Evaluation of the predicted vs. achieved occlusal outcomes with the Invisalign® appliance: A retrospective investigation of adult patients.

INTRODUCTION: The aim of this study was to investigate the relationship between occlusal contacts, overbite, transverse expansion, and the buccolingual inclination of the teeth with reference to the predicted treatment outcomes and achieved outcomes related to the use of the Invisalign® appliance in mild-to-moderate Class I malocclusions. MATERIAL AND METHODS: The occlusal contacts, overbite, the buccolingual inclination and transverse expansion of the maxillary arch of adult patients satisfying inclusion and exclusion criteria were measured at the initial (pre-treatment), predicted, and achieved treatment stages using metrology software. Pearson correlation coefficients and regression equations were calculated to determine the association between the initial, predicted and achieved changes in occlusal contact against the other variables. RESULTS: Thirty-three patients, who commenced treatment between 2013 and 2018 and satisfied inclusion/exclusion criteria were evaluated. An overall loss of posterior contact was recorded and highlighted by a significantly greater loss of contact from the maxillary buccal occlusal surfaces compared to the palatal occlusal surfaces. The mean [SD] achieved overbite outcome (2.94mm [1.17]) was greater than the predicted (1.74mm [0.87), P<0.001). The buccolingual inclination was significantly increased for the lateral incisors and first and second molars despite a predicted decrease (P≤0.007). Achieved transverse expansion showed significant variation from the predicted. The loss of posterior occlusal contact was correlated with the buccolingual inclination (r=0.70) and transverse expansion (r=0.74) of the posterior teeth. CONCLUSIONS: In mild-to-moderate Class I malocclusions, treatment using the Invisalign® appliance resulted in an overall loss of posterior contact. The loss of occlusal contact was correlated with deficiencies in achieved buccolingual inclination and transverse expansion of the posterior teeth. Planned bodily expansion was ineffective as most expansion occurred due to unplanned buccal tipping.

Occlusal contacts and treatment with the Invisalign appliance: a retrospective analysis of predicted vs achieved outcomes.

OBJECTIVES: To quantify the predicted occlusal contact outcomes compared with the clinically achieved occlusal contacts following treatment using the Invisalign aligner appliance. MATERIALS AND METHODS: The occlusal contacts of 33 adult patients presenting with a Class I mild-to-moderate malocclusion (spacing <4 mm or crowding of <6 mm) and treated using the Invisalign appliance were measured at the initial, predicted, and achieved stages of treatment by the metrology software Geomagic Control X. Assessed measurements were related to individual teeth and anterior, posterior, and overall contacts. RESULTS: The mean (standard deviation) difference between the achieved occlusal contact was significantly less than that predicted for overall occlusal contact and posterior occlusal contact (P < .0025). The achieved posterior occlusal contact was also less than pretreatment initial posterior occlusal contact. There was no difference in anterior occlusal contact between the predicted and achieved outcomes (P > .05). The central and lateral incisors displayed no statistically significant difference between the predicted and achieved occlusal contact. The patients with prescribed overcorrection demonstrated a statistically significant difference in predicted occlusal contact compared with those with nonprescribed overcorrection (P ≤ .0025), but no statistically significant difference in achieved occlusal contact. CONCLUSIONS: Treatment by the Invisalign appliance in Class I mild-to-moderate malocclusion resulted in a decrease in posterior occlusal contact. Further research is required to account for the deficiencies between the predicted and achieved clinical outcome related to occlusal contact and to determine the corrective changes required in the treatment protocols.

Exploring the Stories of Parents' Experiences With Infant Hearing-Loss Screening and Diagnosis in the United States.

OBJECTIVES: For the past 20 years, birthing hospitals in the United States have required newborns to undergo a hearing-loss screening before leaving the hospital. Since the initial newborn hearing screening mandates, there has been much outcome research documenting the successes and barriers of the programs. However, we know little about the experiences of their parents during the time between screening and diagnosis. We propose that elucidating the parents' experiences with newborn hearing-loss screening and diagnosis-via their own stories-is a first step toward understanding their varied experiences and has the potential to ultimately improve hearing healthcare for both children and their families. Thus, to better understand the early hearing screening and detection experience from hearing parents' perspectives, we asked the following research question: what are parents' experiences with their newborns' hearing-loss screening and diagnosis in the United States? DESIGN: The present study employed a prospective, cross-sectional qualitative design. Specifically, we gathered stories from 13 hearing parents who each have a child born in the United States and diagnosed with hearing loss no later than 14 mo of age between the years of 2016 and 2020. We used thematic analysis to uncover common themes across parent narratives. Saturation was reached at interview no. 4; thus no further sampling was needed. FINDINGS: Two major themes emerged from the data: (1) hearing healthcare experiences and (2) parents' early experiences during the period between their child's newborn hearing-loss screening and diagnosis. Subthemes were also uncovered. Three emergent subthemes related to health-care experiences included: (1) downplayed newborn hearing screening referrals, (2) clinician-centered care, and (3) medical expenses and health coverage. The three subthemes of the second theme were as follows: (1) parent-to-parent support, (2) "mom guilt," and (3) a new reality. CONCLUSION: The present study's narrative accounts from parents about their infants' early hearing detection experiences revealed several, different subthemes that emerged from the same, mandated newborn experience in US families. These findings highlight important moments throughout the hearing-loss screening and detection process, which could benefit from more effective, family-centered hearing healthcare. This knowledge also facilitates the field's move toward improved education of future and current providers and regarding family-centered approach, which could address concerns and expectations of new parents at the very start of their newborns' hearing-loss journeys.

Exploring the Types of Stories Hearing Parents Tell About Rearing their Children Who Use Cochlear Implants.

OBJECTIVES: The majority of children born in the United States with hearing loss (HL) are born to parents with hearing. Many of these parents ultimately choose cochlear implantation for their children. There are now decades of research showing which these children's speech, language, listening, and education seem to benefit from cochlear implantation. To date, however, we know little about the experiences of the parents who guided these children throughout their journeys. We propose that elucidating the types of stories these parents tell is a first step toward understanding their varied experiences and has the potential to ultimately improve healthcare outcomes for both children and their families. Thus, to better understand parents' experience, we asked the following research question: what types of stories do parents with hearing tell about rearing their children with HL who use cochlear implants? DESIGN: In this prospective qualitative study, we used a narrative approach. Specifically, we conducted narrative interviews with 20 hearing parents who are rearing young children (mean age = 5.4 years) born with HL who use cochlear implants. We then used thematic narrative analysis to identify recurring themes throughout the narratives that coalesced into the types of stories parents told about their experiences. FINDINGS: Thematic narrative analysis revealed five story types: (1) stories of personal growth, (2) proactive stories, (3) stories of strain and inundation, (4) detached stories, and (5) stories of persistence. CONCLUSIONS: In the present study, different types of stories emerged from parents' experiences that share common events-a family's baby is identified at birth with unexpected permanent HL, the family chooses to pursue cochlear implantation for their child, and then the family raises said pediatric cochlear implant user into adulthood. Despite these similarities, the stories also varied in their sensemaking. Some parents told stories in which a positive life narrative turned bad, whereas others told stories in which a narrative of surviving turned into one of thriving. These findings specifically contribute to the field of hearing healthcare by providing professionals with insight into parents' sensemaking via the types of stories they shared centered on their perceptions and experiences following their child's diagnosis of HL and their decision to pursue cochlear implantation.

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa.

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.

The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

Role of four calcium transport proteins, encoded by nca-1, nca-2, nca-3, and cax, in maintaining intracellular calcium levels in Neurospora crassa.

We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca(2+)-ATPase, or NCA-3, a PMC-type Ca(2+)-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca(2+)-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca(2+)/H(+) exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.

Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa.

We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca(2+)-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca(2+)-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca(2+)/H(+) exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 mum from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

A model for the proteolipid ring and bafilomycin/concanamycin-binding site in the vacuolar ATPase of Neurospora crassa.

The vacuolar ATPase has been implicated in a variety of physiological processes in eukaryotic cells. Bafilomycin and concanamycin, highly potent and specific inhibitors of the vacuolar ATPase, have been widely used to investigate the enzyme. Derivatives have been developed as possible therapeutic drugs. We have used random mutagenesis and site-directed mutagenesis to identify 23 residues in the c subunit involved in binding these drugs. We generated a model for the structure of the ring of c subunits in Neurospora crassa by using data from the crystal structure of the homologous subunits of the bacterium Enterococcus hirae (Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G., and Walker, J. E. (2005) Science 308, 654-659). In the model 10 of the 11 mutation sites that confer the highest degree of resistance are closely clustered. They form a putative drug-binding pocket at the interface between helices 1 and 2 on one c subunit and helix 4 of the adjacent c subunit. The excellent fit of the N. crassa sequence to the E. hirae structure and the degree to which the structural model predicts the clustering of these residues suggest that the folding of the bacterial and eukaryotic polypeptides is very similar.

Analysis of strains with mutations in six genes encoding subunits of the V-ATPase: eukaryotes differ in the composition of the V0 sector of the enzyme.

To address questions about the structure of the vacuolar ATPase, we have generated mutant strains of Neurospora crassa defective in six subunits, C, H, a, c, c', and c''. Except for strains lacking subunit c', the mutant strains were indistinguishable from each other in most phenotypic characteristics. They did not accumulate arginine in the vacuoles, grew poorly at pH 5.8 with altered morphology, and failed to grow at alkaline pH. Consistent with findings from Saccharomyces cerevisiae, the data indicate that subunits C and H are essential for generation of a functional enzyme. Unlike S. cerevisiae, N. crassa has a single isoform of the a subunit. Analysis of other fungal genomes indicates that only the budding yeasts have a two-gene family for subunit a. It has been unclear whether subunit c', a small proteolipid, is a component of all V-ATPases. Our data suggest that this subunit is present in all fungi, but not in other organisms. Mutation or deletion of the N. crassa gene encoding subunit c' did not completely eliminate V-ATPase function. Unlike other V-ATPase null strains, they grew, although slowly, at alkaline pH, were able to form conidia (asexual spores), and were inhibited by concanamycin, a specific inhibitor of the V-ATPase. The phenotypic character in which strains differed was the ability to go through the sexual cycle to generate mature spores and viable mutant progeny. Strains lacking the integral membrane subunits a, c, c', and c'' had more severe defects than strains lacking subunits C or H.

V-ATPases as drug targets.

V-ATPases are large, complex enzymes responsible for acidification of many internal compartments in eukaryotic cells. They also occur on plasma membranes of specialized cells, where they acidify the surrounding milieu. Numerous physiological processes depend on the activity of V-ATPases, and V-ATPases are implicated as a contributing factor in multiple diseases, including osteoporosis, deafness, and cancer. Three classes of natural products have been identified as potent inhibitors of V-ATPases. The bafilomycins and concanamycins, which inhibit all known eukaryotic V-ATPases, are the most extensively studied inhibitors. They bind the Vo subunit c and may inhibit the enzyme by preventing rotation of the c subunit ring. The salicylihalamides and lobatamides show remarkable specificity for animal V-ATPases. The chondropsins preferentially inhibit the fungal V-ATPase. Because of the variety of processes and diseases associated with V-ATPases and the possibility of designing selective inhibitors, the V-ATPases are attractive targets for development of therapeutic agents.

The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae.

The vacuolar H+-ATPase is inhibited with high specificity and potency by bafilomycin and concanamycin, macrolide antibiotics with similar structures. We previously reported that mutation at three residues in subunit c of the vacuolar ATPase from Neurospora crassa conferred strong resistance to bafilomycin but little or no resistance to concanamycin (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). We have identified additional mutated sites in subunit c that confer resistance to bafilomycin. Furthermore, by subjecting a resistant mutant to a second round of mutation we isolated strains with increased resistance to both bafilomycin and concanamycin. In all of these strains the second mutation is also in subunit c, suggesting it forms at least part of the concanamycin binding site. Site-directed mutagenesis of the gene encoding subunit c in Saccharomyces cerevisiae showed that single mutations in each of the residues identified in one of the double mutants of N. crassa conferred resistance to both bafilomycin and concanamycin. Mutations at the corresponding sites in the VMA11 and VMA16 genes of S. cerevisiae, which encode the c' and c" subunits, did not confer resistance to the drugs. In all, nine residues of subunit c have been implicated in drug binding. The positions of these residues support a model in which the drug binding site is a pocket formed by helices 1, 2, and 4. We hypothesize that the drugs inhibit by preventing the rotation of the c subunits.

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases.

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.

Lobatamide C: total synthesis, stereochemical assignment, preparation of simplified analogues, and V-ATPase inhibition studies.

The total synthesis and stereochemical assignment of the potent antitumor macrolide lobatamide C, as well as synthesis of simplified lobatamide analogues, is reported. Cu(I)-mediated enamide formation methodology has been developed to prepare the highly unsaturated enamide side chain of the natural product and analogues. A key fragment coupling employs base-mediated esterification of a beta-hydroxy acid and a salicylate cyanomethyl ester. Three additional stereoisomers of lobatamide C have been prepared using related synthetic routes. The stereochemistry at C8, C11, and C15 of lobatamide C was assigned by comparison of stereoisomers and X-ray analysis of a crystalline derivative. Synthetic lobatamide C, stereoisomers, and simplified analogues have been evaluated for inhibition of bovine chromaffin granule membrane V-ATPase. The salicylate phenol, enamide NH, and ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

Synthesis and V-ATPase inhibition of simplified lobatamide analogues.

[structure: see text] Simplified analogues of the lobatamides have been synthesized and evaluated for inhibition of bovine V-ATPase. The salicylate phenol, enamide NH, and the ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

Mutations in subunit C of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site.

Bafilomycin A1, a potent inhibitor of vacuolar H(+)-ATPases (V-ATPase), inhibited growth of Neurospora crassa in medium adjusted to alkaline pH. Ninety-eight mutant strains were selected for growth on medium (pH 7.2) containing 0.3 or 1.0 microm bafilomycin. Three criteria suggested that 11 mutant strains were altered in the V-ATPase: 1) these strains accumulated high amounts of arginine when grown at pH 5.8 in the presence of bafilomycin, 2) the mutation mapped to the locus of vma-3, which encodes the proteolipid subunit c of the V-ATPase, and 3) V-ATPase activity in purified vacuolar membranes was resistant to bafilomycin. Sequencing of the genomic DNA encoding vma-3 identified the following mutations: T32I (two strains), F136L (two strains), Y143H (two strains), and Y143N (five strains). Characterization of V-ATPase activity in the four kinds of mutant strains showed that the enzyme was resistant to bafilomycin in vitro, with half-maximal inhibition obtained at 80-400 nm compared with 6.3 nm for the wild-type enzyme. Surprisingly, the mutant enzymes showed only weak resistance to concanamycin. Interestingly, the positions of two mutations corresponded to positions of oligomycin-resistant mutations in the c subunit of F(1)F(0)-ATP synthases (F-ATPases), suggesting that bafilomycin and oligomycin utilize a similar binding site and mechanism of inhibition in the related F- and V-ATPases.