on the epiphysiology of bees by evaluating the survival, tolerance and body water loss in two bee species ( Honey bees are poikilothermal animal. The ability of bees to maintain and regulate body temperature is poor. The ambient temperature affects the metabolic and physiological responses of bees, and thus, affecting in their life activities ,2,3 suchRelative humidity (RH) has a significant effect on larvae during incubation . It has Honeybees, known for their importance as pollinators in natural habitats, have long-term co-evolution with plants, and their biological characteristics are perfectly harmonious in color, aroma, and structure . In receTemperature stress can induce oxidative stress in insects, and their antioxidase play a major role against oxidative stress . Under hA. cerana and A. mellifera are the two most commonly cultured bee species. They are also widely used for pollination in agricultural facilities [A. cerana and A. mellifera were used as research objects. We explored the changes in the physiological activities of honeybees under high temperature and humidity conditions, and compared their adaptability.cilities . CurrentA. cerana and A. mellifera for the study. For the epiphysiological experiment, we chose foragers that returned to the hive. For the enzyme activity experiment, we selected 1–2 capped brood combs from the colony and placed it in an incubator at 34°C and 75%RH. After emergence from the cell, the bee were marked on the back with non-toxic, odorless paint, returned to the original colony, and then sampled at the age of 20 days.The study was conducted at the Shanxi Agricultural University  from June to August 2018. We selected three healthy colonies of For the experiment, we used a 16cm × 7cm × 7cm wooden box with one side covered by a transparent plastic plate. Thirty workers per each cage per species were used per treatment. 5 mL of water is provided to the bees in each box, 1mL 30% syrup. We chose the temperatures and humidity levels based on the temperature and humidity data of the greenhouse. Temperatures of 35°C, 40°C, and 45°C at constant RH, and RH levels of 50%, 60%, and 70% at constant temperature were tested. The following experiments were conducted under controlled conditions of temperatures and RH in incubators . The number of dead workers was counted every hour under different treatment conditions. Worker survival rate per each cage was calculated as the total number of hours at which all bees had died. Subsequently, the mean survival rate per treatment was calculated by dividing the total number of hours at which all the bees died in the three cages by three.We used the method of Atmowidjojo  to evaluFor the experiment, we used a 10mL centrifuge tube with numerous small holes and one bee was placed in each tube (30 bees per species per treatment). The bees were weighed using a balance  to the nearest 0.1 mg (W1). The temperature and humidity selected for this experiment were consistent with those of the survival rate experiment. The test bees were maintained at a certain temperature and humidity for 2 hours  and then reweighed (W2). The water loss was calculated as (W1)–(W2). To determine the water loss per bee, each weight was divided by 10 and the results are expressed as mg h –1. The rate of water loss was calculated as (W1)–(W2) / (W1).For this experiment, we used 20-day-old bees, which were placed in separate 10-mL centrifuge tubes and treated for 2 h at different temperatures and humidity. We set up four different treatments: control treatment (CK: 25°C 30% RH), high temperature treatment (T: 45°C 30% RH), high humidity treatment (RH: 25°C 80% RH), and high temperature and high humidity treatment (TH: 45°C 80% RH).Ten samples were taken from the whole tissue and ground with liquid nitrogen. To this mixture, a certain amount of PBS (pH = 7.4) was added and mixed by vortexing. The mixture was then centrifuged at 2000–3000 rpm for approximately 20 min and the supernatant was collected carefully. (1) Standard sample loading: standard and sample wells were set (standard wells plus 50μL of different concentrations of standard); (2) Loading: The sample wells to be tested and the blank wells were set separately . Approximately 40 μl of the sample dilution was added to each sample well, and the test was performed on an enzyme-labeled plate, followed by the addition of 10 μl of the sample to be tested . (3) Addition of enzyme: To each well, 100 μl of enzyme labeling reagent was added, except for blank wells. (4) Incubation: After sealing with a sealing membrane, the plates were incubated at 37° C for 60 min. (5) Dosing: The 20-fold concentrated washing solution was diluted 20 times with distilled water and used. (6) Cleaning: The sealing film was carefully removed, the liquid was discarded, and the wells were allowed to dry; thereafter, each well was filled with the cleaning solution, allowed to stand for 30 seconds, then discarded; this step was repeated 5 times. (7) Color development: First, 50 μl of developer A was added to each well, followed by 50 μl of the developer B; thereafter, the mixture was gently shaken and mixed, and colored at 37° C for 15 min. (8) Termination: A 50 μl sample of stop solution was added to each well to stop the reaction. (9) Measurement: Zeroing was performed with blank holes, and the absorbance (OD) of each well was measured sequentially at the corresponding wavelength.The activity of five enzymes, namely, SOD, CAT, POD, GPX, and GSR was tested using their respective assay kits .The same method was followed as described for antioxidant enzyme activities. The activity of five enzymes, DDTase, GST, CES, CYP450, and AChE, was tested using their respective assay kits .The study was designed using the principle of complete randomization. The data were statistically analyzed by the analysis of variance (ANOVA) and the means were compared by the least significant difference test (LSD p<0.05).A. mellifera was slightly higher than that of the A. cerana. A significant difference (P < 0.05) was found between the survival rate of A. cerana and A. mellifera at 35°C and 60% RH, whereas, no significant differences were found among the other treatments . At 60% RH and 70% RH, the survival rate of A. cerana was significantly lower at 45°C than at 35°C and 40°C. At 60% RH, A. mellifera showed a relatively long survival rate at 35°C, followed by that at 40°C, and the survival time of A. mellifera was the shortest at 45°C. Significant differences between the three treatment were observed (P < 0.05). At 50% RH, the survival rate of A. mellifera was significantly lower at 40°C and 45°C than at 35°C. At 70% RH, the survival rate of A. mellifera was significantly lower at 45°C than at 35°C and 40°C.At constant humidity, the survival rate of bees decreased with increasing temperature. A significant (P < 0.05) and strong negative correlation was observed between survival rate and temperature . At 50% A. cerana was significantly lower at 60% RH and 70% RH than at 50% RH. At 45°C, the survival rate of A. cerana was significantly lower at 70% RH than at 50% RH and 60% RH. At 35°C, the survival rate of A. mellifera at 60% RH and 70% RH was significantly lower than at 50% RH. At 40°C, the differences between the three treatment were not significant. At 45°C, the survival rate of bees was significantly lower at 50% RH and 70% RH than at 60% RH.At constant temperature, the survival rate of bees decreased with increasing humidity. A significant (P < 0.05) and moderate positive correlation was found between the survival rate and humidity . At 35°CTemperature had a higher effect on the survival of workers compared to humidity. The results showed that the highest reduction in worker survival rate was at temperatures of 40°C and 45°C, and RH levels of 60% and 70%. However, the reduction effect of temperature on the survival rate was higher than that of humidity.A. mellifera workers become intolerant at 48°C, whereas, A. cerana workers become intolerant at 55°C  between the three treatments  (A. mellifera was significantly higher than that of A. cerana (P < 0.05) . In the .05)Figs  and 11.A. cerana, the SOD activity of TH treatment was significantly higher than the other treatments (P < 0.05)(In P < 0.05); the GPXP < 0.05); and forP < 0.05); the PODP < 0.05) and 13; A. mellifera and A. cerana were consistent. Overall, the detoxification enzyme activity of A. mellifera was higher than that of A. cerana. In the CK treatment, the CES activity of A. mellifera was significantly higher than that of A. cerana (P < 0.05) (We measured the activity of five detoxification enzymes. The results were consistent with those of antioxidant enzyme activities. Enzyme activity in honeybees subjected to different treatments was higher than that of the controls. The results of  < 0.05) .A. cerana, the CES activity of CK treatment was significantly lower than the other treatments (P < 0.05) (A. mellifera. The DDTase activity of RH treatment was significantly higher than the other treatments (P < 0.05) (In  < 0.05) ; and for < 0.05) ; and forA. mellifera and A. cerana) living in different climate regions. These two bees can be used to study other species with long-term adaptation to specific environments in order to determine their particular physiological adaptability [In the long-term evolution process, insects have evolved a series of behavioral responses and physiological mechanisms to adapt to extreme environmental temperatures . Survivatability . The preApis dorsata can survive for 5 days and 48 hours at 38° C and 45° C, respectively. Free and Spencer-Booth [In our study, high temperature environment exhibited a negative effect on the survival of honeybees. This result is consistent with those of previous studies. Mardan and Kevan  found ther-Booth  indicateer-Booth . Abou-Sher-Booth  investigA. mellifera showed higher survival than that of A. cerana at 40°C and 45°C. These results indicate that A. mellifera seems to be more suitable for high temperature and high humidity than A. cerana. According to Joshi, the effect of humidity on the bees was negligible [The present study showed that the average survival rate of worker bees was highest at 35°C, and gligible . HoweverA. cerana seem to be more adaptable to extreme heat than A. mellifera. Abou-Shaara [The results showed that u-Shaara  showed tu-Shaara  found thu-Shaara  reportedA. mellifera was mostly intolerant at 54–60°C, and A. cerana at 57–60°C. In extreme environments, tolerance of A. cerana was stronger than that of A. mellifera. Atmowidjojo [A. cerana was higher than that of the A. mellifera under extreme high temperature, indicating that the cooling efficiency of the A. cerana was better and the tolerance was stronger under extreme high temperature.In this study, owidjojo  found thAs an adaptation to avoid high temperatures, insects first reduce the body and body surface temperature by increasing the evaporation of water from the body to avoid high temperature damage. Evaporative cooling with water droplets is a common behavior of bees to reduce body temperature at high ambient temperatures . HeinricA. mellifera seem to be better adapted to high temperatures and humidity than A. cerana as A. mellifera presented more body water loss than A. cerana under different temperature and humidity treatments. This is consistent with the findings of Al-Qarni [A. mellifera was higher than that of A. cerana exposed to a higher temperature for 2 h. However, considering the difference in body shape between the two bees, we further studied the water loss rate of the two bees, and found that under the extreme high temperatures of 40°C and 45°C, the water loss rate of A. cerana was higher than that of A. mellifera. This can be explained by the observation that extreme high temperature tolerance of the A. cerana in the tolerance experiment was higher than that of the A. mellifera. In addition, in the present study, the water loss rate increased with the increase in temperature. This is consistent with the finding of Roberts and Harrison [On the basis of our results, we believe that Al-Qarni , who fouHarrison , who obsAntioxidant enzymes are a type of defensive enzymes in bees, which can effectively remove the superoxide free radicals produced by the body during the metabolism process, thus eliminating the toxic effects on cells. Antioxidant enzymes play an important role in scavenging free radicals, preventing oxygen free radicals from damaging the composition, structure and function of cells, and protecting cells from oxidative damage. Therefore, they play an important role in maintaining the balance of active oxygen metabolism in bees .Investigating the antioxidant response of insects crucial for understanding their physiological and biochemical aspects in response to temperature stress . To deteWith respect to endogenous and exogenous toxic substances, the detoxification enzyme system can effectively remove them and protect the body from damage. The antioxidant enzyme system and detoxification enzyme system are an important stress-resistance mechanisms for biological response to various environmental stresses; they play an important role in the process of biological adaptation to the environment .Clofenotane dehydrochlorinase (DDTase), glutathione S-transferase (GST), carboxylesterase (CES), cytochromeP450 (CYP450P), and acetyl cholinesterase (AChE) are several important detoxifying enzymes. They have been used as a biomarkers to assess the production of toxic substances in an organism under oxidative stress. In order to understand the response of detoxification enzymes to honeybees under high temperature and high humidity stress, we determined the activity of several detoxifying enzymes in bees. The results showed that high temperature and high humidity can improve the detoxification enzyme activity of bees, which indicates that the high temperature or high humidity can cause oxidative stress and metabolic poisons of bees. This suggests that the honeybee's detoxification enzyme system is responsive to oxidative stress caused by heat stress. A variety of detoxifying enzymes in bees can synergistically eliminate metabolic poisons damage to bees.A. mellifera appeared to have more powerful antioxidant capacity than A. cerana. This is consistent with previous experimental results that A. mellifera survived at high temperatures for a longer period of time.Comprehensive analysis showed that heat stress caused an imbalance of redox metabolism in bees, and caused oxidative stress. This indicates that heat stress has caused damage to bees and may be the most important factor in generating oxidative stress. Under this oxidative stress, antioxidant enzymes exhibit positive resistance to protection. This indicates that they may be an important mechanism of anti-oxidative stress of bees, and also reflect certain physiological adaptability. A. mellifera are more adaptable under greenhouse conditions, high temperatures, and high humidity. We believe that the effect of high humidity on bees is the result of collaboration with high temperatures. The effect of humidity as a single factor is not yet clear. In addition, whether the effect of high temperature and high humidity on young bees is consistent with adult bees requires further investigation. The findings of this study provide a theoretical basis for investigating the stress resistance mechanism of honeybees under high temperature and humidity conditions. The study also highlights the importance of regulating temperature and humidity in agricultural production facilities and the protection of pollination bee colonies.High temperature and high humidity have a negative effect on workers’ survival. It is reasonable to believe that


Synaptic abnormalities, perturbed endosomal recycling mediated by loss of the small GTPase RAB11, and neuroinflammatory signaling have been associated with multiple neurodegenerative diseases including the motor neuron disease, amyotrophic lateral sclerosis (ALS). This is consistent with the neuroprotective effect of RAB11 overexpression as well as of anti-inflammatory compounds. However, most studies were in animal models, and this phenomenon has not been demonstrated in human patients. Moreover, crosstalk between endosomal trafficking and inflammatory signaling pathways in ALS remains enigmatic. Here, we investigated RAB11 expression and MAPK/ERK/AKT signaling in 10 post-mortem spinal cord specimens from patients with sporadic ALS and age-matched controls. All 10 ALS patients showed TDP-43 pathology, whereas two specimens showed an overlapping FUS pathology and one had an acquired Q331K mutation in TDP-43. There was consistent RAB11 downregulation in all ALS cases, while p-AKT and phospho-ribosomal S6 kinase (p-p90RSK) were upregulated. Furthermore, competition between AKT and ERK pathways was observed in ALS, suggesting subtle differences among the TDP-43-ALS subtypes, which may influence patient therapeutic responses. Our findings demonstrate a complex regulation/perturbation pattern of signaling cascades involving MAPK/AKT/RAB11 in spinal cord tissue from ALS patients. These results underscore the relationships between ALS pathology, altered neuronal trafficking, and inflammation.The online version of this article (10.1186/s13041-019-0475-y) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disorder of motor neurons. The major subtype of ALS (~ 97% of cases) is associated with TAR DNA binding protein of 43 kDa (TDP-43) proteinopathy characterized by nucleo-cytosolic mislocalization . SynaptiTARDBP gene that encodes TDP-43  compared with controls (Figs. The inverse correlation between p-AKT and p-ERK1/2 levels is likely dependent on the underlying pathology, including TDP-43 aggregation versus fragmentation phenotype and/or its overlap with either FUS, C9ORF72 or SETX pathology. This reveals the complexity of the disease mechanisms and underscores the dynamic crosstalk involving direct or inverse relationships between these pathways.In conclusion, our study shows overall dysfunction in a complex network of signaling cascades involving MAPK/AKT/RAB11 pathways associated with neurodegeneration in ALS (summarized in Fig. Additional file 1:Figure S1. Relative levels of monomeric TDP-43 and RAB11 in ALS patients spinal cord tissue. Related to Fig. Additional file 2:Figure S2. AKT/MAPK/ERK signaling in TDP-43-ALS patients. Related to Fig.


In the Kaplan–Meier analysis, patients with a larger extent of ground glass opacity (GGO), fibrosis, and traction bronchiectasis experienced an earlier onset of AE-IPF . In the multivariate analysis, a larger extent of GGO and fibrosis on HRCT were independent predictors of AE-IPF . Additionally, Japanese ethnicity was independently associated with the incidence of AE-IPF after adjustment for HRCT findings (p = 0.0074). In conclusion, a larger extent of GGO and fibrosis on HRCT and Japanese ethnicity appear to be risk factors for AE-IPF.Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is a major cause of morbidity and death in IPF. However, sensitive predictive factors of AE-IPF have not been well-investigated. To investigate whether high-resolution computed tomographic (HRCT) abnormalities predict AE-IPF in independent ethnic cohorts, this study included 121 patients with IPF . Two radiologists independently visually assessed the presence and extent of lung abnormalities in each patient. Twenty-two (18.2%) patients experienced AE-IPF during the follow-up. The incidence of AE-IPF was significantly higher in the Japanese patients ( Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrosis of the lung with unknown etiology resulting in a continuous deterioration of pulmonary function and poor outcomes. The clinical course of IPF is usually progressive, and disease progression can occur at different rates. There are even cases that seem to stabilize over many years. Some patients experience abrupt deterioration in their respiratory status with no identifiable cause, which is termed acute exacerbation of IPF (AE-IPF). AE-IPF is one of the most important causes of death in patients with IPF ,2,3.There have been several studies that identified clinical risk factors for AE-IPF, including poor pulmonary function ,4,5, rapIt has been suggested that Asian patients are more likely to experience AE-IPF than other ethnic patients . RecentlThe aim of our study was to investigate the association between HRCT findings and the risk of AE-IPF in a well-defined international cohort.This study was approved by the Institutional Review Board in Ruhrlandklinik (IRB 06-3170) and Hiroshima University Hospital (IRB 1964). Written informed consent was not obtained from each participant, because this was a retrospective study using only clinical and HRCT data from daily practice, which is part of the standard of care for examinations.n = 9). The remaining 121 patients were included in our study (67 Japanese patients and 54 German patients). The median durations of follow-up for the Japanese and the German IPF patients were 36 months and 31 months, respectively.We reviewed the medical records of all patients who were diagnosed as having IPF in the Hiroshima University Hospital  from 2003 to 2012, and the Ruhrlandklinik  from 2006 to 2012. All patients underwent HRCT scanning. The diagnosis of IPF was made according to the American Thoracic Society/European Respiratory Society consensus classification 2002 , and thoDiagnosis of AE-IPF was made according to a modification of the criteria defined by Collard et al. : (1) UnePhysiological assessments included measurements of thoracic gas volume, vital capacity (VC), forced expiratory volume in 1 s, and single-breath diffusing capacity of the lung for carbon monoxide (DLco) according to the American Thoracic Society/European Respiratory Society guidelines . HRCT scans were performed at the time of stable disease and obtained at the end of inspiration using a variety of scanners. The protocols required 1–2 mm collimation sections reconstructed at 1 or 2 cm intervals. Due to the generation of CT scanners used, the examinations were not performed as low-dose CT, and image reconstruction was done without iterative reconstruction. All images were anonymized using Digital Imaging Communications in Medicine at standard window settings for visualization of the lung parenchyma . The images were visually reviewed by two radiologists  who were blinded to all clinical and functional data. The lung was divided into six zones . The reviewers evaluated the extent of the emphysema, ground glass opacity (GGO), and fibrosis area, which consisted of reticulation and honeycombing. They also evaluated the presence or absence of traction bronchiectasis in each zone. The extent of emphysema and lung parenchymal abnormalities was estimated to the nearest 5% for each of the six zones. The score of each finding was calculated by averaging the scores of the six zones. We defined the extent of traction bronchiectasis as the number of zones with the presence of traction bronchiectasis. The average of the two radiologists’ evaluations was used for the analysis. The findings were defined according to the Fleischner Society’s glossary .U-test or chi-square test, as appropriate. The inter-observer agreement was assessed using the intraclass correlation coefficient. Intraclass correlation coefficients of 0.61–0.80 were interpreted as substantial and 0.81–1.00 as excellent agreement, as previously described [p values less than 0.05 were regarded as significant. All statistical analyses were performed with EZR  [Individual variables for two groups were analyzed by Mann–Whitney’s escribed . Correla, Japan) , which ip < 0.01). In this study, no AE-IPF was induced by surgery, lung biopsy, bronchoalveolar lavage, or chemotherapy.(0.60–0.78), ICC = 0.75 (0.66–0.82), ICC = 0.73 (0.63–0.80)), and also for the extent of traction bronchiectasis (ICC = 0.68 (0.58–0.77)) and for the two components of the fibrosis area: reticulation (ICC = 0.74 (0.65–0.81)) and honeycombing (ICC = 0.62 (0.50–0.72)). The inter-observer agreement was substantial for the extent of emphysema, GGO, fibrosis area . The extent of fibrosis area was moderately correlated with the extent of traction bronchiectasis , There was no significant correlation between the other HRCT findings.Firstly, we analyzed the correlation between the various HRCT characteristics. The extent of GGO was weakly correlated with the extent of fibrosis area , and also inversely correlated with %DLco . The extent of emphysema was inversely correlated with %DLco . Secondly, we analyzed the correlation between the various HRCT findings and pulmonary function. The extent of GGO, fibrosis area, and traction bronchiectasis were inversely correlated with %VC   . In the ctively) . Regardictively) .In this study, we investigated whether HRCT abnormalities could predict the occurrence of AE-IPF in the independent two ethnic cohorts by using the same definition criteria of AE-IPF, and demonstrated that a larger extent of GGO and fibrosis area on HRCT and Japanese ethnicity were independent risk factors for AE-IPF. Areas of minor GGO are findings frequently seen on HRCT in patients with IPF ,12. TheyPrevious studies showed that the extent of fibrosis on HRCT was correlated with the extent of fibrotic areas on histopathology. Fibrotic areas, including reticular opacity and honeycombing, were found to be predictors of mortality in patients with IPF ,12,13. CAE may occur at any time during the course of IPF, although it appears to be more common in patients with more advanced IPF. Previous studies demonstrated that low forced vital capacity (FVC) , low DLcp = 0.012) [The presence of emphysema may complicate the evaluation of HRCT findings and also affect the outcome of patients with IPF. Patients with combined pulmonary fibrosis and emphysema (CPFE) have more frequent secondary pulmonary hypertension associated with high mortality . However= 0.012) . In our We demonstrated that the Japanese ethnicity was independently associated with the risk of AE-IPF. This is in contrast with data from the INPULSIS trials, which showed that there was no difference in the risk of AE-IPF between Asian and White patients  but was Our study has several potential limitations. Firstly, the design of this study was retrospective, and the treatments were not identical in the two populations with regard to the use of prednisone and azathioprine . AlthougDespite these limitations, the results from our study appear to be important in that we can demonstrate that the extent of lung parenchymal abnormalities on HRCT and Japanese ethnicity are independent risk factors of AE-IPF.In conclusion, the extent of lung parenchymal abnormalities on HRCT and Japanese ethnicity appear to be independently associated with the incidence of AE-IPF. HRCT may be useful for assessing the risk of AE-IPF in patients with IPF, independent of ethnicity.


Quantitative metal ion doping enables modulation of the device sensitivity and a quasi-quantitative detection of •OH generated in aqueous solution or from living cells. Owing to its high sensitivity, selectivity, real-time label-free response, capability for quasi-quantitative detection and user-friendly portable feature, it is valuable in biological research, human health, environmental monitoring, etc.Due to ultra-high reactivity, direct determination of free radicals, especially hydroxyl radical (•OH) with ultra-short lifetime, by field-effect transistor (FET) sensors remains a challenge, which hampers evaluating the role that free radical plays in physiological and pathological processes. Here, we develop a •OH FET sensor with a graphene channel functionalized by metal ion indicators. At the electrolyte/graphene interface, highly reactive •OH cuts the cysteamine to release the metal ions, resulting in surface charge de-doping and a current response. By this inner-cutting strategy, the •OH is selectively detected with a concentration down to 10 2+ from the electrolyte/graphene interface and selectively detects •OH with a concentration down to 10−9 M.Field effect transistors based on graphene hold promise for future sensing applications. Here, the authors report a free radical sensor in which the •OH radical cuts cysteamine to release Cd For instance, overproduced reactive oxygen species (ROS) cause oxidative stress through the oxidation of biomolecules, such as lipids, proteins, and DNA, in cells and tissues2. Among them, hydroxyl radical (•OH), as one of the most reactive chemical species known, induces large aggression to human health due to its ultra-high reactivity with various biological species compared with other ROS5. The •OH can damage the bases of DNA and mediate redox alteration of cell-membrane Ca2+ channels. Therefore, the fast and real-time monitoring strategies for physiologically important free radicals, especially the •OH, is of great significance. Till now, analytical methods are still the bottleneck for progress in understanding physiological and pathological events such as aging, cancer, ischemia/reperfusion injury, traumatic brain injury, etc.7. In order to understand the role that free radical plays in biological and pathological events, much attention has been paid on monitoring the free radicals in living systems. Traditionally, the free radical is detected by electron spin resonance (ESR) spectroscopy8, fluorescence spectroscopy11, chromatography13, and electrochemical approaches14. However, these techniques suffer from some of the disadvantages such as costly instrumentation, low throughput, complicated sample preparation, need for well-trained operators, or lack of portability. Moreover, these techniques normally require spin traps or fluorescent probes, which introduce additional contaminations that are involved in the detection system and interfere with the desired signals14. The spin traps or fluorescent probes determine the detection limit and selectivity. In some cases, the detection limit of •OH is only 10−6 M (fluorescence)11, while ROS such as •O2− and •HO2 can also induce a response in the fluorescence or ESR signal15. Therefore, it is still a challenge to real-time monitor the free radicals with high sensitivity and selectivity by a low-cost, portable, and user-friendly analytical platform.Detection of the free radical is of great importance for human health, as the free radical is generally believed to play important roles in the pathogenesis of various human diseases17. The channel material with high surface-to-volume ratio is favorable generally since it implies higher adsorption site density available18. Owing to its high carrier mobility and atomic thickness, graphene bears ultra-high sensitivity to electrical perturbations from the external environment22, which has been integrated into FET sensors with advantages such as label-free detection, high stability, flexibility, fast response, biological compatibility, and user-friendship23, compared with other detection techniques. In a graphene-based FET sensor, the graphene is normally surface functionalized, and the specific interaction between the functional group and the analyte allows the selective detection of the pH24, DNA25, RNA26, living cells28, gas29, metal ions32, etc. Although the free radical has a strong doping effect to graphene34, it is still challenging to directly monitor the free radical in the aqueous environment by the FET sensors. One pioneering work35 develops a poly(3-hexylthiophene) FET sensor functionalized with rutin. The oxidation of rutin by superoxide (•O2−) induces current perturbations and enables the detection of •O2−. However, compared with •O2− or other ROS, the highly reactive •OH has ultra-short lifetime. The lifetime of •OH is around 3 × 10−6 s36, 6 orders of magnitude shorter than that of •O2− (~1 s)37. Considering the ultra-short lifetime and the relatively low rate constant of the oxidation reaction 38, the •OH is easy to convert to H2O2 or other ROS without reaction with the functional groups. Till now, a FET sensor that can monitor highly reactive free radicals like •OH is still absent. Moreover, besides the pH value, quantitative analysis of the chemicals is difficult for a FET sensor, hampering its practical applications, especially in the bio-system.As a promising detection technique, field-effect transistor (FET)-based sensor works by transducing and monitoring the absorbate-induced perturbations into the conductance change in the channel materials, typically in terms of the source-drain current10 M−1 s−1)[41. At the electrolyte/graphene interface, metal ions are released from graphene, leading to a surface charge de-doping and a current response in the channel. Thus, by monitoring the current perturbations induced by the metal ions, the inner-cutting strategy enables indirect detection of •OH with a concentration down to 10−9 M. The label-free real-time detection has high selectivity to •OH over metal ions or other ROS interferents. The sensitivity can be easily tuned by modifying the graphene channel with different amounts of metal ions, and then quasi-quantitative detection of •OH is realized when measuring the sample by using FET sensors with different sensitivities. As an application, the FET sensors were used in quasi-quantitative detection of •OH generated from Hela cells, showing great potential of the inner-cutting strategy for practical applications in monitoring free radicals or other analytes with ultra-high reactivity and ultra-short lifetime.Herein we develop a FET sensor that can selectively detect highly reactive free radicals. The FET sensor has a graphene channel functionalized with metal ion indicators via cysteamine as the inner connector. Owing to the ultra-fast reaction between •OH and thiol derivatives (rate constant: ~10 M−1 s−1), the cys2/Si substrate, where graphene acts as the sensing channel, SiO2 as the gate dielectric, and Si as the back gate. Raman spectrum  is patterned on graphene by photolithography as the electrodes. Au nanoparticles (NPs) are deposited onto the graphene channel by vacuum evaporation and thermal annealing (step i). The resulting channel is denoted as graphene/Au. Field-emission scanning electron microscopic (FESEM) and transmission electron microscopic (TEM) images reveal that the Au NPs are uniformly distributed on the graphene surface without agglomeration  through Au−S bonds (step ii). Finally, protoporphyrin IX (PP) is immobilized on the cysteamine (graphene/Au/Cys-PP) (step iii). The corresponding AFM image shows that the height of the Au NPs increases to around 6 nm  and N 1s (400.4 eV) peaks appear after step ii, indicating immobilization of cysteamine onto graphene/Au. A slight increase of N 1s peak and decrease of S 2p are observed after step iii. The result implies the successful assembly of PP, since PP contains high proportion of N without S. In contrast, Cys-PP cannot be directly linked to graphene surface as no N 1s or S 2p peaks appear on bare graphene after step iii 43. In the process, the drain-source current (Ids) rapidly decreases upon successive addition of Cd2+. After coordination with Cd2+ (step iv), the Ids of the graphene/Au/Cys-PP-Cd2+ channel , thus the graphene/Au/Cys-PP-Cd2+ FET works as a real-time label-free •OH sensor. The current changes  upon the same concentration of metal ions or ROS over •OH suggests the high selectivity of the present sensor for detecting •OH against metal ions and other coexisting biological molecules. Moreover, the storage stability of the graphene/Au/Cys-PP-Cd2+ FET sensor is investigated by monitoring the current response to 10−4 M •OH three times daily after storage in a refrigerator, and the current response when adding 10−4 M •OH still retains >94.5% of its initial response after 1 week of storage  in 0.01× phosphate-buffered saline . The linear nature of the Ids versus drain-source voltage (Vds) curve reveals an ideal Ohmic contact between the channel and the electrode, and such reliable contact provides the possibility of the application in sensitive sensors. The Ohmic contact remains at different Vbg, suggesting that the current changes are mainly affected by the electrostatic gate doping effect, rather than the contact resistance. The Ids versus liquid gate voltage (Vlg) 37, •NO (0.09 to >2 s)49, etc. The •OH is easy to convert to H2O2 or other ROS without arising current responses in the channel. Thus it is still difficult to directly monitor the highly reactive free radicals like •OH in the aqueous environment, although these radicals have a doping effect to graphene34. The control experiment also shows that no critical changes in the current are detected 5 and •O2− (104–105 M−1 S−1)50. After the reaction, the XPS spectra of the graphene/Au/Cys-PP-Cd2+  increases stepwise at each concentration. Figure I/I0 plotted as a function of the •OH concentration from 10−9 to 10−3 M, which can be described by the Hill–Langmuir equation54:I/I0)max is the saturated value of the ΔI/I0, C•OH is the •OH concentration, and KD is a constant related to the ability of the •OH to cut the Au-S bonds. The KD value is extracted from the fitted curve to be about 9.95 μM, indicating the relatively high reactivity between •OH and Au-S bonds.To prove the inner-cutting mechanism, we measured the graphene/Au/Cys-PP-Cdrve Fig.  and 13 g3 M Fig. . Percent2− (0.94 V), H2O2 (0.32 V), and ROO• (0.77 V)5. The redox potential is defined compared with H2/H+ to represent the affinity of a substance for electrons. Higher potential means larger inclination to obtain electrons and stronger oxidizability, thus the cysteamine is inclined to react with •OH rather than other ROS. This, as well as higher reaction rate constant of •OH with the cysteamine compared with other ROS, gives rise to selective reaction of •OH and cysteamine and endows the FET sensor with fast response and high selectivity  was added as a stimulus to induce oxidative stress for the production of ROS within the Hela cells9. To verify the intercellular generation of ROS, the cells are incubated in 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) solution, which functions as the fluorescent probe for •OH. The probe is monitored by confocal microscopy upon 488 nm excitation. From the overlay image of the confocal fluorescence image and bright-field image 5. Stable ROS  can penetrate the cell membrane, reach the solution environment5, and generate the extracellular •OH radicals. As a result, the graphene/Au/Cys-PP-Zn2+ FET device (modified by 10−4 M Zn2+) has a response in Ids when the Hela cells are stimulated by LPS  has a small change of Ids when exposing in 10−7 M •OH, while no response is observed in the case of the FET sensor modified by 10−6 M Zn2+. According to this result, the current response of different FET sensors upon the addition of 10 μg mL−1 LPS indicates the extracellular •OH concentration is at a level of ~10−7 M. This amount is lower than the intracellular concentration (10−6 M) measured by other methods11, in correspondence with the fact that the extracellular •OH originates from other ROS released from the cells and the production of •OH is relatively low 56.Quasi-quantitative detection of the •OH generated from the Hela cells is realized by using the FET sensors modified with different amount of Zn2+ from the electrolyte/graphene interface, leading to charge de-doping of the graphene channel. This, as well as the high carrier mobility and atomic thickness of graphene, result in high sensitivity of the FET sensor. By this strategy, •OH with a concentration down to 10−9 M is detectable, comparable or higher than some of the conventional free radical detection techniques 11. The inner-cutting strategy can not only monitor •OH but also has the potential to detect other types of highly reactive free radicals. Compared with other detection technologies like ESR spectroscopy and fluorescence spectroscopy, the inner-cutting graphene FET sensor is a portable and user-friendly analytical platform that realizes label-free real-time detection with both high selectivity and sensitivity. More importantly, based on the controllable charge doping and de-doping by metal ions, the •OH generated from the Hela cells can be quasi-quantitatively measured by using the graphene/Au/Cys-PP-Zn2+ FET sensors with different amount of Zn2+ dopant, indicating that the FET sensor is compatible with living system when non-heavy metal ions are used as the indicator, and the figure of merit still maintains when detecting in living system since 10−7 M extracellular •OH can be readily detected. This work extends the application of FET sensors in monitoring highly reactive free radical and realizes a real-time quasi-quantitative detection in living environment. Such a portable and user-friendly free radical FET sensor has great potential for practical applications in biological research, human health, environmental monitoring, etc.In this article, we realize the detection of •OH by an inner-cutting graphene FET sensor. Highly reactive •OH cuts the cysteamine to release Cd2/Si substrate to fabricate a FET device by photolithography. The device was thermally annealed to improve the contacts between graphene and electrodes (50 nm Au/5 nm Cr), and then Au NPs were deposited onto graphene by vacuum evaporation  and thermal annealing at 200 °C for 30 min. The resulting graphene/Au NPs was submerged in 10−2 M cysteamine (Sigma-Aldrich) solution. The assembly of cysteamine on Au NPs was kept overnight at room temperature, followed by rinsing with ethanol for several times to remove extra cysteamine. The cysteamine-modified device was immersed in 10−5 M PP (Sigma-Aldrich) and N,N-dimethylformamide (DMF) solution for 15 h, by adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich) and N-hydroxysuccinimide (Sigma-Aldrich) as the catalysts. The resulting FET sensor, with immobilized PP, was rinsed with DMF, acetone, ethanol, and distilled water in sequence to remove extra residues. The graphene/Au/Cys-PP was dipped in Cd2+, Zn2+, or Mg2+ solution for 6 h before tests. Owing to the coordination reaction with PP, Cd2+, Zn2+, or Mg2+ was bound to the surface of FET to charge-dope graphene. A solution chamber, which was employed in all solution-based measurements, was fabricated by a three-dimensional printer.A monolayer graphene film was produced by CVD (Supplementary Note The samples were measured by XPS (Perkin-Elmer PHI5300 with 250 W Mg Kα source), Raman , FESEM , TEM , AFM , and optical microscope . The UV-vis absorption spectra (Lambda 750) were measured from the fluorine-doped tin oxide (FTO)-coated glass. The FTO-coated glass was thoroughly cleaned by sonication in the soapy water, ethanol, and 1.0 M KOH solution, followed by a rinse with doubly distilled water, and then it was dried with pure nitrogen stream and treated by the modification processes as graphene.2O2 and Fe2+ (H2O2/Fe2+ = 6:1). The electrolyte was 10−4 M PBS . All the aqueous solutions were prepared using distilled water with a resistivity of 18.2 MΩ cm. Ag/AgCl reference electrode was used as the liquid gate electrode. The Vgs was set lower than ±0.8 V (versus Ag/AgCl) and the Vds was set to 50 mV to avoid any electrochemical reaction on the electrodes.The electrical signal was measured by a semiconductor parameter analyzer  and a probe station  at room temperature in air. The •OH was generated through Fenton reaction by H−6 M DCFH-DA as the fluorescent probe. The device was immersed in 0.01 × PBS to keep the cells in good status and stored in a small culture dish for the measurement. Electrical measurement was performed by a semiconductor parameter analyzer  and a probe station  without applying Vlg or Vbg. After the Ids became stable for 1000 s (Vds = 50 mV), the electrolyte solution was quickly changed to 10 μg mL−1 LPS (0.01 × PBS) solution. The real-time Ids was recorded for 4500 s in air.Hela cells were cultured on the FET sensors (Supplementary Note Supplementary Information


Silver-coated conductive yarn is used as electrode in the sample to improve its flexibility and applicability as wearable textile. These graphene nanoplatelet/PVDF-HFP coated samples with various high-contents of graphene were characterized using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), sheet resistance analysis, and electrical heating performance analysis. Graphene nanoplatelet/PVDF-HFP coated cotton fabric improved the crystallinity and thermal stability with increasing thw high-content of GNP. With an increasing of the high-content of graphene nanoplatelet in the PVDF-HFP composite solution, the sheet resistance of samples tended to gradually decrease. That of, 64 wt% graphene nanoplatelet/PVDF-HFP composite coated sample (64 GR/cotton) was 44 Ω/sq. The electrical heating performance of graphene nanoplatelet/PVDF-HFP composite coated cotton fabric was improved with increasing the high-content of graphene nanoplatelet. When 5 V was applied to 64 GR/cotton, its surface temperature has been indicated to be about 48 °C and it could be used at a low voltage (<10 V). Thus, a horseshoe-pattern type electrical heating textile that is coated by high content of graphene nanoplatelet/PVDF-HFP composite solution sewn with silver-coated conductive yarn is expected to be applied to glove, shoes, jacket, and so on to improve its wearability and applicability.We prepared a horseshoe-pattern type electrical heating textile that was coated with high graphene nanoplatelet (GNP) content (32 wt% to 64 wt%) of graphene nanoplatelet/poly(vinylidene fluoride- Electronic textiles (E-textiles) are emerging and gradually being integrated into our homes and daily life due to their potential applications in conductive fabrics, flexible sensors, and wearable heaters ,2. Recenco-hexafluoropropylene) (PVDF-HFP) composite that was coated on flame retardant cotton fabric. The width of HP was 2.5 mm and lengths of HP were 100 mm, 75 mm, and 50 mm, respectively. The sheet resistance of HP-pattern coated fabric decreased with decreasing the sample length and coated area from 73 kΩ/cm2 to 23 kΩ/cm2. The electrical heating property of HP showed an appearance of locally excess heat at the curved-shape of HP area. For surface temperature of HP samples with sample lengths of 100 mm, 75 mm, and 50 mm were 6.0 ± 2.4 °C, 6.8 ± 4.5 °C, and 3.5 ± 1.7 °C, respectively. It has been confirmed that heating performance is improved due to the collision of electrons in the curved region with decreasing sample size. The highest temperature also showed an increase of heating performance. Shin et al. [Carbon-based nanomaterials are known to exhibit electric heating properties in accordance with Joule heating. Related studies ,22,23,24Thus, in this study, we fabricated a horseshoe-pattern type graphene nanoplatelet/PVDF-HFP composite coated electrical heating textiles that exhibited temperature of a 50 °C at 5 V. This is a harmless electrical heating condition to the human body using a high content of graphene nanoplatelet. It can be applied to heating gloves and clothing in winter or under extreme conditions. The samples were coated on flame retardant cotton fabric with a horseshoe pattern by using the graphene nanoplatelet/PVDF-HFP composite. We tried to apply silver coated conductive yarn sewn in the graphene nanoplatelet/PVDF-HFP coated fabric as textile in order to improve their flexibility and wearability. The electrical and electrical heating properties of these samples were then analyzed.2 was used as substrate material. Silver coated conductive yarn of 140 D  was used as an electrode. Commercial cotton yarn of 280 D was used as lower yarn for sewing. These samples were sewn with an industrial sewing machine .Graphene nanoplatelets  and PVDF-HFP chips  were used in this study. First-grade acetone  was used as the solvent. Flame retardant cotton fabric  with twill structure at thickness of 0.65 mm and weight per unit area of 0.042 g/cmThe high contents of graphene nanoplatelet/PVDF-HFP composite solution with various contents of graphene nanoplatelet were prepared for use in this study. To obtain graphene nanoplatelet/PVDF-HFP composite solution, 15 wt% of PVDF-HFP solution was prepared by placing PVDF-HFP chips in 1st grade acetone. Different contents  of graphene nanoplatelets were then added into the as-prepared PVDF-HFP solution. These prepared graphene nanoplatelet/PVDF-HFP composite solution were stirred for more than one week with a digitally controlled hotplate stirrer  at room temperature to prepare the homogeneously dispersed solutions.The samples were measured with a fabric image analysis system  at ×6.5 magnification and a field emission scanning electron microscope  at ×300, ×1000, ×8000 and ×20,000 magnifications to investigate the morphologies of coated cotton fabrics. The acceleration voltage was applied at 5 kV to measure the FE-SEM. Samples were visualized after a platinum coating process for 60 s.X-ray diffraction (XRD) spectra were obtained with X-ray diffractometer  using Ni-filtered CuKα radiation and measurement range from 5° to 65° to confirm the crystal structure of graphene/PVDF-HFP coated samples with high content of graphene at various graphene loadings.The thermal stabilities of the samples were determined with thermogravimetric analysis  and differential scanning calorimetry . TGA was performed in oxygen at a heating rate of 20 °C/min. The measurement temperature ranged from 30 °C to 700 °C. DSC was conducted under nitrogen atmosphere with a starting temperature of 30 °C, which was increased up to 350 °C at a heating rate of 20 °C/min.sR) is expressed in ohms per square. It was calculated according to Equation (1):R is resistance measured by the multimeter, W is the width of the sample, and D is the distance between the two electrodes.Sheet resistance was measured with a multimeter  based on the AATCC-76 method to determine the electrical properties. Two parallel conductive prove were placed in contact with both edges of the samples. The sheet resistance . Both edges of samples consisting of silver coated conductive yarn were connected to the alligator clips. For application in clothing, this study applied low voltage from 0 V to 6 V with 1 V (DC) intervals for 3 min. A thermal imaging camera  was used to measure the surface temperature after applying different voltages. The current value was measured when the voltage was applied to the sample. Three samples were measured and the mean value was calculated.Time-dependent temperature changes of samples were measured with a temperature data logger  and a temperature sensor  to confirm the part of local area. As shown in trans) and 2nd maximum temperature (2nd Tmax) at 287.1 °C and 444.6 °C. The residue was 49.0% at the 1st Ttrans, 35.1% at the 2nd Tmax, and 11.0% at 650 °C, respectively. The onset temperature of GR/cotton with various high-content of graphene nanoplatelets ranging from 32 wt% to 64 wt% ranged from 230.1 °C to 234.1 °C, and reaching the 1st Ttrans of 277.3 °C to 293.2 °C and 2nd Tmax of 457.1 °C to 468.6 °C. The residue rate at reached 1st Ttrans, 2nd Tmax, and 650 °C for 32 GR/cotton to 64 GR/cotton ranged from 67.7% to 74.4%, 39.4% to 51.3%, and 25.3% to 39.4%, respectively. Yang et al. [The thermal degradation process of GR/cotton with various high-content of graphene nanoplatelets was investigated by thermo-gravimetric analysis in oxygen. The results are shown in g et al.  have alsR2 of 0.9137. The sheet resistance of 32 GR/cotton, 40 GR/cotton, 48 GR/cotton, 56 GR/cotton, and 64 GR/cotton were 1.0 × 102 ± 1.0 × 101 Ω/sq, 7.2 × 101 ± 6.8 Ω/sq, 6.3 × 101 ± 2.9 × 101 Ω/sq, 5.5 × 101 ± 1.7 × 101 Ω/sq, and 4.4 × 101 ± 1.3 × 101 Ω/sq, respectively. Previous studies on electrical properties of films or coated fabrics that were made of graphene/polymer composites have reported that the sheet resistance decreases with increasing graphene contents in the composite [The sheet resistance of GR/cotton coated with various high-content of graphene nanoplatelets was measured to determine the electrical properties. omposite ,12,13,14The electrical properties of GR/cotton coated with various high contents of graphene nanoplatelets were confirmed to be related to the crystal structure. As mentioned above, the electrical conductivity in the direction perpendicular of the graphite/polymer composite to the graphite alignment have been reported to be five to six orders of magnitude higher than that parallel to the graphite alignment, since the increase in the electric anisotropy can be attributed to the flake-like shape of graphite flakes and their higher degree of alignment . The resTo investigate the electrical heating property of GR/cotton coated with various high-content of graphene nanoplatelets, surface temperatures of samples were confirmed while using IR thermal images. The surface temperature of each sample was measured at various applied voltages from 1 V to 6 V with intervals of 1 V for 3 min. To evaluate electrical heating properties of graphene/polymer or carbon-nano material/ polymer composite based heater, copper materials, such as copper plate, copper wire, and copper tape, has been mainly used as electrode ,23,24. TAs shown in Q) yielded from the applied electrical energy is calculated according to Equation (2):I is current, R is resistance, and T is period of the time.As shown in This result indicates that 56 GR/cotton and 64 GR/cotton were more affected than 32 GR/cotton, 40 GR/cotton, and 48 GR/cotton by the current value according to period of time. Therefore, the long-lasting electrical heating behavior was confirmed in the GR/cotton with various high-content of graphene nanoplatelets, with 64 GR/cotton presenting the best electrical heating performance and behavior.In summary, this study prepared horseshoe pattern type of graphene nanoplatelet/PVDF-HFP composite with high-content of graphene nanoplatelet that was coated on flame retardant cotton fabric (GR/cotton) as a fabric heating element GR/cotton was coated with graphene nanoplatelet/PVDF-HFP composite with high-content of graphene nanoplatelet ranging from 32 wt% to 64 wt%, with intervals of 8 wt%. To evaluate the electrical heating properties of fabric heating elements, copper materials, such as copper plate, copper wire, and copper tape, has been mainly used as electrode. However, those are difficult to apply to wearable textile, because they have brittle, corrode, and tarnish over time. In our study, to improve its flexibility and applicability as a wearable textile, silver coated conductive yarn was used as the electrode of GR/cotton. With increasing the high-content of graphene nanoplatelet in GR/cotton, stacked and overlapped graphene nanoplatelet layers were also increased. It improved the crystallinity of graphene nanoplatelet peak 2θ = 26.6° and thermal stability. The sheet resistance of the GR/cotton sample tended to linearly decrease with an increasing high-content of graphene nanoplatelet. It was decreased by half from 32 GR/cotton to 64 GR/cotton. Furthermore, the electrical heating properties of GR/cotton were improved with increasing the high-content of graphene nanoplatelet. When 5 V was applied to 64 GR/cotton, the surface temperature was indicated at about 48 °C. Additionally, samples could reach steady-state temperature within 10 min. and maintained the temperature for 30 min. Therefore, the horseshoe-pattern type of graphene nanoplatelet/PVDF-HFP composite with high-content of graphene nanoplatelet coated on flame-retardant cotton fabric (GR/cotton) is expected to have applications as wearable fabric heating elements, such as gloves, shoes, jacket, and so on for use in winter and extreme conditions.


Candida albican adhesion subsequently decreasing the susceptibility of denture stomatitis incidence. Azadirachta Indica, commonly known as Neem powder has antimicrobial effect but the effect of its addition to acrylic denture base on C. albicans adhesion has not been investigated. The aim of this study was determine whether adding neem powder to acrylic denture base materials could reduce Candida albicansadhesion.Denture with antimicrobial activities is desirable to prevent p≤0.05 was statistically significant).One hundred and twenty acrylic resin denture specimens were fabricated and divided into heat-polymerized (n=60) and auto-polymerized (n=60) groups. Each group was further divided into 6 groups (n=10) based on the neem concentration: 0, 0.5, 1, 1.5, 2 and 2.5 wt% of the polymer. After polymerization, the specimens were polished, stored in distilled water, sonicated, sterilized, submerged in artificial saliva containing C. albicans, and finally, placed in an incubator at 37°C. Slide counting and direct culture methods were used to assess the antifungal effects of the neem addition. An analysis of variance and post hoc Tukey’s test were performed for the data analysis . Moreover, the count decreased as the neem concentration increased (lowest count with 2.5 wt%).Based on the results, the neem addition significantly decreased the C. albicans; therefore, the incorporation of neem could be a possible denture stomatitis prevention method.The results suggest that adding neem powder to acrylic resin denture base materials reduces the adhesion of  Key words:Denture stomatitis, Candida albicans, Azadirachta indica, neem powder, denture base. C. albicans in DS, and the results have suggested that it is the main fungal source and a key factor in the pathogenesis of DS. Moreover, it has the ability to develop into a biofilm  is a widespread disease occurring in both partial and complete denture wearers that tends to affect the mucosa of the palate . Poor or biofilm ,5. The d biofilm ,6. C. albicans growth have been investigated experimentally using natural products, such as henna and thymoquinone (TQ), and mixing them with denture base materials  denture base materials were investigated by Al-Thobity et al. They concluded that the addition of TQ could be effective for preventing C. albicans adhesion and proliferation on the denture surface as one possible method for preventing DS . They were covered with modeling wax  and invested in type III dental stone  within a 61B Two Flask Compress . Next, the wax was melted away to create mold spaces using a wax elimination machine, and a separating medium  was applied to all of the stone surfaces while the stone was still warm. The prepared heat-polymerized acrylic resin  was mixed according to the manufacturer’s instructions. Then, it was packed (in the dough stage) into the previously created mold spaces under pressure. It was kept in the flask clamps for 1 h before processing. The flasks were processed using the conventional curing method by placing them into a thermal curing unit  for 8 h at 74°C, and then, increasing the temperature to 100°C for 1 h.The auto-polymerized acrylic resin  was prepared with the neem concentrations the same way that the heat-polymerized acrylic powder was prepared . A totalAfter the complete polymerization of both materials, the specimens were finished and polished using a tungsten carbide bur  with a thin cross-cut at 18,000 rpm, followed by a coarse grain cylindrical rubber top bur  , and then, a fine grain cylindrical rubber top bur  . In ordeC. albicans adherence assay-C. albicans cells (ATCC 10231)   for two C. albicans cells were removed from the acrylic plates by washing them three times with phosphate-buffered saline (PBS). Then, they were placed in sterile tubes with 1 ml of Sabouraud dextrose broth  for 48 h. The adherent C. albicans cells were dislodged from the acrylic specimen surfaces by scraping them. They were vortexed for 10 min, followed by centrifugation at 4,500 rpm for 5 min. Then, the effectiveness of each disinfecting agent concentration was evaluated using 2 different methods.The nonadherent Direct Culture Method (Colony Forming Units)C. albicans colonies in each quadrant in which acceptable growth was noted was counted using a colony counter pen , with the final number corrected for the dilution factor.First, a 100 µl sample was obtained from each centrifuge tube. The sample was serially diluted, spread onto a petri dish, and incubated for 48 h at 37°C. After incubation, the number of -Slide Counting MethodC. albicans. The living C. albicans is transparent with a blue peripheral line, while the dead C. albicans usually appears blue. A slide counter contains 4 major squares that are each divided into 16 smaller squares. The C. albicans was counted on 2 major squares and multiplied by 2 to obtain the total C. albicans count on each slide, which was observed under a light microscope at low power (10x).A volume of 7.5 μl was acquired from each concentrated pellet and added to 2.5 μl of a trypan blue solution in 0.4% PBS . Then, a microscopic evaluation of the new 10 μl solution was conducted after placing it on a slide counter . The trypan blue stain can distinguish between living and dead -Statistical Analysis C. albicans counts of the different neem concentrations were compared. An analysis of variance and post hoc Tukey’s honestly significant difference test were performed to compare the differences in the means of the observations at the various intervals and the baseline, and a p value ≤ 0.05 was considered to be statistically significant. The software calculated the bias representing the average discrepancy between the methods, and the upper and lower limits of agreement (LoAs) were calculated as the bias ± 1.96 times the standard deviation of the bias. The closer the bias was to zero and the narrower the LoA, the more agreement there was between the methods.The data analysis was conducted using IBM SPSS Statistics for Windows version 19.0 . The fungal growth results were presented as means and standard deviations, and the differences in the live C. albicans count decreased significantly as the neem concentration increased (p<0.000). All of the pairwise comparisons showed significant differences between the means, with the exception of the HD and HE with the direct culture method. The HE showed maximum inhibition against the C. albicans with values of 340.8±11.5 and 1,673.5±110.9 for direct culture and slide counting methods, respectively, when compared to the control group . Figure The means, standard deviations and p values of the heat-polymerized specimens for both tests are presented in C. albicans count as the neem concentration increased in all of the modified groups in both the direct culture and slide counting methods. Figure The means, standard deviations and p values of the auto-polymerized specimens for both tests are summarized in C. albicans colony count for the two methods. The bias is described as follows: as the average difference gets closer to zero, the method becomes more concordant. The upper and lower LoAs represent the bias ± 1.5 standard deviations of the bias. The upper and lower LoAs for the direct culture method and slide counting method in this experiment were -4.4 and 1.5, respectively.The Bland-Altman plot in Figure C. albicans to the denture base materials was then tested. The results showed that the mean number of C. albicans colonies in the tested groups decreased significantly when compared with the conventional heat-polymerized PMMA. Therefore, the null hypothesis stating that the amount of C. albicans adhering to the PMMA plus neem denture materials would not be different from the amount adhering to the conventional heat-polymerized PMMA was rejected A novel DS treatment approach was tested in the present study with the addition of neem powder to denture base materials. Its effectiveness against the adherence of C. albicans adhesion to the heat-polymerized acrylic resin. The relationships between the neem concentrations and the C. albicans counts confirmed the antifungal effects of the neem. One previous study reported that because neem includes the chemical constituents of alkaloids, flavonoids, terpenoids and tannins ( tannins , it has  tannins . The abi tannins ,21,22. A tannins .Because of the antimicrobial effects of neem powder, it has been used as an active ingredient in many toothpastes and tooth powders. Additionally, it is quite beneficial in dentistry for naturally treating gingival problems and maintaining oral health. Neem twigs have been used to relieve toothaches, as an oral deodorant, for cleaning teeth  and as aet al. evaluated the antifungal effects of a neem extract on dental composite resin, and they reported that the neem leaves showed potential antifungal effects on biofilm formation, as well as C. albicans adhesion and colonization (et al. (et al. (C. albicans adhesion at various concentrations.In agreement with the previous study, Polaquini nization . For all (et al. ,12 and A (et al. , who fouC. albicans adhesion because of its surface properties. The importance of studying the antifungal effects of neem powder in auto-polymerized acrylic resin material is based on its versatility in clinical applications that require the use of this resin.An interim removable prosthesis is a prosthesis designed to improve stabilization, esthetics and/or function for a limited duration of time, and it can be substituted later with a final prosthesis . Auto-poC. albicans adhesion due to its weak surface properties, especially its excessive surface roughness when compared to the heat-polymerized resin (When comparing the values of both resins, the auto-polymerized resin showed higher values in the respective neem concentrations when compared to the heat-polymerized resin. However, the results of the two tests were the same, even with the high values reported in the auto-polymerized samples that had greater colony numbers. This is consistent with several previous studies ,33,34, wed resin . These ded resin ,32.C. albicans adhesion to the heat-polymerized and auto-polymerized PMMA plus neem denture base resins. In this study, similar log reductions with robustness and agreement were observed between the direct culture method and the slide counting method in the Bland-Altman plots (Fig. The strengths of this study include the use of two separate antimicrobial assays and two different materials for the assessment and quantification of the ots Fig. .C. albicans adhesion; thus, it can be used as a possible DS treatment and prevention method. Neem’s ability to inhibit C. albicans, as determined by this current study, should launch additional investigations determining the efficacy of neem in combating C. albicans. Moreover, this study may encourage the use of neem as an effective alternative method for the prevention and/or treatment of this pathogen in patients.Neem is cheap, widely available, and easy to handle and apply, in addition to its antifungal activities, which indicates that the use of neem powder can help control C. albicans. Additionally, no aging process was implemented. In future investigations with well-simulated conditions, a wide range of microorganisms, including those forming biofilms, should be assessed. Moreover, comparisons of the effects of neem and different antifungal agents on C. albicans adhesion, as well as investigations of the effects of neem on the physical properties of the acrylic resin denture base materials are needed.This study did have some limitations. For example, the oral environment contains many types of microorganisms, not just C. albicans to denture base materials. PMMA acrylic resin modified with neem could be used for the fabrication of removable prostheses as a possible DS treatment or prevention method. However, further investigations of the physical and mechanical results of adding neem to denture base materials are required.Within the limitations of this study, it can be concluded that neem powder exhibits antifungal activity by decreasing the adhesion of