Cpeb1 expression through the 3’ untranslated region (3’UTR). To investigate the molecular basis of the regulatory potential of the Cpeb1 3’UTR, here we performed reporter analyses that examined expression levels of Gfp reporter mRNA containing the Cpeb1 3’UTR. Our findings indicate that CPEB1 represses the translation of Cpeb1 mRNA and that miR‐145a‐5p and let‐7b‐5p are involved in the reduction in Cpeb1 expression in the absence of AUF1. These results suggest that Cpeb1 expression is post‐transcriptionally regulated by AUF1, CPEB1, and microRNAs.Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates the translation of numerous mRNAs. We previously showed that AU‐rich binding factor 1 (AUF1) regulates  Cpeb1 3’UTR and AUF1, an mRNA decay factor, increased Cpeb1 mRNA levels but reduced the protein levels . In this study, we revealed that Cpeb1 expression is post‐transcriptionally regulated by multiple inhibitory machineries that are regulated by AUF1, CPEB1, and microRNAs.We have previously shown that loss of interaction between AREs in the  AREAU‐rich elementAUF1AU‐rich binding factor 1CPEB1cytoplasmic polyadenylation element binding protein 1SGstress granule4A1‐2U) on target mRNAs [Tex19.1 mRNA, which are essential for mouse oocyte maturation [Cpeb1 expression is downregulated [Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is an RNA‐binding protein that interacts with cytoplasmic polyadenylation elements  in the Cpeb1 3’UTR and AU‐rich binding factor 1 (AUF1), an mRNA decay factor, increased Cpeb1 mRNA levels but reduced the protein levels [Cpeb1 expression is regulated by several distinct mechanisms through the Cpeb1 3’UTR. Interestingly, recent studies have demonstrated that a number of microRNAs (miRNAs), which function as major regulators of post‐transcriptional mRNA expression, can modulate Cpeb1 expression by interacting with the Cpeb1 3’UTR in glioma [Cpeb1 expression is post‐transcriptionally regulated by multiple inhibitory mechanisms. To address this hypothesis, we focused on CPEB1‐mediated negative autoregulation, discordant role of AUF1, and miRNA‐mediated repression in Cpeb1 expression.Considering the physiologic and pathologic significance of CPEB1, it is important to investigate the detailed regulatory mechanisms of n levels . These fn glioma , during n glioma , and in n glioma . We thusCpeb1 mRNA‐specific stealth RNAi siRNAs (Thermo Fisher) were designed based on the coding regions . For inhibition of miRNA, mirVana miRNA inhibitors  were used. Transfection of siRNA and mirVana was carried out using Lipofectamine RNAiMAX (Thermo Fisher) according to the manufacturer’s instructions.Mouse neuroblastoma Neuro2a cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . For plasmid transfection, Lipofectamine 3000 (Thermo Fisher) was used according to the manufacturer’s instructions. For small interfering RNA (siRNA) transfection, Pfu polymerase  and specific oligonucleotide primers. The full‐length mutant 3’UTR was digested using BspEI and BglII and inserted into pAcGFP‐C1. All the expression plasmids contained an SV40 PAS derived from pAcGFP‐C1 downstream of the Cpeb1 3’UTR. Oligonucleotide primers with mutations and their sequences are listed in Table The reporter plasmid GFP‐3’UTR was obtained as previously described . Mutant Hprt) was used as an internal control. For quantification of miRNA, RNA polyadenylation, first‐stranded cDNA synthesis, and qPCR were performed using Mir‐X miRNA qRT‐PCR TB Green Kit (TaKaRa) according to the manufacturer’s instructions. U6 small nuclear RNA was used as an internal control. Oligonucleotide sequences are listed in Table To perform quantitative PCR (qPCR), total RNA was extracted from Neuro2a cells using Sepasol‐RNA I Super G . cDNAs were then synthesized from 1 μg total RNA by using PrimeScript RT Reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. qPCR was carried out using THUNDERBIRD SYBR qPCR Mix  and the Rotor‐Gene Q real‐time PCR system . Hypoxanthine guanine phosphoribosyl transferase  as described previously . Brieflyrabbit polyclonal antibodies against CPEB1  and GAPDH . The secondary antibody was HRP‐conjugated donkey anti‐rabbit IgG antibody . The raw images of western blots are shown in Fig. Neuro2a cells were lysed in radioimmunoprecipitation assay buffer . SDS/polyacrylamide gel electrophoresis (PAGE) was performed using 8% and 10% SDS/polyacrylamide gel to detect CPEB1 and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), respectively. In the case of detecting CPEB1, SDS/PAGE was performed until a prestained molecular marker protein  was fully run to the bottom of the gel to separate CPEB1 from adjacent nonspecific bands. Subsequent western blot analysis was performed using Trans‐Blot Turbo Transfer System . Primary antibodies and horseradish peroxidase (HRP)‐conjugated secondary antibodies were used at dilutions of 1 : 500 and 1 : 1000, respectively, in 5% Blocking One  in Tris‐buffered saline containing 0.1% Tween 20. Proteins were visualized using Luminata Classico Western HRP Substrate  and detected using ImageQuant LAS 4000 mini . The primary antibodies used in this study were The RNA immunoprecipitation assay was performed as previously described . Total Rthe Bonferroni–Dunn test. Significant values (P) are indicated in the figure legends. Error bars indicate standard deviation.Data are presented as the mean + standard deviation of the mean of at least three experimental replicates. Statistical analyses were performed using a one‐way analysis of variance (ANOVA) followed by Cpeb1 3’UTR represses both Cpeb1 mRNA and protein expression levels in Neuro2a cell [Cpeb1 mRNA levels but reduced the protein levels. The opposite expression pattern between mRNA and protein levels suggests that other post‐transcriptional mechanisms that are distinct from ARE‐dependent mRNA decay could be involved in the reduction of protein levels CPEB1. Therefore, we first focused on CPEB1‐mediated negative autoregulation, searched for cytoplasmic polyadenylation elements (CPEs) in the Cpeb1 3’UTR, and identified four candidate CPEs . Endogenous Cpeb1 mRNA and protein levels were significantly reduced by the addition of siCPEB1  and searched for putative miRNAs that could interact with the Cpeb1 3’UTR. We focused on the four miRNAs that were broadly conserved among vertebrates  for miR‐145a‐5p was mutated   Fig. . After tut) Fig. . Gfp mRNely Fig. . We next‐5p Fig. . Gfp mRNrol Fig. a, b. Thelet‐7b‐5p were mutated  AUF1 and CPEB1 in combination promote stability and translation of Cpeb1 mRNA; and (b) an alternative inhibitory mechanism is involved in the expression of Cpeb1 mRNA especially in the absence of AUF1 and CPEB1. Considering that individual knockdown of AUF1 or CPEB1 enhanced RNA level [trans‐acting factors negatively regulate Cpeb1 mRNA expression. Therefore, we hypothesized that an alternative inhibitory mechanism exists and regulates the expression of Cpeb1 mRNA in the absence of AUF1 and CPEB1.Next, we demonstrated that mutations of both ARE and CPE in reporter plasmid significantly reduced RNA and protein levels of GFP compared with those in GFP‐3’UTR‐transfected cells Fig. . In addiNA level  and protmiR‐145a‐5p and let‐7b‐5p repress Cpeb1 expression in Neuro2a cells. In addition to several miRNAs that have previously been reported to interact with the Cpeb1 3’UTR [Cpeb1 3’UTR were identified by in silico miRNA target database analysis by using TargetScan [miR‐145a‐5p and let‐7b‐5p were predominantly expressed in Neuro2a cells  because MRE for miR‐145a‐5p exists immediately adjacent to CPE3 and CPE4  and lead to the precise regulation of Cpeb1 expression , which retain the ability to maintain translationally dormant state of target mRNAs , 33, 34.turation . Furtherturation . AlthougCpeb1 expression is post‐transcriptionally regulated by multiple inhibitory machineries that are regulated by AUF1, CPEB1, and microRNAs.In conclusion, our results suggest a possible mechanism by which The named authors have no conflict of interest to the best of our knowledge, financial, or otherwise.All authors read and approved the final manuscript. MK, HY, and YN conceived and supervised the study; SO and TK designed experiments; SO, RK, and SS performed experiments; SO and YH analyzed data; SO, SH, and ST wrote the manuscript.Fig. S1. siCPEB1 reduced endogenous CPEB1 protein levels.Click here for additional data file.Fig. S2. miRNA inhibitors enhanced endogenous CPEB1 protein levels.Click here for additional data file.Table S1. Oligonucleotide sequences.Click here for additional data file.


This randomized clinical trial compares the clinical response to Gram stain–guided vs guideline-based antibiotic therapy in patients with ventilator-associated pneumonia. Does Gram stain–guided antibiotic therapy restrict the administration of broad-spectrum antibiotic agents for ventilator-associated pneumonia without detrimental effects on patient outcomes?Staphylococcus aureus agents.In this randomized clinical trial that included 206 patients with ventilator-associated pneumonia in the intensive care unit, the clinical response to Gram stain–guided antibiotic therapy was noninferior to that of guideline-based antibiotic therapy (76.7% vs 71.8%). Gram stain–guided antibiotic therapy reduced the use of antipseudomonal agents and anti–methicillin-resistant The findings of this trial suggest that Gram staining can be used in the critical care setting to ameliorate the spread of multidrug-resistant pathogens. Gram staining should provide immediate information for detecting causative pathogens. However, the effect of Gram staining on restricting the initial antibiotic choice has not been investigated in intensive care units (ICUs).To compare the clinical response to Gram stain–guided restrictive antibiotic therapy vs guideline-based broad-spectrum antibiotic treatment in patients with ventilator-associated pneumonia (VAP).This multicenter, open-label, noninferiority randomized clinical trial (Gram Stain-Guided Antibiotics Choice for VAP) was conducted in the ICUs of 12 tertiary referral hospitals in Japan from April 1, 2018, through May 31, 2020. Patients aged 15 years or older with a VAP diagnosis and a modified Clinical Pulmonary Infection Score of 5 or higher were included. The primary analysis was based on the per-protocol analysis population.Patients were randomized to Gram stain–guided antibiotic therapy or guideline-based antibiotic therapy .Staphylococcus aureus (MRSA) agents as initial antibiotic therapies; 28-day mortality, ICU-free days, ventilator-free days; and adverse events.The primary outcome was the clinical response rate; clinical response was defined as completion of antibiotic therapy within 14 days, improvement or lack of progression of baseline radiographic findings, resolution of signs and symptoms of pneumonia, and lack of antibiotic agent readministration, with a noninferiority margin of 20%. Secondary outcomes were the proportions of antipseudomonal agents and anti–methicillin-resistant P < .001 for noninferiority). Reduced use of antipseudomonal agents  and anti-MRSA agents  was observed in the Gram stain–guided group vs guideline-based group. The 28-day cumulative incidence of mortality was 13.6% (n = 14) in the Gram stain–guided group vs 17.5% (n = 18) in the guideline-based group (P = .39). Escalation of antibiotics according to culture results was performed in 7 patients (6.8%) in the Gram stain–guided group and 1 patient (1.0%) in the guideline-based group (P = .03). There were no significant differences between the groups in ICU-free days, ventilator-free days, and adverse events.In total, 206 patients  were randomized to the Gram stain–guided group (n = 103) or guideline-based group (n = 103). Clinical response occurred in 79 patients (76.7%) in the Gram stain–guided group and 74 patients (71.8%) in the guideline-based group (risk difference, 0.05; 95% CI, –0.07 to 0.17; Results of this trial showed that Gram stain–guided treatment was noninferior to guideline-based treatment and significantly reduced the use of broad-spectrum antibiotics in patients with VAP. Gram staining can potentially ameliorate the multidrug-resistant organisms in the critical care setting.NCT03506113ClinicalTrials.gov Identifier:  Therefore, we conducted the Gram Stain-Guided Antibiotics Choice for VAP (GRACE-VAP) trial to compare the clinical response to Gram stain–guided restrictive antibiotic treatment vs guideline-based broad-spectrum antibiotic treatment in patients with VAP.Gram staining of respiratory samples, an inexpensive, simple examination that is used worldwide, including in lower-income countries, may guide the appropriate use of initial antibiotic therapy. Several studies evaluated the accuracy of Gram staining in detecting causative pathogens, but their results were conflicting.20) was concordant with the Declaration of Helsinki21 and the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan and was approved by the institutional review boards of Osaka General Medical Center and each of the participating institutions. Written informed consent was obtained from all participants or their representatives. We followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline.The GRACE-VAP trial was a multicenter, open-label, noninferiority randomized clinical trial with blinded end point assessment that was conducted from April 1, 2018, through May 31, 2020. The trial protocol   of 5 or higher .Data on race and ethnicity were not collected. It was presumed that only Japanese citizens would participate in the trial.Patients were randomized to receive either Gram stain–guided antibiotic treatment or guideline-based standard treatment . Randomi23 Gram staining was performed in the laboratory in each ICU or the microbiology laboratory in each institution just after respiratory samples for sputum culture were collected. Bronchoalveolar lavage was not performed to obtain respiratory samples. Gram stain was performed by the Favor method, in which heat-fixed smears on slides were flooded with 0.2% Victoria blue for 30 seconds and washed with tap water, smears were decolorized with 2% picric acid ethanol, and cells were counterstained with 0.004% fuchsin for 30 seconds and then washed with tap water.15 Respiratory sample quality was assessed by Miller and Jones classification and Geckler classification. We categorized the Gram stain results as gram-positive cocci (GPC) chains, GPC clusters, gram-positive bacilli, gram-negative rods (GNR), or a combination thereof.16 A nonpseudomonal β-lactam antibiotic was administered when Gram stain results showed only GPC chains and/or gram-positive bacilli. An anti-MRSA agent was administered when Gram stain results showed GPC clusters without GNR. An antipseudomonal agent was administered when Gram stain results showed GNR without GPC clusters. The combination of an antipseudomonal agent and anti-MRSA agent was administered when Gram stain results showed both GPC clusters and GNR. The most broad-spectrum antibiotic agent among the categories was administered when Gram stain results showed 2 or more categories. We administered the combination of an antipseudomonal agent and an anti-MRSA agent when the Gram stain did not show any microorganisms.Patients in the Gram stain–guided group received antibiotics according to the results of Gram staining of endotracheal aspirate , and the frequency of MDR GNR was low (<10%) in all participating institutions . The primary outcome was the clinical response rate at the follow-up or test-of-cure visit. Clinical response was defined as meeting the following 4 outcomes: completion of antibiotic therapy within 14 days, improvement or lack of progression of baseline radiographic findings at the end of therapy, resolution of signs and symptoms of pneumonia  at the follow-up or test-of-cure visit, and lack of antibiotic agent readministration owing to pneumonia by the follow-up or test-of-cure visit.Clostridioides difficile infection, skin rash, and seizure. Kidney impairment was defined as both an increase in creatinine level to at least 1.5 times over baseline within the follow-up or test-of-cure period, with the worst creatinine level being 1.0 mg/dL or higher. We considered the therapy appropriate when all pathogens that were isolated with at least 1 semiquantitative growth from respiratory samples were covered by the selected antibiotic agents.The secondary outcomes were 28-day mortality; 28-day ICU-free days; 28-day ventilator-free days; proportions of antipseudomonal agents and anti-MRSA agents as initial antibiotic therapies; coverage rate of initial antibiotic therapies; de-escalation rate; antibiotic therapy days; and adverse events, including kidney impairment, thrombocytopenia, diarrhea, The site investigators collected information that was relevant to the outcomes. Each member of the event adjudication committee, who was blinded to the group randomization, independently judged the primary and secondary outcomes according to the individual data. When any disagreements occurred among the committee members, a consensus was reached by discussion.20 and the statistical analysis plan was fixed before the enrollment of the final patient.The primary efficacy analysis assessed the noninferiority of the clinical response in the Gram stain–guided group compared with the guideline-based group. Details of the statistical analyses were described previously,16 and a 20% noninferiority margin. This trial required 86 patients per group to achieve 80% power with a 1-sided α = 2.5%. Assuming a 10% rate of nonevaluable patients, we decided to enroll 100 patients per group.Sample size was based on an assumed clinical response rate of 67.8% to both the guideline-based treatment and the Gram stain–guided treatment, as determined in a previous study,Categorical variables were expressed as frequencies with percentages, and continuous variables were expressed as medians with IQRs. Because the noninferiority of the primary outcome was assessed, the primary analysis was performed in the per-protocol analysis population, which consisted of all randomized patients who were not lost to follow-up and had no major protocol deviations. We verified the noninferiority of the primary outcome using the normal theory test for binomial proportions. We performed a sensitivity analysis for primary efficacy analysis in the patients with an mCPIS of 5 or higher because some patients who were considered eligible at randomization were found to have an mCPIS of 4 after data monitoring.2 test for categorical variables and Wilcoxon rank sum test for continuous variables.Secondary outcomes were analyzed under a superiority assumption, which was based on the intention-to-treat principle. The cumulative incidence of mortality at 28 days was estimated with the Kaplan-Meier method, and the differences between groups were assessed by log-rank test. The hazard ratios and 95% CIs for mortality in the Gram stain–guided group vs the guideline-based group were estimated with a Cox proportional hazards regression model. Other secondary outcomes were compared using the χPredefined subgroup analyses for the primary outcome were conducted with the same method used in the primary efficacy analysis. The subgroup factors, which affected a priori the bacterial flora of a patient’s oral cavity and airway or were related to patient severity, included the presence of previous antibiotic therapy during hospitalization, length of ICU stay before randomization (≥5 or <5 days), comorbidity of chronic obstructive pulmonary disease, TBI, postcardiopulmonary arrest syndrome, presence of sepsis, development of acute kidney injury, and Acute Physiology and Chronic Health Evaluation II score . Because few patients had septic shock in the trial, we changed the predefined subgroup from septic shock to sepsis.P values were 2-sided, and P < .05 was considered to be significant except in the noninferiority test for clinical response, for which a 1-sided P < .025 was considered to be significant. All statistical analyses were performed with Stata, version 16.1 (StataCorp LLC) and SAS, version 9.4 (SAS Institute Inc).All Staphylococcus aureus (n = 103 [50.0%]) was the most frequently isolated bacteria from endotracheal aspirate, followed by Klebsiella spp (34 [16.5%]) and Haemophilus influenza (20 [9.7%]) . As a result, we randomized 206 patients, 103 of whom were randomized to the Gram stain–guided group and 103 to the guideline-based group . The samP < .001 for noninferiority) (P < .001 for noninferiority). Clinical response rates at the participating institutions are shown in eTable 7 in Per-protocol analysis showed that, at the follow-up or test-of-cure visit, 79 patients (76.7%) in the Gram stain–guided group had a clinical response compared with 74 patients (71.8%) in the guideline-based group  . SensitiP = .39) . We observed reduced use of antipseudomonal agents  and anti-MRSA agents  in the Gram stain–guided group vs the guideline-based group (S. aureus (κ = 0.59) and a sensitivity of 83.0% and specificity of 60.7% in detecting gram-negative pathogens (κ = 0.43). Concordance between the results of Gram staining and culture is shown in eTable 8 in P = .18). Escalation of antibiotics according to culture results was performed in 7 patients (6.8%) in the Gram stain–guided group and 1 patient (1.0%) in the guideline-based group (P = .03). We observed no significant differences between the groups for all other secondary outcomes  in the Gram stain–guided group and 17.5% (n = 18) in the guideline-based group (ed group . Gram stoutcomes .P = .01 for interaction). However, none of the prespecified subgroups showed substantial heterogeneity other than patients with TBI (P = .08 for interaction), although the interaction between an intervention effect and the existence of previous antibiotic therapy did not reach statistical significance. According to the length of ICU stay before randomization, the occurrence of a clinical response was similar between the 2 groups among both the patients with an ICU stay of 5 or more days and those with an ICU stay of fewer than 5 days.Among patients with TBI, the clinical response rate was 100% (n = 11) in the Gram stain–guided group and 61.5% (n = 13) in the guideline-based group. There was substantial heterogeneity in the effect of Gram stain–guided therapy on the clinical response in patients with TBI .There were 69 adverse events in the Gram stain–guided group and 79 in the guideline-based group . The mosThe GRACE-VAP trial addressed the effectiveness of Gram staining to safely restrict overuse of broad-spectrum antibiotic agents in critically ill patients with VAP. Gram stain–guided restrictive antibiotic therapy was noninferior to guideline-based broad-spectrum antibiotic therapy in patients with VAP in terms of the clinical response rate. Gram staining of endotracheal aspirates optimized the use of broad-spectrum antibiotic agents for VAP without detrimental effects on patient outcomes.28 A major approach to optimizing antibiotic choice and minimizing unnecessary administration of antibiotics is molecular point-of-care testing. Several randomized clinical trials have been conducted to evaluate the efficacy of such tests, but the results have been conflicting.28 Moreover, these tests are expensive and require precision measurement instruments, and not all medical facilities can install such expensive equipment. In the GRACE-VAP trial, we used the time-honored Gram stain technique as part of the daily management of infectious diseases. We believe that the trial results are acceptable and have the potential to change the strategy of antibiotic choice worldwide.Antimicrobial resistance resulting from the overuse of antibiotics is arguably one of the greatest threats to global human health. Various procedures have been proposed to improve antimicrobial stewardship in patients with suspected pneumonia, including VAP.29 Although this algorithm can certainly limit broad-spectrum antibiotic use, it might lead to an increase in inappropriate antibiotic choice.30 We estimated antibiotic choices and inappropriate coverage rate using the European guidelines for the patients in the guideline-based group. The European guidelines limited broad antibiotic use; however, the appropriate coverage rate also decreased considerably. If we applied the European guidelines to the patients in the guideline-based group, the lower coverage rate might result in worse outcomes for this group. Therefore, the findings should be considered robust when the European guidelines are used, but this estimation should be verified in clinical settings.Antibiotic choice in the guideline-based group was based on the 2016 VAP guidelines. Alternatively, the European guidelines recommend a different algorithm based on MDR risk factors for selecting antibiotic agents.31 However, in the subgroup analysis of the present trial, we did not observe any adverse effects on the clinical response attributable to previous antibiotic therapy.Previous antibiotic therapy can influence the results of Gram staining. Antibiotic exposure can change bacterial flora in respiratory samples, making it difficult for physicians to identify causative pathogens. An observational study found that culture positivity was reduced by 20% in blood culture specimens that were obtained during antibiotic therapy.32 but the magnitude of 20% may have been too large for clinicians and the sample size was not large enough to be conclusive. The trial was conducted as planned, and the lower confidence limit of the risk difference of clinical response (−0.07) was sufficiently far from the noninferiority margin of −0.2. Moreover, the incidence of the primary outcome was observed as assumed, and all secondary outcomes and subgroup analyses supported the evidence, suggesting that the findings in the trial could be considered robust. Third, the generalizability of the trial should be considered because of the setting. Namely, the frequency of MDR GNR was low (<10%) at all participating institutions, and nonfermenters, including Pseudomonas aeruginosa and Acinetobacter baumannii, that were isolated from respiratory samples were lower than expected. Moreover, few of the trial participants had septic shock or an extremely low partial pressure of oxygen to fraction of inspired oxygen ratio. Therefore, Gram staining may not be suitable in clinical practice in patients with the most severe VAP or in hospitals or regions in which the frequency of MDR GNR is very high.This trial has several limitations. First, the open-label design may have influenced the behavior of the site investigator in charge. However, the schedule of clinical assessments with objective measurements was prefixed, and outcomes were assessed by independent members of the event adjudication committee, who were blinded to the group randomization. Moreover, there was no difference in the secondary end point of 28-day mortality, which was least affected by the open-label design. Second, we set the noninferiority margin at 20%, which was consistent with that in a previous trial,This GRACE-VAP trial found that Gram stain–guided treatment compared with guideline-based treatment significantly restricted the use of broad-spectrum antibiotics without detrimental effects and yielded noninferior clinical responses in patients with VAP in the ICU. This finding reinforces the potential use of Gram staining in the critical care setting to ameliorate the spread of MDR pathogens.


It was shown that 1,3-dimethylimidazolium (phosphonooxy-)oligosulphanide is able to initiate radical polymerization under drying aqueous solutions of acrylamide, even at room temperature. According to XRF data, the synthesized polyacrylamide gel contains 0.28 wt% of sulphur. The formed polymer network has a low crosslinking density and a high equilibrium degree of swelling. The swelling rate of polyacrylamide gel in water corresponds to the first order kinetic equation with the rate constant 6.2 × 10−2 min−1. The initiator is promising for combining acrylamide polymerization with the processes of gel molding and drying.The new initiator of the polymerization of acrylamide, leading to the formation of crosslinked polyacrylamide, was discovered. The structure of the synthesized polyacrylamide was characterized by XRD,  Ac. Ac24]. The synthesized polyacrylamide was only capable of limited swelling in water, which indicates the formation of a three-dimensional polymer network . The lin−2 min−1, and the equilibrium degree of swelling is 7.09. The obtained kinetic data make it possible to quantitatively estimate the average molecular weight of the chain segments between the network nodes according to the Flory and Rehner Equation (3) [The swelling rate constant of polyacrylamide in water was 6.2 × 10tion (3) :(3)M¯c=−−3, the volume fraction of the polymer oligosulphanide as an initiator of acrylamide polymerization under conditions of slow drying of its aqueous solutions allows the formation of a lightly crosslinked polymer network with high water absorption.Taking into account the density of polyacrylamide 1.3 g × cmK (25 ℃) , M¯c is The listed circumstances can be explained if we admit the mechanism shown in 8 [The possibility of initiating polymerization of acrylamide with 1,3-dimethylimidazolium (phosphonooxy-)oligosulphanide is consistent with the increased ability for the formation of S–S bonds to homolytic decomposition, compared to S8 ,29,30,31−2 min−1. Thus, simultaneously with the formation of polyacrylamide chains, the process responsible for their crosslinking develops, which opens the possibility of obtaining polymer networks by drying aqueous solutions of acrylamide. This reaction opens new perspectives and modes of forming articles from crosslinked polyacrylamide.The product of the interaction of elemental sulphur with 1,3-dimethylimidazolium dimethylphosphate is capable of initiating acrylamide polymerization upon the slow evaporation of water at room temperature. The resulting polyacrylamide is capable of limited swelling in water, and the equilibrium mass degree of swelling is 7.09, and the swelling rate constant at a temperature of 298 K (25 °C) is 6.2 × 10Apparently, both the initiation of polymerization of acrylamide and the crosslinking of the chains of the resulting polyacrylamide are a consequence of the low stability of the S–S bonds of linear sulphur chains of 1,3-dimethylimidazolium (phosphonooxy-)oligosulphanide.


The increasing demand for high-strength lightweight concrete (HSLWC) with excellent mechanical properties has inspired the development of nanomaterials in fundamentally solving brittleness and cracking. This work investigated the effects of graphene oxide (GO) on the mechanical properties and microstructure of HSLWC, including the workability, density, compressive strength in different curing regimes, splitting tensile strength, flexural strength, modulus of elasticity and scanning electron microscopy (SEM). Six groups of mixtures were mixed with GO aqueous solution at a dosage of 0.00%, 0.02%, 0.04%, 0.05%, 0.06%, and 0.08% by weight of cement, respectively, and dispersed by ultrasound for 30 min. The test results showed that adding a low volume of GO to the specimens could slightly increase the density, rationally reduce the slump, and significantly improve the mechanical properties. The maximum increase in compressive strength, splitting tensile strength, modulus of elasticity and flexural strength of HSLWC with GO at 28 days was by 24%, 17%, 15%, 20%, respectively, as compared with HSLWC without GO. Simultaneously, the SEM results showed that GO could not only fill nano-scale pores, but also regulate the formation and growth of flower-like crystals, which was an important factor for the further improvement of properties. The research results provided a potential new pathway to improve the mechanical properties of HSLWC. Concrete is considered the most widely used material in construction, and the demand for concrete with more efficient technical specifications is increasing every year . Ordinar3 and performance limitations such as brittleness more prominent than ordinary concrete [3 and a 28-day compressive strength range of 34–79 MPa [LWAC refers to concrete with a dry density of less than 1850 kg/mconcrete ,7,8. In concrete ,10. Receconcrete ,12,13,144–79 MPa . Some re4–79 MPa ,17,18. T4–79 MPa . The add4–79 MPa . In gene2 in cement to produce a 3D network structure [Graphene oxide (GO) is a kind of nanomaterial with high strength, good toughness and large specific surface area, which has attracted attention from various fields due to its good physical and mechanical properties ,22,23. Atructure . Lin et tructure . Pan Z etructure .In recent years, the preparation process of GO has gradually matured, and a common process is the oxidation of graphite and ultrasonic treatment. Several studies on the addition of low-volume GO to cement and cement-based materials have been developed with precise results in terms of improving mechanical properties. Pan Z et al. also reported that the addition of 0.05% GO by weight of cement which was synthesized from natural graphite using the modified Hummers method could increase the compressive strength of cement slurry by 33% and the flexural strength by 59% . Lv et aAt present, many researchers have focused on the effect of GO on the mechanical properties ,37, dura2/g and specific gravity of 3.14 was used as a binder in all mixes. The coarse and fine aggregates were shale ceramsite (SC) with a nominal maximum size of 19.1 mm and shale pottery sand (SPC) with a fineness modulus of 2.96, respectively. The physical properties of aggregates are shown in In this study, ordinary Portland cement type II 42.5R with a specific surface area of 3590 cmTo verify the effectiveness of GO on the mechanical properties of HSLWC, a grade 50 high-strength concrete was designed as the control mix. Typically, the small amounts of GO (about 0.02–0.08% by weight of cement) gave the best gains in the mechanical properties of cement composites . The mixThe contribution of GO is to achieve uniform dispersion. Due to the poor dispersion effect and agglomeration of GO in cement slurry , PCE wasAll the dried aggregates materials were placed into a pan mixer and mixed for 2 min. Then, the cement was added and mixing continued for another 3 min at moderate speed. After that, 70% of the pre-mixed GOS solution was added into the mixture for a further 3 min. Subsequently, the remaining GOS solution was added and mixed for 2 min before conducting a slump test.The concrete specimens were cast in the molds to test mechanical properties and microstructure. Slump and density tests were carried out in accordance with the ASTM C143 procedure and the ASTM 138 procedure, respectively, to evaluate the workability of the concrete. In addition, the mechanical tests of every mixture included 100 mm cubes for compressive strength at 1, 3, 7, 28 and 56 days in accordance with the ASTM C39 procedure; cylinders of 100 mm diameter × 200 mm height for splitting tensile strength at 28 days in accordance with the ASTM C469 procedure; prisms of 100 × 100 × 500 mm for flexural strength at 28 days in accordance with the ASTM C78 procedure; and cylinders of 150 mm diameter × 300 mm height for modulus of elasticity at 28 days in accordance with the ASTM C469 procedure. All specimens were compacted in three layers on a vibration table during casting and demolded after 24 h. To obtain valid values, three samples were tested for each testing phase.In order to investigate the microstructure characteristics, the fracture of the sample was cut into about 10 × 10 × 5 mm after curing in water for 28 days and sprayed with thin gold on the surface of the sample to enhance the electrical conductivity of the sample. Their characteristics regarding crystal morphology were observed with a scanning electron microscope .Concrete should be cured in the curing environment after demolding. The curing environment has a certain influence on concrete performance. Investigating the curing environment of concrete can study the sensitivity of concrete compressive strength in the absence of curing ,12. In sThese results indicate that the incorporation of GO had a negative impact on the workability of HSLWC because of its large specific surface area. A similar conclusion was also reported in previous studies . As can 3. It has been reported that the oven dry density of lightweight concrete was 1440–1840 kg/m3 [3 lower than the saturated density, while the traditional LWAC was about 100–200 kg/m3 [40 kg/m3 . Therefo40 kg/m3 . Owing t00 kg/m3 .The 28-day compressive strength of HSLWC under different curing regimes are given in In addition, for specimens without GO (G0 mix), the compressive strength under the 5W curing regime was 97% of that of the FW curing regime, and reached maturity at 7W. Similar reports in the literature suggested that the 7W curing regime was required to achieve maturity strength ,45. MoreIn addition, the 28-day splitting tensile strength of G0, G2, G4, G5, G6 and G8 under full water curing were 3.44, 3.75, 3.82, 4.01, 3.93 and 3.89 MPa, respectively. It was also found that the specimen with GO (G5) had a maximum increase of 17% in 28-day splitting tensile strength, compared with the specimen without GO. The use of light aggregate made the splitting tensile strength of the mixture lower than that of ordinary weight concrete, but it obviously conformed to the requirement of ASTM C330, stating that the minimum splitting tensile strength of a structural light aggregate concrete is 2 MPa . Holm ret is the 28-day splitting tensile strength (MPa), and fcu is the 28-day cube compressive strength (MPa).Similar to the splitting tensile strength, the trend also presented a strong parabolic fitting relationship, as shown in r is the 28-day flexural strength (MPa), and fcu is the 28-day cube compressive strength (MPa).The effect of GO on HSLWC can be further investigated through the analysis of microstructure characteristics as well as mechanical properties. The hydration reaction of cement naturally produces rod-like crystals, needle-like crystals, lamellar crystals and nano-scale pores and other products, which have adverse effects on the mechanical properties of concrete. These crystals were generally assembled from a complex of AFt, AFm, and CH in a disordered state . In contAlthough the fluidity of high-strength lightweight concrete (HSLWC) decreased with the increasing addition of graphene oxide (GO), the test showed satisfactory workability. At a low volume of GO addition, the density of the mixture did not increase significantly, and was still in the range of structural lightweight concrete.With the addition of GO, the maximum increase in compressive strength, splitting tensile strength, flexural strength and elastic modulus of HSLWC at 28 days was 24%, 17%, 15%, 20%, respectively. The results also indicate that 0.05% is the optimum value of GO content for improving the splitting tensile strength of HSLWC, and keeping in water for 4 days after demolding was recommended as an efficient curing regime.Microscopic tests showed that GO not only filled nano-scale pores, but also regulated the formation and growth of flower-like crystals, which might contribute to the further improvement of mechanical properties of HSLWC.The optimal amount of GO in different types of HSLWC and the mechanism of mechanical properties’ improvement still need to be investigated by a large number of experiments, and thus still require further research in the future.


A case of intractable IgG4-related orbital disease (IgG4-ROD) was successfully treated by debulking surgery combined with low-dose prednisolone and azathioprine as maintenance therapy. A 64-year-old man visited our clinic with progressive bilateral upper eyelid swelling and right eye fullness of a year’s duration. He was previously treated with systemic corticosteroids for the IgG4-ROD and experienced a partial clinical response but relapsed upon prednisolone cessation. The patient underwent debulking surgery of the right lacrimal gland and right upper eyelid. His post-operative medication was oral prednisolone (5 mg) every other day and 50 mg azathioprine per day. The patient’s right eye remained asymptomatic during the 18 months of follow-up. Debulking surgery combined with low-dose prednisolone and azathioprine, as a maintenance therapy, is an effective and alternative treatment for the long-term control of intractable IgG4-ROD. IgG4-related disease (IgG4-RD) is an immune-mediated systemic fibroinflammatory disorder characterized by dense lymphoplasmacytic infiltration with a predominance of IgG4-positive plasma cells, storiform fibrosis, and obliterative phlebitis . IgG4-RDA 64-year-old man with a history of diabetes mellitus and glaucoma under oral Tristar and topical Xalatan and Azagar control presented with bilateral upper eyelid swelling and progressive right eye fullness for one year. The patient previously experienced a partial clinical response with oral prednisolone of 6 months’ duration for IgG4-ROD but relapsed upon prednisolone cessation. The oral prednisolone treatment also worsened his blood sugar control due to glucocorticoid-induced diabetes mellitus. He sought surgical intervention at our clinic after his symptoms worsened and after discontinuation of prednisolone.Regarding presentation, bilateral upper eyelid swelling and a palpable mass over the right sub-brow area were observed a. His beDuring the follow-up after surgery, the patient did not develop dry eye, and the mean intraocular pressure was 18 mmHg in the right eye and 21 mmHg in the left eye with the same medication as before surgery. Reduced right upper eyelid swelling and proptosis were observed three weeks after the surgery a. The paIgG4-ROD is a fibro-inflammatory condition, with tumefactive lesions, which mimics a variety of disorders. In particular, several studies have reported that the IgG4-ROD was associated with the extranodal marginal zone B-cell lymphoma ,3. It isCurrently, the first-line therapy for IgG4-ROD is systemic corticosteroid administration and the optimal initial dose of prednisolone is 0.6 mg/kg/day with a taper of 10% every two weeks ,3,5. TheSince IgG4-ROD is considered an immune-mediated systemic disease, the efficacy of debulking surgery is uncertain. Nevertheless, the tumor-like characteristics of IgG4-RD require surgery in certain cases where one solitary mass is observed, for example, a single pulmonary nodule . In the In conclusion, IgG4-ROD is a systemic disease, and medical treatment with systemic glucocorticoids and immunosuppressants is the first treatment option to be considered. Debulking surgery combined with low-dose prednisolone and azathioprine may offer an alternative therapy for the patients with intractable IgG4-ROD who have an incomplete response or who relapse during prednisolone maintenance therapy.


TPR) with no worse than a 4.12% false positive rate (FPR) in detecting physical tampering events.This paper proposes a practical physical tampering detection mechanism using inexpensive commercial off-the-shelf (COTS) Wi-Fi endpoint devices with a deep neural network (DNN) on channel state information (CSI) in the Wi-Fi signals. Attributed to the DNN that identifies physical tampering events due to the multi-subcarrier characteristics in CSI, our methodology takes effect using only one COTS Wi-Fi endpoint with a single embedded antenna to detect changes in the relative orientation between the Wi-Fi infrastructure and the endpoint, in contrast to previous sophisticated, proprietary approaches. Preliminary results show that our detectors manage to achieve a 95.89% true positive rate ( Security is highly important in Internet of things (IoT) systems especially concerning the communication architecture and the accessibility. Anyone authorised with the right credentials can access the system and make changes accordingly with respect to the level of authorisation. The pervasive deployment of Wi-Fi infrastructures in almost all indoor environments makes Wi-Fi the ubiquitous candidate as a communication protocol for IoT systems in monitoring and sensing localisation, human behaviour, surveillance, people counting, sedentary behaviour analysis, and so on ,2,3,4,5.Judging by the fact that Wi-Fi-based sensing and localisation applications are ever-expanding and becoming more appealing, security and protection against any threats to Wi-Fi infrastructures and communication should be properly addressed for optimal performance. Issues for software-based security of Wi-Fi such as eavesdropping, denial-of-service attacks, and traffic redirection have been intensively tackled and analysed across Open Systems Interconnection (OSI) layers including encryption, virtual private network (VPN), and Wi-Fi Protected Access (WPA) ,10,11. AImagine that an unauthorised user in a restricted area, with malicious intent, tries to sabotage the Wi-Fi access point (AP) by moving or changing the equipment altogether. Applications such as localisation with a fingerprinting technique or a live view of Wi-Fi-based surveillance cameras may lose their functionality due to such changes in location and orientation of the Wi-Fi transmitters. Hence, it is critical to have an additional layer of physical security to detect such attacks by utilising the characteristics of modern Wi-Fi electromagnetic waves.The orthogonal frequency division multiplexing (OFDM) modulation scheme used in modern Wi-Fi and 4G communication networks divides the spectrum band into several small and partially overlapped subcarriers to improve data transmission robustness and bandwidth efficiency. Channel state information (CSI) represents the properties of each subcarrier, specifically, the amplitude and phase of each transmitting and receiving antenna . In singTPRs), with as low as 4.12% and 0.28% false positive rates (FPRs), respectively, on two brands of Wi-Fi access points (APs) based on system-on-chip (SoC) silicon, without leveraging advanced Wi-Fi features such as multiple-input multiple-output (MIMO). Moreover, even after periods of deterioration of the measured signal models, the TPR and FPR in our approach were still retained at the levels of 84.53% and 2.61%, respectively.This paper introduces a physical tampering detection approach using deep neural networks (DNNs) on the CSI readings from inexpensive COTS Wi-Fi devices with a single printed circuitry board (PCB) antenna, in contrast to previous proprietary approaches ,18. PrelThe remainder of the paper is organised as follows: CSI is a fine-grained value from the physical (PHY) layer that details the amplitude and phase of each subcarrier in the radiofrequency domain. Due to its stability and diversity, the amplitude and phase of CSI were leveraged for indoor sensing and localisation applications in ,20,21 anMIMO-enabled Wi-Fi devices can estimate AoA using the phase difference between individual elements of the antenna array. The authors of  utilisedThe orientation of the Wi-Fi device heavily influences the performance of the CSI-based sensing system, as indicated in ,30. MostTPR of 53% in a busy office environment using Intel 5300 NICs with three antennas [TPR of 99.6% using USRP X310 with directional antenna. Regarding other vendors’ Wi-Fi chips, the Nexmon CSI Tool [On the other hand, the sensitivity of CSI to location and orientation may be viable in designing a system that detects changes in the physical location of the Wi-Fi Aps, which is critical in restricted areas. A prior study was able to achieve a antennas , while [antennas  improvedCSI Tool ,34 suppoCSI Tool  and AtheCSI Tool ,37 are aIn the real world, the position and orientation of the Wi-Fi AP or transmitter should be fixed after installation for optimal performance and connectivity. The CSI detector should be able to capture CSI data, as well as compare and match the information with the trained database to verify the position and orientation of the AP. In real cases, the authorised professional holds the detector at the exact predetermined location and orientation to have the same CSI waveforms as during the fingerprinting phase. If, for whatever reason, the detector picks up quite different CSI readings, then it is very likely the Wi-Fi AP was physically tampered with. Hence, this paper aimed to study the feasibility of using a COTS Wi-Fi device as the detector to capture the CSI waveforms for a deep neural network (DNN) analysis instead of sophisticated, proprietary hardware.x for one training sample wasOur DNN model was a fully connected neural network with two hidden layers, as depicted in , where For the experiment, matrix The first and second hidden layers had 64 and 32 neurons, respectively, following the rule of thumb as suggested by  that theWe adopted similar data collection settings to , which aThe activation column vector The activation matrix Each hidden neuron receives a set of The result of z and a across hidden layer As the neuron receives previously predicted value A matrix representation of weight Throughout the experiment, we chose Raspberry Pi 3B+ (RPi3B+) with a Broadcom BCM 43455c0 Wi-Fi chip with a single transmitter/receiver PCB antenna  as the mFor this experiment, the APs used were Synology RT2600ac and Asus RT-AX88U. The former, based on Qualcomm Atheros QCA9984 SoC, supports 802.11ac and MU-MIMO for 2.4 GHz and 5 GHz radio bands and features 4 × 4 transmitter/receiver antennas . The latMeanwhile, a host PC capable of transmitting packets continuously to the AP was used to generate the packet traffic. The host PC consistently generated traffic by sending ‘ping’ requests to the target AP so that the CSI data from the AP responses could be captured and saved by RPi3B+ for further offline analysis. The purpose of the experiment was to determine the effect of orientation of APs on the received CSI waveforms. With the assumption of software-related security concerns being out of the context of this study and that the user could not tamper with the internal hardware of the Wi-Fi device, the physical tampering events were narrowed down to moving and rotating the Wi-Fi device. It was observed that, generally, the amount of clockwise rotation of the AP is equivalent to the amount of counter-clockwise rotation of the mobile detector. For the sake of the experiment, both APs were kept constant, and only the positions and orientations of the mobile CSI detector were changed. Hence, the environment remained intact with no major changes except for the relative orientations between the AP and the mobile CSI detecting device over the course of the experiment.The experiment was conducted in the laboratory where both the Synology and the Asus APs were used one at a time. To monitor the CSI waveforms, the authorised professional held the CSI detector at predetermined RPs where the fingerprints were already collected and processed. The layout of the RPs, as well as the mounting locations of the Wi-Fi Aps, is shown in ACC) and other evaluation metrics.RPi3B+, mounted at the top of a 1.2 m high tripod, captured CSI data from one AP at a time at each RP to formulate a dataset. Each dataset had seven different classes, each matching one RP. During the measurement session, RPi3B+ was placed in the same orientation, facing the same direction for all seven RPs. After successfully capturing CSI data at all RPs with the detector facing the same orientation to create the dataset, the measurement session was iterated with the detector facing another orientation to create another dataset. The same setting was repeated each day for the duration of the experiments to determine the effects of the orientation and any time-varying characteristics on the CSI waveforms for matching accuracy  format for each RP with a total of 3500 samples for each dataset. For this work, we did not utilise a relational database.The extracted CSI data were in the form of complex numbers. MATLAB was used to extract those complex numbers from the raw ACC of RPs. CSI phase data were not used in the DNN, as they behaved extremely randomly and nonrepetitively across the feasible field with COTS Wi-Fi devices [A Python program was used to implement our DNN model to be trained by the CSI amplitude information for further offline prediction on the  devices ,46,47. SFor the experiment with AP 1, the baseline Dataset 1 was used as the training dataset, whereas Dataset 2, Dataset 5, and Dataset 6 were the testing datasets with the mobile CSI detector (the RPi3B+) oriented in the same direction as in Dataset 1 with measuring times of 1 day, 3 days, and 4 days, respectively, after gathering Dataset 1. Dataset 3 and Dataset 4 were generated on the same day and in the same time period as Dataset 1, but with the orientation of the RPi3B+ detector rotating 45° to the left and right from the original one in Dataset 1, respectively.Ten normalised CSI amplitude waveforms for different RPs of Dataset 1 and Dataset 6 are shown in Next, RPi3B+ was turned 45° left and right from the previous measurement to Orientation 2 (OR 2) and Orientation 3 (OR 3). The training data were kept the same as the previous measurement while using the datasets of the new orientation as the testing data. Note that both datasets were taken in the same time period. The CSI amplitude waveforms for different RPs across different orientations are shown in From the comparison of When the system is deployed in a real scenario, our hypothetical trained professional will know exactly at which location (RP) and in which orientation they should stand to check for any physical tampering event. At exactly the same RP and in the predetermined orientation, the model will match the collected signal characteristics so that the detector will recognise it at the exact RP and in the exact orientation, thus deducing no physical tampering of the AP. Datasets 2, 5, and 6 resemble the aforementioned situation.ACC. As the waveforms in Dataset 1 are not transferrable to those detected, the detector can easily trigger an alert or related actions for the detected tampering event. This result shows that physical tampering detection is achievable using COTS Wi-Fi devices with a single embedded antenna.On the other hand, when that trained professional holds the detector in different orientations, as in the cases with Datasets 3 and 4, the signal characteristics would change significantly and the waveforms would be distinguishable from the training dataset, thus decreasing the ACC, TPR, and FPR. ACC can be calculated using the following formula:FP is obtained when the model incorrectly predicts the label as the true label, and FN is obtained when the model incorrectly predicts the label not as the true label. TPR or sensitivity can, thus, be calculated asFPR or fallout can, thus, be calculated asThe performance metrics of interest were ACC, TPR, and FPR results at different RPs across different orientations and different time periods against AP 1 are summarised in TPR or sensitivity is important for detecting any physical tampering so that as many as possible positive instances will be correctly recognised and identified and as few as possible positive cases will go unnoticed. The macro average of TPR from the confusion matrix of Dataset 2 in FPR as low as 0.42%. Meanwhile, from the confusion matrix of Dataset 3 in The TPR obtained with the testing datasets of the same orientation was at least 95.89%, while the FPR was no worse than 4.12%. Moreover, after 96 h (4 days), the model still retained 84.53% TPR and 2.61% FPR. Evidently, even though the ACC deteriorated over time, our detector model still held an adequately high TPR and low FPR as it could still recognise whether the physical Wi-Fi infrastructure was altered.Overall, the TPR and minimises low FPR. According to The receiver operating characteristic (ROC) curve in Next, we go one step further by investigating the influence of different Wi-Fi AP models with different kinds of SoCs. We reiterated the experiment using another AP, namely, the Broadcom-based Asus RT-AX88U (AP 2). Five more datasets, designated Datasets 7 through 11, were generated using AP 2 in a similar manner as in the case of AP 1. Dataset 7, the baseline dataset, was used as the training dataset. Datasets 8 and 11 were the testing datasets with the CSI detector (the RPi3B+) aimed in the same direction as Dataset 7 but measured after 1 and 3 days, respectively. Datasets 9 and 10 were generated on the same day and in the same time period as Dataset 7, but with the orientation of the RPi3B+ rotated 45° left and right from the original one in Dataset 7, respectively.Ten CSI amplitude waveforms of AP 2 at different RPs after normalisation are shown in TPR from Dataset 7 was 98.34% with FPR as low as 0.28%. Meanwhile, at RP 6 in the direction of OR 2, the detector could 100.00% correctly identify the occurrence of a physical tampering event on AP 2. The AP 2 results resonated with the observations for AP 1, which further validated the capability of simple single-antenna COTS mobile devices including RPi3B+ as a physical tampering detector, for multiple brands of Wi-Fi infrastructure devices based on various SoCs.When the relative orientation between AP 2 and the mobile CSI detector was altered from the original direction of the training dataset, the trained DNN model was still sensitive enough to detect the change, thus triggering an action for any possible physical tampering event. The evaluation metrics on different RPs across different orientations and time periods using AP 2 are summarised in TPR and no worse than 4.12% FPR in detecting physical tampering events using Wi-Fi APs from different vendors, clearly demonstrating the potential of using a COTS Wi-Fi board with a single embedded antenna, together with our proposed DNN model, as an effective physical tampering detector for Wi-Fi infrastructure without using sophisticated proprietary hardware or advanced solutions.This paper recognised that the orientations of Wi-Fi transceivers have a significant effect on the captured CSI waveforms; thus, it proposed a practical real-world usage based on such a feature, i.e., physical tampering detection. The channel stationarity characteristic of CSI can be observed in which the waveforms are repetitive under the same orientation, as can be inferred from the classification accuracies by machine learning methods such as a DNN. Our preliminary approach achieved at least 95.89% ACC in the event of dynamic changes and environmental noise, which hampered the tampering detection.The time-varying trends of CSI readings were observed across various APs based on different Wi-Fi SoCs even in stationary environments. Nevertheless, our experimental results indicated that our proposed approach was relatively robust even after days of degeneration in the captured CSI signal models. In the long run, calibration and transfer learning of CSI waveforms are recommended to mitigate the declining trend of CSI has become part and parcel of daily Wi-Fi communication standards with the advancement of the Wi-Fi protocol; thus, future research can be performed on harvesting its potential, especially using COTS Wi-Fi devices. For example, it might be feasible to compare the results using the current implementation to real-time CSI extraction and monitoring on, e.g., modern smartphone devices, to further verify the feasibility of physical Wi-Fi infrastructure tampering detection as a smartphone IoT application. The 0.50 m resolution of RPs clearly shows the potential of this inexpensive solution in extracting CSI for successfully localisation with submeter level unit length.


The knowledge of sustainable development believes that natural resources should be treated limited, and waste must be managed rationally. This idea and the constant striving to reduce production costs make the use of waste materials potential substitutes for traditionally used raw materials. In cement concrete technology, there are many possibilities to use waste materials either as cement replacement or aggregate in concrete production. The basic aim of this research work is to study the impact of wooden ash (WA) as binding material in proportions 10%, 20%, and 30% by weight of cement on high strength ductile cementitious composite concrete. The fresh property was evaluated through the slump cone test, while the mechanical property was evaluated through compressive and split tensile strength test. Load deflection curve, ductility index, and maximum and minimum crack were also studied to find flexure cracking behaviors of reinforced cement concrete (RCC) beam. The durability of high-strength ductile concrete was studied through water absorption and acid attacks test. Pozzolanic activity of wooden ash was studied through XRD analysis. However, there is no exact definition of high-strength concrete. Each country defines high-strength concrete in its specific way. For example, Americans define high-strength concrete as concrete having a compressive strength of more than 40 MPa2. Manufacturers evaluate the strength of the aggregates, the optimal size of the aggregates, the bond between the cement paste and aggregates, and the surface properties of the aggregate when giving more picking aggregates for high-strength concrete. Any of these characteristics may restrict the ultimate strength of high-strength concrete3.The production of high-strength concrete requires the optimal use of the key components that may be used to produce normal-strength concrete. Manufacturers of high-strength concrete are aware of the factors that influence the compressive strength, and they have the know-how to control them to obtain the desired compressive strength. In addition, to choose high-quality Portland cement, manufacturers often prefer to optimize the materials and the combination of materials by changing the quantities of cement, water, aggregates, and admixtures in the final product6, a study reported that Shell Sunflower Ash (SSA) and Shell Pumpkin Ash (SPA) can be successfully utilized as partial replacement of cement in concrete7. A variety of commercial end products have been found much effective in concrete. Various studies were conducted to manufacture concrete from waste materials to lower the cost and reduce the scarcity of standard materials9. The methods of reusing waste material in concrete make it inexpensive, and reuse is considered the most environmentally friendly alternative for dealing with the problem of trash disposal10.In addition to silica fume, ground granulated blast furnace slag, and fly ash13. The typical amount of ash produced by wood-burning is 6–10 percent of the total weight of wood burned. The composition of this ash may be extremely varied depending on geographical location and industrial operations12. In 2007, leftover ash was utilized as fertilizer in 150,000 tons14. As the cost of ash disposal continues to rise due to the increased quantity of ash produced, it is necessary to implement a sustainable ash management strategy that incorporates ash by natural processes15. Landfilling is the most common way for the disposal of ash, accounting for 70 percent of all ash produced, while the remainder being utilized in the soil as a supplement (20 percent) or for other miscellaneous purposes (10 percent)16. The majority of the remaining ash (54 percent) is utilized in agricultural production. The rest of the ash was used for various purposes, including revegetation of damaged areas, soil mix manufacture, composting, and other applications14. Approximately half of the wood ash resource generated yearly is still disposed of in landfills which cause environmental pollution.Environmental concerns and economic pressure are among the factors propelling industrialization's growth today. For this reason, several research projects are being carried out across the globe on the use of waste materials to minimize environmental risks while also streamlining the current disposal of wastage and recycling processes because of affordability16. Researchers18 have conducted tests showing the promising results of wooden ash in terms of mechanical performance and proved that wood ash is a suitable material to use as a partial substitution of cement in concrete production. Subramaniam19 quantified a 15% optimum dosage of wood ash that replaced the cement (by weight) with an acceptable high compressive strength limit for casting blocks in concrete production. Abdullahi20 found a 20% optimum replacement rate and showed that the wood ash content is increased when the water requirement increases. A study reported that Tagouk ash (TA) improved compressive strength up to 15% substitution by weight cement21.The requirement of hydraulic cement is reduced by using wood ash partial cement replacement in concrete to a large extent1.Although wood ash is considered very effective in concrete, there are still some pores of wooden ash cement concrete in hardening concrete due to its physical nature (surface area and high-water absorption), which adversely affects the mechanical performance of concrete. Therefore, it is essential to add filler to wooden ash cement concrete to attain high strength durable concrete. So, other materials can be used as filler materials such as waste glass, stone dust, waste tile, etc., where wooden ash cement concrete may be used as a waste and a filling material22. Reduction of coarse particles and enhanced fine particles results in more cement leading to increased cost of high-strength ductile cementitious composite but more importantly the heat of hydration increased which results in more shrinkage cracks leading to low durability and mechanical performance.According to micromechanics design theory, coarse particles should be removed while fine particles should be increased for high strength ductile cementitious composite for better dispersion of fibersAlthough WA can be successfully used in concrete production, but concrete has still voids which adversely effects mechanical and durability performance of concrete. Therefore, it is essential to add filler material in concrete which fills the voids among concrete ingredient, leading to more dense concrete. Furthermore, although WA improved mechanical performance of concrete but still concrete is weak in tension which results brittle failure. Therefore, it is necessary to add some of tensile reinforcement in order to achieved high strength ductile durable concrete. Additionally, majority of the researchers focus on mechanical performance of concrete while less information is available on durability aspect of concrete with WA as binding material. Therefore, this study aims to partially substitute wooden ash as binding in the proportion of 10 to 30% in increment of 10% by weight of cement. Marble waste was used as filler material while nylon fibers was used as tensile reinforcement. The impact of wooden ash (WA) on mechanical performance, including flexure cracking behaviors and durability, were investigated. Pozzolanic activity of wooden ash was also discussed in detail. The successful utilization of wooden ash in HSDFRC gives multiple benefits, including cost benefits environmental protection by making concrete eco-friendly, sustainable, and non-aggressive to the environment.23. The physical and chemical properties of cement are given in Table Ordinary Portland cement (OPC) type I cement was used as per ASTM C15024. The physical properties of WA as a concrete ingredient significantly rely on burning conditions especially the time of burning and the temperature effect on the crystallinity and microstructure of WA25. The physical and chemical properties of wooden ash was provided in Table Wooden ash (WA) can be used as a pozzolanic material according to ASTM C311Natural river sand was used as a fine aggregate (F.A) which had a fineness modulus of 2.7, and crushed stone with a maximum of 25 mm size was utilized as a coarse aggregate (C.A). Both fine and coarse aggregates were used in saturated surface dry (SSD) conditions before blending in typical concrete properties of aggregate were provided in Table Nylon fibers used in this study were procured from sika company Islamabad Pakistan, having 35 mm length with 0.55 mm diameter. The physical properties of nylon fibers are given in Table Waste marble was procured from Pak marble factory industrial zone Peshawar Pakistan and ground at PCSIR lab Peshawar. Table 26. Compressive strength was evaluated on a standard size cylinder 150 × 300 mm through the compressive testing machine as per ASTM standard27. Similar cylindrical samples were prepared for split tensile strength of size 150 × 300 mm as per ASTM28. The beam size (150 × 150 × 700 mm) was casted and tested to find their flexure strength as per ASTM29. 100 mm cubes samples were used as per ASTM30. A 100 mm cubical sample was casted to evaluate the acid resistance of concrete which was cured of 4% sulfuric acid for a specified period. To maintain 4% concentration acid, it had to be changed once a week. Acid attacks were calculated in terms of mass loss in percentage due to the attack of sulfuric acid. All tests were conducted after 14 days, 28 days, and 56 days of curing. At least three samples were casted for each batch, and their average value was considered as the actual result of that test. ASTM C 3131 procedure was used to make the samples, and compaction was performed by hand by Roding in three different levels giving twenty blows with tamping rod to each level. Constant water to binder ratio of (0.38) was used for all batches. Four mix proportion of HSDFRC-A with varying percentage of wooden ash as show in Table The workability of fresh concrete was measured through the slump cone test as per ASTM standardFigure Figure Figure 38. Therefore, it is recommended to use wooden ash up to 20% of the substation by weight of cement.Figure Relative analysis was also carried out in which 28 days control compressive strength was considered as reference concrete, from which another dose of wooden ash was measured. Figure 3. Similar to compressive strength, split tensile strength of HSDFC increased with addition of wooden ash up to 20% substitution of wooden ash and then decreased gradually giving maximum split tensile strength when the substitution rate of wooden ash is 20% while minimum workability is achieved at 0% substitution of wooden ash (control). The average value of control split tensile strength of concrete at 28 days of curing is 6.4 MPa and increased to 10.8 MPa at 20% substitution of wooden ash, about 68% higher than reference concrete. It is worth mentioning that wooden ash improved split tensile more effectively than the compressive strength of HSDFRC. The positive effect of wooden ash of split tensile strength of HSDFC is due to the pozzolanic reaction, which gives additional binding property which improves the bond between concrete ingredient (aggregate) and fibers which result in more split tensile strength. It has also been reported that fiber increased split tensile strength more effectively than compressive strength due to the prevention of cracks33. Fiber prevents the crack formation of concrete which results to enhance split tensile strength. A study shows that even if a crack occurs in fiber reinforced concrete, the fiber can prevent the propagation of the crack's length and width35. Also ,the micro filler of wooden ash fills the voids between concrete ingredients, leading to more dense concrete, which results in more split tensile strength. However, at a higher dose, split tensile strength gradually decreased due to lack of workability, which results in more compaction effort being required and hence more voids in hardened concrete which ultimately decreased the split strength of HSDFRC.Figure The flexural test measures the force required to bend a beam under the action of the applied load. Figure Figure Figure Figure 36 can be used to determine the ductility of the beams, which is the ratio of the deflection at peak load to the yield point deflection. Concrete is a brittle material that gives no warning or deformation prior to failure, making it unsuitable for use as a construction material. The ductility index of various dosages of wooden ash was demonstrated in Table 37.Ductility refers to a material's ability to withstand significant plastic deformation before failure, which can be expressed as percent elongation or percent area reduction in a tensile test. The ACI 36332.Water absorption is one of the simple tests to detect durability of concrete. Higher water absorption results in lower durability of concrete. More water absorption also led to freezing and thawing action, which resulted in the degradation of concrete. A study shows that higher water absorption of concrete causes freezing and thawing of concrete, particularly when it is placed in abruptly changing temperatureFigure 3 (nitric acids), H2SO4 (sulfuric acids), etc. In this study, H2SO4 (sulfuric acids) is considered as an acid attack on the concrete specimens with different proportions of wooden ash.Several aggressive acids are available, like HCL (hydrochloric acids), NHO38. Therefore, it is recommended to use wooden ash up to 20% substation by weight of cement.Figure θ is held steady to a range of 20 to 60 degrees with the angular step of 0.02 degrees. A step time of 0.5 s is set down, and a 0.5-degree deviation slit was used. XRD patterns of wooden ash and marble waste are given in Fig. 2 and CSH  gel in reference and WA replaced concrete. Peaks of C-S-H gel at 30° and 45° were chosen for evaluation. For the reference concrete without WA, C-S-H gel is less than SiO2. C-S-H gel is formed during the hydration process of cement due to the chemical reaction of CH with SiO2. The amount of SiO2 is less than CH, which converts all SiO2 into C-S-H gel, and hence no more SiO2 is accessible for the reaction with calcium hydrate. All Silica SiO2 was consumed and converted into C-S-H gel. Calcium hydrate rests unreactive, creating weak pockets causing the lower mechanical performance of concrete. A study also concluded that calcium hydrate is active to reactive with other chemical compounds available in the cement causing less strength39. It has also been observed that Pozzolanic materials should be a substitute in concrete to utilize calcium hydrate, which is a by-product form through the hydration process of cement to achieve durable concrete39. WA was partially substituted as cement replacement to neutralize calcium hydrate. It can be observed by XRD analysis that peak of SiO2 decreases while the peak of C-S-H is enhanced as the proportion WA is enhanced. Maximum C-S-H peaks were examined when the replacement rate of WA was 30%. It is due to the pozzolanic reaction of WA, which converts calcium hydrate into calcium silicate (C-S-H) gel.To examine the mineralogy of the experiment, hardened pasts of wooden ash were ground lightly and later exposed to XRD investigation when throwing around sieve #200. The experiment was evaluated in the powder form glass concrete, which is ground into fine powder form by trampling glass by employing the diffractometer apparatus accessible in Peshawar university MRL lab. The test procedures were planned so that copper (cu) and Kα rays were utilized as supply to the process by using 30 mA and 40 kV requirements. A gap of 2The workability of HSDFRC is decreased with the substitution of wooden ash. It is due to physical nature of wooden ash (irregular particle shape and larger surface area) which enhances the water requirement for lubrication.Compressive strength and split tensile strength of HSDFRC is increased up to 20% substitution of wooden ash. It is due to pozzolanic reaction and micro filler of wooden ash. Flexure strength characteristics and load–deflection were considerably improved with the substitution of wooden ash. The ultimate peak load of 20% substation wooden is about 27% higher than reference concrete.Morphology of fibers analysis displayed positive outcomes in terms of the mechanical performance and durability of HSDFRC.Durability aspects such as water absorption and acid resistance of HSDFRC is of 20% substitution is much better than reference concrete. It is due to pozzolanic reaction and micro filler of wooden ash, which gives more dense concrete with fewer voids which ultimately decreases water absorption and acid penetration through concrete.The effect of wooden ash on mechanical performance, flexure strength characteristics, durability and microstructure analysis of HSDFRC were studied in this research. The substitution ratio of wooden ash was 10%,20%, and 30% by weight of cement were employed. Based on experimental results, the following conclusions have been drawn.The overall study demonstrates that wooden ash is a better substitution of cement for concrete production because it enhances the concrete microstructure and bonds among fibers and concrete ingredients.