IntroductionCandida albicans has been detected together with Streptococcus mutans in high numbers in plaque-biofilm from children with early childhood caries (ECC). The goal of this study was to examine the C. albicans carriage in children with severe early childhood caries (S-ECC) and the maternal relatedness.MethodsSubjects in this pilot cross-sectional study were recruited based on a convenient sample. DMFT(S)/dmft(s) caries and plaque scores were assessed during a comprehensive oral exam. Social-demographic and related background information was collected through a questionnaire. Saliva and plaque sample from all children and mother subjects were collected. C. albicans were isolated by BBL™ CHROMagar™ and also identified using germ tube test. S. mutans was isolated using Mitis Salivarius with Bacitracin selective medium and identified by colony morphology. Genetic relatedness was examined using restriction endonuclease analysis of the C. albicans genome using BssHII (REAG-B). Multilocus sequence typing was used to examine the clustering information of isolated C. albicans. Spot assay was performed to examine the C. albicans Caspofungin susceptibility between S-ECC children and their mothers. All statistical analyses (power analysis for sample size, Spearman’s correlation coefficient and multiple regression analyses) were implemented with SAS 9.4ResultsA total of 18 S-ECC child-mother pairs and 17 caries free child-mother pairs were enrolled in the study. Results indicated high C. albicans carriage rate in the oral cavity (saliva and plaque) of both S-ECC children and their mothers (>80%). Spearman’s correlation coefficient also indicated a significant correlation between salivary and plaque C. albicans and S. mutans carriage (p<0.01) and caries severity (p<0.05). The levels of C. albicans in the prepared saliva and plaque sample (1ml resuspension) of S-ECC children were 1.3 ± 4.5 x104 cfu/ml and 1.2 ± 3.5 x104 cfu/ml (~3-log higher vs. caries-free children). Among 18 child-mother pairs, >60% of them demonstrated identical C. albicans REAG-B pattern. C. albicans isolated from >65% of child-mother pairs demonstrated similar susceptibility to caspofungin in spot assay, while no caspofungin resistant strains were seen when compared with C. albicans wild-type strain SC5314. Interestingly, the regression analysis showed that factors such as antibiotic usage, birth weight, inhaler use, brushing frequency, and daycare attendance had no significant effect on the oral carriage of C. albicans in the S-ECC children.ConclusionsOur results reveal that both the child with S-ECC and the mother were highly infected with C. albicans, while most of the strains were genetically related, suggesting that the mother might be a source for C. albicans acquisition in the oral cavity of children affected by the disease.

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.

Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidyindependent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1 336 ) was previously shown to possess similar affinity for A2 and retain ϳ60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys 36 that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1 37-336 ) showed little if any affinity for A2 (K d >2 M), whereas factor VIIIa reconstituted with A2 plus A1 37-336 /A3-C1-C2 dimer demonstrated significant cofactor activity (ϳ30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1 37-336 showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K m for factor X when A1 was cleaved at Arg 336 . These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K m for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasmabased assays.Factor VIII, a plasma protein that participates in the blood coagulation cascade, is deficient or defective in individuals with hemophilia A. Factor VIII functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa. Factor VIII is synthesized as a multi-domain, single chain molecule (A1-A2-B-A3-C1-C2) (1) with a molecular mass of ϳ300 kDa (2, 3). Factor VIII is processed to a series of divalent metal ion-linked heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains. This procofactor is activated by cleavage at Arg 372 , Arg 740 , and Arg 1689 by thrombin and factor Xa, converting the dimer into the factor VIIIa trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with a weak affinity by electrostatic interactions (5, 6). Factor VIIIa is unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (5-7). Under physiological conditions, the K d for this interaction is ϳ260 nM (8, 9); however, at slightly acidic pH and low ionic strength, this interaction is facilitated by an ϳ10-fold increase in the affinity (K d ϭ ϳ30 nM) (8).The role of factor VIIIa in the intrinsic factor Xase is to bind factor IXa, which increases the k cat for factor Xa formation by several orders of magnitude compared with factor IXa alone (10). Interactive sites for factor IXa are localized to A2 and A3 domains (11-13). Recent studies have shown that modulation of factor IXa by the isolated A2...

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ϳ100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ϳ1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is synthesized as a 300-kDa precursor (1, 2) with the domain structure A1-A2-B-A3-C1-C2 (3). It is processed to a series of divalent metal ion-dependent heterodimers after cleavage at the B-A3 junction, generating a HC 1 (A1-A2-B domains) and a LC (A3-C1-C2 domains). Additional cleavage sites within the B domain result in variably sized HCs minimally represented by contiguous A1-A2 domains. The two chains can be separated by chelating reagents (4, 5) and isolated after ion exchange and/or immunoaffinity chromatography. Factor VIII activity can be reconstituted from the separated chains by combining them in the presence of a divalent metal ion (6).Factor VIII functions in the intrinsic factor Xase complex as a cofactor for the serine protease, factor IXa in the surface-dependent conversion of factor X to Xa. This activity is dependent upon conversion of factor VIII to the active cofactor form, factor VIIIa, by thrombin or factor Xa. These enzymes cleave factor VIII HC at Arg-740, removing the B domain (or fragments) and at Arg-372, bisecting the HC into the A1 and the A2 subunits (7). The proteases also cleave factor VIII LC at Arg-1689 (7), liberating an acid-rich region and creating a new NH 2 terminus. Thus, factor VIIIa is a heterotrimer of subunits designated as A1, A2, and A3-C1-C2 (8, 9). The A1 and A3-C1-C2 subunits retain the divalent metal ion-dependent linkage, whereas the A2 subunit is weakly associated with the A1-A3-C1-C2 dimer by primarily electrostatic interactions (9, 10).Two regions of factor VIII have been identified as interactive sites for factor IXa. A high affinity site (K d ϳ15 nM (11)) was localized to the A3 domain of the LC in and around residues 1811-1818 (...