Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation
© Hollanda et al; licensee BioMed Central Ltd. 2011
Received: 12 March 2011
Accepted: 25 July 2011
Published: 25 July 2011
This study aimed the use of mesoporous silica under the naturally transformable Neisseria meningitidis, an important pathogen implicated in the genetic horizontal transfer of DNA causing a escape of the principal vaccination measures worldwide by the capsular switching process. This study verified the effects of mesoporous silica under N. meningitidis transformation specifically under the capsular replacement.
we used three different mesoporous silica particles to verify their action in N. meningitis transformation frequency.
we verified the increase in the capsular gene replacement of this bacterium with the three mesoporous silica nanoparticles.
the mesouporous silica particles were capable of increasing the capsule replacement frequency in N. meningitidis.
Freshly isolated Neisseria meningitidis are naturally competent and exchange genetic information with each other by this process. They are also known as a commensal bacterium of the human upper respiratory tract that may occasionally provoke invasive infections such as septicemia and meningitis. This natural competence has been directly correlated to pilliation of these organisms  as well as a specific uptake sequence contained multifold within the genome of these bacteria. Pilliated strains are easily transformed by direct incubation with a plasmid containing the uptake sequence or chromosomal DNA . The advantages of doing genetic manipulations within these well-known strains are numerous. Development of systems to construct specific genomic mutations has been used to study their pathogenesis [3–5].
The use of the mutations for the study of the capsular polysaccharide of N. meningitidi s allowed the advances in the meningococci pathogenesis understandings [6–8]. The capsular polysaccharide is a major virulence factor and a protective antigen. Meningococcal strains are classified into 12 different serogroups according to their capsular immune specificity, among wich the serogroups A, B, C, Y and W135 are the most frequently found in invasive infections. The capsule of serogroups B, C, Y and W135 strains is composed of either homopolymers (B and C) or heteropolymers (Y and W135) of sialic acid-containing polysaccharides that are specifically linked, depending on the serogroup [9–11]. This polymerization is mediated by the polysialyltransferase, encoded by the siaD gene in strains of serogroups B and C (also called synD and synE, respectively) and by synG in serogroup W135. Capsule switching after replacement of synE, in a serogroup C strain, by synG may result from the conversion of capsule genes by transformation and allelic recombination [3, 12, 13]. The capsule switching from serogroup C to B N. meningitidis was observed in several countries after vaccination campaigns [3, 14–17]. It might explain the emergence and the clonal expansion of strains of serogroup W135 of N. meningitidis in the year 2000 among Hajj pilgrims who had been vaccinated against meningococci of serogroups A and C [11, 18]. These W135 strains belong to the same clonal complex ET-37/ST-11 as prominent serogroup C strains involved in outbreaks worldwide [12, 19]. Hence, the emergence of these W135 strains in epidemic conditions raised the question about a possible capsule switching as an escape mechanism to vaccine-induced immunity. Also, these events are expected to occur continuously and can be selected by immune response against a particular capsular polysaccharide . However, the interference of immune response with transformation efficacy has not yet been evaluated. Specific capsular antibodies are expected to bind to the bacterial surface and hence the interference in DNA recognition and uptake.
In addition, environmental interference on the transformation process of this bacterium is also unknown. This work aimed at the use of different mesoporous silica SBA-15, SBA-16 and [SBA-15/P(N-iPAAm)], an organic-inorganic hybrids systems based on mesoporous materials and stimuli-responsive polymers, for the study of these nanostructures effect on the transformation process of meningococci, specifically their functions on capsular switching process. Mesoporous silica materials are a fairly new type of material that has pores in the mesoscopic range of 2-50 nm. The characteristic features of ordered mesoporous materials are their monodispersed and adjustable pore size in an inert and biocompatible matrix with an easily modified surface. The intrinsic uniform porous structure of this class of compounds with their large specific surface area and pore volume, associated with surface silanol groups, makes these materials suitable as an adsorbent model for studies involving surface phenomena. The methods used in this work verified the effect of mesoporous silica SBA-15, SBA-16 and [SBA-15/P(N-iPAAm)] on the transformation of the serogroup C N. meningitidis against two different donor DNA obtained from mutants of this microorganism (M2 and M6).
Bacterial Strains used in this work
Escherichia coli F-, end A1, hsd R17 c, sup E44, thi- 1, gir A96, rel A1
Plasmid containing ΔNMB0065::ΩaaDA
Plasmid containning the fusion of synG::ermAM
Neisseria meningitidis serogroup C, BIOMERIEUX
Neisseria meningitidis serogroup W135, ATCC35559
N.meningitidis isogenic mutant ΔNMB0065:: ΩaaDA
W135ATCC transformed with pLAN13 to generate a fusioned strain synG:ermAM
The mesoporous silica nanoparticles SBA-15, SBA-16 and [SBA-15/P(N-iPAAm)] were characterized by Sousa et al. . Both, SBA-15 and SBA-16 are composed of SiO2 but the characteristic features of SBA-15 are the presence of channels arranged in a two-dimensional hexagonal structure and wheat like macroscopic morphology with mean sizes in micrometer scale which consist of many ropelike aggregates. On the other hand, SBA-16 is an example of ordered mesoporous silica with a three dimensional cubic cage structure with three dimensional channel connectivity. Also, in SBA-16 the arrays of the ordered and uniform pores can be observed for which each spherical particle is a single crystal arranged in cubic structure.
N2 adsorption results.
SBA-15/P( N -iPAAm)
Oligonucleotides used in this work
Sequence 5'- 3'
A preliminary analysis of the action of the mesoporous silica was performed to determine the influence of this nanostructure under Neisseria meningitidis growth. The results did not show any influence on bacterial growth of the presence of DNA in addition of SBa15, SBa16 or SBA-15/P(N-iPAAm) (data not showed).
The first mutant referent to NMB0065 sequence mutants was the strain M2, this mutant had the NMB0065 sequence from N. meningitidis C2135 amplified using 03.12-3 and 03.12-4 oligonucleotides (Table 3). This fragment was cloned into the pGEM-T Easy Vector System II (Promega Corporation, Madison, WI, USA), to generate the plasmid pLAN6. E. coli strain Z501 was transformed with plasmid pLAN6 resulting in the plasmid pLAN7. The ΩaaDA cassette was inserted into the BclI site of pLAN7 to generate plasmid pLAN45, which was transformed into the C2135 strain to generate the strain M2 (Figure 3).
The construction of serogroup W135 mutants with transcriptional fusion synG:: ermAM was initiated by amplifying the region of synG gene using the 98-30 and 03-12-5 oligonucleotides (Table 3) on DNA from the serogroup W135atcc strain. The amplified fragment was cloned into the pGEM-T Easy Vector System I (Promega, Madison, WI, USA), to generate the plasmid pLAN11. Another fragment was amplified using the 04-02-2/galECK29A from synG downstream sequence, cloned into pGEM-T Easy Vector, to generate pLAN52. The ermAM cassette was amplified by ERAM1/ERAM3 and insered into Nco I site of pLAN52 to generate pLAN53. The fragment amplified from pLAN53 with the ERAM1 and galECK29A  was inserted into Pst I site of pLAN11 to generate pLAN13-2. This plasmid was linearised by the enzyme SphI and transformed into W135ATCC strain to generate the synG::ermAM fusion strain M6, erythromycin resistant (Figure 4).
Values obtained from C21 35 Transformation using the donor DNA from M2 and M6 mutants.
Donor DNA (1 μg)
Mean of the UCF transformants obtained in 1.108 UFC
Ratio (means obtained exposed to silica/mean of negative control)
P values (one way Tukey's test)
Negative Control (without mesoporous silica) M2
932 ± 175,50
1,00 ± 0,11
SBa 15 + DNA M2
1696 ± 73,30
1,52 ± 0,25
(P < 0,05)
SBa 16 + DNA M2
1840 ± 423,32
1,97 ± 0,22
(P < 0,05)
SBa 15 (P( N -iPAAm) + DNA M2
1544 ± 358,10
1,38 ± 0,05
(P < 0,05)
Negative Control (without mesoporous silica) M6
106 ± 10,00
0,92 ± 0,06
SBa 15 + DNA M6
364,33 ± 117,11
3,17 ± 0,80
0,0475 (P < 0,05)
SBa 16 + DNA M6
558,70 ± 59,56
4,50 ± 0,43
0,0008 (P < 0,05)
SBa 15 (P( N -iPAAm) + DNA M6
598,67 ± 107,56
5,20 ± 0,80
0,0058 (P < 0,05)
The graphic of Figure 5 shows significant increase of transformation frequencies using M2 and M6 donor DNA and the mesoporous silica SBA-15, SBA-16 and SBA-15/P(N-iPAAm). The use of a different DNA donor had as aim the certification of the independence of mesoporous silica effect on the same bacterial strain-N. meningitidis C2135. The analysis of the PCR had demonstrated the transfer of the gene synG from M6 donor strains to C2135 receptor strain (data not showed).
The data analyses were made by ratio values between the numbers of transformants CFU obtained with mesoporous silica action by the median value of transformants CFU obtained without silica treatment. The values were analyzed by ANOVA one-way analysis of variance (Tukey test compared each treatment to control without mesoporous silica in transformation). The meningococci growth was not affected by the presence of mesoporous silica (data not shown).
As showed in table 4, the significant values of P < 0.05 obtained in the ratio values between transformation using the donors M2 and M6 mutants DNA, respectively. These values are considered significant when compared with the transformation frequency obtained from negative control without silica action. Thus, the actions of mesopourous silica under the meningococci transformation increased the capacity of the C2135 strains, specially using the construction M6, directly implicated in the capsular switching outbreaks.
Despite the exact mechanism of the capsular switching is still under investigation, we proposed that this process is related to the action of mesoporous silica structures in the transformation frequencies in 1.108 cfu, with a significant increase when mesoporous silica was used. The behavior of SBA-16, regarding to transformation process of C2135 strain with donor DNA from M2 mutant, was different from that observed for the others. This nanoparticle showed increase of transformation frequency more than SBA-15, and SBA-15/P(N-iPAAm) mesoporous silica. Besides the differences in the textural properties showed in Table 2, a probable cause for the different responses is the presence of singular morphological arrangements, as they are hierarchically organized in a special way. Moreover, it is worth noticing that the three-dimensional interconnected pore structure of sample SBA-16 can facilitate the occurrence of adsorption.
The important information is the chromosomal localization of the NMB0065 and synG gene. Both are gene of bacterial chromosome and their biological characteristics determined in Neisseria meningitidis when these genes are recombined onto chromosomes level. Nevertheless, N. meningitidis rarely replicate the plasmids provided from E.coli constructions, as those performed in these work (plasmids from pLAN series), exceptionally when in the plasmid carrier antibiotic resistant from another species of Neisseria as N. gonorrhoeae[24–26].
Also the practical implications of the silica action under meningococci are very important to the workers that usually are exposed at these nanoparticles [27–29]. The careful action of adopting the safety measures not only the silicosis [30–33] but also for adopting safety mesures to prevent not only silicosis but also changing pathology and host adaptation of N. meningitidis, will be important in places where silica nanoparticles are present, especially in aerosols. This work is the first to cite the relationships between the silica risks of health caused by meningococcal capsular switching or capsular replacement. This neglected process is described just as an immunologically controlled phenomenon not involving the environmental influences such as the presence of the nanostructures in the atmosphere.
Nevertheless, the capsular switching is described in regions as the sub Saharan Africa [11, 34–36] and Saudi Arabia (Hajj pilgrimage) [34, 37–43] in desert zones where probably silica nanostructures are present that facilities the capsular switching process. New experiments using the animal models could confirm this hypothesis and has been performed by the research group for Neisseria meningitdis and other natural competent bacteria as Streptococcus pneumoniae and Haemophilus influenzae.
This study has been financier supported by CAPES, FAPESP, CNPq and FAPEMIG. These supports help us to reagent supply and equipments for all this research development. FAPESP (number 2008/56777-5) and CNPq (number 575313/2008-0) funding the Laboratory of Biotechnology (Coordinated by M.L.). FAPEMIG funding the laboratory coordinated by E.M.B.S. CNPq and CAPES funding with the personal fellowships for students: R.F.C.P., AS and GAF. Thanks for the English revision for Luiz Paulo Manzo, Júlia N. Varela and Maria Cecília T. Amstalden.
- Tonjum T, Koomey M: The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships--a review. Gene. 1997, 192: 155-163. 10.1016/S0378-1119(97)00018-8.View ArticleGoogle Scholar
- Goodman SD, Scocca JJ: Factors influencing the specific interaction of Neisseria gonorrhoeae with transforming DNA. J Bacteriol. 1991, 173: 5921-5923.Google Scholar
- Swartley JS, Marfin AA, Edupuganti S, Liu LJ, Cieslak P, Perkins B, Wenger JD, Stephens DS: Capsule switching of Neisseria meningitidis. Proc Natl Acad Sci USA. 1997, 94: 271-276. 10.1073/pnas.94.1.271.View ArticleGoogle Scholar
- Zhou D, Stephens DS, Gibson BW, Engstrom JJ, McAllister CF, Lee FK, Apicella MA: Lipooligosaccharide biosynthesis in pathogenic Neisseria. Cloning, identification, and characterization of the phosphoglucomutase gene. J Biol Chem. 1994, 269: 11162-11169.Google Scholar
- Stephens DS, McGee ZA, Melly MA, Hoffman LH, Gregg CR: Attachment of pathogenic Neisseria to human mucosal surfaces: role in pathogenesis. Infection. 1982, 10: 192-195. 10.1007/BF01640777.View ArticleGoogle Scholar
- Alonso JM, Guiyoule A, Zarantonelli ML, Ramisse F, Pires R, Antignac A, Deghmane AE, Huerre M, van der Werf S, Taha MK: A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice. FEMS Microbiol Lett. 2003, 222: 99-106. 10.1016/S0378-1097(03)00252-0.View ArticleGoogle Scholar
- Nassif X, So M: Interaction of pathogenic neisseriae with nonphagocytic cells. Clin Microbiol Rev. 1995, 8: 376-388.Google Scholar
- Spinosa MR, Progida C, Tala A, Cogli L, Alifano P, Bucci C: The Neisseria meningitidis capsule is important for intracellular survival in human cells. Infect Immun. 2007, 75: 3594-3603. 10.1128/IAI.01945-06.View ArticleGoogle Scholar
- Frosch M, Muller D, Bousset K, Muller A: Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression. Infect Immun. 1992, 60: 798-803.Google Scholar
- Taha MK, Parent Du Chatelet I, Schlumberger M, Sanou I, Djibo S, de Chabalier F, Alonso JM: Neisseria meningitidis serogroups W135 and A were equally prevalent among meningitis cases occurring at the end of the 2001 epidemics in Burkina Faso and Niger. J Clin Microbiol. 2002, 40: 1083-1084. 10.1128/JCM.40.3.1083-1084.2002.View ArticleGoogle Scholar
- Taha MK, Antignac A, Renault P, Perrocheau A, Levy-bruhl D, Nicolas P, Alonso JM: Clonal spread of Neisseria meningitidis W135. Presse Med. 2001, 30: 1535-1538.Google Scholar
- Lancellotti M, Guiyoule A, Ruckly C, Hong E, Alonso JM, Taha MK: Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex. Microbes Infect. 2006, 8: 191-196. 10.1016/j.micinf.2005.06.012.View ArticleGoogle Scholar
- Zarantonelli ML, Lancellotti M, Deghmane AE, Giorgini D, Hong E, Ruckly C, Alonso JM, Taha MK: Hyperinvasive genotypes of Neisseria meningitidis in France. Clin Microbiol Infect. 2008, 14: 467-472. 10.1111/j.1469-0691.2008.01955.x.View ArticleGoogle Scholar
- Kriz P, Kriz B, Svandova E, Musilek M: Antimeningococcal herd immunity in the Czech Republic--influence of an emerging clone, Neisseria meningitidis ET-15/37. Epidemiol Infect. 1999, 123: 193-200. 10.1017/S095026889900285X.View ArticleGoogle Scholar
- Alcala B, Salcedo C, Arreaza L, Abad R, Enriquez R, De La Fuente L, Uria MJ, Vazquez JA: Antigenic and/or phase variation of PorA protein in non-subtypable Neisseria meningitidis strains isolated in Spain. J Med Microbiol. 2004, 53: 515-518. 10.1099/jmm.0.05517-0.View ArticleGoogle Scholar
- Perez-Trallero E, Vicente D, Montes M, Cisterna R: Positive effect of meningococcal C vaccination on serogroup replacement in Neisseria meningitidis. Lancet. 2002, 360: 953-View ArticleGoogle Scholar
- Stefanelli P, Fazio C, Neri A, Sofia T, Mastrantonio P: First report of capsule replacement among electrophoretic type 37 Neisseria meningitidis strains in Italy. J Clin Microbiol. 2003, 41: 5783-5786. 10.1128/JCM.41.12.5783-5786.2003.View ArticleGoogle Scholar
- Taha MK, Bichier E, Perrocheau A, Alonso JM: Circumvention of herd immunity during an outbreak of meningococcal disease could be correlated to escape mutation in the porA gene of Neisseria meningitidis. Infect Immun. 2001, 69: 1971-1973. 10.1128/IAI.69.3.1971-1973.2001.View ArticleGoogle Scholar
- Zarantonelli ML, Antignac A, Lancellotti M, Guiyoule A, Alonso JM, Taha MK: Immunogenicity of meningococcal PBP2 during natural infection and protective activity of anti-PBP2 antibodies against meningococcal bacteraemia in mice. J Antimicrob Chemother. 2006, 57: 924-930. 10.1093/jac/dkl066.View ArticleGoogle Scholar
- Taha MK, Morand PC, Pereira Y, Eugene E, Giorgini D, Larribe M, Nassif X: Pilus-mediated adhesion of Neisseria meningitidis: the essential role of cell contact-dependent transcriptional upregulation of the PilC1 protein. Mol Microbiol. 1998, 28: 1153-1163. 10.1046/j.1365-2958.1998.00876.x.View ArticleGoogle Scholar
- Souza KC, Ardisson JD, Sousa EM: Study of mesoporous silica/magnetite systems in drug controlled release. J Mater Sci Mater Med. 2009, 20: 507-512. 10.1007/s10856-008-3592-1.View ArticleGoogle Scholar
- Giorgini D, Taha MK: Molecular typing of Neisseria meningitidis serogroup A using the polymerase chain reaction and restriction endonuclease pattern analysis. Mol Cell Probes. 1995, 9: 297-306. 10.1016/S0890-8508(95)91540-0.View ArticleGoogle Scholar
- Dolan-Livengood JM, Miller YK, Martin LE, Urwin R, Stephens DS: Genetic basis for nongroupable Neisseria meningitidis. J Infect Dis. 2003, 187: 1616-1628. 10.1086/374740.View ArticleGoogle Scholar
- Dillon JR, Pauze M, Yeung KH: Spread of penicillinase-producing and transfer plasmids from the gonococcus to Neisseria meningitidis. Lancet. 1983, 1: 779-781.View ArticleGoogle Scholar
- Ikeda F, Tsuji A, Kaneko Y, Nishida M, Goto S: Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis. Microbiol Immunol. 1986, 30: 737-742.View ArticleGoogle Scholar
- Naessan CL, Egge-Jacobsen W, Heiniger RW, Wolfgang MC, Aas FE, Rohr A, Winther-Larsen HC, Koomey M: Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae. J Bacteriol. 2008, 190: 387-400. 10.1128/JB.00765-07.View ArticleGoogle Scholar
- Abraham JL, McEuen DD: Inorganic particulates associated with pulmonary alveolar proteinosis: SEM and X-ray microanalysis results. Appl Pathol. 1986, 4: 138-146.Google Scholar
- van den Brule S, Misson P, Buhling F, Lison D, Huaux F: Overexpression of cathepsin K during silica-induced lung fibrosis and control by TGF-beta. Respir Res. 2005, 6: 84-10.1186/1465-9921-6-84.View ArticleGoogle Scholar
- Barboza CE, Winter DH, Seiscento M, Santos Ude P, Terra Filho M: Tuberculosis and silicosis: epidemiology, diagnosis and chemoprophylaxis. J Bras Pneumol. 2008, 34: 959-966. 10.1590/S1806-37132008001100012.View ArticleGoogle Scholar
- Ding M, Chen F, Shi X, Yucesoy B, Mossman B, Vallyathan V: Diseases caused by silica: mechanisms of injury and disease development. Int Immunopharmacol. 2002, 2: 173-182. 10.1016/S1567-5769(01)00170-9.View ArticleGoogle Scholar
- Harrison J, Chen JQ, Miller W, Chen W, Hnizdo E, Lu J, Chisholm W, Keane M, Gao P, Wallace W: Risk of silicosis in cohorts of Chinese tin and tungsten miners and pottery workers (II): Workplace-specific silica particle surface composition. Am J Ind Med. 2005, 48: 10-15. 10.1002/ajim.20175.View ArticleGoogle Scholar
- Hearl FJ: Industrial hygiene sampling and applications to ambient silica monitoring. J Expo Anal Environ Epidemiol. 1997, 7: 279-289.Google Scholar
- Linch KD: Respirable concrete dust--silicosis hazard in the construction industry. Appl Occup Environ Hyg. 2002, 17: 209-221. 10.1080/104732202753438298.View ArticleGoogle Scholar
- Alonso JM, Bertherat E, Perea W, Borrow R, Chanteau S, Cohet C, Dodet B, Greenwood B, LaForce FM, Muros-Le Rouzic E: From genomics to surveillance, prevention and control: new challenges for the African meningitis belt. Bull Soc Pathol Exot. 2006, 99: 404-408.Google Scholar
- Caugant DA, Nicolas P: Molecular surveillance of meningococcal meningitis in Africa. Vaccine. 2007, 25 (Suppl 1): A8-11.View ArticleGoogle Scholar
- Zombre S, Hacen MM, Ouango G, Sanou S, Adamou Y, Koumare B, Konde MK: The outbreak of meningitis due to Neisseria meningitidis W135 in 2003 in Burkina Faso and the national response: main lessons learnt. Vaccine. 2007, 25 (Suppl 1): A69-71.View ArticleGoogle Scholar
- Dull PM, Abdelwahab J, Sacchi CT, Becker M, Noble CA, Barnett GA, Kaiser RM, Mayer LW, Whitney AM, Schmink S: Neisseria meningitidis serogroup W-135 carriage among US travelers to the 2001 Hajj. J Infect Dis. 2005, 191: 33-39. 10.1086/425927.View ArticleGoogle Scholar
- Taha MK, Giorgini D, Ducos-Galand M, Alonso JM: Continuing diversification of Neisseria meningitidis W135 as a primary cause of meningococcal disease after emergence of the serogroup in 2000. J Clin Microbiol. 2004, 42: 4158-4163. 10.1128/JCM.42.9.4158-4163.2004.View ArticleGoogle Scholar
- Wang JL, Liu DP, Yen JJ, Yu CJ, Liu HC, Lin CY, Chang SC: Clinical features and outcome of sporadic serogroup W135 disease Taiwan. BMC Infect Dis. 2006, 6: 7-10.1186/1471-2334-6-7.View ArticleGoogle Scholar
- Wilder-Smith A: W135 meningococcal carriage in association with the Hajj pilgrimage 2001: the Singapore experience. Int J Antimicrob Agents. 2003, 21: 112-115. 10.1016/S0924-8579(02)00355-2.View ArticleGoogle Scholar
- Wilder-Smith A: Meningococcal vaccine in travelers. Curr Opin Infect Dis. 2007, 20: 454-460. 10.1097/QCO.0b013e3282a64700.View ArticleGoogle Scholar
- Wilder-Smith A, Barkham TM, Chew SK, Paton NI: Absence of Neisseria meningitidis W-135 electrophoretic Type 37 during the Hajj, 2002. Emerg Infect Dis. 2003, 9: 734-737.View ArticleGoogle Scholar
- Wilder-Smith A, Barkham TM, Earnest A, Paton NI: Acquisition of W135 meningococcal carriage in Hajj pilgrims and transmission to household contacts: prospective study. Bmj. 2002, 325: 365-366. 10.1136/bmj.325.7360.365.View ArticleGoogle Scholar
- Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983, 166: 557-580. 10.1016/S0022-2836(83)80284-8.View ArticleGoogle Scholar
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