SIFT, PROVEAN, Condel, and PolyPhen-2, which together increased the accuracy of prediction and identified 18 highly pathogenic non-synonymous SNPs (nsSNPs) out of 607 SNPs. Subsequently, these 18 high-confidence pathogenic nsSNPs were analysed for BLM protein stability, structure–function relationships and disease associations using various bioinformatics tools. These 18 mutants of the BLM protein along with the native protein were further investigated using molecular dynamics simulations to examine the structural consequences of the mutations, which might reveal their malfunction and contribution to disease. In addition, 28 SNPs were predicted as “stop gained” nonsense SNPs and one SNP was predicted as “start lost”. Two SNPs in the 3′UTR were found to abolish miRNA binding and thus may enhance the expression of BLM. Interestingly, we found that BLM mRNA overexpression is associated with different types of cancers. Further investigation showed that the dysregulation of BLM is associated with poor overall survival (OS) for lung and gastric cancer patients and hence led to the conclusion that BLM has the potential to be used as an important prognostic marker for the detection of lung and gastric cancer.The BLM helicase protein plays a vital role in DNA replication and the maintenance of genomic integrity. Variation in the BLM helicase gene resulted in defects in the DNA repair mechanism and was reported to be associated with Bloom syndrome (BS) and cancer. Despite extensive investigation of helicase proteins in humans, no attempt has previously been made to comprehensively analyse the single nucleotide polymorphism (SNPs) of the BLM gene. In this study, a comprehensive analysis of SNPs on the BLM gene was performed to identify, characterize and validate the pathogenic SNPs using computational approaches. We obtained SNP data from the dbSNP database version 150 and mapped these data to the genomic coordinates of the “NM_000057.3” transcript expressing BLM helicase (P54132). There were 607 SNPs mapped to missense, 29 SNPs mapped to nonsense, and 19 SNPs mapped to 3′-UTR regions. Initially, we used many consensus tools of  In a complex with topoisomerase Topo IIIa and Rmi1/Rmi2, BLM helicase repairs, double-strand DNA breaks (DSBs) through a homologous recombination (HR) pathway4. Consequently, cells lacking functional BLM show an about tenfold increase in chromatid breaks, mitotic recombination, and sister chromatid crossover formation5. Bloom syndrome (BS) is a rare autosomal recessive genetic disorder caused by pathogenic variants in the BLM gene. BS belongs to OMIM  entry 210900, which is characterized by genome instability that includes increased crossovers between homologous chromosomes6. The BLM gene is transcribed to a 97.93 kb-long precursor-mRNA with 21 exons, which encode a 1,417 amino acid protein. The literature shows that a large number of BS patients show insertion, deletion and missense mutations that change the amino acid sequence or nonsense mutations that introduce a premature stop codon in the BLM gene and thus inactivate the BLM helicase9. Symptoms of BS include low birth weight, dolichocephaly , congenital short stature, growth retardation, sun-sensitive facial rash, an elevated risk of diabetes mellitus, reduced fertility and immune deficiency12. The absence of BLM protein activity causes a defect in DNA repair with a consequent increased rate of mutations and thus poses an elevated risk of cance15. The average life span of BS patients is approximately 27 years, with the most common cause of death being cancer (https://weill.cornell.edu/bsr/).The BLM gene encodes an important nuclear protein, BLM helicase, which is involved in DNA replication and the maintenance of genomic integrity. BLM is a 3′ to 5′ DNA helicase that belongs to the evolutionarily conserved RecQ helicase family. Most mammals have five RecQ helicases . Helicases are crucial for unwinding duplex DNA to produce the transient single-stranded DNA (ssDNA) intermediates necessary for replication, recombination, and repair6. SNPs can alter the functional consequences of proteins. In the coding region of genes, SNPs may be synonymous, non-synonymous (nsSNPs) or nonsense16. Synonymous SNPs change the nucleotide base residue but do not change the amino acid residue in the protein sequence due to the degeneracy of the genetic code. The nsSNPs, also called missense variants, alter amino acid residues in protein sequences and thus change the function of proteins through altering protein activity, solubility and protein structure. Nonsense SNPs introduce premature termination in the protein sequence.Single nucleotide polymorphisms (SNPs) are a common genetic variation contributing greatly to phenotypic variation in the general populationcis-elements, such as miRNA binding sites, that can also affect the regulation of gene expression20. SNPs have emerged as genetic markers for diseases, and there are many SNP markers available in public databases. Previous reports have shown the value of defining mutations as deleterious or non-deleterious and their connection with certain diseases, thus identifying pathogenic SNPs that are functionally compromised due to structure-damaging properties27.The non-coding region of the gene contains several regulatory 34.In recent decades, the computational approach has become established as an effective method that streamlines time consuming, laborious experimentation and allows researchers to shortlist the most critical or pathological SNPs. This ability leads us to focus on selectively targeted SNPs instead of scanning full genes for the identification of pathological SNPs by experimental mutational analysis. Computational studies have also evaluated and analysed genetic mutations for their pathological effects and are effective in establishing the underlying molecular mechanism11, no attempt was previously made to comprehensively and systematically analyse and establish the functional consequences of SNPs of the BLM gene. The aim of this study was to identify the high-confidence pathogenic SNPs of the BLM gene and to determine their structural, molecular and functional consequences using computational approaches. This work may be useful in the development of precision medicine-based treatment for diseases caused by these genomic variations. In the future, this report can be used for biomarker discovery by establishing the importance of SNPs for the diagnosis of Bloom syndrome, as well as cancer, and will further aid in targeted therapies.With recent advances in high-throughput sequencing technology, hundreds of new SNPs have been mapped to human BLM genes. However, not all SNPs are functionally important. Despite extensive studies of helicase proteins in humans and the effect of their polymorphisms in cancer41. Earlier studies support the notion that the application of various powerful tools and algorithms leads to increased prediction accuracy45. To ensure that the results are of the highest accuracy, we utilized several computational algorithms that can be used for the prediction of nsSNPs of BLM helicases because of their disease-related properties. For this purpose, we used tools such as SIFT15, PROVEAN46, Polyphen-2.047, SNAP2-248, Muscle49, Weblogo51, SNP&GO52, nsSNPAnalyzer53, and Mutpred-254. This approach enabled greater accuracy for the prediction of most disease-associated mutations in BLM genes and their structural consequences.Application of computational methodological approaches for acquiring biological insight is well established12. We used “BLM” as our search term and filtered for SNPs (https://www.ncbi.nlm.nih.gov/variation/view/?q=BLM). Furthermore, we mapped these SNPs to the genomic coordinates of the “NM_000057.3” transcript expressing the BLM helicase (P54132) for computational analysis of the effect of missense variants and nonsense SNPs as well as SNPs in the UTR region. The protein sequences of the genes for BLM (P54132) were retrieved from the UniProt database (https://www.UniProt.org). We employed various sets of computational tools that together increased the accuracy and reliability of the identification of pathogenic SNPs and their effects on the structural and functional consequences of BLM , which predicted an amino acid substitution score from zero to one. SIFT predicted substituted amino acids to be damaging at a default threshold score < 0.05, while a score ≥ 0.05 was predicted to be tolerated.An algorithm that predicts tolerant and intolerant coding base substitutions based upon amino acid properties and sequence homologyhttps://provean.jcvi.org/) that uses an alignment-based scoring method for predicting the functional consequences of single and multiple amino acid substitutions and in-frame deletions and insertions46. The tool has a default threshold score, i.e., -2.5. If a protein variant is below the threshold, it is predicted as deleterious; above that threshold, a protein variant is considered neutral.An online tool (https://bbglab.irbbarcelona.org/fannsdb/) that predicts the consequences of non-synonymous SNPs as neutral or deleterious55. It uses a consensus deleteriousness (Condel) score calculated by integrating the normalized score of five predictive tools: SIF15, PolyPhen-247, Logre56, MAPP57, and MutationAssessor58. The Condel score could vary between 0 to 1, where a higher score indicates SNPs as deleterious.A tool (47. The prediction of the PolyPhen-2 server (https://genetics.bwh.harvard.edu/pph2/) is based on a number of features, including structural and sequence comparison information. Its score varies between 0.0 (benign) and 10.0 (damaging). The PolyPhen-2 prediction output categorizes SNPs into three basic categories: benign (score < 0.2), possibly damaging (score between 0.2 and 0.96), and probably damaging (score > 0.96).A tool that predicts the structural and functional consequences of a particular amino acid substitution in a human protein49, and all the respective sites for human BLM helicase SNPs were extracted from the alignment using an in-house Perl script. The sequence logo for the SNP positions was prepared using Weblogo v.2.8.251. A sequence logo is a graphical representation of a multiple sequence alignment, whereby a stack of amino acid symbols corresponds to a column position in the alignment. Within a stack, the varying heights of amino acid symbols show the relative frequency of each amino acid at that position.BLM helicase sequences were retrieved from the UniProt protein sequence database using a search option with the key words ‘BLM helicase’. The sequences containing the key words ‘Bloom’ and/or ‘Blm’ with the names were considered, and then the fragments and irrelevant sequences were removed. Finally, 153 sequences were obtained and considered for multiple sequence alignment. The multiple sequence alignment was performed by Muscle v.3.8.31https://snps.biofold.org/snps-and-go)52. It is a support vector machine (SVM)-based tool that considers protein sequence features, evolutionary information, and functional annotation according to Gene Ontology terms. We input the Swiss-Prot Code of BLM helicase (P54132) and provided the list of amino acid mutations. The results predicted whether helicase polymorphisms would be disease associated or not by three methods: (a) SNPs&GO, (b) PhD-SNP, and (c) PANTHER. A probability score > 0.5 is predicted as a disease-associated variation.A webserver that predicts whether an amino acid substitution is associated with a disease or not 53. It can predict (a) the structural environment of SNPs; (b) the normalized probability of substitution in the multiple sequence alignment; and (c) the similarity and dissimilarity between the variant and the original amino acid.A random forest classifier developed using curated SNP datasets from SwisProt to predict the phenotypic effects of nsSNPs (https://rostlab.org/services/snap/)48. SNAP2 incorporates various features, including evolutionary similarity from multiple sequence alignments and secondary structure and solvent accessibility, to predict whether a substitution is likely to alter the protein effect. It predicts a score from -100, considered strongly neutral, to + 100, considered as a strong effect. A threshold score > 0 is considered an effect. We input the protein sequences of BLM helicases and obtained the score of SNPs.A tool used to predict the functional consequences of non-synonymous SNPs using a neural network (https://mutpred.mutdb.org/)54. It is programmed on 53,180 pathogenic and 206,946 putatively benign amino acid substitutions from HGMD, Swiss-Prot, dbSNP, and orthologous alignments. It also incorporated the impact of amino acid substitutions on over 50 different local structural and functional protein properties and thus helped to infer the molecular mechanisms of pathogenicity. The outcome of MutPred2 includes the following: (A) a general pathogenicity score (g), which is the likelihood that a substituted amino acid is pathogenic; (B) predicted molecular mechanism; (C) property score (pr) of molecular mechanism and its P-value (P); and (D) affected PROSITE and ELM Motifs. General scores vary from 0.0 (benign) to 1.0 (pathogenic), where with g ≥ 0.5 would suggest pathogenicity; however, g ≥ 0.68 yields a false positive rate (fpr) of 10%, whereas g ≥ 0.80 yields an fpr of 5%. The higher the property score (Pr), the more likely that the molecular mechanism of the disease involves the alteration of the property.A neural network-based method to predict the molecular and phenotypic impact of amino acid variants as pathogenic or benign in humans . The WT structure consists of the BLM protein  bound to 1 ADP molecule, 1 Ca2+ ion, 1 Zn2+ ion, and a 3′-overhang DNA duplex. A few residues were missing in the crystal structure: 799–807, 1,011–1,013, 1,069–1,071, 1,093–1,104, 1,195–1,206, and 1,292–1,298. We modelled these residues using Modeller9v1561. The modelled structure was minimized and refined using simulations. Corresponding point mutations were introduced in the obtained structure using Modeller9v1561 to generate mutant structures. For the simulations, the protein and nucleic acids were represented by Amber forcefields: ff14SB62 and DNA (OL15)63, respectively. For bound ADP and two ions, GAFF charges provided with the AMBER forcefields were used. The modelled systems were solvated in a cubic box with TIP3P potential at a 10 Å marginal radius and neutralized with Na+ or Cl− ions. Long-range interactions were modelled using particle mesh Ewald (PME) with a tolerance of 1e-05 and a grid spacing of 1.2 Å. A non-bonded cut-off of 12 Å was applied to the Lennard–Jones potential, and the cut-off for the direct-space part of the Coulomb forces was a switching function starting at a distance of 10 Å and reaching zero at 12 Å. A 2 fs time step was used for all simulations while constraining bonds involving hydrogens with the Lincs algorithm. Periodic boundary conditions were employed to eliminate surface effects. To reach the desired temperature and pressure, minimized systems were simulated for 5 ns while coupled to a v-rescale thermostat and Parinello-Rahman barostat implemented in Gromacs2016.564. The equilibrated structures of the WT and 18 mutants were simulated further for 20 ns each.The coordinates of the native human BLM helicase were retrieved from the PDB database with PDB id ‘4O3M’65. Root mean square deviations (rmsd) and radius of gyration values for all backbone atoms were calculated with respect to the crystal structure. Root mean square fluctuations (rmsf) of Cα atoms in all trajectories were also calculated. The changes in the secondary structure of proteins during the course of simulation were analysed using the DSSP program as implemented in the Bio3D package written in R68. Domain-wise rmsd values were calculated for each domain after aligning the rest of the protein.Structural analyses were performed using built-in programs of Gromacs2016.5 and VMD-1.9.3https://kmplot.com/analysis/)69. The tool analyses the effect of 54,675 genes on the survival outcome of patients using 10,293 cancer samples from the Affymetrix microarray data in the Gene Expression Omnibus (GEO: https://www.ncbi.nlm.nih.gov/geo/), the European Genome-phenome Archive (EGA: https://ega.crg.eu/) and The Cancer Genome Atlas (TCGA: https://cancergenome.nih.gov/) databases. We analysed the potential effect of BLM gene expression on overall survival in a large number of cancer patients, including patients with gastric , ovarian (1816), lung , and breast  cancer. The hazard ratio (HR) with 95% confidence intervals and log rank P-value (below 0.05 were considered significant) were calculated. Biased arrays were excluded for quality control.Kaplan–Meier plots were analysed using online KM plotter software (https://gepia.cancer-pku.cn/index.html)70. The tool uses expression data from 8,587 normal and 9,736 tumour samples obtained from the TCGA and the Genotype-Tissue Expression (GTEx) projects. The mRNA expression on box plot was calculated using “Expression DIY” module using parameters: Gene “BLM”; |Log2FC| cut-off “1.5”; p-value cut-off “0.01”, and match TCGA normal and GTEx data.A web-based tool for analysing the differential expression of mRNA in tumour and normal cells ((https://compbio.uthsc.edu/miRSNP/)71. It was used to examine the impact of SNPs on miRNA-target (BLM) binding and on BLM gene expression.A database of variants in miRNA and miRNA target sites 72 and is frequently used to filter for the most potential deleterious nsSNPs in target genes to understand the aetiology of various diseases75. Computational analysis can provide molecular insight into the changes in protein structure and function due to point mutations in the target genes. In the current study, we report nsSNPs in BLM genes, which are most likely associated with pathogenic conditions involving BLM and other BS-associated diseases, such as cancers. Our findings suggest that the application of multiple powerful algorithms to SNP datasets identifies the most deleterious SNPs, which might be associated with diseases. The accuracy of nsSNP prediction was also supported by various reports discussed below.The application of computational biology in genome research is well establishedFor this study, we retrieved 11,983 rsIDs of SNPs mapped in the human BLM gene from dbSNP Table . Howeverhttps://www.ensembl.org/Tools/VEP). VEP has advantages, such as using the latest human genome assembly, GRCh38.p10, and predicting thousands of SNPs from multiple tools, including SIFT, PROVEAN, Condel, and PolyPhen-2, to retrieve accurate results. We uploaded the 597 nsSNP accession numbers to the VEP tool, and the prediction results were used for further analysis.To predict the damaging nsSNPs, we employed multiple consensus tools. Initially, we used the online tool VEP (Condel (score > 0.522), which showed 136 nsSNPs as damaging (Table SIFT (score = 0), PROVEAN (score < -8.0) and Condel (score > 0.9), we obtained 18 nsSNPs (Table We found 28 nsSNPs predicted as “stop gained” and one nsSNP as a “start lost” mutation Table . The remng Table . To obtaPs Table .Table 1L). The reported 18 nsSNP variants were looked into the sequence logo, and three of them, W803R, H805L, and G891V, were found to naturally occur in other sequences; however, their frequency was very low. The two positions G952 and G978 were found to be 100% conserved, as shown in the sequence logo. The complete sequence alignment of BLM helicases is also provided in Supplementary_file1. Afterwards, these 18 high-confidence nsSNPs were further assessed for functional and structural consequences using different bioinformatics tools.The sequence logo for nsSNP positions in BLM helicase sequences show that these positions are highly conserved among various species Fig. . The rep77. Therefore, we analysed these nsSNPs in terms of amino acid substitutions and their effect on the stability of mutant BLM proteins by IMutant2 (https://folding.biofold.org/i-mutant/i-mutant2.0.html) and PoPMuSiC v3.1 (https://soft.dezyme.com) tools79.Studies have shown that most disease-associated missense mutations change the stability of proteinsBoth IMutant2 and PoPMuSiC predict the effect of mutations on proteins based on protein 3D structures. Because the whole protein 3D structure of the BLM gene is not yet available, partial protein structures, such as PDB id 4O3M with a length of 640–1,290 amino acids, i.e., 613 amino acids long, were used for analysis. We submitted the 18 amino acid substitutions to IMutant2 and PoPMuSiC, which predicted the stability of the BLM protein variants. We found that three mutants  and two mutants (P702L and G891V) were predicted to stabilize the mutant proteins by IMutant2 and PoPMuSiC, respectively (Table https://snps.biofold.org/snps-and-go/snps-and-go.html). The predicted disease associations from three different tools were analysed. The output of (a) SNPs&GO and (b) PhD-SNP predicted that all the tested SNPs were associated with diseases, while (c) PANTHER predicted 16 SNPs as disease associated and two as neutral  at the default threshold for cancer-promoting mutations and disease-causing mutations80. The output results of these tools showed that two (G952V and G978V) were associated with cancer , as well as in RecQ helicases in bacteria and yeast. BLM mutants lacking the HRDC domain possess core helicase and ATPase activities similar to those of the wild-type protein84 but are defective in both strand annealing74 and double Holliday junction dissolution83.Bloom syndrome proteins comprise three domains: ATPase domain , RecQ family-specific C-terminal (RQC) domain , and Helicase and RNase D C-terminal (HRDC) domain . The C-terminus of the ATPase domain, known as the Zn subdomain, consists of Zn-binding residues . One ADP molecule is observed to be bound to the inter-subdomain cleft between two ATPase subdomains, 1A (642–857) and 2A . In the BLM WT crystal structure, the duplex region of DNA is bound to the RQC domain surface via an unconventional winged-helix domain. This interaction differs from that known in conventional winged-helix domains, where the minor and major grooves of dsDNA are recognized by one recognition helix and β wingWe evaluated the 18 predicted deleterious amino acid substitutions for possible damage to the 3D structure of the BLM helicase using molecular dynamics simulations. When these SNP positions were mapped onto the structure of the BLM helicase Fig.  and rmsf85. Modelling studies of the BLM residues confirm that P690 along with R717, W803, and Y811 are located in the first lobes of the helicase domain. Among the point mutations of BLM, P690L does not affect the extremely conserved GK(T/S) residues of motif I . Another report from Shashtri et al. showed, using a HU-based hypersensitivity-based study, that the P690L mutant of the BLM protein was unable to rescue the DNA damage response in the BS cell line compared to the WT5. The reason behind the loss of function could be due to leucine (hydrophobic) replacing proline (helix breaker), which may culminate in reduced flexibility and hydrophobicity, causing weakened dsDNA binding and altering the location of the dsDNA within the motif, which leads to failure to bind ATP and/or Mg2+85.Mutations P690L and P702L are present in ATPase domain 1A, near the ADP binding pocket. Our computational results suggest that their properties are similar to those of the WT. However, Mirzaei et al., reported six mutant residues  that cause loss of function for the BLM protein89. Because it is situated as a key residue in a loop region, it plays a crucial role in various functions. Another set of mutations, W803L, W803R, H805L, Y811C, P825L in ATPase domain 1A show similar dynamics as WT. W803, a residue that is conserved in all five human RecQ homologs , suggests that this residue plays a key role in helicase activity. Hence, the W803R mutation might hamper helicase activity, which is one of the major causes of BS development. Though our molecular dynamics simulation-based study suggests similar dynamics to those of the WT, other studies suggest that these residues are crucial for the above-mentioned activities. Hence, we also speculate that this mutation may lead to a deterioration in BLM activity, which may lead to the development of BS. The downstream Y811C and P825L mutations showed no structural differences as WT. Though there is no evidence for P825 or Y811, it is identified as a key residue for the functioning of BLM proteins98. Another report5 suggested based on HU hypersensitivity that, similar to P602L, the Y811C mutant of the BLM protein was unable to rescue the DNA damage response in the BS cell line compared to the WT.Studies from Mirzaei et al. showed that the W803 mutation leads to impaired BLM function associated with Bloom syndrome. This mutation site is located in the highly conserved region called the aromatic-rich (AR) loop and is crucial for ATP binding, hydrolysis, DNA binding, ssDNA binding and unwinding90, also endorsed our results, which suggests that the G891 mutation along with Q672R, I841T and C901Y inactivates the helicase domain8. There are also point mutations, such as K803A, Q680P and I849T, in mouse BLM91. All these point mutations hamper ATPase and DNA unwinding activity. G972V mutation, in the N terminal region of BLM, may hamper the replicative role of BLM as reported by Selak et al.92, a major issue in Bloom Syndrome97. 972 is proximal to the nucleic acid overhang. Mutating G972 to valine pushes the nearby K968, which is directly interacting with the nucleic acid overhang, and this further affected the downstream RCQ domain and to some extent to the Zn subdomain, as observed from the domain-wise rmsd values. To depict that the nsSNP leads to full inactivation of the BLM protein, Mirzaei et al.85, exploited a yeast model system by designing a chimera BLM gene consisting of the yeast and human BLM genes. The introduction of nsSNP into the chimera BLM leads to impaired BLM function, which was examined and confirmed by a hypersensitivity assay of cells to hydroxyurea (HU), a DNA-damaging agent85. The hypersensitivity assay showed that six-point mutations  cause total loss-of-function of BLM, and three cause partial loss-of-function . The location of G972 is in close proximity to the arginine finger; therefore, this mutation might impair coordination among the ATP binding of lobe 1 with lobe 2. G978V mutation resulted in a change in the backbone orientation of the ATPase domain 1B, induced structural deviations in this interface area, as well as in the Zn subdomain, and influenced the downstream HRDC domain . The structural influence of these mutants can be observed by larger variations in domain-wise rmsd plots Fig. . A repor98.Another set of mutations, Y974C and Y1044C in ATPase 1B domain, showed no significant destabilizing structural effects during the sub-nano seconds simulations. Y974 is present at the interface, pointing towards the cleft between ATPase domains 1A and 1B and forming stacking interactions with F1045 of the nearby helix. In the native structure, Y1044 forms hydrogen-bonding interactions with E971 of the nearby helix, and this interaction is maintained throughout the simulations of the WT. Mutating tyrosine to cysteine disrupts these hydrogen-bonding interactions but results in another set of stable interactions with S1025 of a nearby helix, thus maintaining the structural dynamics of the BLM. Although our computational results suggest that Y1044C is similar to the WT, other studies suggest that Y1044C is present along with S897C in Japanese male patients and white female patients with metachronous colon cancer in the C-terminal of BLM in CRC. This issue can be further explored in the future using BS cells and model organisms. Highly conserved BLM variants were reported to be found in the C-terminal helicase domain, which may lead to a predisposition to hereditary CRC99. This lower binding of BLM with counterparts of the DNA region may lead to disruption in the functional outcome. This may result in a predisposition to many unwanted mutations and thus to Bloom syndrome. A comprehensive site-directed mutational analysis by Guo et al.100, suggests that there is a significant reduction in zinc binding ability, which further indicates that these highly conserved cysteine residues are essential for Zn2+ ion binding and thus for BLM activity. The effect of cysteine residue mutation on DNA binding ability was shown by Guo et al.100, using a gel shift assay, which indicates that there was a significant reduction in DNA binding. This result suggests that conserved cysteine residues are key to the function of zinc binding domains, which are essential for the DNA helicase and ATPase activity of BLM. Therefore, the study suggests that DNA-binding ability is compromised due to this mutation100. Another study also showed, using the chimera mutational approach, that mutating C1055 leads to hypersensitivity to hydroxyurea (HU)100.In the Zn subdomain, highly conserved residues C1036, C1055, C1063, and C1066 form interactions with the Zn atom in the BLM protein. C1036F mutation influenced the RQC domain. In the native structure, C1055 interacts with the Zn atom, along with C1063. For the C1055R mutation, when the conformations of mutants after simulation are visualized with respect to the native structure, arginine being positively charged shows repulsion with positively charged Zn and destabilization of the domain Figs. , 5, 6. T85. C1066 is one of the four cysteines binding the Zn atom in the Zn subdomain. Our in silico results suggest that there is no effect because of the C1066Y mutation, but another report using site-directed mutational analysis suggests  that there is a significant reduction in the zinc-binding ability, which further indicates that these highly conserved cysteine residues are essential for Zn+2 ion binding for subsequent BLM activity100. A report from Shashtri et al. noted that C1066Y is crucial for Zn coordination, which is required for helicase activity and consequently for the development of BS5.D1046 is present within the Zn subdomain and forms hydrogen-bonding interactions with the nearby R1037. This interaction is maintained intermittently during the simulations of the native structure. Though hydrophilic and negatively charged residues have been mutated to neutral and hydrophobic valines, the mutation occurred at the periphery of the protein, thus inducing local perturbations in the structure, and did not significantly affect the overall structural dynamics of the helicase except for the RQC domain Fig. . The los100. For some mutations, however, local disruptions are observed in subdomains but can result in a complete loss of function. For instance, the mutation of cysteines  and glutamate (D1064V) in the Zn binding subdomain, which results in the loss of Zn binding upon mutation and thus hinders the functioning of BLM helicase, was also supported by other reports100. However, in all the mutations, we did not find considerable changes in the binding of DNA. This result may be attributed to the fact that the mutations are performed in the ATPase domain, and the simulation timescales in this study are not long enough to observe large allosteric effects. For the mutations G891V, G952V and G978V, however, we did find larger deviations in RCQ domain binding and in the C-terminal HRDC domain, as indicated by rmsd values, thus suggesting the possibility of allosteric effects of mutations in the ATPase domain in nucleic acid binding. This conclusion was further strengthened by reports suggesting a role for cysteine residues in the Zn-binding domain in helicase activity. The mutation of cysteine hampers BLM helicase activity, which may be responsible for the development of BS100. The overall secondary structure remained consistent throughout the simulations of mutants as well as of the native structure within the simulated timescale  is associated with lower overall survival for lung and gastric cancer patients, whereas we did not find a significant association between expression of the BLM gene and overall survival for breast and ovarian cancer patients Fig. .Figure 8101. However, another study found that BLM mRNA overexpression is associated with poor survival of breast cancer patients102. Interestingly, the same study reported that subcellular localization of the BLM protein is found to be high in the cytoplasm compared to the nucleus, although the analysis of normal breast tissue revealed that BLM protein is strongly localized in the nucleus and not the cytoplasm102. It might be possible that variants of the BLM gene express a truncated BLM, which lacks signals for protein localization.Mutation in the BLM gene in BL cells showed a reduction in the expression of its mRNA and protein, resulting in excessive chromosome instability and a high frequency of sister chromatid exchanges (SCEs)Therefore, this study also indicates the importance of BLM gene expression as a better prognostic marker for the detection of gastric and lung cancers. Studies have found that nsSNPs can affect the functional activity of proteins; therefore, we expect that the 18 nsSNPs identified here have functional effects, as in BLM deregulation.To understand the role of the BLM gene in cancer, we studied the its expression levels in different cancers. A box plot was generated using GEPIA , which showed that the expression of BLM significantly increased in different cancer patients compared to normal expression Fig. . Therefo19. On the other hand, SNPs in the 3′UTR region of mRNA can introduce new binding sites for other new miRNAs. We analysed the impact of gene regulation by miRNA due to SNPs in BLM mRNA using PolymiRTS v3.0. A list of 17 #rsIDs mapped to the 3′UTR and was fed into the PolymiRTS server, which yielded a list of binding miRNAs affected by these polymorphisms. The results showed that the binding of hsa-miR-6507-5p and hsa-miR-3976 to BLM mRNA was abolished due to polymorphisms rs116293756 and rs28363374, respectively  against disease-causing SNPs would be a very promising therapeutic approach to suppress the overexpression of the BLM mutant gene105.Earlier studies have shown the role of SNP in the 3′UTR of mRNA, which may lead to the partial or complete attenuation of complementary binding of miRNAs to the 3′UTR region106. Subsequently, RAD51 recombinase assembles at the ssDNA breakpoint and catalyzes the HR and DNA repair. During the HR process, invasion of ssDNA into the homologous sequence, typically a sister chromatid that works as a template, synthesizes the damaged DNA. At the last stage of HR, BLM forms a complex along with other enzymes and helps to separate the repaired DNA from the template DNA strand, and in this way BLM helps to maintain the genomic integrity107.DNA damage during the replication process is corrected by the HR pathway. The HR starts with the degradation of 5′-terminal of DSBs, which generates 3′-overhang ssDNA109. Therefore, BLM works as a ‘anti-recombinase’ at early and late stages of HR by: (i) preventing HR by disrupting the RAD51 assembly on the ssDNA of DSBs to make sure that HR only occurs between sequences with high homology; and (ii) preventing SCEs by separating the repaired DNA from the template DNA strand that arises at the final stages of HR111.Biochemical studies showed that the deletion of BLM enhances the assembly of RAD51 at the ssDNA break site, while overexpression of BLM disrupts the RAD51 assembly through its helicase activity112. The loss of RAD51 leads to insufficient HR resulting in genomic instability and DNA damage. However, defective BLM increases the hyperaccumulation of RAD51 at DNA damage site resulting in a high level of HR and high frequency of SCEs112. Therefore, our study supports previous findings that an aberration in the activity of BLM through pathogenic nsSNPs or high expression of BLM  have been reported in the literature to have a partial effect on helicase activitySince the MD simulation-based results suggest that G978V is pathogenic but no experimental evidence suggests its role in BS, future studies may sequence this pathogenic mutation site, especially in BS patients and other related diseases, and/or study it using mutational approaches using various models. Most of the pathogenic nsSNPs listed in the BLM protein suggest hampering the activity, such as ATP hydrolysis, DNA binding, and DNA unwinding, which is associated with various cellular procedures, such as replication, recombination, and DNA repair. BLM protein interacts with various factors for proper DNA replication and repair of DNA damage using the HR pathway. Turbulence during such a procedure always leads to the incorporation of erroneous copying of genomic information, giving rise to genomic instability and consequently leading to neoplastic transformation. Further future studies of BLM protein-related pathological nsSNPs will allow us to manage BS and its associated diseases. Figure Our study found 28 nsSNPs as “stop gained” and one nsSNP as “start lost” mutations, which can express a truncated form of BLM with the loss of important domains and nuclear signals and thus strongly localized BLM in the cytoplasm instead of the nucleus. As other studies have found, cancer cells have high cytoplasmic BLM, and it would be interesting to experimentally validate the nsSNPs with BLM protein localization in cancer cells. Furthermore, our investigation found that two SNPs, rs116293756 and rs28363374, at the 3′UTR of BLM abolished the binding affinity with the miRNAs hsa-miR-6507-5p and hsa-miR-3976, respectively, and thus enhanced the expression of BLM. Interestingly, we also found that BLM expression was significantly higher in some cancers including BLCA , COAD (Colon adenocarcinoma) and LUSC (Lung squamous cell carcinoma) (Fig. In the future, our selected nsSNPs in the BLM gene can be further studied in different populations to explore and validate the contribution of these variants in BS and cancer, which may further lead to the design and development of potential drugs for the better management of BS and other associated diseases. Furthermore, our study provides key support to investigators to conduct future studies on pathological mutations and their structural consequences on BLM. The SNPs predicted from our study can be further validated by wet-lab scientists to investigate the evidence of BLM protein mutations in association to BS and develop a potential drug target for BS. These findings may enrich the available SNPs databases and then can be utilized for further research.Supplementary informationSupplementary FiguresSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7


The article presents results obtained during testing of concrete based on CEM I 42.5R Portland cement, fine and coarse aggregate, glass, volatile ash, and superplastifier. The concrete mixture was modified using filler consisting of bromosilicate heat resistant cullet. Recycled aggregate was added to the batch. Samples for the need of testing were produced as (100 × 100 × 100) mm cubes. Before commencing proper tests, samples have been heated within the temperature range of 20–800 °C. Tests carried out during the proper testing procedure included tests of compressive strength, elevated temperature, impact strength, as well as macroscopic tests of the contact area. The obtained test results have provided proof of there being a possibility of producing special concrete, modified by products obtained from heat resistant cullet. This type of is generally characterized by satisfactory performance parameters. The average compressive strength for concrete modified by a 10% of heat resistant cullet was determined as 43.6 MPa and 48.3 MPa respectively after 28 and 180 days of curing. A particularly challenging issue facing different countries worldwide is to find ways to stop the degrading impact of the population on the surroundings. Two phenomena that arise from uncontrolled economic and civilizational growth and development are exerting a hazardous impact on the natural environment. The first disquieting phenomenon is the generation of all types of waste, and the second one is the depletion of natural resources. Both phenomena require global attention to allow innovative and sustainable applications being worked out .Mass-scale consumptionism goes back to the 19th and 20th centuries. It has caused an uncontrolled economic growth worldwide and wasting of natural resources ,4.The latest Eurostat results from May 2017 determine the percentage of particular economic sectors in total generation of waste in the European Union (EU). The building sector generated 34.7% of the total amount of waste in 2014. At that time, the EU generated 2503 million tonnes of waste. The subsequent positions were occupied by mining and quarrying (28.2%), production (10.2%), services related to water supply and sewage disposal management (9.1%), and households (8.3%). The remaining 9.5% comprised waste generated by other types of economic activity, primarily service (3.9%) and power sectors (3.7%) [The availability of natural raw materials is of a great social importance. The strategy of rational resource management is an interesting research issue based on the sustainable development principle. Recycling and re-use of natural raw materials are the main focus here. Researchers show considerable interest in possibilities of using recovered aggregate. The absence of clear guidance in this respect limits the usage of those materials and causes their marginalisation. According to standard  aggregatIn its support of sustainable development in the construction sector, in 2015 the European Commission adopted a plan relating to a closed loop economy. The innovative concept consisted in introducing a closed service life of a product. The product is not thrown out, and as an effect to the landfill after the end of its service life it is subject to recovery and recycling. Stockpiled waste may to a large extent be used as secondary raw materials. Processing of waste can reduce the generated amounts ,13,14,15Numerous research studies have provided proof of the suitability of using aggregate obtained from recovery. According to estimations of the Polish Association of Aggregate Producers the value of this production is ca. 4.5 million tonnes, which comes up to 2.5% of all aggregate amount used in Poland . The useIn Denmark, designing of ecological concrete was initiated already in 1998, thanks to which the waste from other processes may be used as a raw material. This can limit the volume of production waste and reduces the obtaining of aggregate from natural deposits ,19,20.The process of obtaining recycled aggregate from concrete is complicated, and it comprises assessing concrete source, selective facility demolishing, crushing larger elements, removal of any present contamination, initial and elementary concrete crushing, fractionation, and the improvement process .Cullet waste stored in piles does not decompose ,23. It iNumerous studies ,39,40,41For needs of the testing procedure use was made of cullet coming from diverse sources, such as for example float type window glazing, car windows, tempered glass, crystal glass, mirrors, colorless, green and brown packaging glass, and kinescope glass. The cullet may be used as a substitute of cement, sand, and coarse aggregate. Research of available literature has shown that if cement is replaced by cullet, after 28 days of curing concrete composite has a lowered compressive strength as compared to reference concrete. On the other hand, after 90 and 180 days of curing a much smaller impact of the presence of cullet is observed on lowering the strength of composites , which iObservations of the topography of studied material under a scanning electron microscope (SEM) allow the presumptions that recycled waste material that contain highly resistant glass is characterized by a denser microstructure as compared to a common sample . FurtherConcrete structures may be subjected to dynamic loads caused by such phenomena, as earthquakes, waves, wind, and machinery. This necessitates making a correct assessment of dynamic response of the structure to this type of load. Researchers have studied the major part of strength properties of concrete, however, literature still contains insufficient research concerning tests of such dynamic features, as the vibration damping factor .Researchers ,47,48,49To date, literature has not provided any studies pertaining to cement composites with heat resistant cullet as a substitute of coarse aggregate, in the context of their durability under the influence of elevated temperature and dynamic loads.The main objective of this study was to determine possibilities of reusing waste obtained from heat resistant cullet as recycling aggregate in the production of concrete composites. This justifies the legitimacy of the undertaken issue and emphasises the cognitive and interdisciplinary nature of the study. The conducted analysis comprised selected mechanical features of the designed composite, and the compressive strength was studied.The concrete mix was designed ,55,56,57Volatile ash used in the testing came from coal burning in the Elektrownia PGNiG Termika . The plant in Siekierki transforms this ash into two types of products, consequently prolonging its life cycle and bringing it back in usage. The used mineral additive was certified ProAsh cat. An ash with confirmed pozzolanic properties, which meets requirements of the standard , and is Use was made of volatile ash, since it was found that concrete with this addition (contents of 20%) offers better strength, resistance to cracking, corrosion, and high temperature .The concrete was modified by a chemical admixture of 18 BVC consisting of a superplastifier based on lignosulfonates.Test trials comprised three mixtures having different contents of glass recyclate . The original mixture was modified by adding heat resistant cullet to the trial batch, and consequently the amount of water and aggregate has been optimized appropriately to the volume of replaced materials, moisture contents of fine and coarse aggregate and of recyclate. When selecting recycled aggregate for needs of producing the composite material, physical parameters, and ecologic requirements were taken into consideration. Bromosilicate cullet  comes maSamples for the needs of experimental testing were produced as cubes having a side length of 100 mm and stored in laboratory conditions in accordance with the standard . The samCompressive strength testing was conducted in line with requirements of the standard  after 28The resistance of the composite to high temperature was tested in a special furnace PK 1100/5. The samples were heated up to the temperatures of 200, 400, 600, and 800 °C, which were then maintained for 60 min until finishing of the heating process. The samples remained in the furnace until their complete cooling down.First the samples were dried in a laboratory drying device to achieve a constant mass with temperature of 105 ± 5 °C. The signal from the thermocouples was registered by a computer furnished with dedicated software. The temperature distribution was monitored with the use of four thermoelements furnished in the specially drilled witness sample . The opeThe testing comprised selected elements of the fire environment and the projected thermal process were programmed. Thermal fire conditions in the furnace chamber have been illustrated by the standard curve “temperature–time” . TestingThe energy absorption testing of concrete samples was carried out at the laboratory temperature and dynamic load. Type DP FEST 1000 drop weight would hit the samples with identical energy of 150 J, from the height of 595.4 mm. The test consisted of the breaking a (100 × 100 × 100) mm cubic sample by a single hammer blow. Samples used for the testing first had to undergo thermal processing. The objective of the experiment was to measure the work, which corresponded to the energy used to break it and enabled finding ways in which the loading rate affects the fragility of material.The scanning electron microscope (SEM)  was used to carry out stereologic tests of the surface of concrete composites. The testing was conducted on specially prepared microsections obtained from places where damage had been sustained. The appropriately prepared surface of the microsections was then polished with the use of sandpaper. Tests comprised studies of microstructure and phase composition of hardened concrete. In addition, an EDS (energy dispersive X-ray spectrometry) microanalysis was implemented of the chemical composition, which comprised dispersion of the X-ray energy. Chemical elements comprised by the tested material have been identified.The subject of the experiment was a population of hardened concrete composites designed by the authors. Source concrete was modified by heat resistant cullet having different cullet contents. The population was divided into three series  each with one batch. Next, nine reference samples have been selected from each batch. To be able to obtain representative samples, the selection of samples for needs of the studies was random. Concrete composites were analyzed with respect to selected mechanical properties, i.e., compressive strength, resistance to elevated temperature, and dynamic impact. In addition, the structure of composites was determined using the scanning electron microscopy SEM method.Results of tests pertaining to compressive strength were presented in The rate of increase of compressive strength of the concrete mix depends on the composition of the mix. A slight increase was recorded of the average compressive strength at extended curing time of concrete composites produced with the use of recyclate. The average compressive strength B5 and B10 after 180 days amounted to 42.8 MPa and 48.3 MPa, respectively. The replacement of natural aggregate with heat resistant cullet caused a reduction of compressive strength by 23.57% and 13.75%. The target strength determined on control samples was found to be 56 MPa. In addition, the impact of recyclate on the strength parameters of the designed composite was noticed. Introducing it into the concrete mix at the level of 5% causes a decrease in the concrete strength of approx. 25% and 31% after 28 and 180 days of maturing compared to the control concrete. In the case of increasing the amount of recyclate to 10%, a smaller decrease in strength was observed, i.e., approx. 23% and 16%. The obtained test results indicate that the increase in the amount of re-rectal in the composite translates into an increase in the compressive strength of concrete.Destructive tests were carried out on samples which had not been heated up. The conducted tests suggest that it is possible to gain a better strength of composite material by increasing the amount of recyclate in the concrete mixture.Base samples as well as those heated with 200 °C show no changes in the internal structure. This confirms their high compressive strength. Samples of the B5 and B10 series loaded with 200 °C even showed increases in compressive strength compared to the base samples. There is some strengthening of the concrete interface. The heating of samples at a temperature of 400 °C causes a decrease in the pozzolanic activity of the binder, the formation of pores and the formation of microcracks. Increasing the soaking temperature of the samples (600 °C and 800 °C) causes the C-H-S gel to break down, increasing the size of pores and scratches, especially between the glass aggregate and the binder. You can see the crumbling of the top layer of concrete samples caused by dehydration of the C-S-H gel and the expansion of the volume of concrete caused by high temperatures. The effect of the above-mentioned factors is a change in the morphology of the concrete and, with increasing temperature, a gradual weakening of the concrete interface. Papers ,66,67 paThe highest strength was recorded after heating up of the samples to the temperature of 200 °C after 180 days of curing, i.e., 56.9 MPa for B0 samples, 53.0 MPa for B5 samples, 50.6 MPa for B10 samples. The compressive strength was reduced by 6.85% and 11.07% respectively. Samples heated at higher temperatures, i.e., 400, 600, and 800 °C have been found to have a bigger reduction of compressive strength as compared to samples made of control concrete. Heating up of composite in the above specified temperatures caused a decrease in strength by 12.39–11.23–0% respectively (concrete B5) and 11.76–18.84–30.51% (concrete B10). A smaller content of recyclate in the mixture has a more advantageous impact on the strength of samples heated up at fire temperatures.Pursuant to adopted assumptions, the testing was carried out in a chamber furnace in accordance with the temperature–time standard curve, conforming to the scenario of a developed fire in a closed premise, during which rescue and extinguishing actions may be carried out. Thermocouples distributed in the pilot sample allow modelling of the progress of a standardized fire. Six samples were placed at a time in the furnace and next heated up at high temperatures: 200, 400, 600, and 800 °C. The heating time depended on the height of programmed temperature.The actual distribution of temperatures presented on The experiment has proven that the biggest temperature increase occurs over time, and in different moments of the fire the concrete composite is characterised by different strength parameters. The heated concrete goes through three phases: initial phase, temperature growth phase, and the cooling phase. In the first phase, heat from the surroundings permeates into the composite much quicker and is absorbed by the sample over its entire external surface (the air-concrete contact border). Heat that permeates inside the sample causes heating up of its subsequent layers. In the next phase, temperature grows slower. The moment when temperatures become identical proves even heating up of the sample over its entire volume. During cooling down, a temperature increase of internal layers is found to take place.The samples were hit by a drop hammer with energy of 150 J. The impact caused motion of particles in the concrete medium in relation to the balance situation. Results obtained from the impact test were obtained on As shown on As a result of stand testing b the maxWhen analyzing the time courses of vibrations on The analysis of the structure and gaseous phase are performed in the SEM scanning electron microscope, generally making use of zooms from 200 to 10,000×. The testing comprised in the first place sample fractures heated at the temperature of 20, 200, 400, 600, and 800 °C. The samples were taken from material left over from the process of impact stress testing.The identification of microthermic cracking and an analysis of the morphology microthermal cracking and of the morphology of concrete composites were presented on The obtained results enabled the qualitative assessment of the structure and phase composition of hardened composite. The reference concrete contains grains of hydrated calcareous silicate that is interconnected in the form of compact gel. According to Diamond’s classification  it confoFurthermore, during structural tests of the planned composite the EDS microanalysis has been carried out . Example2 and limestone oxide CaO. In the tested samples also tested were peals which originated from crystalline intrusions of portlandite and calcite. Metakaolinite generated in the dehydration process, at a temperature of 800 °C, consists of reactive silica SiO2 and aluminium oxide Al2O5. When reacting with calcium hydroxide, oxides form watered calcium silicates and calcium aluminosilicates. The EDS analysis pointed to a reduction in the contents of Al2O5 by as much as 77.76% /12.93% /54.71%  as compared to the sample of reference concrete. The SiO2 contents fell by 28.82%/24.15%/15.28% respectively. In an indirect way, the growth of Ca(OH)2 may be proven by the increased amounts of CaO equalling to 57.28%/36.84%/33.76%.An analysis of the above presented X-ray photograms implies that the main hydration product is the amorphous phase consisting of watered calcium silicates (gel C-S-H). Hardened concrete is characterized by high contents of silica SiOIn the tests, an attempt has been made to make a statistical assessment of results of compressive strength. Basic statistical parameters were delimited, such as mean value, median, range, standard deviation, and variability coefficient. The conducted r-Pearson analysis suggests that increasing of the amount of recyclate and the concrete causes proportional changes  of the mean compressive strength. An increased amount of recyclate used to modify the amount of recyclate applied for modifying concrete in at a quantity that exceeds 10% would most probably cause an increase of its average compressive strength as compared to reference concrete.The research of Mahesh , pertain2). The variability of the average compressive strength of concrete is explained by a temperature variability of 90.60, 88.67, 77.68% respectively for the following correlation coefficients 0.95; 0.94; 0.88.The objective of the next test was to verify the subsequent research presumption. The presumption assumed a connection of temperature at which the element with compressive strength of concrete composite is heated up. The obtained correlation was illustrated on To verify the directional hypothesis, the r-Pearson analysis was performed. The regression lines were found to have a negative deviation. This proves highly negative correlation of variables, in other words a negative relation takes place between the two variables. The correlation coefficient proves the force of the interdependence. The higher the temperature, the lower the compressive strength of composites. Results of the negative correlation were found to be statistically significant, which has provided proof for the hypothesis. The test strength was determined on the basis of the adopted significance level of 0,05. The results were provided in One of the characteristic features of concrete composite is an increase of strength after heating up to the temperature of 200 °C. Also recorded was a considerable decrease in compressive strength by ca. 50% of concrete within the range of 200–600 °C. A further increase of temperature over 600 °C leads to a significant strength decrease. The composite achieves a load bearing of ca. 8–10 MPa. As has been shown by the conducted literature analysis, higher temperatures weaken the structure of composite, with surface cracking appearing. The higher the temperature affecting the material, the bigger is the decrease in the strength. Thanks to good thermal insulation, high temperature (200–800 °C) occurs only in the surface zone of the element, and does not reach the section core. The sample becomes heated up in a uniform way, which is proven by the temperature equalization moment.The compressive strength of concrete is strongly correlated with the temperature acting on the given element. A negative relation takes place between the two variables. The result is decreasing curves of regression, determined by a linear function. The average compressive strength of concrete composite as compared to temperature change varied by 90.60% (B0), 88.67% (B5), and 77.68% (B10) respectively. The correlation coefficients amount to 0.95 (B0), 0.94 (B5), 0.88 (B10).Heating up of the samples to a high temperature causes changes in the sample structure; the higher the temperature, the bigger the impact speed, and in such a situation weakened concrete is less resistant to impact loads. A complex mechanism of energy dispersion in the concrete becomes noticeable. Higher sample heating temperature causes a higher level of material damping.During a comparison of structure of B0 concrete subjected to the impact of elevated temperatures to 800 °C under the SEM microscope with reference concrete at 20 °C, no scratches or delaminations have been observed in aggregate (sand) and binder. The experience of other researchers indicates that if the quantitative description of the structure is known, it is possible to define the dependence between the structure and parameters of the technological process. Measurements and an analysis of the image of concrete B5 and B10 pointed to the existence of scratching of the element. Taking into account extreme temperature loads (20 °C and 800 °C) in samples with a supplement consisting of crushed glass (5% and 10%) in the concrete mixtures, considerable differences in scratch sizes have been observed.Differences in chemical composition of concrete subjected to the EDS analysis may arise from local differences in the material structure. The addition of volatile ash leads to a reduction in the crack opening size. The concrete compressive strength tends to drop with the increase of crack opening.The authors are planning to undertake further research studies and carry out additional tests with the objective of learning the impact of crushed cullet on properties of concrete composites. A more extensive scope of studies would allow extending the acquired knowledge and the determination of dynamic characteristics of the planned concrete not comprised by the program under the present study and those that lack references in publications of other authors.From the ecological viewpoint, the reuse of waste brings about significant economic advantages. Appropriate disposal and processing allow their re-use. Consequently, there is a need of further development of the ecological awareness of the society.Interest in the application of cullet is growing, as it is a material that does not undergo biodegradation, and as a result this raw material may be processed many times. The studies have shown that this recyclate may constitute, in some cases, a perfect supplementing of aggregate of natural origin, as it does not require improvement and purification processing.All studied concrete types achieved different classes after 180 days of curing i.e., C45/55 (B0), C30/37(B5), C35/45 (B10).The average compressive strength for concrete modified by 10% of heat resistant cullet was determined for 43.6 MPa and 48.3 MPa respectively after 28 and 180 days of curing. The increase in strength over time contributed to increase the concrete class from C30/37 to C35/45.The increase of temperature to 800 °C caused in all composites a change of the structure and a reduction of strength to 8–10 MPa.When the sample was subjected to the temperature of 200 °C, strength has been found to grow from 1.61% to 23.83%.Temperature caused damage to the concrete structure, and cracking was visible under the microscope was detected. The biggest discontinuity of the material had 90.7 µm.Based on the conducted tests and analyses, it has been proven that waste of heat resistant cullet may be used again as recycled aggregate for concrete.The conducting testing of the structure has pointed to minimum concentrations of selected elements. No admissible environmental standards have been exceeded, which allows the presumption that the concrete composite modified by heat resistant cullet may be considered as being a full value building material.An analysis of the results and observations made during the executed experiment allow the following presumptions:The concrete composite modified by waste products obtained from heat resistant cullet is found to offer satisfactory performance parameters, which has been proven during the conducted experiments.Source materials contain little information which would describe the impact of heat resistant cullet on physical, mechanical, and chemical properties of concrete composite. Also offered information pertaining to concrete impact strength is limited. Given the absence of standard regulations concerning the undertaken research, in-depth interpretation of obtained results would require the implementation of supplementary studies. Findings presented in this article serve as a basis for further research.


This paper proposes binary and ternary combinations of sewage sludge ash (SSA) with fly ash (FA), marble dust (MD) and rice husk ash (RHA) as partial replacements of Portland cement in concretes with a similar dosage to that used in precast blocks, with very dry consistency. Several physical-mechanical tests were carried out on concrete specimens with curing ages of 28 and 90 days: density, water absorption, capillary water absorption, ultrasonic pulse velocity and compressive strength. The combinations of residues significantly improve the properties of the cementitious systems: 30% replacement of Portland cement provides strength values similar to the reference sample, showing the synergetic effects of the combination of the mineral additions. The significance of this research relies on the combined use of the mineral additions as well as the use of them for the precast block industry. The results show synergies among the additions and even that some of them showed relevant improvements when they are used in combination, performing better than when used individually. The management of the unavoidable waste generation in modern societies has become one of the most serious environmental challenges. Thus, negligent or improper waste management produces a substantial effect on the receiving environments and can cause contamination in the water, ground, and air, and contributes to climate change, affecting ecosystems and human health. However, appropriate waste management becomes a resource that contributes to the reduction in raw material consumption, produces remarkable benefits for the preservation of natural assets, reduces the impact on climate change and boosts sustainable development .The construction field, encompassing civil works and the building industry, is responsible for 60% of the raw materials extracted from the lithosphere . The proThis paper is a feasibility study for the use of four waste products generated from industrial processes to be applied as mineral additions for cement in concrete. Moreover, three of them originate from outside the construction activity. These were sewage sludge ash (SSA) coming from wastewater treatment plants, fly ash (FA) generated from thermal coal plants, marble dust (MD) derived from the stone cutting industry and rice husk ash (RHA) from rice processing plants. Mineral additions are added to cementitious materials such as concrete or Portland cement-based mortars in order to achieve additional properties or improve other features, especially in terms of durability or mechanical properties . The addResearch dealing with the use of mineral admixtures began in the 1950s, mainly concentrated on their possible use in the production of Portland cement, mortars or special concrete types. At the time of writing, there has been an exponential growth in the use of all types of mineral additions, with global production estimated to have been as follows (in million t/year): 370 FA, 35 blast furnace slag, 20 RHA and 2 of silica fume .Two of these residues, SSA and MD, represent a serious environmental problem at the local level due to the volume produced and gathered. Regarding SSA, around 1.06 million tons of dry material are produced annually in Spain . This tyRegarding MD, it is worth noting that it is not a contaminant waste, being composed of 98% calcium carbonate. The main impacts of such an industry remain at the local scale. In the case of Spain, Alicante province produces 70% of the domestic marble, with Spain being one of the most relevant producers in the world . This industry produces approximately 500,000 sludge tons from polishing and cutting natural stones .As an example, in the case of SSA, it has been shown to be adequate to partially replace cement up to proportions of 10%, or with 2% substitution of the mix sand. In such cases, the specimens showed analogous compressive strengths compared with control specimens. Regarding this leaching study, results indicate that the mixture of mortar or concrete with ash, cement and sand induced the stabilization of Mo and Se, so in this way, this constitutes an effective treatment of the ashes . Previou2 and Al2O3 [The use of FA as a mineral admixture has been widely known for more than three decades. The most significant properties rely on its pozzolanic activity for the medium and long term, with spherical particles and high amounts of vitreous SiOnd Al2O3 . In addind Al2O3 .SSA has been shown to possess significant pozzolanic activity for ternary combinations for the production of cement/SSA/FA binders. This pozzolanic activity increased the mechanical properties of mortars in the age time-period from 7 to 28 days. In these ternary combinations, SSA harmed the fluidity of the mixes while the addition of FA enhanced it ,23.Published research has shown that MD in cementitious composite materials, employed as an addition to substitute up to 10% of sand, enhances the cohesion of the mixture, maintaining the compressive strength, reducing permeability, and even improving the mechanical performance in comparison with the effect of the same amount of limestone filler ,25. RegaRHA has demonstrated a significant positive impact on both the mechanical and durability properties in cementitious matrices ,28. ReseThe effect of applying these wastes—either individually or combined—for the partial substitution of cement in conventional matrixes and binders is well known:It is also worth summarizing the recent advances as well as the limitations of the SCM used in this paper . SSA wasA previous work about concrete blocks, where the fine aggregate was substituted by fly ash and the coarse aggregate was substituted by bottom ash, is relevant to the specific application analyzed in this paper, namely concrete for block manufacturing. The results show that they provide fire passive protection and that no loss was found in terms of the mechanical properties of the product. Furthermore, according with the results of the leaching tests, there was no evidence of environmental problems . At the This paper reports on the effect of combining SSA with diverse mineral residues on the properties of concrete to be used—with characteristics in accordance with the manufacturing process for precast block production. The significance of this research relies on the replacement of Portland cement by several materials proceeding from waste that will expand knowledge about future possibilities to improve the properties of cementitious composites in both fresh and hardened states. Moreover, the waste materials were used individually and combined in binary and ternary combinations. The synergies among the mentioned wastes, considering with special care SSA, were assessed and will boost the future exploitation of them. It is worth noting that this study is the continuation of previous works published by the authors related to the replacement of cement mortars and concretes by binary and ternary combinations of waste materials .This paper shows the results of several concrete cubic specimens with different sizes, configurations and manufacturing processes compared with the concrete blocks produced in a plant. That is to say, the results are not directly applicable to the blocks, although the same mix proportioning as the one that would be used in the regular concrete block manufacturing in the plant was used. Having said that, this study could serve as the initial phase for the production of pilot precast blocks with the use of the mineral additions that have been shown to yield the most effective results in this initial trial stage. This issue is covered in more detail in the experimental section.The CE mark for construction and building materials is well known given that it is required in order to commercialize any product. In order to obtain this quality label, manufacturers have to establish and guarantee certain characteristics of their products although it does not refer to compliance with any explicit and minimal requirements. That is to say, some of the additions studied could be considered unsuitable in terms of quality for certain uses, while their qualities could be perfectly suitable for other uses.The significance of this research relies on the combined use of the mineral additions as well as the use of them for the precast block industry. In order to do so, compressive strength, water absorption and density of concrete mixtures were assessed in order to meet the requirements for the CE marking of the blocks. In addition, this study was focused in the binary and ternary combination of such mineral residues, assessing not only the results but also the synergetic effects that appear. Indeed, certain materials showed that they improve their properties when they are used in combination, performing better than when used individually.In this study, the following mineral additions were used: SSA, FA, MD and RHA. SSA was obtained from the wastewater treatment plant of Pinedo . The product was supplied in bulk and was obtained from the release of a fluidized bed incinerator, subjected to a maximum temperature of 800 °C. The FA was also supplied in bulk and came from the coal power plant of Andorra-Teruel . Regarding MD, it came from a refuse dump in Novelda  that accumulates the waste from various local industries. RHA originated in an energy recovery plant that uses rice husk for fuel. The product was ground to reduce particle size and internal porosity before it was supplied by the company DACSA, .2 high early strength class. The composition was in accordance with Spanish Standard RC-16 (by mass): (a) clinker, 65–79%; (b) a mix of blast furnace slag and limestone, 21–35%; (c) 0–5% of other minor components. Concerning the aggregates, they were two crushed limestone aggregates: 0–4 mm size (F-0/4) and 2–8 mm size (F-2/8). These aggregates were supplied by the blocks manufacturing plant.Spanish Portland cement type CEM II/B-M (S-LL)-42.5R (supplied by Cemex) employed in this study was chosen in order to be the same used for the production of the blocks in the plant. This cement type is a blended cement of 42.5 N/mmThe control specimen was manufactured with an analogous mix proportioning of the concrete blocks produced in the plant. The consistency was dry with null slump in the test. This is a key-point in the manufacturing process of the precast blocks given that the concrete is poured into the molds for a very short time-period before it is removed for curing.It can be observed that the predominant addition is SSA, as it appears in almost all combinations. The control specimen (C) is accompanied by the following samples: substitutions of 10, 20 and 30% of cement by SSA ; 20% substitution of cement by the binary combination (10 + 10%) of two residues: SSA + FA, SSA + MD and MD + FA ; and cement replacement by 30% of residue combinations: mix 31 (10%SSA + 10%FA + 10%RHA) and mix 32 (20%SSA + 10%RHA).In order to assess the variations of the properties, the weight of binder was kept constant as the cement plus the mineral addition. Therefore, the water/binder ratio was 0.68 in all the formulations. If The raw materials were characterized by means of a particle and size distribution of the aggregates and sand equivalent test. In addition, X-ray fluorescence analysis (XRF) and scanning electron microscopy (SEM) of the mineral additions used in the study  were performed.Dmd), water absorption (Abs), capillary water absorption (Cap), ultrasonic pulse velocity (Upv) and compressive strength (Cs). This test program was designed in accordance with the standard EN 771-3 Specification for masonry units—Part 3: Aggregate concrete masonry units (Dense and lightweight aggregates), applied for precast concrete blocks. In the standard, all the previously mentioned tests (except Upv) are listed in the initial tests. SEM was also used on the concrete specimens.For every concrete type, six specimens were cast: three were used for mechanical testing and the other three for physical tests. Thus, with these specimens, the following test program was conducted: dry mass density  following the standard UNE-EN 933-8 was completed for the fraction 0/2 mm.The X-ray fluorescence technique (XRF) was performed with a sequential rhodium tube and beryllium window X-ray spectrometer . This technique was applied in order to achieve some additional data from the chemical composition of the mineral additions that could enhance the comprehension and discussion of the results.Regarding the scanning electron microscope (SEM), it was performed by a JEOL JSM-840 equipment, Tokyo, Japan.In the XRD technique, applied for the qualitative identification of the mineralogical composition of the concrete samples, a JSO-Debyeflex Seifert 2002  equipment was used, provided with a copper cathode and nickel filter.The aim of this study was to reproduce as much as possible the concrete mix design and pouring procedures used in the real industry. Thus, the sequence and procedures followed by the local plant for the manufacture of the blocks were carefully studied before any batch was performed in the laboratory. These procedures were assumed as a reference for the manufacture of the blocks: concrete was poured into the molds and a single blow of compaction-vibration was carried out; then, the molds were removed and the blocks were transported to the curing chamber.3 were manufactured by pouring the concrete on PVC molds following the standard UNE-EN 12390-1. Before the molds were filled, a release agent was sprayed on the walls and they were protected with plastic sheets in order to ease the specimen removal. This operation allowed for extracting the concrete element despite the dry consistency of the mixture.Cubic specimens of dimensions 150 × 150 × 150 mmIn order to perform an adequate mix of the components, the humidity was carefully controlled. This is of great importance given the strict consistency requirement of the mix design. Some of the components such as cement and the mineral additions were dry in origin. In the first step of the mix sequence, those dry components were mixed in absence of water. Then, the mixing of aggregates and water was conducted. The concrete was poured into the mold and it was compacted for 30 s at low speed by means of a pneumatic hammer (Milwaukee Kango-900). The specimens were stored in a moisture room for 24 h and were demolded by means of compressed air. After this, the specimens were stored in a curing chamber at 90% RH 20 °C until the testing age. In specimen “30” two forms of curing were used , Equation (1):Cap is given in kg/m2min0.5, fQ is the mass after 4 h testing (g), Q0 is the initial mass (g) before starting the test (the samples were previously dried to constant mass in an oven), A is the section of the specimen (225 cm2), and t is the duration of the test (240 min).The density and absorption were assessed following the standard UNE-EN 12390-7. Regarding the water absorption, it was carried out in agreement with the standard UNE-83982, measuring at intervals of 5, 10, 15, 30, 60, 120, 180 and 240 min for 4 h. Equation (1) was used to compute the capillary absorption (Q) respect to the initial mass:Q is given in %, tQ is the final mass (g) after the t test time and Q0 is the mass (g) before the test starts (specimen dried in an oven to constant mass and cooled at room temperature into a desiccator).Equation (2) was used to determine the mass increase (Q) of concrete samples versus time, during state 1, water filling would occur by absorption through the capillary pores of specimens. When the time corresponding to According to the theoretical model formulated by Fagerlund , with reUpv tests were performed by means of a Steinkamp BP-5 instrument and following the standard UNE-EN 12504-4. After the Upv test, compressive strength tests were carried out on the specimens. The direct measurement method was applied: transducers were placed on the transverse axis, in the direction in which the concrete was poured.On three specimens of each concrete type, the The compressive strength tests were completed on a Suzpecar CMP-150 t press, according to UNE-EN 12390-3, being the load applied in the same direction at which the concrete was poured when filling the molds.In order to perform the statistical analysis, the software Statgraphics was used.Concerning the Sand Equivalent Index, the results obtained show that the fine aggregate met the requirement of the Spanish Structural Code EHE-08 for concrete elements under general environmental exposures such as class I, IIa or IIb and not subjected to other explicit type of exposure. The requirement is to be equal or higher than 70% and the result of SE test of the fine aggregate was 68.4%.XRF) results are shown in The X-ray fluorescence (2 (17.27%) and Al2O3 (9.64%). With this composition, SSA is expected to behave as an active mineral addition for concrete. It merits mentioning that the amounts of SO3, P2O5 and Fe2O3 were also remarkable. In the case of FA, the amounts of silica (36.70%) and alumina (25.57%) were much higher than in the previous case of SSA. Therefore, it could be considered as a pozzolanic mineral addition that could be classified in class F Fly Ash following the ASTM C618. Regarding MD, 64.25% of the CaO was found to be calcium carbonate (CaCO3). Thus, the composition of MD showed that it was likely to behave as an inert mineral addition. For RHA, it can be seen that it is essentially SiO2, exceeding 80% of the total composition of the addition. Subsequently, RHA is highly expected to behave as a pozzolanic addition.SSA presented a significant content of CaO (30.24%), SiOMicrographs  show theAbs) and dry mass density (Dmd) of the mixtures cured for 28 and 90 days are shown in The average values of absorption  produced denser concrete: probably, the water demand of SSA and RHA produced lower concrete compaction.In general terms, Abs and Dmd can be established, and more precisely, a linear, very strong relationship between the variables analyzed, with R2 equal to 0.96 for 28 and 0.97 for 90 days of curing. With the results obtained from water absorption test, the opposite tendency marked by the density is met , which means that higher absorption values coincide with lower densities. For example, the group conformed by reference sample and samples “10”, “20” and “30”, with SSA, for higher percentage of residue, higher absorption is recorded, with an increase in relative value of up to 42% (in sample “30”), compared to reference sample, cured for 90 days. Based on Table 8 (correlation matrix) a statistically significant relationship between Cap) average values for samples cured for 28 and 90 days, obtained after four hours of testing.Cap by increasing the amount of cement replaced by SSA, with significant increases observed between these mixtures, reaching a maximum of 371.4% compared to the control specimen cured for 90 days.When analyzing the results of reference sample and the three samples containing only SSA as a mineral admixture , we noticed an increase in Δ in Cap occurred in reference sample (−34.4%); a remarkable decrease also occurs, but to a lesser extent, in sample 10 (10% SSA) and those containing FA. Apparently, the flattering effect of Cap caused by SSA in small quantities, as in sample “10”, happens under the age of 90 days, and for additions in larger amounts but combined with FA, this effect is cancelled to some extent.Regarding the increases obtained between the two curing ages of each sample Δ in , the higCap in sample “21” (SSA + FA) was similar (0.83 versus 0.84 kg/m2min0.5 at 90 days). Lower values compared to reference sample were found in Samples “22” (SSA + MD) and “23” (MD + FA). For all the mixtures studied, Sample “23” showed the closest value to reference sample, with a relative value of 124.4% at 90 days. This result is in line with what was observed in the previous section, where sample “23” also showed the nearest values regarding to density (99.4%) and absorption (105.6%) for combinations of mineral admixtures after 90 days of curing. For ternary combinations of additions at 90 days, lower Cap values than those achieved by sample “30” (30% SSA) are obtained, but in the best case (Sample “31”) it is nearly three times the one found for the reference sample (280.5%).In binary combinations of additions, considering sample “20” (20% SSA) as a reference, the value resulting from (Q) during the four-hour test is shown in The behavior regarding the mass gain average by capillary absorption In the bottom of the graphs, therefore with lower values of capillary absorption, including the reference sample, samples “10” and “20”, and the binary combinations of mineral admixtures . These mixtures show lines parallel to each other, which indicates similar behavior during the test. After four hours of testing, the upslope remains, which means that water do not reach the upper surface in any of these samples, so that the filling is produced by water absorption through the capillary pores (state 1 of the Fagerlund model);n, which would correspond to the continuity of the capillary refill through pores of air by the process of diffusion and dissolution of air, will be approximately 120 min in sample “32” and between 180–240 min in samples “30” and “30’ ” (specimens cured for 90 days).Upper half of the graphics, therefore with higher capillary absorption values, including samples “30” and “30’ ” (cured under water), and the ternary combinations of mineral additions (“31” and “32”). A similar behavior between the specimens of this series is observed , but the main novelty of the parameters analyzed so far is that the slope is very low when the test is finished, which means that the water front reaches the top of the specimens, saturating the specimens faster. The time tTwo zones are observed:Cap and Abs. By examining Based on the correlation matrix and statistical study shown in Table 8, it is possible to conclude that there exists statistical correlation between 2 = 0.60). For the 90-day curing samples, the correlation coefficient increased up to 0.83 and, therefore, improving the classification to very strong. This linear fitting of the 28-day curing specimens could be classified as moderate-strong (R(Cs) of the blocks for 28 and 90 day curing ages are shown in The compressive strength 30 and 30’, this increase is of approximately 60%, but the values of Cs at 28 days are the lowest of all the mixtures tested.As can be observed, the compressive strengths of this type of concrete was low given the high porosity of the concrete. The results of the control specimens, used as reference, were for the 28-day compressive strength equal to 7 MPa, while it reached 9.7 MPa after 90 days. Furthermore, this increment of compressive strength with the curing time was observed for all the concrete mixtures. For example, in samples SSA show a decrease in Cs compared to the reference sample with the same curing age: sample “30” (90 days) does not exceed the 77% of the reference. This behavior can be related to the low pozzolanic reactivity of this mineral admixture. Sample “30’” suffers a significant drop compared to its counterpart, 5.6 vs. 7.4 MPa, possibly due to the washing effect of a porous matrix, produced during its curing under water.Samples with replacement of cement by SSA in all cases, with values close or equal to reference sample in several mixtures cured for 90 days: 22 (SSA + MD), 23 (MD + FA) or 32 (SSA + RHA). Sample 32 should be highlighted because, with a high replacement of cement (30%) by 20%SSA and 10%RHA, the compressive strength achieved is equal to the reference sample for the two curing ages: 7 MPa at 28 days for both cases and 9.5 versus 9.7 MPa for 90 days. Therefore, a significant saving of Portland cement is achieved with excellent mechanical response. This behavior in terms of mechanical strength may be attributed to the high reactivity of RHA. This mineral admixture usually presents a good pozzolanic reaction rate, and the chemical reaction of portlandite and reactive silica from RHA produces an extra quantity of calcium silicate hydrate (C-S-H). Mixtures containing MD also showed high compressive strength values: this can be related to the ability of carbonates to chemically react towards reactive alumina (present in SSA or in FA). This reaction produces cementing calcium carboaluminate hydrates [Combinations of waste improve the results of the simple additions of hydrates .Upv ranged between 95 and 100% of the reference sample. According to the correlation matrix (Table 8), it can be observed that there is no statistically significant relationship between Cs and Upv, since the p-value in the ANOVA table is greater than 0.05 (0.80). The correlation coefficient (R2) is equal to 0.01, which indicates a very weak correlation between variables. This behavior may be due to the high porosity of the matrix studied, making that Upv technique is not appropriate for analyzing the development of compressive strength.The relative Cs and two parameters: CaO/SiO2 and CaO/(SiO2 + Al2O3). With the available data, no clear correlation appears.This might occur because in addition to the reactivity of the mineral additions, the degree of compaction (density) is also an influencing factor. Moreover, it should be considered that the reactivity of the additions is different. In some cases, there was a large amount of reactive silica available (RHA). In some others, there was a combination of silica and alumina (FA). In both cases, RHA and FA, there is a significant pozzolanic contribution, much higher than the case of SSA. In the case of MD, there was a large quantity of carbonates that can ease the development of mechanical strengths by the formation of calcium carboaluminates hydrates. This was found for recent studies dealing with LC3 ,40.The following images were obtained by the scanning electron microscopy (SEM) technique on concrete samples cured for 90 days.In micrographs In The presence of portlandite and fibrillar C-S-H is observed in In SSA/FA/RHA, with a rough texture. The crystal formation shown in SSA/RHA.It is possible to see in A multivariate statistical analysis is summarized in Cs-28 days of curing age) with a value of bias (−2.1) in the limit of the range of −2 to +2, can be observed, which indicates significant deviations from normality. By extending the former analysis, it is observed that this atypical value is due to the low values of Cs achieved by sample “30′ ”. However, since the p-value for the Grubbs test is greater than 0.05 (0.41), it can be listed as a non-significant value with a significance level of 5.0%, assuming that the others values follow a normal distribution, so it was decided to be included in the correlation matrix (The measurements of bias (degree of symmetry) and kurtosis (degree of sharpness or flatness), which can be used to determine if the sample comes from a normal distribution (bell curve), are of particular interest. A variable (n matrix . This an(a)p-value shown is derived from the analysis of variance (ANOVA). ANOVA tests the statistical significance of the estimated correlations ;The Spearman correlations between each variable pair are shown at the top, on the diagonal matrix. These values, ranking between +1 and −1, measure the strength of the linear relationship between the variables. At the bottom of the cell, the (b)2 between variables are located below the diagonal matrix. These variables are colored according to the scale of Evans [The determination coefficients Rof Evans , (1–0.8 A matrix of correlations among all the variables studied is shown in The replacement of 30% of cement by SSA involves a decrease in density and compressive strength compared with the ones in the control specimen. Nevertheless, replacing 10% of cement by SSA or by other binary or ternary combinations of residues showed a similar response to that of the reference samples. The binary combination of 20% replacement of SSA/RHA and the ternary combination of 30% replacement of SSA/FA/RHA showed even higher compressive strength than the reference samples. These combinations could be commercially feasible. In addition, it is a valid solution for the sustainability regulations proposed by the directives of the Spanish National Plan of Sewage Sludge.There is a direct correlation between the concrete density and the general and capillary absorption values of this type of concrete: when density decreases, both the general and the capillary absorption values increase.