More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, pro- viding insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of funda- mental mechanisms regulating eukaryotic gene expression.
Matrix Attachment Regions are DNA sequences that mediate binding of chromatin to the nuclear matrix or scaffold and have been shown to increase and stabilize transgene expression in animal and plant systems. The mechanisms by which they do this remain unknown. One hypothesis states that MARs can increase transgene expression by preventing read-through transcription between multi-copy tandem and/or inverted repeats and adjacent endogenous promoters, which can trigger RNA-mediated silencing. Four constructs were designed that placed the tobacco Rb7 MAR or a λ spacer control sequence between a CaMV 35s promoter-driven sense copy and promoterless antisense copy of the LUCint coding region. Two constructs contained a Nos polyadenylation signal following the transcribed LUCint before the MAR or λ spacer, while the other two did not. Previous studies of MAR effects have compared different populations of random integration events, and expression levels have typically shown very high variability. To reduce this variation, we used the FLP- FRT site-specific recombination system from the 2 µm yeast plasmid to remove the MAR sequence in planta from single-copy plant lines after integration, permitting us to compare constructs with or without a MAR at the same genomic locus. Gene expression was
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Two nuclear matrix attachment regions (MARs) bracket a 550-bp segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type 16 (HPV- 16). One of these MARs is located in the 5 ⴕ third of the LCR (5 ⴕ -LCR-MAR); the other lies within the E6 gene (E6-MAR). To study their function, we linked these MARs in various natural or artificial permutations to a chimeric gene consisting of the HPV-16 enhancer-promoter segment and a reporter gene. In transient trans- fections of HeLa cells, the presence of either of these two MARs strongly represses reporter gene expression. In contrast to this, but similar to the published behavior of cellular MARs, reporter gene expression is stimulated strongly by the E6-MAR and moderately by the 5 ⴕ -LCR-MAR in stable transfectants of HeLa or C33A cells. To search for binding sites of soluble nuclear proteins which may be responsible for repression during transient transfections, we performed electrophoretic mobility shift assays (EMSAs) of overlapping oligonucleotides that represented all sequences of these two MARs. Both MARs contain multiple sites for two strongly binding proteins and weak binding sites for additional factors. The strongest complex, with at least five binding sites in each MAR, is generated by the CCAAT displacement factor (CDP)/Cut, as judged by biochemical purification, by EMSAs with competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in CDP/Cut. In analogy to poorly understood mechanisms acting on cellular MARs, activation after physical linkage to chromosomal DNA may result from competition between the nuclear matrix and CDP/Cut. Our observations show that cis-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent transcriptional enhancers, and activated expression of HPV-16 oncogenes dependent on chromosomal integration may positively select tu- morigenic cells during the multistep etiology of cervical cancer.
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Accumulating evidence indicates that retroviral integra- tion site selection is influenced by properties of cellular DNA structure [11,21-24]. A recent large-scale study found that DNA structural features such as bendability and A-philicity served as preferred integration sites . The present study was performed to assess the role of matrix attachment regions (MARs) in retroviral integra- tion site selection. MARs are DNA sequences located at the bases of DNA loops that attach to the nuclear matrix, and are thus positioned near the machinery for DNA replica- tion, transcription, RNA processing and transport (reviewed in ). There is no consensus sequence that defines a MAR; however, MARs are commonly found to have intrinsic DNA bending properties, to contain tran- scription factor binding sites, AT-rich stretches, sites for topoisomerase I and II binding and cleavage, and high unwinding potential [26,27]. MARs function as structural regulatory elements by organizing the DNA into loop domains. Studies have shown that MARs influence the expression of cellular genes, and can enhance viral gene expression when in the vicinity of viral promoters and enhancers [28-30]. This property has made the inclusion of MARs in gene therapy vectors attractive for enhanced and prolonged expression of the transgene in a specific cell-type or developmental stage [31-33]. MARs have been implicated in virus-mediated malignancies, particularly as targets of integration by small DNA tumor viruses. Specif- ically, integrated SV40, HBV, HPV16 and HPV18 have been found within or in close proximity to MARs in tumors or transformed cell lines . Other reports indi- cate that HTLV-1 and HIV-1 may integrate preferentially near MARS [34,35].
The E5 gene and the early-late intergenic region contain the strongest MAR of HPV-16, which is contiguous with weak MARs in E2 and E1 gene sequences. From Fig. 4F it is appar- ent that an 802-bp DdeI fragment had the highest affinity to the nuclear matrix preparations. This fragment is between nucle- otide positions 3536 and 4337 and includes the 39 end of E2, the E5 gene, and the early-late intergenic region. Figure 6 shows a refined analysis of this genomic region. The E5 gene is included in a 3,650-bp Asp718-Bsp120 fragment, which also contains the E1 and E2 genes and the 59 terminus of L2. After digestion with either SspB1, MunI, or Ppu10I, this region can be subdivided into four, five, or three smaller fragments, re- spectively. As documented in Fig. 6A to C and summarized in Fig. 6D, the strongest MAR activity can be seen in fragments including the E5 gene and a small part of the 39 terminus of E2. Critical information can be gathered from a 428-bp SspB1 fragment which includes sequences between positions 3681 and 4108, including all of E5. As judged by densitometric analyses with a PhosphorImager, all fragments 59 of E5 with E1 and E2 gene sequences have relatively weaker MAR activity. The unsatisfactory analysis of the 777-bp fragment, which was poorly separated from a 783-bp fragment, was later clarified by inclusion in another experiment (see Fig. 9). In Fig. 6A, a 422-bp SspB1 fragment with the 59 end of L2, also including part of the early-late intergenic region, is not well resolved from the 428-bp fragment with strong MAR activity. From the comparison of the positions of the respective bands in the precipitate and the supernatant fractions, it seems that the 422-bp fragment is very poorly bound. This would place the 39 border of this MAR in the intergenic region, with an extremely A-T-rich segment in the middle of this region apparently not contributing much to the MAR property.
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mentioned previously (Bode and Maass, 1988). MARs are operationally defined by their ability to bind isolated nuclear matrices in vitro and have been isolated from a variety of organisms, including fungi (Amati and Gasser, 1988), animals (Mirkovitch et al., 1984), and plants (Hall et al., 1991). MARs are generally AT rich (greater than 65% AT), which is thought to facilitate DNA unwinding at base unpairing regions (BURs) that may be involved in matrix binding (Bode et al., 1996). A database of MAR sequences and nuclear matrix proteins involved in DNA attachment has been assembled for public use called “S/MARt DB” (Liebich et al., 2002). Although MARs may contain several different sequence elements, it has been a challenge to use sequence information alone to identify DNA fragments with the capacity to bind to the nuclear matrix. Relying on the cumulative effect of sequence elements such as AT tracts, Drosophila topoisomerase II sites, DNA bending properties and others, algorithms have been produced to identify potential MAR sequences (Boulikas, 1995; Benham et al., 1997; Singh et al., 1997; Glazko et al., 2001). It is not surprising that these attempts have met with limited success, as not all of these MAR-related motifs correlate well with the ability to bind nuclear matrices in vitro (Michalowski et al., 1999).
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We found that chr4 genes associated with TSS S/MARs were significantly enriched for transcription factors (as annotated in the AGRIS database; Table 12), consistent with a prediction by Tetko et al. (2006) using in silico prediction of S/MARs in Arabidopsis, who anticipated transcription factors genes to contain a significant proportion of S/MARs. Van Drunen et al. (1997) identified three S/MARs upstream of the transcription factor ATH1 (At4g32980), one of which appeared to be in the 5’ proximal region (van Drunen et al., 1997). Our results show that the 5’ proximal ATH1 S/MAR is in cluster D and primarily exonic. ATH1 is an important light-regulated homeobox protein (Quaedvlieg et al., 1995). It is also interesting that while ATH1 is “light-regulated”, the mRNA is absent from our cell culture even though it is grown in continuous light. In plants, ATH1 expression is both light- responsive and dependent on AGAMOUS expression, which also not active in the cultured cells. Perhaps in the absence of AGAMOUS, the ATH1 S/MAR remains bound to the nuclear matrix and transcription is repressed.
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In conclusion, it should be pointed out that although a lot has been learned about the function of MARs, the precise mechanism through which they influence gene expression is still obscure and probably diverse. One possible model involves the MAR-binding protein HMGI(Y) that could depelete histone H1 and cause chromatin opening. MARs also mediate the targeting of DNA to the nuclear matrix, which is enriched in RNA polymerases and splicing complexes (Berezney et al., 1995). Another mechanism could rely on the MAR-induced changes in DNA topology, since matrix attachment regions have been shown to interact with topoisomerase II and contain sequences, which unwind easily under superhelical tension (Bode et al., 1992; Benham et al., 1997). Yet another pathway may involve MeCP2 - a ubiquitously expressed protein that interacts with both methylated CpG dinucleotides and MAR sequences. MeCP2 associates with histone deacetylases and might therefore confer repression upon MAR-containing regions of chromatin. Finally, certain MAR sequences are also recognized by tissue-specific transcriptional regulators, such as Bight and SATB1, which can modulate transcription at the sites of binding.
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The regeneration of periodontium requires restitution of periodontal attachment apparatus, new bone formation, new cementum deposition upon denuded root surface and insertion of functionally oriented new collagen fibers of periodontal ligament into new bone and new cementum . The selective migration, proliferation and differentiation of the cells derived from periodontal ligament and alveolar bone are essential in achieving successful periodontal regeneration . The characteristics, biological problems, and technical complications associated with periodontal wound healing and tissue regeneration have been reviewed extensively [3-6]. There are several techniques used alone or in combination to achieve periodontal regeneration, including root surface modification, bone grafts or substitutes, guided tissue regeneration, and biological mediators. The biological mediators include extracellular matrix proteins and cell attachment factors, mediators of cell metabolism and activity, and growth and differentiation factors.
ABSTRACT Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulﬁlimine cross-links between type IV basement membrane collagens. Loss of function in the Caenorhabditis elegans peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we ﬁnd that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of let-805, encoding the trans- membrane protein myotactin, which suppress phenotypes of pxn-2 null mutants and of other basement membrane mutants such as F-spondin/spon-1. These let-805 suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (emb-9, let-2) and perlecan (unc-52), as well as a mutation affecting spectraplakin (vab-10), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of- function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix.
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nuclear matrix, while the rest of the protein is distributed throughout the chromatin- bound and soluble nuclear and cytoplasmic compartments of transfected cells. Inter- estingly, it was shown previously that a conserved patch of basic amino acids within the hinge region of HPV11 E2 is essential for strong attachment to the nuclear matrix (28). Mutation of this patch of basic amino acids in HPV11 E2 to alanine residues resulted in an increase in cytoplasmic E2 localization. Also, there was a loss of nuclear E2 foci, which are thought to be important for HPV DNA replication. It was suggested from these studies that the nuclear matrix provides a structural scaffold for E2 compartmen- talization and that attachment to the nuclear matrix is important for viral DNA repli- cation and viral mRNA transcription. Our ﬁndings suggest that the regulation of nuclear distribution of the HPV16 E2 protein is more complex and that ChlR1 binding solubilizes a pool of E2 by reducing nuclear matrix association. However, it is important to note that the differences in subcellular localization of E2 WT and E2 Y131A overexpressed in
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Geltrex Matrix (Thermo Fisher Scientific). Each dish was coated with 500 μ L Geltrex Matrix diluted to a final concen- tration of 1 mg/mL in DMEM without FBS. Plates were incu- bated for 1 h in 37 ° C and Geltrex Matrix solution was aspirated and dishes were air-dried (Nest Scientific, Rahway, NJ, USA). After 24 h of incubation, cells were treated with ND, NG, or nGO nanoparticles at the concentration of 20 μ g/mL for the next 24 h. Subsequently, cells were washed twice with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich). Actin cytoskeleton was stained with phalloidin conjugated with Atto 633 (Sigma-Aldrich). Imaging was performed using Olympus FV1000 confocal microscope (Olympus Soft Imaging Solu- tions) equipped with 60 × oil immersion objective.
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“ABPD” may be another mechanism by which parents execute “context-specific” maladaptive socializing practices in children’s achievement domains (especially in sport) (e.g., Tofler & Butterbaugh, 2005a; Tofler et al., 2005b). As an example, sport can be a competitive and reward/evaluation-focused context in which the demonstration of ability is important and emphasized by significant others. The unique characteristic and atmosphere of sport is an open door to aggressive and ambitious parents, vulnerable to ABPD pressures, especially when parents place their self-worth on a child’s success and failure in sport. Objectification of a child is one of the mechanisms of parental “achievement by proxy” in Tofler at al.’s proposed ABPD spectrum. That is, parents may come to regard their children as an object, rather than a person, as a means to indirectly satisfy their own needs for achievement. This controlling parental behaviour may drive a child to succeed to please parents or feel valued. However, it may also lead children to feel guilt or lose self-value if they cannot meet parents’ expectations and requirements. This introjection of parental objectification, thwarting one’s psychological needs for autonomy, competence and relatedness in sport, could serve as an influential contextual cue to activate insecurely “sport-specific” attachment representations.
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Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP
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The APXS instruments have discovered evidence of varied fluid alteration regimes at three different landing sites located thousands of kilometers apart [e.g., Ming et al., 2008; Squyres et al., 2012; McLennan et al., 2013; Thompson et al., 2016]. Variations in Al/Si and Mg/Si (Figure 1-1) demonstrate diverse alteration conditions, including silica enrichments that are linked to alteration by acidic fluids [Ming et al., 2008; Schmidt et al., 2008; Yen et al., 2017], Al enrichments indicative of smectite formation [Ming et al., 2008], and high Al, Na, and K in alkalic rocks in Gale Crater consistent with mantle metasomatism [Stolper et al., 2013; Schmidt et al., 2014; Treiman et al., 2016]. The APXS also quantifies geologically significant trace elements (Ni, Zn, Br, Ge, ± Rb, Sr, Pb, Cu, Se), which have been used to track geochemical processes. For example, Ni and Zn indicate multiple processes (Figure 1-2), including the scavenging of Ni in Mg- sulfates in acidic fluids [Yen et al., 2017], depletion of Ni and Zn by leaching in acidic fluids [Gellert et al., 2015], concentration of Zn by Cl-rich diagenetic fluids in sandstones and veins (see Chapter 5), and Ni enrichment by meteoritic input to the surface [Yen et al., 2006]. By studying these trends in tandem with other rover instrument data (e.g., X- ray diffraction, imaging), the geochemical evidence from APXS data provides constraints for the history of each of the rover sites. In general, the rover missions have revealed that a complex interplay of volcanic, impact, and sedimentary processes on the Martian surface occurred with fluid conditions ranging from hydrothermal [e.g., Schmidt et al., 2008; Squyres et al., 2008, 2012] to low temperature and circumneutral [McLennan et al., 2013] to concentrated acidic brines [e.g., Morris et al., 2006]. The data acquired from the Mars surface by rovers is complementary to detailed studies of meteorites that also point to fluid history on Mars [e.g., Filiberto et al., 2014; McCubbin et al., 2016].
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Most of this variation comes from changes in the charge and structure of the V3 loop, but variability in the conserved co- receptor binding surface, which displays some interisolate dif- ferences in electrostatic potential (Fig. 8 and results not shown), may also influence polyanion binding. The data that we have obtained from kinetic and MAb inhibition studies of gp120-heparin binding strongly suggest a phenomenon based on an initial high-affinity association via the V3 loop followed by weaker binding via a second site, most probably the con- served coreceptor binding region. Although we do not have unequivocal evidence of a direct interaction between polyan- ions and the conserved coreceptor binding surface, the data presented here provide a strong case for this. Thus, (i) polya- nions potently inhibit the attachment of MAbs specific for the conserved coreceptor binding surface and regions of the V3 loop but, with the exception of one V2 loop-specific MAb, have little or no effect on MAbs to other exposed regions of gp120; (ii) CD4i-specific and V3 loop-specific MAbs inhibit the hep- arin-gp120 interaction in an additive manner; (iii) heparin binds mutants of gp120 from which the NH 2 and COOH ter-
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Do the ECM and CMAC features of the MZtmem2 phenotype represent two separate functions of Tmem2, or are these roles of Tmem2 inter-related? Although composition of the ECM is not likely to have a direct impact on α DG glycosylation, prior studies have found that laminin organization can be influenced by DGC glycosylation state (Kanagawa et al., 2005; Michele et al., 2002). Alternatively, Tmem2 could influence the ECM and DGC through two independent mechanisms. In this regard, it is intriguing that the Tmem2 ectodomain can fulfill some, but not all, aspects of Tmem2 function: although the ectodomain can improve fiber attachment in MZtmem2 mutants, it seems less effective than full-length Tmem2 and it cannot rescue α DG glycosylation. Thus, our data suggest that Tmem2 can function in the extracellular environment, consistent with our prior finding that myocardial expression of tmem2 can non- autonomously rescue MZtmem2 endocardial phenotypes (Totong et al., 2011). At the same time, our results suggest that functions of Tmem2 at distinct subcellular locations are relevant to its influence on post-translational modification of α DG. We therefore favor a model in which independent activities of Tmem2, affecting ECM organization and α DG glycosylation, collaborate to enforce muscle fiber attachment.
The most appropriate way to determine community attachment is still a point of debate in tourism and sociology. Mixing of CA and PA items in the name of community attachment is commonplace and occurs even when sociologists and tourism researchers collaborate. Brehm, Eisenhaur and Krannich (2004b) purposely mix CA and PA, (though they do not call them as such) in order to ―examine more closely the connection between two dimensions of community attachment—social and natural environment—and attitudes about local environmental issues‖ (p. 143). Citing McCool and Martin‘s (1994) finding that newcomers had a higher attachment level than long-term residents, Brehm et al. (2004b) sought to further examine the differences between the two resident groups with regard to community attachment and environmental concerns. After collecting data in Wyoming and Utah, Brehm et al. (2004b) concluded that CA was comprised of two distinct factors: social attachment, and natural environment attachment.
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A simple and easy way to construct platinum nanoparticle (PtNPs) arrays on a carbon matrix is presented and the catalytic properties of the developed materials are evidenced with electrochemical studies. Technically, the key point resides in being able to get metallic nanoparticles of homogeneous size that are well dispersed on a carbon substrate. Specific efforts in this direction led us, here, to obtain, for the first time, well-defined and well-spread PtNPs of 100 ± 40 nm on carbon microspheres. The resulting material was used for the development of a low cost practical Pt nanoelectrode array sensor for aqueous proton detection. In addition, methanol oxidation was studied to exemplify potential uses in fuel cells. The procedures described are very simple to apply and represent an original strategy for the development of well-defined catalytic surfaces.
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Biofilm formation could be defined as one of leading causes for bacteria developing multi-drug resistance. One biofilm life cycle contains four stages, the initial attach- ment of bacteria, microbial colonies formation, bacterial growth and ECM generation and biofilm matures as the latest stage, followed by the dispersal of the bacteria to find new niches (Fig. 1). The surface of the substratum presents host polymeric matrix, which is mainly com- posed of exopolysaccharides, proteins, nucleic acids, and other substances, facilitating irreversible attachment of the bacteria. It was reported that cell surface-associated proteins such as Aap and SasG were involved in Staphylo- coccus epidermidis initiating attachment and Aap protein contains G5 domain, which was responsible for bacterial intercellular cell adhesion . Extracellular components, including the surface-exposed protein, the extracellu- lar glucan-binding protein and the glycosyltransferases (GtfE, GtfG and GtfH), also play an important role in cell adhesive abilities . Sortase A (SrtA), a transpeptidase that can anchor cell surface proteins, also elicits extracel- lular localization and biofilm formation during infection of Gram-positive bacteria, such as Staphylococcus aureus
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