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Genetic constructs with different leader sequences for intra- and extracellular expression of the target protein were generated and an original method for effective selection of clones with maximum expression was developed. For intracellular expression in the Pichia pastoris system, seprin content in cells was 6 mg/liter. [ABSTRACT FROM AUTHOR]

Type I-IV proximal spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseases, which are characterized in the majority of cases by a severely disabling course. Proximal SMA results from mutations in the telomeric copy of SMN1 gene. Major SMN1 gene mutation types are deletions in the exons 7 and/or 8, which were revealed to be in the homozygous state in 95% of patients. Deletions in the indicated exons of SMN1 gene were revealed in a compound-heterozygous state in combination with intragenic point mutations in the remainder 5% of proximal SMA cases. In the present study, we performed an analysis of point mutations in eight patients with type I-III proximal SMA phenotype, which had a deletion in 7-8-exons of SMN1 gene in the heterozygous state. We revealed seven different mutations, two of which (c.824G>C (p.Gly275Ala) and c.825-2A>T) are described here for the first time. In addition, mutation c.824G>C (p.Gly275Ala) was observed twice in the examined sample. In seven cases a heterozygous carrier of point mutations was one of the parents of the affected children (in six cases, the father; in one case, the mother). Only one mutation, c.43C>T (p.Gln15X), emerged de novo in a generative cell or male cell of the child's father. [ABSTRACT FROM AUTHOR]

The Bacillus pumilus 3-19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The glutamyl endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations. [ABSTRACT FROM AUTHOR]

Thomsen's and Becker's diseases are the most prevalent nondystrophic myotonias. Their frequency varies, according to different sources, from 1: 100000 to 1: 10000. Thomsen's myotonia is autosomal dominant, and Becker's myotonia is autosomal recessive. Both diseases result from mutations of the CLCN1 gene encoding chloride ion channels of skeletal muscles. Molecular genetic analysis of the CLCN1 gene has been performed in patients with diagnoses of nondystrophic Thomsen's and Becker's myotonias living in the Russian Federation. A sample of 79 unrelated probands with nondystrophic Thomsen's and Becker's myotonias and 44 their relatives has been formed in the Laboratory of DNA Diagnosis of the Medical Genetic Research Center of the Russian Academy of Medical Sciences. Forty CLCN1 gene mutations have been found in a total of 118 chromosomes of 66 probands, including 21 familial and 45 sporadic cases. About half the mutations detected (45%) have been found for the first time; they are not described in the SNP database (ncbi.nlm.nih.gov). The following mutations (substitutions) have been detected in more than one chromosome, accounting for a total of 59.3% of chromosomes with mutations: Gly190Ser (5.9%), c.1437_1450del14 (9.3%), Ala493Glu (5.1%), Thr550Met (3.4%), Tyr686Stop (5.1%), and Arg894Stop (30.5%). [ABSTRACT FROM AUTHOR]

novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K and k values are 0.06 mM and 1210 sec, respectively, and the p I value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease. [ABSTRACT FROM AUTHOR]

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers. [ABSTRACT FROM AUTHOR]

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. [ABSTRACT FROM AUTHOR]

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37°C, and the proteins were stable within the pH range of 7.2–9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds. [ABSTRACT FROM AUTHOR]

Two subtilisin-like serine proteinases of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 (pCS9) on the 28th and 48th h of culture growth (early and late proteinase, respectively) have been isolated by ion-exchange chromatography on CM-cellulose and by FPLC. Molecular weights of both proteinases were determined. The N-terminal sequences of the recombinant protein and mature proteinases of the original strain were compared. Kinetic parameters and substrate specificities of the early and late proteinase were analyzed. Physicochemical properties of the enzymes were studied. [ABSTRACT FROM AUTHOR]

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosyntheis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5–1%, gelatin at concentrations of 0.5–1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V–VII stages of sporulation). [ABSTRACT FROM AUTHOR]

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases. [ABSTRACT FROM AUTHOR]

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th hours of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30–150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50–100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different. [ABSTRACT FROM AUTHOR]

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The Km for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4–Gln5, Glu17–Asp18, and Cys11–Ser12 bonds in the oxidized A-chain of insulin and Glu13–Ala14, Glu21–Arg22, Cys7–Gly8, and Cys19–Gly20 bonds in the oxidized B-chain of insulin. [ABSTRACT FROM AUTHOR]

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes. [ABSTRACT FROM AUTHOR]

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites. [ABSTRACT FROM AUTHOR]

Sarcoglycanopathies encompass four distinct forms of limb-girdle muscular dystrophies (LGMD), denoted as LGMD R3-R6, arising from mutations within the SGCA, SGCB, SGCG, and SGCD genes. The global prevalence of sarcoglycanopathies is low, making it challenging to study these diseases. The principal objective of this study was to explore the spectrum of mutations in a cohort of Russian patients with sarcoglycanopathies and to ascertain the frequency of these conditions in the Russian Federation. We conducted a retrospective analysis of clinical and molecular genetic data from 49 Russian patients with sarcoglycan genes variants. The results indicated that variants in the SGCA gene were found in 71.4% of cases, with SGCB and SGCG genes each exhibiting variants in 12.2 % of patients. SGCD gene variants were detected in 4.1% of cases. Bi-allelic pathogenic and likely pathogenic variants were identified in 46 of the 49 cases of sarcoglycanopathies: LGMD R3 (n = 34), LGMD R4 (n = 4), LGMD R5 (n = 6), and LGMD R6 (n = 2). A total of 31 distinct variants were identified, comprising 25 previously reported and 6 novel variants. Two major variants, c.229C>T and c.271G>A, were detected within the SGCA, constituting 61.4% of all mutant alleles in Russian patients with LGMD R3. Both LGMD R6 cases were caused by the homozygous nonsense variant c.493C>T p.(Arg165Ter) in the SGCD gene. The incidence of sarcoglycanopathies in the Russian Federation was estimated to be at least 1 in 4,115,039, which is lower than the reported incidence in other populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A fifth world case of autosomal recessive Siddiqi syndrome (SIDDIS) related to FITM2 g ene is presented. In a consanguineous Lezgin (a Dagestan ethnicity) family, there were two affected brothers aged 28 yrs (proband, personally examined) and 32 yrs. Whole-exome sequencing followed by familial Sanger sequencing detected a novel FITM2 missence variant c.452A>G, p.Asp151Gly homozygous in both patients and heterozygous in parents and unaffected brother. SIDDIS typical features in the patients were physical and motor delay with neuropsychiatric regress, loss of speech and walking, early-onset progressive sensorineural deafness, cognitive deficiency, skin lesion. However, there was no dystonia, which is a characteristic SIDDIS feature, while at least the proband had spastic paraparesis not described in SIDDIS earlier. Another distinction was mental state: behavioral disorders in both patients, positive symptoms in the proband and severe cognitive impairment in the brother. The case expands our understanding of the clinical and molecular-genetic spectrum of SIDDIS.

A case of DEE98, a rare developmental and epileptic encephalopathy related to previously reported the de novo missense mutation p.Arg908Gln in the ATP1A2 gene, is described. A girl examined first time in 11 months had microcephaly, severe mental and motor delay, strabismus, spastic paraparesis and pachypolymicrogyria on brain MRI that is atypical for DEE98. Epilepsy with polymorphic seizures started at the age of 15 months. There was a remission lasting 9 months, after which seizures renewed. DEE98 was diagnosed at the age of 2 years 9 months by exome sequencing verified by trio Sanger sequencing. Another finding from high-throughput exome sequencing were two previously undescribed heterozygous variants of uncertain pathogenicity in the SPART gene, which causes autosomal recessive spastic paraplegia type 20 (SPG20); Sanger sequencing confirmed the trans position of the variants. The common clinical sign with typical SPG20 was early spastic paraparesis with contractures; other symptoms did not coincide. Considering the phenotypic diversity of SPG20 and the possibility of a combination of two independent diseases, we performed an additional study of the pathogenicity of SPART variants at the mRNA level: pathogenicity was not confirmed, and there were no grounds to diagnose SPG20.

Gerstmann-Sträussler disease (GSD) is a very rare autosomal dominant late-onset neurodegenerative disorder related to prion protein gene PRNP . Mutation p.Pro102Leu produces about 80% of cases, which are often named GSD-102. DNA testing provides exact diagnosis. In the presented Russian family there were 3 patients: a female index case, age 32 years, her brother, age 37 years (age of onset in both is 27 years) and their deceased father (onset in 35 years, death in 44 years). GSD was not suspected until whole exome sequencing in the female detected PRNP mutation p.Pro102Leu confirmed in her and in the brother by Sanger sequencing. Atypical features of the case are: early onset in siblings, absence of mental and behavioral problems in the female and in the father and mild disturbances in the brother; epilepsy in the brother; atypical onset with transient signs in the brother. Other intrafamilial differences are prevailing spastic paraparesis in the female in contrast to predominant ataxia in the brother and dysarthria absence in the female. The case illustrates GSD-102 variability, complicating clinical diagnostics.

A rare case of autosomal dominant spastic paraplegia in a 36-year-old female with two reported earlier mutations of most common spastic paraplegia forms: SPG4 (mutation p.Cys28Leufs*20 in SPAST gene) and SPG3 (mutation p.Val405Met in ATL1 gene) is presented. The mutations detected by massively parallel sequencing (MPS) panel were inherited from affected mother and clinically unaffected father, respectively. The proband, her 61-year-old mother and deceased grandfather had 'uncomplicated' paraplegia with onset in 4 th decade. The 67-year-old father had no even minimal subclinical signs of the disease and no affected relatives, detection of his low-penetrating ATL1 mutation was unexpected. MPS methods are the most informative for identifying a patient and/or family members with a combined hereditary neurological pathology, especially a combination of similar forms of heterogeneous groups, such as spastic paraplegia.

A familial case of a rare autosomal dominant Alzheimer's disease (AD), related to PSEN1 gene (AD3, OMIM 607822), differing from common multifactorial form by earlier onset and, in part of cases, by accompanying neurological signs, spastic paraparesis particularly, is presented. The first sign in a female proband and in her son was paraparesis manifested at the age of 29 and 21 years, respectively. Cognitive disturbances developed soon; the former diagnosis was hereditary spastic paraplegia with cognitive impairment, In the proband examined in 2008 at 33 years old the diagnosis was not established. In the son examined in 2022 at 27 years old whole-exome sequencing detected a novel PSEN1 missense mutation p.Thr421Ala. The mutation was confirmed by Sanger sequencing in him, found out in the proband (who was severely disabled by that time) and excluded in her unaffected mother. Except for different age of onset, AD3 in two patients was similar, though in whole it is variable, also in relatives. The variability and rareness of the disease hampers clinical diagnostics. Massive parallel sequencing is a most reliable diagnostic method.

GNE myopathy (GNEM) is a rare hereditary disease, but at the same time, it is the most common distal myopathy in several countries due to a founder effect of some pathogenic variants in the GNE gene. We collected the largest cohort of patients with GNEM from Russia and analyzed their mutational spectrum and clinical data. In our cohort, 10 novel variants were found, including 2 frameshift variants and 2 large deletions. One novel missense variant c.169_170delGCinsTT (p.(Ala57Phe)) was detected in 4 families in a homozygous state and in 3 unrelated patients in a compound heterozygous state. It was the second most frequent variant in our cohort. All families with this novel frequent variant were non-consanguineous and originated from the 3 neighboring areas in the European part of Russia. The clinical picture of the patients carrying this novel variant was typical, but the severity of clinical manifestation differed significantly. In our study, we reported two atypical cases expanding the phenotypic spectrum of GNEM. One female patient had severe quadriceps atrophy, hand joint contractures, keloid scars, and non-classical pattern on leg muscle magnetic resonance imaging, which was more similar to atypical collagenopathy rather than GNEM. Another patient initially had been observed with spinal muscular atrophy due to asymmetric atrophy of hand muscles and results of electromyography. The peculiar pattern of muscle involvement on magnetic resonance imaging consisted of pronounced changes in the posterior thigh muscle group with relatively spared muscles of the lower legs, apart from the soleus muscles. Different variants in the GNE gene were found in both atypical cases. Thus, our data expand the mutational and clinical spectrum of GNEM.

Intellectual development disorder (IDD) is characterized by a general deficit in intellectual and adaptive functioning. In recent years, there has been a growing interest in studying the genetic structure of IDD. Of particular difficulty are patients with non-specific IDD, for whom it is impossible to establish a clinical diagnosis without complex genetic diagnostics. We examined 198 patients with non-specific IDD from 171 families using whole-exome sequencing and chromosome microarray analysis. Hereditary forms of IDD account for at least 35.7% of non-specific IDD, of which 26.9% are monogenic forms. Variants in the genes associated with the BAF (SWI/SNF) complex were the most frequently identified. We were unable to identify phenotypic features that would allow differential diagnosis of monogenic and microstructural chromosomal rearrangements in non-specific IDD at the stage of clinical examination, but due to its higher efficiency, exome sequencing should be the diagnostic method of the highest priority study after the standard examination of patients with NIDD in Russia.

A case of spastic paraplegia type 4 (SPG4) due to SPAST p.Arg499His mutation de novo in a child, aged 2 years 8 months, is presented. The differences of this first Russian case with the mutation and of a number of reported cases from typical SPG4 are very early onset, severe disabling spasticity and additional signs, cognitive disturbances in particular; SPAST mutations de novo are also infrequent. Specific patterns point to the relationship between genotype and phenotype. Methods of exome sequencing are particularly informative in atypical cases difficult for clinical diagnostics.

Merosine deficient congenital muscular dystrophy is one of the most common forms of congenital muscular dystrophy. This disease is caused by a primary deficiency or a functionally inactive form of the protein merosin in muscle tissue. The type of inheritance of this disease is autosomal recessive. De novo variants with this type of inheritance are rare, and it is quite possible that the de novo variant may hide a mosaic form in the parent of an affected child. We present a birth family with two affected children who inherited a previously undescribed pathogenic variant c.1755del from their mother and a previously described pathogenic variant c.9253C > T in the LAMA2 gene from their mosaic father. LAMA2 gene mutation analysis was performed by mass parallel sequencing and direct sequencing of genomic DNAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chausova, Ryzhkova, Rudenskaya, Chernykh, Shchagina and Polyakov.)

Introduction: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a rare neurological syndrome caused by pathogenic variants in the C19orf12 and is characterized by iron deposition in the basal ganglia and substantia nigra. Only a limited number of cohort studies were published to date and the prevalence of MPAN remains uncertain., Methods: Recruited subjects with MPAN in Russia were diagnosed by whole-exome sequencing or Sanger sequencing of the C19orf12 gene. Data of over 14000 whole exome sequencing analyses was used to calculate the estimated disease frequency. RNA analysis was performed by RT-PCR. QSVanalyzer software was used to quantify the allelic disbalance., Results: We describe the clinical and molecular characterizations of 17 patients with MPAN. DNA analysis detected three previously undescribed pathogenic/likely pathogenic variants in the C19orf12 gene. The estimated disease frequency was calculated to be 1:619150. We describe unusual clinical observations in several cases. One patient showed severe neurogenic muscle weakness along with a lack of marked spasticity or optic nerve atrophy. In another mild clinical case with the NM_001031726.3:c.204_214del (p.(Gly69Argfs*10)) variant in a heterozygous state, a marked allelic disbalance was observed on the RNA level with reduced expression level of the wild-type allele. Thus, this case became the first one of a possible regulatory variant causing MPAN., Conclusion: We reported a detailed clinical and molecular characterization of the third-largest MPAN cohort. We expanded the mutational and clinical spectrum of MPAN. Moreover, we calculated the estimated MPAN frequency in the Russian population for the first time., (Copyright © 2021 Elsevier Ltd. All rights reserved.)