Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired (HGT) and the redeployment of existing genes and pathways in novel contexts. Here we address the latter aspect in Paulinella micropora KR01  that has independently evolved spliced leader (SL) trans‐splicing (SLTS) of nuclear–derived transcripts. We investigated the role of this process in gene regulation, novel gene origination, and endosymbiont integration. Our analysis shows that 20% of KR01 genes give rise to transcripts with at least one  sites of SL addition. This process, which often occurs at canonical cis‐splicing acceptor sites , results in shorter transcripts that may produce 5′‐truncated proteins with novel functions. SL–truncated transcripts fall into four categories that may show: (i) altered protein localization, (ii) altered protein function, structure, or regulation, (iii) loss of valid alternative start codons, preventing translation, or (iv) multiple SL addition sites at the 5′‐terminus. The SL RNA genes required for SLTS are putatively absent in the heterotrophic sister lineage of photosynthetic Paulinella species. Moreover, a high proportion of transcripts derived from genes of endosymbiotic gene transfer (EGT) and HGT origin contain SL sequences. We hypothesize that truncation of transcripts by SL addition may facilitate the generation and expression of novel gene variants and that SLTS may have enhanced the activation and fixation of foreign genes in the host genome of the photosynthetic lineages, playing a key role in primary endosymbiont integration.Plastid primary endosymbiosis has occurred twice, once in the Archaeplastida ancestor and once in the  CDScoding DNA sequencecrTPchromatophore transit peptideEGTendosymbiotic gene transferGOgene ontologyGSPgene–specific primersHGThorizontal gene transferKEGGkyoto encyclopedia of genes and genomesORFopen reading frameSLspliced leaderSLTSsplice leader trans‐splicingUTRuntranslated regionPaulinella. Of the nine described Paulinella species, three (which form a monophyletic clade) harbor an organelle (the chromatophore) derived from an alpha‐cyanobacterial endosymbiont  occurred ca. 1.5 Ga . Although the spliceosome has a strong preference for SLTS at acceptor‐first sites and cis‐splicing at donor‐first sites, in Schistosoma mansoni, 43% of SL sequences are added at donor‐first sites , Chungnam Province, South Korea. KR01 cells were grown at 23°C in Fernbach flasks containing 0.75 L DY‐V medium on a 12:12 h light:dark cycle at a light intensity of ~15–20 μmol photons · m−2 · s−1.The g for 1 min at 4°C. The pellets were resuspended in the supernatant, transferred to ice‐cold 1.5 mL tubes, pelleted by centrifugation at 10,000g for 1 min at 4°C, frozen in liquid nitrogen, and stored at −80°C. RNA was extracted using the Qiagen RNeasy Plant Mini Kit according to the manufacturer’s instructions . RNA was eluted with 30 μL RNAase–free water, quantified by absorbance (Nanodrop), and quality controlled using an Agilent Bioanalyzer.Two samples (1 h after lights on and 1 h after lights off) were processed to capture a broad transcript diversity. The cells were swirled in liquid nitrogen for ~30 s before being transferred to ice‐cold 50 mL tubes and centrifuged at 100To enrich for 5′‐complete transcripts (with or without SLTS), first‐strand cDNA synthesis was done using the SMARTer PCR cDNA Synthesis Kit  according to the manufacturer's instructions. This procedure resulted in single‐strand (ss) cDNA containing the complete 5′‐end of the mRNA with a sequence complementary to the SMARTer primer.The resulting ss cDNA was amplified by long distance (LD) PCR (Clontech Laboratories), generating ds DNA. Each PCR consisted of a 100 µL reaction volume containing 74 µL of nuclease–free water, 10 µL of 10× Advantage 2 PCR buffer, 2 µL of 50× dNTP (10 mM), 2 µL of 5′ PCR Primer II A, 2 µL of 50× Advantage 2 DNA Polymerase Mix, and 10 µL of the first‐strand reverse transcript product. Initial denaturation was carried out at 95°C for 1 min, followed by 23 cycles of the following thermal‐cycle profile: denaturation at 95°C for 15 s, annealing at 65°C for 30 s, and extension at 68°C for 6 min. About 5 µL of the resulting products were visualized on an agarose gel with ethidium bromide (EtBr).Preparation of cDNA libraries using TruSeq Nano and Sequencing: libraries of ds cDNA were prepared using the TruSeq Nano DNA Sample Preparation Kit (Illumina) following the manufacturer’s instructions and sequenced on a MiSeq instrument (2 × 150 bp reagents) by Genewiz.Paulinella SL sequence ; ORF prediction was guided by homology information produced by a hmmscan (HMMER v3.1b2) search against the Pfam database . GO terms were assigned to the predicted proteins using PANNZER2  from the standard and cap–switching assemblies were aligned against the KR01 genome using CLC Genomics Workbench. Transcripts that aligned to the genome were used to predict open reading frames (ORFs) with TransDecoder v5.2.0  of the SL sequence that was most often recovered by  using BLASTn  and hits between reference genes, and their overlapping SL–containing transcripts (identified by gffcompare) were extracted and analyzed. Hits were retained if they: (i) started ≤2 bp along the SL–containing transcript; (ii) ended >95% along the SL–containing transcript or >95% along the reference gene CDS ; and (iii) started > 100 bp along the reference gene CDS. The point along the CDS where the hit to the SL–containing transcript starts is hereinafter referred to as the "SL–truncation point." This resulted in a set of genes with transcript evidence of an SL sequence being added part way along the reference CDS.Paulinellachromatophora (NCBI SRA: SRX1624577) were trimmed using Trimmomatic and aligned against the available assembled transcripts  against the KR01 reference genome. Only hits with a query coverage of >70% were retained and compared against the locations of predicted reference genes. Genes with SL sequence hits to the genome <100 bp upstream of the start of the gene were considered candidates for mRNA recycling. Candidate genes were manually checked for consistency between the RNA‐seq data  were identified using the bowtie2 locally aligned RNA‐seq reads filtered such that only read pairs where each read was “properly paired” , had ≤5 bp of soft‐clipping at their 3′‐end, and that had an edit distance  of ≤5 bp were retained. This extra filtering was performed so only SL addition sites supported by concordantly aligned reads were identified; soft clipping at the 5′‐end of reads was allowed because this could be caused by the presence of the SL sequence in one or both of the reads in a pair. Aligned reads that passed the filtering criteria were checked for the short SL sequence (CCGGCTTTTCTG); if found, the first position along the CDS, after the identified SL sequence was reported. If the SL sequence was encoded in a soft clipped region of the read, then the position along the CDS of the first mapped base was reported. Only positions with >10 reads ; (ii) transmembrane domains from Phobius and TMHMM (retaining only annotations with the keywords "TRANSMEMBRANE" or "TMhelix"); and (iii) coil regions from COILS. Within each set, overlapping features were merged using bedtools . The following primers were used: SL (F: TGGATAATCCGGCTTTTCTG), g63591, g81093 (R: AGAGGTAAACCATCAGCAAGAC), g19409 , and g851 . Original uncropped gel images are shown in Figures Expression level quantification was calculated for the control light samples only using RSEM v1.3.3  or second (for P. chromatophora) position and finished at position 20 along the SL; the difference in the start position of the different species is because the P. chromatophora SL has a cytosine at its first position rather than a thymine  in the mean TPM levels of the genes with and without SL addition sites. The mean expression level of genes with SL addition sites was higher (62.95) than genes without SLs (22.72).RNA‐seq reads  of a KR01 CDS, 2,237 (57.34%) were at an exon junction. In the majority  of these sites, the SL sequence was added precisely at the exon splice site: i.e., the end of the SL sequence is followed by the first base of the exon to which the SL is attached. Of the 1,664 SL addition sites that did not overlap with exon boundaries , 1,358 did not occur close to the end of the transcript and could therefore be used to construct a sequence logo. These logos predictably showed a preference for addition at sites with the canonical splice site acceptor pattern AG  were found in 3,921 CDSs ; 63 SL addition sites in 59 CDSs (0.097%) were positioned >100 bp from the 5′‐end of each sequence. There is a significant difference in the amount of RNA‐seq data available from these two Paulinella species. A single RNA‐seq library comprising 8,557,030 trimmed and filtered read pairs exist for P. chromatophora, whereas for KR01, there are 42 libraries with 620,702,822 such read‐pairs.In Paulinella, then most, but not all, of the conserved SL addition sites produce non‐functional transcripts that have minimal evolutionary impact. We find that in KR01, SL addition sites with higher‐than‐average read support are positioned closer to the  5′‐end of transcripts than sites with lower than average read support. The latter may represent transcriptional noise because they are located further away from the 5′‐end of CDSs and are more likely to disrupt functional domains: observed in 9.59% of SL–containing transcripts.Given the complex KR01 gene structures . The consequences of this competition for gene expression remain to be explored. Many of the SL addition sites we found may reflect transcriptional noise. This does not however invalidate our hypothesis that SLTS is capable of generating alternative transcripts, giving rise to novel proteins that can be acted upon by natural selection.The “noisy splicing” model also predicts that splicing rate error will be lower for highly expressed genes to prevent loss of resources on non‐functional transcripts. Although our analysis finds that transcripts of genes with SL addition sites had higher expression levels, we would need to identify all SL addition sites along each gene, including those in the UTR, before we can assess if genes with a higher expression level have fewer SL–truncated variants. We are also more likely to identify SL addition sites in genes with higher expression levels, which might explain why SL–containing genes have higher expression levels in KR01, and why we identified more SL addition sites in KR01 (42 RNA‐seq libraries) than Paulinella may have facilitated the activation and fixation of foreign genes in the host genome. To test this hypothesis, we searched for SL sequences in Paulinella genome assemblies to determine if SL RNA genes, required for SLTS, are present in each species. There were 151 hits that cover the full SL sequence in KR01, 52 hits in P. chromatophora, and none in the single‐cell draft genome assemblies  to find evidence of their existence in the heterotrophic sister lineage. These observations suggest that the SL RNA genes in photosynthetic Paulinella are absent in P. ovalis. Therefore, P. ovalis either does not perform SLTS or uses SL RNA genes with divergent sequences. The former would suggest that the SLTS mechanism evolved de novo in photosynthetic Paulinella, whereas the latter might be a result of the fragmented and incomplete nature of the P. ovalis single‐cell genome assemblies and the lack of a comprehensive RNA‐seq dataset from which novel SL RNA genes can be characterized.In KR01, 50 EGT–derived genes were identified by Lhee et al. , 17 of wPaulinella is unknown  and the fragmented nature of the assembled transcripts makes it difficult to discern the biological significance of these results . About eleven genes had SLTS transcripts that may result in translation suppression  transcript variants in the genome. We hypothesize that these two mechanisms (SL truncation and mRNA recycling) provide pathways for generating and fixing novel variants of highly expressed or adaptive genes in the Paulinella genome , is limited to detecting SL sequences that are added to transcripts within the coding region or immediately (less than the length of a full read) upstream of the predicted start codon. Our other approach, which involved assembling the RNA‐seq data de novo before extracting transcripts with SL sequences, would be able to identify SL addition sites in the UTRs of genes. However, this approach identified fewer genes with SL addition sites than the read–based approach, likely because transcript assembly programs are not designed to handle SL–containing transcripts, so it cannot be assumed that they will recover all variants present in the data, particularly, if they are in relatively low abundance.In a number of genes with SL–truncated transcripts, we observe regions (particularly at the 5′‐end) that show evidence of misprediction. This is likely a result of the challenges faced when predicting the correct start codon using an ab initio approach (as done for the KR01 reference genome). In many cases, the N‐terminus of the predicted reference proteins was erroneously extended into and beyond the 5′‐UTR region (resulting in poor read mapping). The use of short‐read RNA‐seq data can also result in ambiguous placement of SL addition sites caused by reads that align to homologous gene regions. In our analysis of genes with SL–truncated transcripts, we verified that the region around the SL addition site of interest was not mispredicted and that both reads in a read pair were aligned in the correct orientation, reducing the chances of detecting false or ambiguous SL addition sites within coding regions. These issues make it more challenging to discern the functional consequences of SL–based transcript truncation. We are nonetheless confident in our results because the observed deviations from the expected positioning of SL addition sites along transcripts provide strong evidence that multiple transcripts are derived from the same gene as a result of SLTS, irrespective of whether SL addition occurs within the coding region or UTR. The only way to identify the true start codon for each full‐length and SL–truncated protein is through proteomics or through advanced translation start site capture techniques that have not yet been established in our model. There is also a need for deep sequencing of expressed transcripts using long‐read–based technologies  which produce reads that can be unambiguously aligned against the available gene models, thereby accurately revealing the structure and location of SL addition sites along a transcript. Our analysis focuses on the potential effects of SL addition on resulting proteins. We did not assess the potential effects that removal of the 5′ untranslated region from a transcript may have on translational efficiency or stability.‐splicing of nuclear–derived transcripts in Paulinella can generate novel or altered proteins that can be acted upon by natural selection, facilitating the integration of the novel photosynthetic organelle, the chromatophore.Our work suggests that the addition of SL sequences at sites within the coding region of KR01 transcripts occurs frequently and may result from “noisy trans‐splicing.” SLTS is implicated in the integration of novel “dark” genes and genes of foreign origin acquired via EGT or HGT. These results lead us to hypothesize that transAG, TGS, and DB were supported by a grant from the National Aeronautics and Space Administration (80NSSC19K0462) awarded to DB. DB was supported by a NIFA‐USDA Hatch grant (NJ01180). We declare no conflicts of interest.A. Gabr: Conceptualization ; Formal analysis ; Investigation ; Methodology ; Writing – original draft ; Writing – review & editing . T.G Stephens: Conceptualization ; Formal analysis ; Investigation ; Methodology ; Writing – original draft ; Writing – review & editing . D. Bhattacharya: Conceptualization ; Funding acquisition ; Project administration ; Supervision ; Writing – original draft ; Writing – review & editing .Figure S1. Examples of alternatively spliced transcripts in the KR01 genome.Click here for additional data file.Figure S2. Examples of alternatively spliced transcripts in the KR01 genome.Click here for additional data file.Figure S3. A maximum likelihood phylogenetic tree constructed using sequences extracted from partial SL hits to the Paulinella  ovalis genome .Click here for additional data file.Table S1. EGT and HGT derived genes in KR01 with info about SL addition sites shown when present.Click here for additional data file.Table S2. Annotation of genes that contained SL transcripts.Click here for additional data file.Table S3. GO terms enriched in SL‐containing transcripts compared against all transcripts that aligned to the KR01 genome.Click here for additional data file.Table S4. List of genes that passed the stringent filtering criteria and were subject to manually inspection.Click here for additional data file.Table S5. Candidate genes for mRNA recycling.Click here for additional data file.


Pseudomonas aeruginosa that complicates treatment. Anti-virulence agents can be viewed as alternative options that cripple virulence factors of the bacteria to facilitate their elimination by the host immunity. The use of nanoparticles in the inhibition of P. aeruginosa virulence factors is a promising strategy. This study aims to study the effect of metformin (MET), metformin nano emulsions (MET-NEs), silver metformin nano emulsions (Ag-MET-NEs) and silver nanoparticles (AgNPs) on P. aeruginosa virulence factors’ expression. The phenotypic results showed that MET-NEs had the highest virulence inhibitory activity. However, concerning RT-PCR results, all tested agents significantly decreased the expression of quorum sensing regulatory genes of P. aeruginosa; lasR, lasI, pqsA, fliC, exoS and pslA, with Ag-MET-NEs being the most potent one, however, it failed to protect mice from P. aeruginosa pathogenesis. MET-NEs showed the highest protective activity against pseudomonal infection in vivo. Our findings support the promising use of nano formulations particularly Ag-MET-NEs as an alternative against multidrug resistant pseudomonal infections via inhibition of virulence factors and quorum sensing gene expression.Long-term antibiotic treatment results in the spread of multi-drug resistance in  Pseudomonas aeruginosa is a frequent etiological agent of opportunistic and hospital acquired infections  infections  and other reactive oxygen species (ROS). This leads to altered redox balance of the host tissues leading to the injury of cells and may lead to death , in addition to the Pseudomonas quinolone signal (PQS) system, and the integrated QS (IQS) system  and mueller hinton broth (MHB), tryptone soya agar (TSA), tryptone soya broth (TSB), and MacConkey agar were obtained from Oxoid . Other chemicals were of pharmaceutical grade. Metformin (MET) and silver nitrate were purchased commercially from Sigma Chemical Company, St. Louis, Mo, USA.P. aeruginosa ATCC 27853 was used in this study. It was provided from the stock culture collection of Microbiology and Immunology Department, Faculty of medicine, Zagazig University. Six clinical MDR P. aeruginosa isolates  were obtained from the stock culture collection of Microbiology and Immunology Department, Faculty of pharmacy, Zagazig University. They were obtained from patients with burn, surgical wound, respiratory tract and urinary tract infections. All isolates were maintained in MHB with 10–15% glycerol and kept at − 80 °C.The standard strain In order to prepare both metformin nano emulsion (MET-NEs) and silver metformin nano emulsions (Ag-MET-NEs), the modified ultra-sonication method was used as referenced , both coconut oil oily phase and tween 80 emulsifier were added drop wise to aqueous phase of either MET in a concentration of 100 mg/mL or MET and already prepared AgNPs (100–0.05 mg/mL) using homogenizer at 10,000 rpm for 30 min for continuous stirring. Then, the ultrasonic sonicator was used to sonicate the emulsion for 1 h. For characterization of the prepared nano emulsions, different physicochemical parameters such as particle size and distribution in addition to zeta potential were measured at the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt. The charge of the particles determines the stability of the nano emulsions. Zeta potential was used to quantify the particle charge and it is detected by using the electrophoretic motion of the particles in an electrical field. DLS Zeta Sizer Technique  was used to measure zeta potential of the optimized formulation. The particle sizes of the prepared nano emulsions were performed by Transmission Electron Microscopy (TEM) using (JEOL electron microscope JEM-100 CX) at an accelerating speed of 80 kV.P. aeruginosa were assessed using the broth micro-dilution method using 96-well microtiter plate according to the clinical and laboratory standards institute (CLSI) guidelines (Wikler The minimum inhibitory concentrations (MICs) of MET, MET-NEs , Ag-MET-NEs (100–0.05 mg/mL) and AgNPs (0.05 mg/mL) against s Wikler .. ; The inhibitory activities of the tested agents against pyocyanin was assessed according to the method described by Das and Manefield .P. aeruginosa isolates, Krishnan et al. method was performed  for 1 h at 37 °C. The decrease in optical density of skimmed milk was estimated at 600 nm using Biotek spectrofluorometer (USA) and considered as measure of proteolytic activity. The test was performed twice  according to the manufacturer instructions. In order to synthesize cDNA, QuantiTect Reverse Transcription Kit was used and it was amplified by Thermo Scientific Maximas SYBR Green/Fluorescein qPCR Master Mix. The primers used are presented (Table rpoD) using the 2−ΔΔCt method , Egypt . The bacterial burden in mice was detected as previously reported by Deshmukh et al. with some modifications  to reach cell density equal to 2.5 × 107 CFU/mL. Seven random groups of 5–6 weeks old healthy albino mice (Mus musculus) with equal weights were included in the experiment. Each group consists of five mice was used for P. aeruginosa. In group 1, untreated bacteria in sterile PBS (100 µL) were used for intraperitoneal injection of the mice. In group 2, 100 µL of bacteria treated with MET were injected in mice, group 3 was injected with 100 µL of MET-NEs-treated bacteria, while group 4, was injected with 100 µL of Ag-MET-NEs-treated bacteria and group 5 was injected with 100 µL of AgNPs-treated bacteria. Two additional groups were used as negative controls; group 6, mice were intraperitoneally injected with sterile PBS (100 µL), while in group 7, mice were uninoculated. Normal feeding and aeration were given to all groups at room temperature. After 24 h postinfection, mice were anaesthetized, sacrificed, livers and kidneys were harvested, weighed and homogenized for enumeration of live bacterial cells as colony forming unit per gram (CFU/g).The effect of the tested agents on the pathogenicity of P < 0.05 for signifcance.The inhibitory activity of the tested agents against virulence factors was analyzed using GraphPad Prism 8 software (One Way ANOVA followed by Dunnett’s multiple comparison tests or Bonferroni’s multiple comparison test) at An important idiosyncratic property of nanoemulsion is its nanoscale particle size. The size distribution analysis of MET-NEs and Ag-MET-NEs was performed using DLS Zeta Sizer Technique. The illustration of the comparative particle size distribution of initially prepared AgNPs, MET-NEs and Ag-MET-NEs is shown in Fig. Figure Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. There was no difference between the MICs of MET and MET-NEs against the tested bacteria. However, the MICs were markedly lowered upon using the combination of MET and AgNPs (Ag-MET-NEs) compared with either MET, MET-NEs or AgNPs alone. Considering the increase in sensitivity to either MET or MET-NEs, the MICs were decreased by 16 to 128 folds, while for AgNPs (16–64) folds among the tested isolates  as shown in  as shown in  as shown in  of the tested agents significantly affected the swarming motility of all treated bacteria as compared to the untreated controls at P < 0.0 as shownThe ability of the tested agents to inhibit proteolytic activity was measured using the modified skimmed milk broth method. It was found that the inhibitory activity of MET-NEs (77.48–99.15%) was higher than MET 35.48–66.32%). Also, Ag-MET-NEs (21.76–98.52%) exhibited higher proteolytic activity than AgNPs (17.90–48.88%). No significant inhibition of protease production was observed with either MET or AgNPs in some tested bacteria. However, AgNPs increased protease production in standard strain (49.19%) as compared to controls , respectively, Ag-MET-NEs exhibited higher reduction (73.08%) than MET-NEs (56.62%). In addition, the expression level of pqsA gene was also significantly reduced; MET-NEs and Ag-MET-NEs showed higher reduction (41.56% and 55.41%), respectively than either MET or AgNPs that exhibited lower activities approximatey (27%) each. Moreover, Ag-MET-NEs showed the highest reduction in the expression of fliC gene (49.63%), however, MET-NEs (28.89%) and AgNPs (14.07%) had lower reduction. No significant reduction was observed after MET treatment. Furthermore, significant reduction in exoS gene expression was observed with MET, AgNPs and MET-NEs  with the highest reduction found with Ag-MET-NEs (73.84%). Concerning the relative expression of pslA, it was significantly diminished with MET-NEs and Ag-MET-NEs nearly (40%) each. However, no significant reduction was found with MET and AgNPs.The influence of the tested agents on the relative expression of the genes that regulates the virulence factors’ production in the standard strain P < 0.05). The results were expressed as log CFU reduction in viable counts per gram of organ tissue. It was found that as compared to MET-NEs that completely eradicated bacteria in liver tissues, the mean log CFU reductions of viable counts decreased from 4.625 in untreated group to 3.090 and 3.895 in MET and AgNPs treated groups, respectively, while no significant reduction of the bacterial burden was observed with Ag-MET-NEs treated group (4.685). Similarly, MET-NEs successfully removed all live bacteria from kidney tissues, surpassing MET, Ag-MET-NEs and AgNPs with mean log CFU reductions from 6.120 in untreated group to 4.255, 5.265 and 3.080, respectively . This ensures that high energy barriers were created to prevent coalescence of the dispersed droplets  exhibited high synergistic activity that than that of MET-NEs or AgNPs alone  greater reduction of MRSA USA300 biofilm bacterial cell counts. In addition, several studies reported antibacterial and anti-biofilm activities of Polyhexamethlene biguanide (PHMB) against a variety of bacterial species . Pyocyanin can reduce Ag+ to Ag0, thus it protects the bacteria from the damage caused by silver ions emitted from nanoparticles. Similarly, this result was also in good agreement with Muller and Merrett . Ag-MET-NEs was the most potent inhibitor against QS regulatory genes followed by MET-NEs, and both were more effective than either the bulk MET or AgNPs, which had relatively similar QS inhibitory activity. This confirms that the quorum quenching activity of the nanoform surpassed that of its bulk one and also suggests some kind of synergism between MET and AgNPs in the combination (Ag-MET-NEs). Hegazy et al. , AgNPs and even Ag-MET-NEs combination which may require further research. In addition, the formulation of metformin in nanoform has much lower accumulation than its bulk form or metal nanoparticles, thus reducing cytoxicity that occurs after exposure. Similarly, NCL formulated as nanosuspensions had lower toxicity in a rat lung infection model involving P. aeruginosa ATCC 27853 infection from collected mice tissues, Hegazy et al. (P. aeruginosa PAO1 (ATCC BAA47B1). Escárcega-González et al. (P. aeruginosa as compared to the untreated group.In the current study, on the contrary to MET that caused approximately 30% clearance of y et al. , reportez et al.  reportedP. aeruginosa. In the present study, it was found that the formulation of metformin in nanoform was promising because it exhibited distinct physical, chemical and bioligical properties as compared to its bulk. In addition, the combination between MET and AgNPs showed synergistic antibacterial effect as well as it greatly inhibited the QS regulatory genes of P. aeruginosa.In conclusion, targeting bacterial virulence and QS offers an alternative strategy because it curbs the bacterial ability to harm the host rather than affecting their growth, and reduces the emergence of MDR pseudomonal infections. This benefit can be maximized via repurposing of FDA approved medications. Metformin is FDA approved antidiabetic drug with QS inhibitory activity against MDR


Diffraction from a lattice of periodically spaced crystals is a topic of current interest because of the great development of self-organised superlattices (SL) of nanocrystals (NC). The self-organisation of NC into SL has theoretical interest, but especially a rich application prospect, as the coherent organisation has large effects on a wide range of material properties. Diffraction is a key method to understand the type and quality of SL ordering. Hereby, the characteristic diffraction signature of an SL of NC—together with the characteristic types of disorder—are theoretically explored. We will explore the diffraction characteristics of supercrystals (SCs) as superlattices (SLs) of nanocrystals (NCs) where the periodic entity is itself a small crystal. There is a widespread current interest on such materials, driven by the changes in properties that the periodic organisation of NCs into an SC yields, mainly for applications in optics , electioWith the advances in SL synthesis methods, characterization tools such as small and wide-angle scattering methods that allow for probing the SLs in their real sample environments and, possibly, in real time, become increasingly important. X-ray scattering experiments allow for obtaining structural information on the ordering state of the assembly along with their lattice parameters and symmetry. A general and comprehensive review of the application of the small-angle scattering techniques has been given by Li et al. , and morThe use of wide-angle X-ray scattering data (WAXS) is far less common in the study of SLs, although it has been shown that information about structural coherence along two and three directions, degree of orientational order, and coherent sizes can be retrieved .Here, we deepen the discussion of diffraction theory of SCs. In particular, we will focus on the diffraction signature of imperfectly ordered SCs, especially concerning the effect of NCs of different sizes in the SL nodes, and the effect of slight rotation of the component NCs with respect to each other. We derive expressions for the powder diffraction total scattering intensity, which includes the size dispersion effect and allow a faster evaluation of Debye Scattering Equation. The orientational disorder will be developed only partly in this paper, delegating the full discussion to an upcoming technical paper. The results presented here will be useful for the quantitative interpretation of diffraction data from SLs.The numerical simulations of powder diffraction patterns hereby presented were computed using the DEBUSSY software suite  and ad hA perfect superlattice of identical nanocrystals, perfectly equioriented in space and periodically arranged without any defect, can be dealt with as a conventional crystalline structure with a large unit cell. Several papers deal with diffraction from SCs, especially within some experimental framework, with a comprehensive review in Jiang et al.  includinna,nb,nc:naa_,nbb_We abstain in the following from describing the atomic content of the unit cell; we will assume that each NC unit cell contains just one point scatterer of unit scattering power in the origin. Generalisation to real NCs with a specified unit cell content is straightforward but able to unnecessarily complicate the notation. The NC’s scattering density is described formally in Equation .The unit cell of the superlattice contains instead a single NC. We can arbitrarily set each superlattice node in the NC’s scattering barycentrumtterers: FNC(q_)=∫(x) .X) isY,We still assume a paralleloidal shape. The dimensional constants Equation ) may somSimilarly, for X is distributed according to a given probability density This is a simple set of nanocrystals on a line, hence forming a rod. Notation: if a variable Consider first the fully ordered case, wherek-spaced nodes isFirstly, for a finite sequence of equispaced points of length k superlattice sites will be simplyevery variable segment. Supposing now every segment is variable, thus* functions are normalised to 1]The average distance between two nodes spaced by Equation , we haveThe fully ordered case is described in In the ordered case,Consider an SL formed by a parallelogramEquation  for the The total distance between two point scatterers belonging each to one of the two NC centered at the If we consider instead the We then have the vector averageEquation  is constWe cannot be too specific on the form of the 3D distribution of Powder average is the average of the diffraction pattern over all possible orientations in space with a uniform distribution. The result will be a function only of N atoms (simplified as point scatterers) with coordinates d-values, each with a multiplicity ℓ-indexed groups of ℓ-groups and instead evaluate the group average For a system of equation  (hereaftan writeI(q)=∑j=1We recall briefly that the DSE is just the spherical average  of the 3D scattering equationSuppose now that we have a distribution for 3D vector distance with a vector average and a covariance matrix (as in Equation ). KnowinEquation ) and theEquation ), and beEquation , we mustEquation .(35)dK__ yieldsdK_,g_=dKquations  in placeWe also explore—partly—the case when the NCs arranged on the SL are all equal sized (no SDE) but not perfectly aligned with each other. This we name the alignment disorder effect (ADE).We develop this case very briefly because of the extensive theoretical analysis involved that suggests dedicating a specific manuscript to it. However, we want to give at least a feeling of the effect on diffraction of alignment disorder.Take two SL sites separated by As for the size disorder case, we imagine an equivalent mechanism where nearest-neighbour only interactions are involved. As such, every NC has a small rotational degree of freedom with respect to its nearest neighbours. The variances of the rotation angles then increase linearly with the number of steps in each SL direction between two SL sites. It is reasonable that each SL direction influences differently the rotation angle around itself than the other SL directions. Then, we have a simple matrix equation for evaluating the angular variances:The two NC spaced by Equation . For theHere, we want to show some numerical calculations of SC diffraction patterns with size disorder effect (SDE). We will start with a system that produces truly 1D scattering . Then, we will have SCs with small NCs and different degrees of disorder and also different SL dimensionality .squared variance due likely to the concomitant effect of rotation, or weaker  can be ignored.z, q is the scattering vector along the same axis, and The scattering equation of this system readsEquation  where tha, Each bunch of planes is equispaced, so we can write, for an isolated bunch of height The average SL is the periodic average of the arrangement of bunches, with an average spacing Example 1D patterns. We consider bunches of equispaced planes (representing the NCs) stacked with dead space on top of each other, see We also set We also calculated the diffraction pattern in the case where every plane bunch (or NC) is substituted by a single scattering plane. This shows directly  the SL sWe constructed cubic NCs  in the We have explored the peculiar disorder effects of NC-based SCs that constitute a growing trend because the SL order introduces or modifies physical properties in ways that are interesting for applications. To explore the underlying mechanism, a great importance is attached to the fine structural features of the superorder. As a rule of thumb, structural effects that modify the X-ray diffraction are also modifying the electronic properties through the band structure. This makes it interesting to explore the quality requirements—in terms of NC size and shape uniformity, and also in terms of co-alignment of the NCs until their periodic arrangement on an SL truly forms an SC. To this aim, we have investigated the diffraction footprint of SC whose constituting NCs have a small size dispersion that must affect the quality of the periodic SL order. It turns out that a small size dispersion (4–5%) is already able to severely affect (up to canceling) the SL coherence, whilst NC misalignment is also very effective at this task, but its destructive effect is higher for larger NC sizes. Therefore, in order to achieve SL interference effects—if they are connected to desirable changes in the electronic properties—great care must be taken to ensure a very sharp size distribution (with relative dispersion at the % order) and a great uniformity and regularity of shape .


In a recently published study combining transcranial magnetic stimulation and electroencephalography (TMS-EEG), an early component of TMS-evoked potentials (TEPs), i.e., M1-P15, was proposed as a measure of transcallosal inhibition between motor cortices. Given that early TEPs are known to be highly variable, further evidence is needed before M1-P15 can be considered a reliable index of effective connectivity. Here, we conceived a new preregistered TMS-EEG study with two aims. The first aim was validating the M1-P15 as a cortical index of transcallosal inhibition by replicating previous findings on its relationship with the ipsilateral silent period (iSP) and with performance in bimanual coordination. The second aim was inducing a task-dependent modulation of transcallosal inhibition. A new sample of 32 healthy right-handed participants underwent behavioral motor tasks and TMS-EEG recording, in which left and right M1 were stimulated both during bimanual tasks and during an iSP paradigm. Hypotheses and methods were preregistered before data collection. Results show a replication of our previous findings on the positive relationship between M1-P15 amplitude and the iSP normalized area. Differently, the relationship between M1-P15 latency and bimanual coordination was not confirmed. Finally, M1-P15 amplitude was modulated by the characteristics of the bimanual task the participants were performing, and not by the contralateral hand activity during the iSP paradigm. In sum, the present results corroborate our previous findings in validating the M1-P15 as a cortical marker of transcallosal inhibition and provide novel evidence of its task-dependent modulation. Importantly, we demonstrate the feasibility of preregistration in the TMS-EEG field to increase methodological rigor and transparency. The combination of transcranial magnetic stimulation and electroencephalography (TMS-EEG) provides a unique perspective on effective connectivity  Validating M1-P15 as a measure of transcallosal inhibition: we expect to replicate the positive relationship between M1-P15 amplitude and the magnitude of iSP; (ii) Evaluating the behavioral relevance of M1-P15: we expect to replicate the relationships between M1-P15 latency and bimanual coordination during the sequential thumb-to-finger opposition task; if the replication was successful, we aimed at exploring the potential cost of TCD asymmetry on unimanual performance; (iii) Validating the M1-P15 through the modulation of the transcallosal inhibition: by manipulating the activity of the hand contralateral to the stimulation, we expect to modulate indexes of transcallosal inhibition, i.e., the iSP and the M1-P15.https://osf.io/pg78j/), aiming at increasing the transparency of the research process, avoiding the risks of undisclosed analytic flexibility and providing an unbiased picture of the results, overall contributing in improving scientific replicability  before data collection  and a sequential mirror-symmetrical thumb-to-finger opposition task (Sequence), as in Bonzano et al. , while EEG was continuously recorded with a TMS-compatible system . The motor hotspot for APB muscle was localized as the scalp site eliciting the highest and most reliable motor-evoked potentials (MEPs) with the same TMS intensity. Coil orientation was kept ~45° from the midline, inducing an anterior-to-posterior and posterior-to-anterior (AP-PA) current direction in the brain. Then, the resting motor threshold (rMT) was estimated using the maximum-likelihood threshold hunting algorithm  and right (RTMS) M1 using a Magstim RapidAwiszus, , 2011, aAwiszus, . This prAwiszus,  (mean rMTapping and 3 blocks for Sequence), and they were performed twice, once during LTMS and once during RTMS . TMS pulses were randomly delivered in the intervals between sounds, based on the results of a pilot study showing that M1-P15 could be recorded at any time of TMS delivery during bimanual movements  or contracted at the same level of strength (Contracted), during LTMS and RTMS in separate blocks in counterbalanced order. To this aim, at the end of the bimanual tasks during TMS-EEG recording, sensors were removed from the participants' fingers. Instead, a pressure sensor was applied on the second phalanx of their left and right index finger. Participants were asked to press on the sensor with their thumb, thus inducing a contraction of APB muscle. First, we estimated the maximal strength: participants were asked to press as strong as possible with their thumb on the sensor for 10 s, with the left and the right hand in separate runs. For each hand, the pressure values were averaged over a sliding window of 500 ms, and the 500 ms bin with the highest average value in the 10 s interval was selected as the maximum strength. Then, the main blocks started: in the Contracted conditions participants were asked to press on the sensors with both hands, while in the Relaxed conditions they had to press on the sensor only with their hand ipsilateral to TMS, keeping the contralateral hand relaxed. Participants were asked to press between 40 and 60% of their maximal strength. Every time the pressure level fell out of the required range, a visual feedback appeared on the screen indicating the actual pressure compared to the target range. Once the pressure level was reached, visual feedback disappeared and only the fixation cross remained in the center of the screen, to avoid eye movements during TMS-EEG recording. TMS was delivered only when the pressure on the sensors was within the target range, for a total of 90 pulses per condition. A break of ~30 s was provided in the middle of the blocks . Moreover, participants were instructed to relax their hands anytime they needed an additional break; in this way, the TMS delivery was automatically interrupted until the pressure level had reached again the requested level.During the iSP paradigm , particiPerformance in the bimanual tasks was evaluated as the inter-hand interval  between the onset of finger tap with the left hand and onset of the corresponding finger tap with the right hand . This approach minimized the risk that differences between conditions that we compared could arise from dissimilarities in the preprocessing steps. The effect of the main steps of the preprocessing on the TMS-EEG data of a single participant are shown in TEPs data analysis was performed in MATLAB R2020b  with custom scripts using EEGLAB v.2020.0 , except for the high-pass filter on the continuous TMS-EEG data, which was modified into 1 Hz instead of 0.1 Hz for computational demands. Although in the present work we were not interested in slow potentials, we preliminary checked on the Pilot data that high-pass filtered TEPs at 1 or 0.1 Hz were qualitatively comparable , downsampled at 4800 Hz and epoched from 200 ms before to 500 ms after the TMS pulse. Then, the source-estimate-utilizing noise-discarding (SOUND) algorithm . From the grand-average of all participants we identified the channels contralateral to TMS with the highest amplitude in the time window between 7 and 25 ms: F4-FC4 for LTMS, F3-FC3 for RTMS. Then, we identified the maximum on the pooled signal of the two channels in the time window between 7 and 25 ms; in case more than one peak was present or no peak could be detected, we manually restricted the time-window to match the M1-P15 topography . Then, we divided the data into the different experimental conditions, and the M1-P15 peak was automatically detected within 10 ms around the individual peak identified previously. M1-P15 amplitude was calculated by averaging over 10 ms around the peak. Overall, this procedure allowed us to perform a semi-automatic peak detection, avoiding subjective bias between the experimental conditions that we aim to compare.The M1-P15 identification was performed as follows. For each subject we first averaged over all conditions following the stimulation of the same hemisphere to increase the signal-to-noise ratio . Then, EMG was band-pass filtered between 100 and 1000 Hz (default FieldTrip filters) and rectified. For each participant, for each trial we checked whether the signal was lower than the mean of prestimulus baseline  for at least 10 ms in the time window between 25 and 75 ms after the TMS pulse; trials which did not satisfy this criterion were discarded. On average, 58.5 trials (64.9%) were considered in the average of each condition . Then, for each participant and condition we averaged over trials, and calculated the iSP with the same parameters as the ones applied on single trials. One subject was excluded in this step because no iSP was detected, leaving 29 participants for the statistical analyses involving the iSP. Finally, we calculated the normalized iSP area using the following formula [(area of the rectangle defined as mean EMG x iSP duration)–(area underneath the iSP)]. Then, the normalized iSP area was defined as the ratio between the iSP area and the area underneath EMG from - 150 to 50 ms preceding TMS . Details on the statistical models and results can be found in If not otherwise specified, the statistical analyses followed what planned in the preregistration ((i) To validate M1-P15 as a measure of interhemispheric inhibition, we run a linear mixed model (LMM) testing whether M1-P15 amplitude predicted the iSP normalized area. We also performed two explorative  control analyses to exclude that the relationship between M1-P15 amplitude and the iSP normalized area could be explained by a common factor, namely the TMS intensity: For this we ran two LMMs testing whether rMT was predictive of M1-P15 amplitude and of iSP normalized area. LMMs were chosen for consistency with our previous work . In separate models, we first considered as predictor the M1-P15 latency measured during the iSP paradigm and during the bimanual Sequence task. Separate LMMs tested M1-P15 latency during LTMS, RTMS and the ratio between the two (LTMS/RTMS) as predictors . Since no significant relationships were observed between M1-P15 and bimanual performance in the Sequence (see Results), the preregistered analyses on the relationships between M1-P15 and bimanual performance in the Tapping, as well as between M1-P15 and unimanual performance, were not performed. We also ran additional control analyses on the behavioral tasks. We compared the bimanual performance between Sequence and Tapping by means of a two-tailed t-test for dependent samples, and we checked for an effect of time by comparing the bimanual performance in the three blocks, as indexed by the inter-hand interval measured during Sequence, by means of one-way repeated-measures analysis of variance (rm-ANOVA) with the 3-level factor Block. We also explored possible modulations of M1-P15 latency, both during the bimanual tasks (2 × 2 rm-ANOVAs with factors Hemisphere and Task) and during the iSP paradigm (2 × 2 rm-ANOVAs with factors Hemisphere and Contraction). ANOVA were performed because we were interested in testing differences between means taking into consideration the repeated-measure design.(ii) LMMs were also applied to assess the behavioral relevance of M1-P15. Bimanual coordination performance in the Contracted, Relaxed) and Hemisphere  as predictors. This statistical model was chosen because we were interested in the effect of Contraction on the two dependent variables. Considering the 2 × 2 design of the rm-MANOVA, a semi-parametric model was performed (modified ANOVA-type statistic—MATS). Post-hoc analyses were performed by separate 2 × 2 rm-ANOVAs on M1-P15 amplitude and the normalized iSP area, respectively. As additional control analysis, the rm-ANOVA was performed to test the effect of Contraction on the  iSP area, as this was the quantification method used in the original study by Giovannelli et al. . Finally, we compared the rMT for the left and the right hemisphere by means of a two-tailed dependent sample t-test.(iii) To assess the modulation of the interhemispheric inhibition, we first tested the effects of contraction levels of the hand contralateral to the stimulation. We performed multivariate analysis of variance for repeated measures (rm-MANOVA) on M1-P15 amplitude and iSP normalized area, with Contraction  = 3.3, p = 0.001), so that the larger the M1-P15, the stronger the interhemispheric inhibition in the ipsilateral APB (t(29) = 1.26, p = 0.22; t(28) = 0.83, p = 0.42; t(28) = 2.85, p = 0.006).(i) As predicted, LMMs showed that M1-P15 amplitude positively predicted the normalized area of the iSP , RTMS , nor the ratio between the two . From the control analysis on the behavioral measure of performance, we excluded an effect of fatigue or learning throughout the behavioral task, because one-way rm-ANOVA with the 3-level factor Block on the inter-hand interval showed a non-significant effect  = 0.75, p = 0.48). Overall, the M1-P15 latency was unaffected by the factors we manipulated, i.e., Hemisphere, both during bimanual paradigm  = 0.02, p = 0.9) and during iSP paradigm  = 2.13, p = 0.16), Task during bimanual paradigm  = 0.56, p = 0.46) and Contraction during iSP paradigm  = 0.47, p = 0.5); no interaction were observed  = 1.91, p = 0.18; Hemisphere X Contraction: F = 0.01, p = 0.92).(ii) No evidence was found that bimanual performance in (2) = 0.64, p = 0.22) nor Contraction X Hemisphere (MATS(2) = 1.00, p = 0.53) on the iSP normalized area and M1-P15 amplitude during the iSP paradigm. We observed a main effect of Hemisphere (MATS(2) = 6.66, p = 0.023): Post-hoc analyses by means of rm-ANOVAs revealed a significant main effect of Hemisphere on iSP only  = 4.24, p = 0.049), with a larger iSP after RTMS compared to LTMS; this effect did not reach the level of significance on M1-P15 amplitude  = 3.69, p = 0.065). The control analyses confirmed a non-significant effect of Contraction on the  iSP area  = 0.21, p = 0.65) and indicated that left and right rMT did not differ (t(29) = 0.11, p = 0.91), suggesting that the effect of Hemisphere cannot be explained by a difference in TMS intensity. Importantly, the M1-P15 amplitude in the bimanual tasks was modulated according to the movements performed  = 6.43, p = 0.017), with a larger M1-P15 recorded during Sequence (5.17 ± 0.5 μV) compared to Tapping  = 0.03, p = 0.87) nor interaction Task X Hemisphere  = 1.79, p = 0.19) were observed. Finally, the inter-hand interval was significantly higher  in the Sequence (25.7 ± 8.6 ms) compared to the Tapping task  = 8.07, p < 0.001; (iii) The iSP paradigm showed no modulation of interhemispheric inhibition by hand contraction for both iSP and M1-P15. The rm-MANOVA revealed no significant main effect of Contraction  to 4 (“very loud noise”), suggesting that the use of noise-canceling earphones playing white noise did not mask the TMS click completely.In our previous work ; In the present study, they were asked to maintain a certain level of muscle contraction (Contracted). Moreover, the relationship between M1-P15 and bimanual coordination was investigated for the M1-P15 recorded during the same bimanual movements performed in the behavioral tasks. Nevertheless, even in this case we did not observe a significant relationship between M1-P15 latency and behavioral performance. It has to be noted that in Bortoletto et al. , but during the behavioral tasks, instead. Second, the reafference of the motor twitches on TEPs is expected to occur approximately at 60 ms after the TMS pulse , the within-subject design including a task modulation represents a powerful strategy to face the problem of confounds deriving from sensory contamination in TMS-evoked potentials. Indeed, the experimental conditions only differ on the task the participants were performing, but they are identical in the stimulation parameters , therefore excluding that possible differences in TEPs between conditions may be due to differences in TMS-induced artifacts or sensory contaminations.Contracted compared to the Relaxed condition . The negative finding of the present work cannot depend on the quantification method for the iSP, i.e., the normalized iSP area vs. the iSP area used in Giovannelli et al.  compared to the repetitive (Tapping) bimanual movements. This result represents the first evidence of a task-dependent modulation of M1-P15, further suggesting the physiological, non-artifactual nature of this early TEP component. The presence of the M1-P15 during the bimanual tasks, with topographical patterns comparable to the ones observed during the iSP paradigm, suggests that the transcallosal information transfer conveys an inhibitory function also during the execution of bimanual movements, supporting the hypothesis that functional inhibition of contralateral M1 may be needed to ensure neural cross-talk at the cortico-spinal level . The patients/participants provided their written informed consent to participate in this study.AZ, GB, and MB conceived and designed the study. GB wrote the scripts for the experimental paradigms and built custom devices. AZ, GB, and EM collected the data. AZ and GB performed the signal processing. AZ and CF planned and performed the statistical analyses. AZ took care of data visualization. AZ and MB managed data curation, supervised the research activity, and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This study was supported by the Italian Ministry of Health funding Ricerca Corrente and by the Department of Philosophy Piero Martinetti of the University of Milan with the Project Departments of Excellence 2018–2022 awarded by the Italian Ministry of Education, University and Research (MIUR) to GB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Foam-filled honeycombs have been widely applied due to their excellent load transfer mitigation and energy absorption capacity. In the present study, a layered graded foam concrete-filled auxetic honeycomb was proposed by tuning its overall compression deformation mode to layer-by-layer deformation mode to realize multi-level structural protection. The effect of the honeycomb cell-wall thickness gradient  and the foam concrete filler density gradient  on the response mode, load transfer, energy absorption, and Poisson’s ratio of the proposed composite was systematically investigated. The results showed that the graded composite exhibited an obvious layered deformation mode and a negative Poisson’s ratio effect under relatively low and moderate loading rates , especially with the foam concrete density gradient. Under a high loading rate (100 m/s), the graded composite demonstrated progressive collapse initiating from the loading end with a layer-by-layer crushing mode, regardless of the thickness and density gradient. In the response of the composite with a 0.2:0.2:0.2 thickness ratio and a 408:575:848 foam concrete gradient subjected to 1 m/s crushing, the first-layer, second-layer, and third-layer foam concrete absorbed 94.62%, 88.72%, and 86.94% of the total foam concrete energy absorption in the corresponding crushing stage, respectively. Compared with the counterpart homogeneous composites, although the graded composite had an insignificant improvement on energy absorption (less than 5%), it was able to significantly reduce the peak load (as high as 30%) to mitigate the load transfer to the protected structure. The effective Poisson’s ratio of the first layer in the composite with positive gradient (408:575:848) increased to −2 then converged to −0.6 under 2 m/s and 10 m/s crushing, and ranged from −0.4 to −0.1 under 50 m/s and 100 m/s crushing, respectively. The effective Poisson’s ratio of the middle and bottom layers increased to −2 initially and converged to range −0.4 to −0.1, regardless of the crushing speed. The staged response mode of the graded composite facilitated the realization of multi-level structure protection with significantly reduced peak load transferred to the protected structure and tuned energy absorption. The accidental industrial explosions and terrorist attacks have posed significant threats to important structures and infrastructures. Therefore, understanding the response of structures is necessary to facilitate structure protection against extreme loading. The response of existing structures, especially reinforced concrete (RC) structures, subjected to blast ,2, impacWhile damage of RC structures is almost inevitable when subjected to blast or impact, applying sacrificial cladding to the structure exterior is a promising protection method. Sandwich structures are widely applied in the aerospace, vehicle, ship, and packaging industries owing to their advantages of being lightweight, having a high specific strength, a strong crashworthiness, and a high energy absorption efficiency. In recent years, due to their excellent energy absorption capacity, they have received increasing attention in the field of structure protection ,7,8,9,10Impact and blast are two major strong dynamic loads, possibly leading to severe damage or strong vibration ,24,25. FThe foam materials and honeycomb are usually applied as the sandwich core due to their excellent energy absorption performance, but their individual use has exhibited unfavorable performances due to their relatively low plateau stress. To take advantage of foam materials and honeycomb, lightweight foam-filled honeycomb was proposed by a simple combination of these two components with significantly improved energy absorption capacity . TherefoOn one hand, due to the characteristics of outward expansion under uniaxial tension and inward shrinkage under uniaxial compression, the concave hexagonal negative Poisson’s ratio honeycomb has a higher yield strength , strongeOn the other hand, foam material, including polymeric foam, metal foam, and foam concrete, is an ideal filler with large densification strain and excellent energy absorption capacity. The polymer foam has the advantage of lightweight but the energy absorption capacity is comparatively unfavorable. Metal foam exhibits excellent energy absorption capacity, but it suffers from high cost and inconvenient construction. In comparison, foam concrete shows more potential to be applied in the field of structural protection due to its merits of being lightweight, low-cost, and easy to manufacture ,44. A seIt has been noted that the mechanical properties and energy absorption capacity of the foam material are mainly determined by its density, and the failure always initiates at the weakest parts which are somewhat random if the loading rate is lower than a certain value . For theAs to impact or collision mitigation, composites with soft core seem promising. Take the bridge pier collision mitigation in the field of civil engineering as an example. The composites can be used as protection cladding attached to the pier. If a vehicle out of control collides with the pier, the composite cladding could dissipate a large amount of energy of the system consisting of the pier, the vehicle, and the cladding, with major deformation concentrated in the cladding. At the same time, the loads to the pier as well as to the vehicle are significantly reduced. Consequently, both the pier and the vehicle with its occupants are effectively protected. After the incident, replacing the damaged cladding could immediately resume the protection capacity.From the viewpoint of practical application, the protection performance could be improved if the gradient was introduced to the core, as the load (per unit area) transferred to the protected structure is the plateau stress of the crushed core. If the collision is moderate, the cladding response likely only occurs in the part of the core with the transferred load as the plateau stress of the crushed part, while the other part remains elastically deformed. Alternatively, in a severe collision, the gradient with direction could be designed to reduce the load transferred to the protected structure compared to that of the homogenous counterpart.Among different foams being filled into hollow structures in the literature, foam concrete is promising in its application in civil engineering. On one hand, foam concrete is cost-effective and highly durable in typical service conditions such as water and oxygen. On the other hand, it can be conveniently cast into hollow structures of almost all typical geometries, which is more convenient than other foams needing cutting before filling, such as metallic foams. In the present study, layered gradient auxetic honeycombs filled with foam concrete are proposed, in which the gradient effect was realized by tuning the cell-wall thickness of the honeycomb and foam concrete density. The influence of the honeycomb cell-wall thickness gradient, the foam concrete density gradient, the loading rate on the response mode, load transfer, and energy absorption of the proposed composites was investigated with numerical models verified by test data. In addition, the effective Poisson’s ratio was employed to evaluate the concave deformation of each layer in the proposed gradient composites subjected to different loading rates.3 foam concrete were also prepared for the quasi-static and dynamic compression tests.The prepared foam concrete-filled auxetic reentrant honeycomb specimens are shown in It is worth noting that the dimension of the honeycomb was designed based on the loading capacity and the dimension of the crosshead and anvil of the INSTRON VHS160/100-20 high-speed testing system. On one hand, the crosshead and anvil were both round in shape with a diameter of 120 mm. Therefore, the honeycomb core was designed to be 80 mm* 80 mm to be within the area of the crosshead and anvil. On the other hand, with this specimen dimension, combined with the selected honeycomb material and thickness and typical foam concrete property, the crushing resistance was lower than the loading capacity of the high-speed testing system (100 kN), which was described in the following section.The quasi-static compression tests with a displacement control of 5 mm/min were conducted with an MTS Exceed E45 testing system with a loading capacity of 300 kN, shown in 3 foam concrete subjected to quasi-static and dynamic uniaxial compression, respectively. For the hollow aluminum honeycomb, there was a negligible difference in the measured stress–strain curves between the quasi-static compression and dynamic test except that the initial peak stress was slightly higher in the dynamic test. As shown in 3 foam concrete under quasi-static and dynamic compression in In addition, with the loading rate of 0.05 mm/min, the quasi-static tensile test of the dog-bone aluminum sheet was carried out according to the standard ASTM E8M-04 to investigate the tensile mechanical properties of the cell-wall material, and the true stress–strain curve is shown in The numerical simulation was carried out with the finite element software LS-DYNA 971. As solid structures with contact interaction were concerned in the present study, the Lagrangian approach was adopted to ensure computational accuracy. Specifically, in the specimen, the auxetic honeycomb, top sheet, and bottom sheet were typical thin-walled structures; therefore, they were modeled with Shell elements to balance computational cost and accuracy. Moreover, the foam concrete filled in the auxetic honeycomb was typically solid; thus, it was modeled with Solid elements. The numerical model fully represented the specimen geometry except for the shorter horizontal edges, which were designed slightly longer for adequate bonding in the test. In the numerical model, the foam concrete-filled auxetic honeycomb was sandwiched by the anvil and the crosshead. All degrees of freedom of the anvil, as well as all the rotations and the in-plane translations of the crosshead, were constrained. As shown in As shown in the numerical model in The crosshead and the anvil were simplified as a cylindrical base and loading plate, which were modeled by the eight-node solid element SOLID164. Since the strength and stiffness of the crosshead and anvil were significantly higher than that of the composite, the deformation of the crosshead and anvil was neglected in the numerical model. Therefore, the loading device, consisting of the crosshead and the anvil, was assumed to be rigid in this numerical simulation by the material model keyword *MAT_RIGID. The material properties of the filled foam concrete were defined by the keyword *MAT_CRUSHABLE_FOAM in the numerical model. This material model was a macroscopic equivalent model, and it was assumed that the foam concrete was an isotropic homogeneous material with the provided macroscopic mechanical properties. In this model, the mechanical properties of foam concrete were defined by the damping, tensile cut-off stress, and the measured stress–strain curve in the compression test in The components in the auxetic honeycomb, including the inner concave folded plates, and the upper and lower surface plates, were connected through a common node contact. The automatic single-to-surface contact algorithm with the keyword *AUTOMATIC_SINGLE_SURFACE was applied to simulate the self-contact of auxetic honeycomb and foam concrete. Meanwhile, an automatic face-to-face contact algorithm with the keyword of *AUTOMATIC_SURFACE_TO_SURFACE was applied between the loading platens and the face sheets, auxetic honeycomb cell wall, and foam concrete filler. The static and dynamic friction coefficients were set to 0.3 and 0.2, respectively. Furthermore, the mesh size sensitivity study of the numerical model was carried out, and it was found that a 2 mm mesh size was a reasonable choice balancing the simulation accuracy and computational efficiency. In the model, around 680,000 elements are used.Before investigating the response of the foam concrete-filled auxetic honeycomb subjected to impact, the numerical model was validated in terms of the nominal stress–strain curve for both hollow honeycomb and that filled with foam concrete, respectively.As shown in 3 foam concrete was further verified by the test data, with the composite and numerical model shown in The numerical model of the aluminum honeycomb filled with 408 kg/mThe results in From the experimental studies in 3, 575 kg/m3, and 848 kg/m3 provided their mechanical characteristics in The quasi-static compressive test on the foam concrete specimen with the density of 408 kg/m3, and each layer had a different thickness of cell-wall but the averaged cell-wall thickness of the graded composite remained the same as the homogenous composite.With the established numerical model of the graded composite, the numerical simulation was carried out to investigate the dynamic response of concave hexagonal auxetic honeycomb filled with foam concrete with layered cell-wall thickness gradient subjected to low-velocity impact, including the response mode, stress–strain characteristics, and energy absorption. 3. In this numerical simulation, three different cell-wall thickness gradients were considered, and the cell-wall thickness of each layer was assigned as 0.2:0.25:0.3, 0.15:0.25:0.35, and 0.1:0.25:0.4, respectively. In addition, the gradient direction was also considered in this study. The average thickness of the single layer of the honeycomb cell wall was set to 0.25 mm, while the thickness of the upper and lower face sheet was set to 0.5 mm, with a plane size of 118 mm × 112 mm. The total mass of the composite was 789 g and its average density was 580 kg/mThe stress–strain relationship of the composite with different cell-wall thickness gradients was compared in In addition to the mechanical characteristics of the homogenous or graded composites with different cell-wall thickness gradients in The stress–strain curves and energy absorption performance of the composites with different cell-wall thickness gradient directions are shown in Based on the results and discussions above, it was concluded that a reasonable arrangement of the cell-wall thickness gradient of the auxetic honeycombs filled with foam concrete was beneficial to achieving the expected staged response mode, providing an alternative approach for specific multi-level protection simply and conveniently.3, 575 kg/m3, and 848 kg/m3 for the top, middle, and bottom layers, respectively. To investigate the effect of foam concrete density gradient on the response mode, load transfer, and energy absorption capacity of the composite, the thickness of the cell wall of these three layers was set identical. Moreover, two separation plates were introduced between different layers, whose geometries were the same as those of the face plates. As shown in 3 foam concrete filler, resulting in an obvious negative Poisson’s ratio effect in the top layer, as shown in The keyword *DATABASE_RCFORC output was adopted to obtain the contact force between the composite and the upper loading or lower supporting plates in The plateau stress, densification strain, and effective energy absorption of the composite at different compression stages are listed in The absorbed energy by the auxetic honeycomb, foam concrete filler, and the composite is shown in The energy absorption performance of the foam concrete filler in each layer with respect to the nominal strain is shown in In summary, it was found that the graded composite with foam concrete density gradient exhibited superior performance to the homogenous composite from the viewpoint of structural protection. It is known that the composite filled with high-density foam concrete provided high plateau stress, resulting in strong energy absorption. However, it also brings high initial peak stress for the protected structure, which is unfavorable for structure protection. The proposed graded composites with foam concrete density gradient could realize low initial peak stress and long stress plateau, implying that it may effectively mitigate the load transfer to the protected structures and reduce the potential damage. In addition, the sequential compression response mode could achieve the purpose of multi-level protection, for instance, only the damaged outer layer is required to be replaced after a small collision in practice.3, 575 kg/m3, and 848 kg/m3, respectively. From the response mode in The previous results in The stress–strain relationships of the composites with foam concrete density gradient and different thicknesses of cell-wall are shown in With cell-wall thicknesses of 0.2 mm, 0.3 mm, 0.4 mm, and 0.5 mm, 3 foam concrete in its top, middle, and the bottom layer was chosen to investigate the influence of the loading rate herein. The deformation mode of the composite under low (1 m/s), medium (10 m/s), and high compression velocities (100 m/s) were illustrated in Cellular materials and structures exhibited different response modes and energy absorption at different compression rates. Therefore, different impact velocities, namely, 1 m/s, 2 m/s, 5 m/s, 10 m/s, 20 m/s, 50 m/s, and 100 m/s were applied to the graded composites to investigate the failure mode, load transfer, and energy absorption characteristics. Due to its comparatively superior performance in the aspects of its deformation mode, load transfer, and energy absorption compared with other composites, the composite with cell-wall thickness of 0.4 mm and filled with 408, 575, and 848 kg/m3 from top to bottom layer, the composite exhibited layered deformation mode due to the different strengths of these layers under quasi-static loading. In contrast, with medium loading rates, all these three layers started to deform almost simultaneously while the composite still showed layered compression deformation. With a high loading rate, the composite underwent progressive collapse, while the deformation initiated and propagated with the loading plate, exhibiting top-to-bottom deformation mode despite the negative foam concrete density gradient arrangement. In summary, it was found that the response mode of the composite with foam concrete density gradient was significantly affected by the loading rate. Under low loading rates, the deformation sequence of each layer was determined by its strength. With medium loading rates, the strength of these layers and the loading speed both influenced the deformation sequence of each layer. With high loading rates, it was observed that the deformation sequence was mainly affected by the loading speed.Furthermore, the deformation mode of the composite with negative foam concrete density gradient is shown in Subjected to high loading speeds, such as 50 m/s and 100 m/s, it was difficult to avoid the high initial peak stress, which was unfavorable for structural protection. In practice, the impact velocity of vehicles is lower than 50 m/s in most collision accidents. Therefore, in the present study, low and medium loading rates were considered to investigate the performance of the composite with foam concrete density gradient, shown in The energy absorption capacity of the composite with positive and negative foam concrete density gradients under different loading rates is also presented in As shown in Compared with positive Poisson’s ratio structures, negative Poisson’s ratio structures demonstrated excellent performance in the aspects of stress distribution, shear modulus, indentation resistance, impact resistance, and energy absorption . In the 3, 575 kg/m3, and 848 kg/m3 foam concrete from the top to the bottom layer, was adopted herein to investigate the effective Poisson’s ratio of these three layers. The composite with cell-wall thickness of 0.4 mm as well as filled with 408 kg/mIn the present study, a novel graded auxetic foam concrete-filled honeycomb was proposed to exhibit layered response with staged load transfer and energy absorption, which was promising to be applied as sacrificial cladding to realize multi-level protection for important structural components. In the proposed layered composite, the strength gradient of different layers was realized by tuning the thickness gradient of honeycomb cell-wall and density gradient of foam concrete filler, whose effect on the deformation mode, initial peak stress, plateau stress, energy absorption, and effective Poisson’s ratio was systematically investigated with a validated numerical model. The main conclusions were drawn as follows,(1)The composite with cell-wall thickness gradient exhibited layered response mode under low or medium loading rates . Increasing the cell-wall thickness gradient led to a more obvious layer-by-layer compression response, lower initial peak stress, and a more fluctuated stress plateau. Compared with the homogenous cell-wall thickness counterpart, the improvement of the energy absorption of the composite with cell-wall thickness gradient was minor, less than 5%.(2)The foam concrete density gradient and direction had a significant effect on the performance of the composite, reflected by the observed three-stage stress plateau corresponding to the layered compression. In the first, second, and third deformation stages, the top layer, middle layer and bottom layer foam concrete of the composite with positive gradient absorbed 94.62%, 88.72%, and 86.94% of the total foam concrete energy absorption, respectively. The layered architecture filled with foam concrete of different densities was able to realize a controllable sequential compression response, convenient to realize multi-level protection.(3)The layered gradient composite exhibited different response modes under different loading rates. Subjected to a low or medium loading rate (less than 10 m/s), the composite underwent an obvious layered response and negative Poisson’s effect, while it exhibited progressive collapse mode from the loading end subjected to a high loading rate (greater than 10 m/s).(4)The composite with positive foam concrete density gradient (408:575:848) produced higher initial peak stress compared with that with negative gradient (848:575:408) by 21%, implying that the former was inferior to the latter in the aspect of initial transferred load to the protected structure.(5)The effective Poisson’s ratio of the proposed composite first increased and then decreased, and finally remained stable with increasing compression stroke. Specifically, the effective Poisson’s ratio of the first layer in the composite with positive gradient (408:575:848) increased to −2 then converged to −0.6 under 2 m/s and 10 m/s crushing, and ranged from −0.4 to −0.1 under 50 m/s and 100 m/s crushing, respectively. The effective Poisson’s ratio of the middle and bottom layers increased to −2 initially and converged to range of −0.4 to −0.1, regardless of the crushing speed.(a)The theoretical model for auxetic honeycombs without and with filler may be established, to provide a quick preliminary prediction.(b)Other configurations of hollow structures with auxeticity under compression may be investigated, especially with foam concrete filler.(c)The theoretical model incorporating the blast load, the graded protective layer, and the protected structural member such as a beam or slab, may be established.The following aspects may be considered for future study:


Ultrasonic pulse velocity (UPV) and rebound hammer tests are accepted as alternatives to destructive testing to determine the compressive strength, dynamic modulus of elasticity, and Poisson’s ratio, which are needed for structural design. Although much work has been conducted for plain concrete, the research data for fibre-reinforced concrete (FRC) is insufficient. In this regard, this study explains the correlations between compressive strength, rebound hammer, and UPV tests for plain concrete and FRC contains 0.25%, 0.50%, and 1.00% of 30 mm and 50 mm long steel fibres. A total of 78 concrete cube and beam specimens were tested by direct, semi-direct, and indirect UPV and rebound hammer test methods. The study found that the rebound hammer test is more suitable for measuring the compressive strength of matured FRC than young concrete. The UPV test revealed that the volume fraction does not, but the length of steel fibres does affect the UPV results by the direct test method. The UPV direct method has the highest velocity, approximately two times the indirect velocity in FRC. UPV measurements can be effectively used to determine the dynamic modulus of elasticity and Poisson’s ratio of FRC. The dynamic elastic modulus increases while the Poisson’s ratio decreases for the same steel fibre length when at increasing FRC fibre content. The results of this study will be significant for non-destructive evaluations of FRC, while additional recommendations for future studies are presented at the end of the paper. The flexural and shear stiffness of concrete elements should be calculated to assess the lateral displacements and deflections. Steel fibres provide great advantages in high-rise concrete buildings and long-span concrete bridges . The modThe equations for estimating the elasticity modulus and Poisson’s ratio of FRC are not included in modern building codes, despite wider applications of steel FRC . An in-dIt has been observed through the literature that there are existing experimental works to investigate the mechanical properties of fibre-reinforced concrete. However, there is minimum available experimental work to evaluate the effect of length and volume fraction of steel fibre on the concrete compressive strength, modulus elasticity, and Poisson’s ratio, which is investigated using destructive and non-destructive tests. The results of this study can be a reference for future studies and provide wide data for fibre-reinforced concrete studies.This paper aims to propose empirical relationships for estimating the compressive strength, modulus of elasticity, and Poisson’s ratio of concrete reinforced with steel fibres, using measured data of UPV tests. This research is also intended to evaluate the effect of test setup , and establish a relationship between the strength of FRC and the characteristics of steel fibres (length and fibre fraction). The experimental study shown in this paper includes non-destructive tests and destructive tests. The non-destructive properties were analyzed based on variables such as fibre length, fibre content, and test setup . The length of steel fibres was 30 mm and 50 mm. Fibre volume fractions in the concrete varied between 0, 0.25, 0.50 and 1.00%. The non-destructive tests used the ultrasonic pulse and rebound hammer tests which were performed on 15 specimens (beam type). The destructive were performed on the 65 cube specimens. The recorded pulse rates allowed for calculating the values of the modulus of elasticity and Poisson’s ratio.Portland cement (42.5N) was used in this research. Cement was obtained from a single supplier to ensure the quality of the material is the same for all concrete mixes.The coarse and fine aggregate grading and characteristic are given in Steel fibres were used in this investigation as steel fibres are the most widely used fibres in FRC. Steel fibre is very good at increasing the most of properties of concrete. The length of steel fibre used in this research is 30 mm and 50 mm. The steel fibre type and properties such as strength and modulus are shown in The final mixture proportion is shown in The mixing procedure was based on ASTM manual . Concrete was mixed in a drum mixer which can mix up to 100 kg of concrete each time. Coarse and fine aggregates were first to dry and mixed for one minute, and then cement was mixed with aggregates for another minute. Thirdly, water was added and mixed until a uniform mix was achieved. Fibre was added to the mix slowly and uniformly, and the concrete was then mixed for five minutes. The water to cement ratio is relatively high, and therefore, admixtures were not added to the concrete mix.The experimental work of the present study consists of 63 cubes and 15 beams with different dimensions, as shown in According to the ASTM C192, the concrete specimens were kept in their moulds for 24 h after mixing and then cured in water at a constant temperature of 20 °C until test. The test age of the specimen is 3, 7, and 28 days and the specimens were taken out of water for UPV testing for less than an hour during the curing days. The cubes were tested for compressive strength at 3, 7, and 28 days. The beam specimens were tested for the UPV measurements at 3, 7, and 28 days.The test age of compressive strength is 3, 7, and 28 days for all specimens (ASTM C39). The compression tests were subdivided into four major groups, i.e., cube specimens with no fibre, cube specimens contained 0.25% 30/50 mm fibre, cube specimens contained 0.50% 30/50 mm fibre, and cube specimens contained 1.00% 30/50 mm fibre. To decrease the error of the test, three cubes for each group were tested.The rebound hammer test is non-destructive testing that can measure the strength of finished concrete structures. The rebound hammer equipment is easy to carry, and the test procedure is simple. The rebound hammer can only estimate the compressive strength on the surface of concrete, and it is not very accurate in predicting the actual compressive strength of concrete. The rebound hammer test is also used to correlate the compression strength for UPV measurements .This method can only determine the relative strength of concrete, which is based on the hardness of the specimen at its surface. If the concrete is exposed to the atmosphere for more than three months, the results may overestimate as much as 50% because of carbonation at the concrete surface. In this research, the rebound hammer test was conducted at 3, 7, and 28 days. The UPV technique is one of the widely used and convenient methods that can provide test results at the lowest cost and rapid measurements. The UPV can determine the quality of concrete, as shown in The UPV is constant during the travel through the specimen unless the lateral dimension is too small and less than a certain minimum value. If the path is below a certain minimum value, the pulse velocity may be decreased, and the data may not be reliable. The ratio of the wavelength and the path dimension of the specimen relate to the reduction of velocity. Based on Age of concrete .Testing procedure .Fibre percentage .Fibre length (30 mm and 50 mm).Several factors can affect the compressive strength of concrete and some of those factors also influence the UPV. The relationship between these variables and the UPV will be investigated in this study. These variables are:Direct UPV measurements.The direct tests were conducted at the long side of each beam specimen as shown in B.Semi-direct UPV measurementsGenerally, the accuracy of semi-direct UPV measurements is less than the direct measurements. Still, the concrete members such as walls and columns can only use semi-direct or indirect UPV measurements due to the limitations in transducer placement. The path length between transducers was calculated using the Pythagorean theorem. The transducer in the length direction was placed in 100 mm spacing along the beam, as shown in C.Indirect UPV measurementsAlthough the indirect UPV measurements are poor sensitivity compared to the direct test, it is necessary to find the relationship between the UPV and the concrete properties by indirect UPV test because sometimes it is not possible to use the direct UPV measurements under some conditions. Both transducers are placed on the same surface of the FRC in this method, as shown in In this method, one transducer (Tx) was placed at position A and the other transducer (Rx) was placed at a distance of X1 away from position A, as shown in Compressive strength was measured at 3, 7, and 28 days as shown in After calculating the average of the six readings of each type of specimen on a particular test day, the rebound hammer test result is shown in The percentage change in strength relative to the plain concrete was calculated in each FRC specimen as shown in It is stated that when the compressive strength increases, the UPV increases. The UPV result follows approximately the same pattern for each kind of FRC at a given test age as shown in According to According to The semi-direct UPV is approximately constant at different ages of concrete, although there is a significant difference in the measured compressive strength, as shown in Direct, semi-direct, and indirect UPV measurements are important measuring methods, and they are directly affecting the pulse velocity values. The average direct UPV is higher than the average semi-direct and indirect UPV on all days. The average semi-direct UPV is higher than the average indirect UPV on all days for all kinds of specimens. The surface velocity (indirect measurements) is about half of the direct velocity for the beam with and without steel fibres. This relationship can be used to determine the Poisson’s ratio and dynamic elastic modulus of concrete.When a material is under compression, the length reduces in the longitudinal direction , and the length increases in the lateral direction (transverse) as shown in 3), v) Poisson’ ratio, The dynamic elastic modulus ( (3)–(5) . CombiniAccording to Equation (6), the dynamic Poisson’s ratio can be determined by measuring the UPV of compression and surface wave propagation. The UPV will change with the different properties of concrete such as its elastic stiffness and mechanical strength.It has been reported that the correlation between the static modulus value and the dynamic modulus value determined by UPV is good and the error oscillates between 10–15%. The error for the resonant test is about 11–14%, indicating the UPV is more accurate . Therefo3 was used for the FRC which is reasonable for normal aggregate concrete with steel fibres. In The density of 2500 kg/m(1)To obtain a certain relationship between the compressive strength of FRC, the number of experimental cases should be increased. It is recommended to use larger dimensions of specimens to increase the accuracy.(2)It is recommended to find correlation equations from the statistical analysis of direct, semi-directed, and indirect methods to determine the compressive strength of FRC.(3)The semi-direct results are low in confidence level. Still, good correlations between semi-direct tests and compressive strength are needed as the semi-direct test could be applicable in most situations.(4)Different steel fibre lengths and fibre types should be explored in UPV tests as there are various types available and applied in real concrete structures.(5)To increase the confidence level of UPV to measure the properties of FRC, fibre content could be decided more widely and also matched with real applications. 0.25%, 0.5%, 0.75%, 1% and 1.25% steel fibre content.In this paper, empirical results have been developed using the ultrasonic pulse velocity (UPV) test to estimate the values of the modulus of elasticity and Poisson’s ratio of concrete reinforced with steel fibres. The results of the study showed that it is essential to select the setup of the non-destructive test that should be applied  since the expected results depend on this setup. It is also necessary to identify the wave types logged by the test equipment because (1)In the long term, the effect of fibre on the rebound hammer result is minimal, while in the short term, the effect is significant. This can confirm that the rebound hammer test is unsuitable for obtaining compressive strength data in FRC early.(2)The volume fraction of steel fibres seems ineffective in increasing the UPV. However, longer steel fibres can slightly increase the UPV measurements. The UPV in specimens with 50 mm long fibres is larger by 5.7%, 3.8%, and 2.0% at the age of 3, 7, and 28 days, respectively, in comparison to the specimens with 30 mm long fibres. This implies that the fibre content does not affect the measured UPV in matured concrete.(3)The UPV and rate of strength gain in FRC have a significant difference at an early age of concrete. Both UPV and compressive strength increase when the fibre content increases without changing the fibre length. For the same fibre content, 50 mm FRC shows a higher UPV value and compressive strength than FRC with 30 mm fibre.(4)The sequence of UPV from largest to smallest is direct UPV, semi-direct UPV, and indirect UPV, respectively. The direct UPV is approximately two times that of indirect UPV. The accuracy of semi-direct UPV is relatively lower.(5)The ratio between surface velocity and direct velocity can be used to determine the dynamic modulus of elasticity and Poisson’s ratio of the concrete. If the fibre content increases, the dynamic elastic modulus increases, and the Poisson’s ratio decrease for the same length of steel fibre. The modulus of elasticity is higher for the 50 mm steel FRC than the 30 mm steel FRC at the same percentage of steel fibre.


A 76-year-old man receiving maintenance therapy with oral steroids for immunoglobulin G4 (IgG4)-related disease presented to our hospital with the chief complaint of visual disturbance. His best corrected visual acuities of the right and left eye were 1.2 and 0.7, respectively. Humphrey visual field test revealed inferior auriculotemporal one-quarter blindness in the left eye. After detailed history-taking for IgG4-related disease, clinical diagnosis based on imaging revealed the marked pituitary/pituitary stalk enlargement with associated optic chiasm compression. Based on the history and initial evaluation findings, a diagnosis of IgG4-related ophthalmic disease was made. Intensified steroid therapy was performed, which led to symptom resolution. IgG4-related diseases are considered in the differential diagnosis when bilateral hemianopsia is observed. When unilateral visual acuity and visual field defects are present, IgG4-related diseases and other organ disorders should be considered. Immunoglobulin G4 (IgG4)-related disease is an autoimmune disease of unknown etiology in which IgG4-positive plasma cells infiltrate the body organs and cause swelling, nodules, and hypertrophic lesions in target organs . IgG4-reTwo major pathological conditions can cause optic neuropathy in this disease: compression or encasement of the optic nerve by an enlarged tissue in the orbit and compression of the optic chiasm by the enlarged pituitary gland. In the former case, compression or encasement of the optic nerve by the enlarged extraocular muscle or localized or diffuse tissue proliferation in the orbit has been reported . The latA 76-year-old man visited his family physician at the end of April 2021 due to a visual disturbance in the left eye and was referred to our department on May 27, 2021. Ophthalmological history included bilateral cataract surgery, left ptosis (both in 2017), intrascleral fixation of left intraocular lens dislocation (in 2020), Irvine-Gass syndrome, and ocular hypertension. Detailed history-taking revealed that the patient received treatment for IgG4-related disease  from his primary care physician.At the initial visit on May 27, 2021, there was no conjunctival hyperemia in both conjunctivae, and anterior chamber cells were occasional in the left eye. Pupillary reflexes were not disturbed and relative afferent pupillary defect (RAPD) was not noted in the left eye. The left optic nerve papilla was mildly erythematous and swollen. Best corrected visual acuities were 1.2 in the right eye and 0.7 in the left eye, and intraocular pressures were 18 mmHg in the right eye and 16 mmHg in the left eye. Critical flicker frequency (CFF) was decreased in the left eye , and Humphrey visual field test revealed inferior arcuate quadrantanopia in the left eye  showed no abnormal signal in the intraorbital optic nerve; however, marked pituitary/pituitary stalk enlargement with associated optic chiasm compression was observed Figure .Based on the history and initial evaluation findings, a diagnosis of left optic neuropathy associated with pituitary/pituitary stalk enlargement caused by IgG4-related disease was made. He was introduced to the hospital of endocrinology and metabolism internal medicine expert and was diagnosed with panhypopituitarism due to pituitary funnel inflammation. Then, the dose of prednisolone was increased to 20 mg/day. On June 10, visual acuity and visual field defects improved Figure .Upon examination on July 7, the size of the pituitary gland and pituitary stalk decreased Figure .The CFF decreased  (39 Hz). The steroid dose was gradually decreased, and maintenance therapy with 2.5-mg prednisolone daily was resumed on August 11.In this case, no optic nerve swelling or signal changes were evident on imaging, but fundus examination revealed mild erythema of the optic nerve papilla. Although this finding may be attributed to inflammatory cytokine production in the eye by iritis, optic neuritis was likely to be caused by an autoimmune mechanism in IgG4-related disease. However, the visual field findings were consistent neither with those commonly observed in optic papillitis nor pituitary enlargement. Moreover, brain MRI findings suggested optic chiasm compression due to pituitary enlargement. However, judging from our MRI data, the relationship of the optic chiasma to the pituitary gland was unclear and remained to be elucidated. The patient’s visual acuity and visual field defects improved after steroid treatment, suggesting that enlargement of the pituitary gland and pituitary stalk was the main pathophysiology of vision loss and visual field defects.The ophthalmologic clinical hallmark of IgG4-related ocular disease is bilateral lacrimal gland enlargement with three features: suborbital nerve enlargement , exophthAlternatively, as other studies have shown infiltration of IgG4-positive plasma cells around the trigeminal nerve in the same disease [IgG4-related disease, an autoimmune disease of unknown etiology, leads to vision loss and visual field defects. Compression or encasement of the optic nerve by an enlarged tissue in the orbit is reported to be the major cause of visual disturbance. However, the possibility of optic chiasm compression by the enlarged pituitary gland should be kept in mind.