Metatranscriptome and Resistome of the Endodontic Microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

De novo ALX4 variant detected in child with non-syndromic craniosynostosis.

Current understanding of the genetic factors contributing to the etiology of non-syndromic craniosynostosis (NSC) remains scarce. The present work investigated the presence of variants in ALX4, EFNA4, and TWIST1 genes in children with NSC to verify if variants within these genes may contribute to the occurrence of these abnormal phenotypes. A total of 101 children (aged 45.07±40.94 months) with NSC participated in this cross-sectional study. Parents and siblings of the probands were invited to participate. Medical and family history of craniosynostosis were documented. Biological samples were collected to obtain genomic DNA. Coding exons of human TWIST1, ALX4, and EFNA4 genes were amplified by polymerase chain reaction and Sanger sequenced. Five missense variants were identified in ALX4 in children with bilateral coronal, sagittal, and metopic synostosis. A de novo ALX4 variant, c.799G>A: p.Ala267Thr, was identified in a proband with sagittal synostosis. Three missense variants were identified in the EFNA4 gene in children with metopic and sagittal synostosis. A TWIST1 variant occurred in a child with unilateral coronal synostosis. Variants were predicted to be among the 0.1% (TWIST1, c.380C>A: p. Ala127Glu) and 1% (ALX4, c.769C>T: p.Arg257Cys, c.799G>A: p.Ala267Thr, c.929G>A: p.Gly310Asp; EFNA4, c.178C>T: p.His60Tyr, C.283A>G: p.Lys95Glu, c.349C>A: Pro117Thr) most deleterious variants in the human genome. With the exception of ALX4, c.799G>A: p.Ala267Thr, all other variants were present in at least one non-affected family member, suggesting incomplete penetrance. Thus, these variants may contribute to the development of craniosynostosis, and should not be discarded as potential candidate genes in the diagnosis of this condition.

Vertucci's Root Canal Configuration of 14,413 Mandibular Anterior Teeth in a Brazilian Population: A Prevalence Study Using Cone-beam Computed Tomography.

INTRODUCTION: This cone-beam computed tomographic (CBCT)-based study evaluated the root canal configurations of mandibular anterior teeth in a large Brazilian population, along with the influence of sex and the frequency of symmetry between the contralateral teeth. METHODS: Overall, 2543 CBCT scans, including those of 4773 lower central incisors (LCIs), 4835 lower lateral incisors (LLIs), and 4805 lower canines (LCs), were analyzed. The scans were obtained from a private radiologic clinic using the PreXion 3D device (Yoshida Dental Mfg Co, Ltd, Tokyo, Japan) with a 0.14-mm voxel size; exposure parameters of 90 kV, 4 mA, and 19 seconds; and a field of view of 80 × 80 mm. Root canal anatomy was analyzed according to the Vertucci classification. Sex influence on the canal distribution and symmetry between the contralateral teeth were analyzed. The data were analyzed statistically and were considered significant when P < .05. RESULTS: All LCIs and LLIs had 1 root, whereas 2.4% of LCs had 2 roots. Two root canals were observed in approximately 20% of LCIs and LLIs and in approximately 11% of LCs. The most common configuration was type I followed by type III. Bilateral symmetry occurred in a majority of the individuals (ie, 91% in LCIs, 87% in LLIs, and 87% in LCs). No sex influence was noted with LLIs and LCs (P > .05). However, LCIs showed a higher prevalence of 2 canals in men (P < .05). CONCLUSIONS: The prevalence of 2 root canals in the mandibular anterior teeth in Brazilians was approximately 20% for central and lateral incisors and 10% for canines.

De novo ALX4 variant detected in child with non-syndromic craniosynostosis

Current understanding of the genetic factors contributing to the etiology of non-syndromic craniosynostosis (NSC) remains scarce. The present work investigated the presence of variants in ALX4, EFNA4, and TWIST1 genes in children with NSC to verify if variants within these genes may contribute to the occurrence of these abnormal phenotypes. A total of 101 children (aged 45.07±40.94 months) with NSC participated in this cross-sectional study. Parents and siblings of the probands were invited to participate. Medical and family history of craniosynostosis were documented. Biological samples were collected to obtain genomic DNA. Coding exons of human TWIST1, ALX4, and EFNA4 genes were amplified by polymerase chain reaction and Sanger sequenced. Five missense variants were identified in ALX4 in children with bilateral coronal, sagittal, and metopic synostosis. A de novo ALX4 variant, c.799G>A: p.Ala267Thr, was identified in a proband with sagittal synostosis. Three missense variants were identified in the EFNA4 gene in children with metopic and sagittal synostosis. A TWIST1 variant occurred in a child with unilateral coronal synostosis. Variants were predicted to be among the 0.1% (TWIST1, c.380C>A: p. Ala127Glu) and 1% (ALX4, c.769C>T: p.Arg257Cys, c.799G>A: p.Ala267Thr, c.929G>A: p.Gly310Asp; EFNA4, c.178C>T: p.His60Tyr, C.283A>G: p.Lys95Glu, c.349C>A: Pro117Thr) most deleterious variants in the human genome. With the exception of ALX4, c.799G>A: p.Ala267Thr, all other variants were present in at least one non-affected family member, suggesting incomplete penetrance. Thus, these variants may contribute to the development of craniosynostosis, and should not be discarded as potential candidate genes in the diagnosis of this condition.

Distance and Bone Density between the Root Apex and the Mandibular Canal: A Cone-beam Study of 9202 Roots from a Brazilian Population.

INTRODUCTION: Accidental damage to the inferior alveolar nerve is of concern during endodontic treatment of the posterior mandible teeth, and a careful evaluation of the relationship between the apices and the mandibular canal (MC) is mandatory to prevent iatrogenic complications. This study aimed to assess the distance between the root apices of posterior teeth and the MC and the bone density in such areas. METHODS: One thousand retrospective cone-beam computed tomographic scans were randomly selected from a private oral radiologic clinic database. The images were acquired with a limited field of view and analyzed by 2 evaluators who measured the shortest distance between the root apices and the upper cortical bone of the MC using Dental Slice software (Bioparts Biomedical Prototyping, Brasília, DF, Brazil). In addition, bone density was estimated using the tool "profile line of Hounsfield" and expressed in Hounsfield units. RESULTS: The distal root of the right second molars was the closest to the MC (mean = 3.41 mm), and the right first premolar root was the farthest from the MC (mean = 5.87 mm), respectively. The root apices were closer to the MC in women than in men (P < .05). In 1.79% of the cases, the roots were in contact or invading the MC, whereas 8.35% of the root apices were close to the MC (<1.00 mm), and 89.85% were distant from the MC (>1.00 mm). In 85.55% of the cases, the bone was dense. CONCLUSIONS: The results suggest that the risk of damage to the inferior alveolar nerve is higher for second molars, especially in female patients. The higher density of the adjacent bone may act as a protective factor against nerve damage arising from endodontic procedures.

Access and Intensity of Mechanization: Empirical Evidence of Rice Farmers in Southern Ghana

ABSTRACT The medium term development plan of Ghana proposed modernization of agriculture to lead the way in transforming the economy. Providing irrigation infrastructure and enhancing farmer access to farm machinery were major interventions proposed. In line with this, the government has been investing in irrigation infrastructure as well as importing farm machinery under various programmes in recent years. This study analyzed access and intensity of mechanization by rice farmers in southern Ghana. The Shai-Osudoku and Ketu North Districts were purposively selected and a total of 360 farmers were randomly sampled from 16 rice growing communities. In general, the results of the descriptive statistics revealed that about 74 % of farmers were still cultivating rice with considerably low level of mechanization. The double hurdle model was employed to estimate the determinants of access to mechanization and the intensity of mechanization. The empirical results of tier one of the double huddle model revealed that size of land, access to credit, availability of farm machinery, expenditure on labour, agrochemical expenditure, the square of age, and gender positively influenced access to mechanization. Seed expenditure, age and district locations negatively influenced access to mechanization. The empirical results of the tier two of the double hurdle model revealed that distance from farm to nearest mechanization centre, rice income, non-farm income and experience were significant variables that positively influenced intensity of mechanization. Land ownership and household size negatively influenced intensity of mechanization. These results have implications for capacity building and government support for rice farmers in southern Ghana.

A lysosome targetable luminescent bioprobe based on a europium ß-diketonate complex for cellular imaging applications.

Herein, we report a novel lysosome targetable luminescent bioprobe derived from a europium coordination compound, namely Eu(pfphOCH3IN)3(DDXPO) 4 [where HpfphOCH3IN = 4,4,5,5,5-pentafluoro-3-hydroxy-1-(1-(4-methoxyphenyl)-1H-indol-3-yl)pent-2-en-1-one and DDXPO = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene oxide]. Notably, the newly designed europium complex exhibits significant quantum yield (Φoverall = 25 ± 3%) and 5D0 excited state lifetime (τ = 398 ± 3 µs) values under physiological pH (7.2) conditions when excited at 405 nm. Hence the developed europium complex has been evaluated for live cell imaging applications using mouse pre-adipocyte cell lines (3T3L1). Colocalization studies of the designed bio-probe with commercial Lysosome-GFP in 3T3L1 cells demonstrated the specific localization of the probe in the lysosome with a high colocalization coefficient (A = 0.83). Most importantly, the developed bioprobe exhibits good cell permeability, photostability and non-cytotoxicity.

Complex Apical Intraradicular Infection and Extraradicular Mineralized Biofilms as the Cause of Wet Canals and Treatment Failure: Report of 2 Cases.

This article describes 2 cases that showed persistent intracanal exudation (wet canal) even after several visits of antimicrobial endodontic treatment. Histologic and histobacteriologic investigation was conducted for determination of the cause. The 2 cases involved teeth with apical periodontitis lesions, which presented persistent exudation refractory to treatment after several visits. In case 1, it was not possible to achieve a dry canal, and surgery had to be performed. In case 2, attempts to dry the canal succeeded and the canal was filled, but follow-up examination showed an enlarged apical periodontitis lesion and extraction was performed. Biopsy specimens consisting of the root apex and apical periodontitis lesion for case 1 and the whole root for case 2 were subjected to histologic and histobacteriologic analyses. Both cases showed complex bacterial infection in the apical root, affecting both the intraradicular space and the outer root surface. Case 1 showed bacterial biofilms in ramifications, on untouched walls, and extending to the external root surface to form a thick and partially mineralized structure with high bacterial density. Different bacterial morphotypes were evidenced. Case 2 had a ledge on the apical canal wall created during instrumentation, which was filled with necrotic debris, filling material, and bacteria. The walls of the apical portion of the canal were covered by a bacterial biofilm, which was continuous with a thick extraradicular biofilm covering the cementum and dentin in resorptive defects. The extraradicular biofilm showed areas of mineralization and was dominated by filamentous bacteria. The 2 cases with wet canals and treatment failure were associated with complex persistent infection in the apical part of the root canal system extending to form thick and partially mineralized biofilm structures (calculus) on the outer apical root surface.

Eight-year follow-up of autogenous tooth transplantation involving multidisciplinary treatment.

Although autogenous tooth transplantation is a widely reported procedure, its success is dependent on a number of factors. Here we describe the surgical technique, endodontic treatment and rehabilitation employed for a patient in whom a lower right third molar was transplanted to substitute an adjacent second molar with extensive caries. During an 8-year follow-up period, normal periodontal healing was observed and no infection, ankylosis or progressive resorption occurred. It may be concluded that transplantation of a third molar is a practicable approach for replacement of a lost permanent tooth, with restoration of esthetics and function.

Whole genomewide linkage screen for neural tube defects reveals regions of interest on chromosomes 7 and 10.

Neural tube defects (NTDs) are the second most common birth defects (1 in 1000 live births) in the world. Periconceptional maternal folate supplementation reduces NTD risk by 50-70%; however, studies of folate related and other developmental genes in humans have failed to definitively identify a major causal gene for NTD. The aetiology of NTDs remains unknown and both genetic and environmental factors are implicated. We present findings from a microsatellite based screen of 44 multiplex pedigrees ascertained through the NTD Collaborative Group. For the linkage analysis, we defined our phenotype narrowly by considering individuals with a lumbosacral level myelomeningocele as affected, then we expanded the phenotype to include all types of NTDs. Two point parametric analyses were performed using VITESSE and HOMOG. Multipoint parametric and nonparametric analyses were performed using ALLEGRO. Initial results identified chromosomes 7 and 10, both with maximum parametric multipoint lod scores (Mlod) >2.0. Chromosome 7 produced the highest score in the 24 cM interval between D7S3056 and D7S3051 (parametric Mlod 2.45; nonparametric Mlod 1.89). Further investigation demonstrated that results on chromosome 7 were being primarily driven by a single large pedigree (parametric Mlod 2.40). When this family was removed from analysis, chromosome 10 was the most interesting region, with a peak Mlod of 2.25 at D10S1731. Based on mouse human synteny, two candidate genes (Meox2, Twist1) were identified on chromosome 7. A review of public databases revealed three biologically plausible candidates (FGFR2, GFRA1, Pax2) on chromosome 10. The results from this screen provide valuable positional data for prioritisation of candidate gene assessment in future studies of NTDs.

The pathology of extracranial scalp and skull masses in young children.

OBJECTIVE: Extracranial subcutaneous masses involving the scalp and/or skull in young children are uncommon lesions that get excised by the neurosurgeon. Although the most common reported lesion is the dermoid cyst, our experience suggests that the spectrum of pathology in these lesions can present diagnostic challenges to the pathologist. MATERIAL: We reviewed 30 consecutive extracranial masses from 29 patients between July 1998 and June 2003. METHOD: Hematoxylin and eosin-stained sections were reviewed in all cases, and immunohistochemistry was performed in select cases. RESULTS: Twenty-three were within the scalp, 5 involved the scalp and skull and 2 were within the limits of the inner and outer tables of the skull. There were 8 dermoid cysts, 2 epidermoid cysts, 6 post-traumatic lesions including 3 calcified cephalhematomas and 3 pseudocysts, 5 vascular lesions including 3 capillary hemangiomas, 1 venous angioma and 1 lymphangioma, 2 cases of cranial fasciitis and 1 case each of benign teratoma, deep granuloma annulare, benign fibrous histiocytoma, congenital melanocytic nevus, hamartoma with ectopic meningothelial elements, cutaneous hyalinised ectopic meningioma and a meningocele with a fibrohistiocytic reaction. No lesions have recurred or exhibited malignant features. CONCLUSIONS: Surgical pathologists and neuropathologists should be aware that the differential diagnosis of "lumps and bumps on babie's heads" is quite varied and can be histologically challenging.

Updated investigations of the role of methylenetetrahydrofolate reductase in human neural tube defects.

Folate supplementation appears to reduce the risk for neural tube defects (NTDs). Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene in the folate metabolism pathway that has been extensively studied in different human populations. We examined the risk associated with having the thermolabile variant (TT) of MTHFR in a study of 175 American Caucasians with NTDs and their families. We found a significant association in patients compared with 195 unrelated controls [odds ratio (OR) = 2.13, 95% confidence interval (95% CI) = 1.11-4.09)], but not in mothers (OR = 1.29, 95% CI = 0.622-2.67) or in fathers (OR = 1.45, 95% CI = 0.681-3.09). We found no evidence for unequal transmission from parents to an affected child (p > 0.10). We failed to find a previously reported association for a combined haplotype for MTHFR and cystathionine beta-synthase, except in subjects with NTDs compared with 559 pooled controls (OR = 2.87, 95% CI = 1.03-8.03). We found no evidence for an association for a novel CA-repeat polymorphism identified in a gene closely linked to MTHFR (p > 0.10). Our studies continue to suggest that additional candidate genes other than MTHFR may be responsible for an increased risk to NTD in some American Caucasian families.

Acute abdominal symptoms and signs in children and young adults with spina bifida: ten years' experience.

BACKGROUND/PURPOSE: Diagnosis and management of the acute abdomen in patients with spina bifida (SB) can be problematic. There are at least 4 clinical factors that can predispose to the development of acute abdominal symptoms and signs, and patients with a thoracic level lesion can have a partially insensate abdomen. The authors analyzed their accumulated experience to determine the annual incidence of acute abdominal signs and symptoms in children and young adults with spina bifida, the differential diagnosis, the operative management, and the outcome. The pertinent literature was reviewed. METHODS: Cases were ascertained during a 10-year period at 1 institution and reviewed retrospectively. RESULTS: Twenty-two episodes of acute abdominal symptoms and signs in 19 children and young adults with SB were ascertained over 10 years at 1 institution, for an annual incidence of 0.74%. More patients had a thoracic level lesion (n = 12; 60%) than in the clinic population as a whole (27%; P =.04), but the gender distribution was similar (58% girls), as was the prevalence of ventriculoperitoneal shunts (VPS; 95%). The median age was 13 years (range, 1 year to 26 years). Hospitalization was necessary for 19 (86%) of the 22 episodes. The duration of symptoms before diagnosis was a median of 3 days (range, 1 to 14 days). Most patients (82%) presented with abdominal pain. Fever was present in 27%, shock in 23%, and peritoneal signs in 23%. There were 14 different final diagnoses, 10 (71%) of which were associated with a predisposing factor. Of the 22 episodes, 18 (82%) could be attributed to an underlying factor: (1) neurogenic bladder (9; 41%); (2) neurogenic bowel (3; 14%); (3) VPS (4; 18%); (4) complications from previous surgery (2; 9%). Thirteen patients (59%) underwent a total of 20 surgical procedures of 12 different kinds. Despite awareness of the complexities involved, 3 patients (14%) died: 1 from complications resulting from bladder perforation; 1 from urosepsis and shock; and 1 from peritonitis caused by VPS infection. CONCLUSION: The differential diagnosis of the acute abdomen in patients with SB is broad, conditions requiring surgery are frequently diagnosed, and the mortality rate is substantial, despite aggressive management.

Clinical outcome differences for lipomyelomeningoceles, intraspinal lipomas, and lipomas of the filum terminale.

Failure to differentiate between the different types of lumbosacral lipomas may lead to inaccurate assumptions and inappropriate management of patients. The goal of this study was to determine whether there is a difference in clinical outcome between patients with lipomyelomeningocles, intraspinal lipomas, and lipomas of the filum terminale. One hundred and fourteen patients with spinal dysraphism were seen at Duke University Medical Center between 1995-1999. All patients who had undergone previous operative intervention for these lesions were excluded. Twenty-two patients with intradural lipomas were identified. Of these, 14 (64%) had lipomyelomeningoceles and 8 (36%) had intraspinal lipomas. Twenty-five patients had filum terminale lipomas. Operative management consisted of lumbosacral laminectomies with microsurgical resection of the lipoma and division of the fatty filum. Average age at presentation in symptomatic patients with lipomas of the filum terminale was 17.7 years, and 23 years in the symptomatic intraspinal lipoma group. Patients with lipomyelomeningoceles ranged in age from 1 day to 18 years, with the majority being younger than 2 years. After an average follow-up of 8 months all patients showed improvement in motor strength following operative intervention. Greater improvements in sensory, bladder, and pain scores were associated with filum terminale lipomas. The least improvements in these categories were seen in the lipomyelomeningocele group. Motor strength is the most likely deficit to improve following operative intervention. Lipomyelomeningoceles, intraspinal lipomas, and filum termniale lipomas have different clinical outcomes following operative intervention.

Genetic studies in neural tube defects. NTD Collaborative Group.

Neural tube defects (NTD) are one of the most common birth defects and are caused by both environmental and genetic factors. The approach to identifying the genes predisposing to NTD, through linkage analysis and candidate gene analysis, is reviewed along with characteristics of a large, nationally ascertained cohort of families. Results from specific assessments of p53, PAX3 and MTHFR failed to suggest that these genes play a major role in NTD development in these families. Advances in genetic laboratory and statistical techniques have made this a prime opportunity for investigation into the causes of complex disorders, such as NTD. However, traditional approaches may prove to be challenging due to the difficulty of ascertaining samplable multiplex families.

Genetic and embryological approaches to studies of neural tube defects: a critical review. NTD Collaborative Group.

Experimental embryological models have suggested that the morphology and quantity of neural tube defects may be governed by their position along the anteroposterior axis of the embryo. Inductive interactions and genetic regulation during axis development may play a role in the patterning of neural tube defects. A major challenge in the study of human neural tube defects is determining whether the spectrum of developmental neural tube anomalies found in individuals and their families mirror experimental models and are regulated by similar processes. We have found that the various neural tube defect phenotypes can be clustered according to their position along the anteroposterior axis. The findings correlate well to the pattern of early genes expression, inductive models of the embryonic axis, and mutant NTD animal models. We suggest that NTD should be studied by their location along the anteroposterior axis and that specific mutant genes may be identified by the observed pattern of NTD in an individual or a family.

A genetic hypothesis for Chiari I malformation with or without syringomyelia.

In several reports the authors have suggested occasional familial aggregation of syringomyelia and/or Chiari 1 malformation (CM1). Familial aggregation is one characteristic of traits that have an underlying genetic basis. The authors provide evidence for familial aggregation of CM1 and syringomyelia (CM1/S) in a large series of families, establishing that there may be a genetic component to CM1/S in at least a subset of families. The authors observed no cases of isolated familial syringomyelia in their family studies, suggesting that familial syringomyelia is more accurately classified as familial CM1 with associated syringomyelia. These data, together with the cosegregation of the trait with known genetic syndromes, support the authors' hypothesis of a genetic basis for some CM1/S cases.

Possible interaction of genotypes at cystathionine beta-synthase and methylenetetrahydrofolate reductase (MTHFR) in neural tube defects. NTD Collaborative Group.

Neural tube defects are a common, complex disorder with genetic and environmental components to risk. We investigated the previously reported interaction between homozygosity for the thermolabile variant at the methylenetetrahydrofolate reductase and heterozygosity for the 844ins68 allele at the cystathionine beta-synthase loci in cases with lumbosacral myelomeningocele and their parents. Using control allele frequencies from our sample pooled with those published in the literature, we confirm a marginally significant interaction at these two loci. This finding suggests that additional, larger studies are warranted to investigate this possible interaction in more detail.

A simple and safe technique for endoscopic third ventriculocisternostomy.

A simple and safe technique for performing an endoscopic third ventriculocisternostomy is described using a small-diameter semirigid neuroendoscope in conjunction with a perforated ventricular catheter to bluntly fenestrate the floor of the third ventricle. All previous descriptions involve the initial use of an introducer sheath that in our experience lends to loss of cerebrospinal fluid, consequently distorting the anatomic landmarks of the third ventricle as well as compromising the crucial concave shape of its floor. Our technique limits the loss of cerebrospinal fluid volume, therefore, reducing the chance of basilar artery complex perforation as a consequence of distortion of third ventricular landmarks and loss of third ventricular floor concavity.