Presynaptic neurons self-tune by inversely coupling neurotransmitter release with the abundance of CaV2 voltage-gated Ca2+ channels.

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability.

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release.

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of ß/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary ß and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/ß or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary ß and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

UNC-2 CaV2 Channel Localization at Presynaptic Active Zones Depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans.

Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel localization by active zone proteins is not completely understood. In a Caenorhabditis elegans (C. elegans) forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic localization of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel puncta at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel localization, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that although SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 localization by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel localization. In addition, we find that core active zone proteins are unequal in their abundance. Although the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel localization in redundant yet distinct manners.SIGNIFICANCE STATEMENT Precise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM, our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, a core active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

Alcohol induces mitochondrial fragmentation and stress responses to maintain normal muscle function in Caenorhabditis elegans.

Chronic excessive ethanol consumption has distinct toxic and adverse effects on a variety of tissues. In skeletal muscle, ethanol causes alcoholic myopathy, which is characterized by myofiber atrophy and the loss of muscle strength. Alcoholic myopathy is more prevalent than all inherited muscle diseases combined. Current evidence indicates that ethanol directly impairs muscle organization and function. However, the underlying mechanism by which ethanol causes toxicity in muscle is poorly understood. Here, we show that the nematode Caenorhabditis elegans exhibits the key features of alcoholic myopathy when exposed to ethanol. As in mammals, ethanol exposure impairs muscle strength and induces the expression of protective genes, including oxidative stress response genes. In addition, ethanol exposure causes the fragmentation of mitochondrial networks aligned with myofibril lattices. This ethanol-induced mitochondrial fragmentation is dependent on the mitochondrial fission factor DRP-1 (dynamin-related protein 1) and its receptor proteins on the outer mitochondrial membrane. Our data indicate that this fragmentation contributes to the activation of the mitochondrial unfolded protein response (UPR). We also found that robust, perpetual mitochondrial UPR activation effectively reduces muscle weakness caused by ethanol exposure. Our results strongly suggest that the modulation of mitochondrial stress responses may provide a method to ameliorate alcohol toxicity and damage to muscle.

Reduced IGF signaling prevents muscle cell death in a Caenorhabditis elegans model of muscular dystrophy.

Duchenne muscular dystrophy, a fatal degenerative muscle disease, is caused by mutations in the dystrophin gene. Loss of dystrophin in the muscle cell membrane causes muscle fiber necrosis. Previously, loss-of-function mutations in dys-1, the Caenorhabditis elegans dystrophin ortholog, were shown to cause a contractile defect and mild fiber degeneration in striated body wall muscle. Here, we show that loss of dystrophin function in C. elegans results in a shorter lifespan and stochastic, age-dependent muscle-cell death. Reduction of dystrophin function also accelerated age-dependent protein aggregation in muscle cells, suggesting a defect in proteostasis. Both muscle cell death and protein aggregation showed wide variability among the muscle cells. These observations suggest that muscle cell death in dys-1 mutants is greatly influenced by cellular environments. Thus, the manipulation of the cellular environment may provide an opportunity to thwart the cell death initiated by the loss of dystrophin. We found that reduced insulin-like growth factor (IGF) signaling, which rejuvenates the cellular environment to protect cells from a variety of age-dependent pathologies, prevented muscle cell death in the dys-1 mutants in a daf-16-dependent manner. Our study suggests that manipulation of the IGF signaling pathways in muscle cells could be a potent intervention for muscular dystrophy.

Presynaptic BK channel localization is dependent on the hierarchical organization of alpha-catulin and dystrobrevin and fine-tuned by CaV2 calcium channels.

BACKGROUND: Large conductance, calcium-activated BK channels regulate many important physiological processes, including smooth muscle excitation, hormone release and synaptic transmission. The biological roles of these channels hinge on their unique ability to respond synergistically to both voltage and cytosolic calcium elevations. Because calcium influx is meticulously regulated both spatially and temporally, the localization of BK channels near calcium channels is critical for their proper function. However, the mechanism underlying BK channel localization near calcium channels is not fully understood. RESULTS: We show here that in C. elegans the localization of SLO-1/BK channels to presynaptic terminals, where UNC-2/CaV2 calcium channels regulate neurotransmitter release, is controlled by the hierarchical organization of CTN-1/α-catulin and DYB-1/dystrobrevin, two proteins that interact with cortical cytoskeletal proteins. CTN-1 organizes a macromolecular SLO-1 channel complex at presynaptic terminals by direct physical interaction. DYB-1 contributes to the maintenance or stabilization of the complex at presynaptic terminals by interacting with CTN-1. We also show that SLO-1 channels are functionally coupled with UNC-2 calcium channels, and that normal localization of SLO-1 to presynaptic terminals requires UNC-2. In the absence of UNC-2, SLO-1 clusters lose the localization specificity, thus accumulating inside and outside of presynaptic terminals. Moreover, CTN-1 is also similarly localized in unc-2 mutants, consistent with the direct interaction between CTN-1 and SLO-1. However, localization of UNC-2 at the presynaptic terminals is not dependent on either CTN-1 or SLO-1. Taken together, our data strongly suggest that the absence of UNC-2 indirectly influences SLO-1 localization via the reorganization of cytoskeletal proteins. CONCLUSION: CTN-1 and DYB-1, which interact with cortical cytoskeletal proteins, are required for the presynaptic punctate localization of SLO-1 in a hierarchical manner. In addition, UNC-2 calcium channels indirectly control the fidelity of SLO-1 puncta localization at presynaptic terminals. We suggest that the absence of UNC-2 leads to the reorganization of the cytoskeletal structure that includes CTN-1, which in turn influences SLO-1 puncta localization.

Time-dependent periimplant bone reaction of acidic monomer-treated injection molded zirconia implants in rabbit tibiae.

PURPOSE: The aim of this study was to evaluate and compare the osseointegration of zirconia implants in rabbit tibiae with and without 10-methacryloyloxydecyl dihydrogen phosphate (MDP) treatment. MATERIALS AND METHODS: Twenty-eight rabbits received a total of 112 external hex-type implants made by the powder injection molding technique with and without MDP treatment before installation in the tibiae. The contact angles on the zirconia implants and zirconia discs before and after MDP application were evaluated. Removal torque (RT) and bone-implant contact (BIC) ratios were measured. RESULTS: The MDP treatment markedly enhanced the hydrophilicity and seemed to alter the topography and chemical composition of the implant and disc surface. As the healing time increases, the BIC and RT were increased in both groups. The MDP-treated implants exhibited higher BIC values than the control implants after 2 and 4 weeks of healing. The RT was higher in MDP-treated implants after 2 weeks of healing but not after 4 weeks of healing. CONCLUSION: The 10-MDP treatments made the surface more hydrophilic and enhanced the osseointegration of the implants in the early healing phase.

Peri-implant bone formation of non-thermal atmospheric pressure plasma-treated zirconia implants with different surface roughness in rabbit tibiae.

OBJECTIVES: The aim of this study was to evaluate and compare the osseointegration of powder-injection molded (PIM) zirconia implants in rabbit tibiae with or without He plasma treatment. MATERIAL AND METHODS: Twenty-five rabbits received 4 types of external hex implants with identical geometry in the tibiae: PIM zirconia implants, roughened PIM zirconia implants, plasma-treated PIM zirconia implants, and plasma-treated roughened PIM zirconia implants. The contact angles of the four types of implants were evaluated. Removal torque tests and histomorphometric analyses were performed. RESULTS: The plasma treatment markedly enhanced the hydrophilicity, but did not seem to change the surface topography of the PIM zirconia implants. There were statistically significant differences in the bone-to-implant contact (BIC) ratios, bone volume (BV/TV), and removal torque values (RTQ) among the tested implant types (P < 0.001). The plasma-treated implants exhibited significantly higher BIC and BV/TV values than the untreated implants. However, the removal torque values favored the rough surface. CONCLUSIONS: The He plasma treatments on PIM zirconia implants made the surface more hydrophilic and enhanced the osseointegration of the implants without changing the micro-topography.

Submorphotypes of the maxillary first molar and their effects on alignment and rotation.

INTRODUCTION: The aim of this study was to explore the shape differences in maxillary first molars with orthographic measurements using 3-dimensional virtual models to assess whether there is variability in morphology that could affect the alignment results when treated by straight-wire appliance systems. METHODS: A total of 175 maxillary first molars with 4 cusps were selected for classification. With 3-dimensional laser scanning and reconstruction software, virtual casts were constructed. After performing several linear and angular measurements on the virtual occlusal plane, the teeth were clustered into 2 groups by the method of partitioning around medoids. To visualize the 2 groups, occlusal polygons were constructed using the average data of these groups. RESULTS: The resultant 2 clusters showed statistically significant differences in the measurements describing the cusp locations and the buccal and lingual outlines. The rotation along the centers made the 2 cluster polygons look similar, but there was a difference in the direction of the midsagittal lines. CONCLUSIONS: There was considerable variability in morphology according to 2 clusters in the population of this study. The occlusal polygons showed that the outlines of the 2 clusters were similar, but the midsagittal line directions and inner geometries were different. The difference between the morphologies of the 2 clusters could result in occlusal contact differences, which might be considered for better alignment of the maxillary posterior segment.

The growth trends of Korean adolescents with bialveolar protrusion: a nine year longitudinal cephalometric study.

Bialveolar protrusion and dental crowding seems to have common features in terms of aetiology and treatment method, although they result in different facial profiles and tooth alignment. The aim of this longitudinal study is to determine when children begin to show bialveolar protrusive traits in order to get more clue about the aetiology of bialveolar protrusion. Longitudinal lateral cephalometric data of children followed from 6 to 14 years of age were used. A total of 155 children (81 girls and 74 boys) with showing Class molar relationships at the age of 14 were assigned to either the protrusive group (PG) or the non-protrusive group (NPG), based on 5 cephalometric measurements. The cephalometric measurements of these 2 groups were compared at each age separately in both sexes by independent t-tests. Throughout the entire observation period, there were differences between PG and NPG in both sexes in measurements which were used for classification at 14 years of age. There were differences between sexes in both PG and NPG in several measurements of at several different ages. Individuals with bialveolar protrusive traits at the age of 14 exhibited the signs early in life, at least from the early mixed dentition.

Interaction of α-catulin with dystrobrevin contributes to integrity of dystrophin complex in muscle.

The dystrophin complex is a multimolecular membrane-associated protein complex whose defects underlie many forms of muscular dystrophy. The dystrophin complex is postulated to function as a structural element that stabilizes the cell membrane by linking the contractile apparatus to the extracellular matrix. A better understanding of how this complex is organized and localized will improve our knowledge of the pathogenic mechanisms of diseases that involve the dystrophin complex. In a Caenorhabditis elegans genetic study, we demonstrate that CTN-1/α-catulin, a cytoskeletal protein, physically interacts with DYB-1/α-dystrobrevin (a component of the dystrophin complex) and that this interaction is critical for the localization of the dystrophin complex near dense bodies, structures analogous to mammalian costameres. We further show that in mouse α-catulin is localized at the sarcolemma and neuromuscular junctions and interacts with α-dystrobrevin and that the level of α-catulin is reduced in α-dystrobrevin-deficient mouse muscle. Intriguingly, in the skeletal muscle of mdx mice lacking dystrophin, we discover that the expression of α-catulin is increased, suggesting a compensatory role of α-catulin in dystrophic muscle. Together, our study demonstrates that the interaction between α-catulin and α-dystrobrevin is evolutionarily conserved in C. elegans and mammalian muscles and strongly suggests that this interaction contributes to the integrity of the dystrophin complex.

An SLC6 transporter of the novel B(0,)- system aids in absorption and detection of nutrient amino acids in Caenorhabditis elegans.

Nutrient amino acid transporters (NATs) of solute carrier family 6 (SLC6) mediate uptake of essential amino acids in mammals and insects. Phylogenomic analysis of the Caenorhabditis elegans (Ce) SLC6 family identifies five genes paralogous to an insect-specific NAT subfamily. Here we cloned and characterized the first representative of the identified nematode-specific transporters, SNF-5. SNF-5 mediates broad spectrum cation-coupled transport of neutral amino acids with submillimolar affinities and stoichiometry of 1 AA:1 Na(+), except for 1 l-Pro:2 Na(+). Unexpectedly, it transports acidic l-Glu(-) and l-Asp(-) (1 AA(-):3 Na(+)), revealing it to be the first member of a new B(0,-) system among characterized SLC6 transporters. This activity correlates with a unique positively charged His(+) 377 in the substrate-binding pocket. snf-5 promoter-driven enhanced green fluorescent protein labels intestinal cells INT1-9 and three pairs of amphid sensory neurons: ASI, ADF and ASK. These cells are intimately involved in control of dauer diapause, development, metabolism and longevity. The snf-5 deletion mutants do not show apparent morphological disorders, but increase dauer formation while reducing dauer maintenance upon starvation. Overall, the present study characterized the first nematode-specific NAT and revealed important structural and functional aspects of this transporter. In addition to the predictable role in alimentary amino acid absorption, our results indicate possible neuronal roles of SNF-5 as an amino acid provider to specific neuronal functions, including sensing of amino acid availability.

Peri-implant bone formations around (Ti,Zr)O(2) -coated zirconia implants with different surface roughness.

AIM: The aim of this study was to evaluate and compare the osseointegration in rabbit tibiae of smooth and roughened powder injection moulded (PIM) zirconia implants with or without (Ti,Zr)O2 surface coatings. MATERIAL AND METHODS: Twenty-five rabbits received four types of external hex implants with identical geometry on the tibiae: PIM zirconia implants, roughened PIM zirconia implants, (Ti,Zr)O2 -coated PIM zirconia implants and (Ti,Zr)O2 -coated roughened PIM zirconia implants. The surface characteristics of the four types of implants were evaluated. Removal torque tests and histomorphometric analyses were performed. RESULTS: The (Ti,Zr)O2 coatings substantially changed the surface topography and chemical composition of the both type of PIM zirconia implants. There were statistically significant differences in the bone to implant contact ratios and removal torque values (RT) among the tested implant types (p < 0.001). The histological response favoured the coated surface at smooth PIM zirconia implants. The removal torque values favoured the rough surface whether coated or uncoated. CONCLUSIONS: Within the limit of this study, the (Ti,Zr)O2 coated PIM zirconia implants, both smooth and rough, showed enhanced histological response (bone to implant contact) compared with uncoated ones. On the other hand, the mechanical anchorage (RT) was higher for rough surface implants, coated or uncoated.

An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

Patterns of vertical facial growth in Korean adolescents analyzed with mixed-effects regression analysis.

INTRODUCTION: To understand the growth patterns of skeletal open bite and deepbite, we present observations from 9 years of pure longitudinal data based on lateral cephalometric radiographs using mixed-effects regression model analysis. METHODS: In total, 51 children (14 years old) with extreme values for the ratio of lower anterior facial height to total anterior facial height were assigned to 1 of 2 groups: a skeletal open-bite group (11 boys, 14 girls) or a skeletal deepbite group (14 boys, 12 girls). Measurements of total anterior facial height, upper anterior facial height, lower anterior facial height, total posterior facial height, ramus height, and ratio of lower anterior facial height to total anterior facial height were obtained for all subjects. All data were analyzed and interpreted using a mixed-effects regression model analysis with random effects. RESULTS: From these 4 groups at 14 years old, statistically significant differences were observed between the groups when subjects of the same sex were compared; however, statistical significance was not reached between subjects of opposite sexes in each group. Morphologic differences were clearly evident from the start and became more pronounced with age. There were statistical significances in the initial values and increases with age in all 6 variables except for increases with age in the ratio of lower anterior facial height to total anterior facial height. Statistical significance was also reached for morphologic differences between the annual increases in the ratio of lower anterior facial height to total anterior facial height and lower anterior facial height. In general, individual random variability was high in all variables when compared with the annual changes over time. CONCLUSIONS: Divergent patterns were established early and became more pronounced with age, with anterior facial height dimensions primarily contributing to these differences. Individual variations were so pronounced that caution is recommended for all clinical decisions.

A three dimensional observation of palatal vault growth in children using mixed effect analysis: a 9 year longitudinal study.

The understanding of palatine vault growth in normal subjects is important to orthodontists. The aim of this study was to evaluate three dimensional (3D) longitudinal changes in the palatal vault from 6 to 14 years of age. Complete dental stone casts were biennially prepared for 50 subjects (25 girls and 25 boys) followed up from 6 to 14 years of age. Virtual casts were constructed using 3D laser scanning and reconstruction software. The reference gingival plane was constructed. The palatal heights were measured from a total of 12 quadrisectional points between the most gingival points of the palatal dentogingival junctions from the canine to the first molar. In addition, the palatal heights were measured from a total of 12 lateral and medial endpoints of the palatine rugae. The measurement changes over time were analyzed using a mixed-effect analysis. There were significant annual increases in all of the variables related to palatal height. However, the individual random variability at baseline was quite large. There was no significant sexual dimorphism in the linear measurements or in the annual increases as fixed effects in the model. During the observation period, increases in palatal vault height were significant in all regions. The growth pattern seemed to differ between genders even though it was not significant. More elaborate methodology is necessary to gain a better understanding of 3D palatal growth.

Different mandibular first molar shapes according to groove and cusp configuration in relation to suggested bracket position.

The aim of this study was to explore the shape differences in the mandibular first molars through orthographic measurements using three-dimensional (3D) virtual models, and study the possibility of new morphologic categories that require more than subjective visual inspection. A total of 164 mandibular first molars with five cusps were selected for classification. Using 3D laser scanning and reconstruction software, virtual casts were constructed. After several linear and angular measurements on the virtual occlusal plane, the teeth were clustered using the partitioning around medoids methods-an unsupervised classification. The cluster analysis presented two clusters that showed statistically significant differences in the measurements over the cusp locations and groove configurations. However, gender differences were not shown in the angular groove and cusp configurations. Two clusters were found in the population of the present study, and this result suggested the existence of a diverse morphologic trait in the mandibular molar even in the same origin and could be considered in positioning orthodontic brackets that have built-in prescriptions.