Whole-genome and transcriptome analysis enhances precision cancer treatment options.

BACKGROUND: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies. PATIENTS AND METHODS: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA. DNA-based data, including mutations, copy number and mutation signatures, were combined with RNA-based data, including gene expression and fusions, to generate comprehensive WGTA profiles. A multidisciplinary molecular tumour board used WGTA profiles to identify and prioritize clinically actionable alterations and inform therapy. Patient responses to WGTA-informed therapies were collected. RESULTS: Clinically actionable targets were identified for 83% of patients, of which 37% of patients received WGTA-informed treatments. RNA expression data were particularly informative, contributing to 67% of WGTA-informed treatments; 25% of treatments were informed by RNA expression alone. Of a total 248 WGTA-informed treatments, 46% resulted in clinical benefit. RNA expression data were comparable to DNA-based mutation and copy number data in aligning to clinically beneficial treatments. Genome signatures also guided therapeutics including platinum, poly-ADP ribose polymerase inhibitors and immunotherapies. Patients accessed WGTA-informed treatments through clinical trials (19%), off-label use (35%) and as standard therapies (46%) including those which would not otherwise have been the next choice of therapy, demonstrating the utility of genomic information to direct use of chemotherapies as well as targeted therapies. CONCLUSIONS: Integrating RNA expression and genome data illuminated treatment options that resulted in 46% of treated patients experiencing positive clinical benefit, supporting the use of comprehensive WGTA profiling in clinical cancer care.

Laboratory simulation of longitudinally cracked teeth using the step-stress cyclic loading method.

AIM: To simulate in a laboratory setting longitudinal cracking in root filled premolar teeth, using cyclic mechanical fatigue. METHODOLOGY: Mesial-occlusal-distal (MOD) cavities were prepared in twenty root filled, single-rooted, mandibular premolars restored with fibre posts and resin composites. The samples were randomly divided into two groups based on the loading approaches: static loading with a crosshead speed of 0.5 mm/min and step-stress cyclic loading (1 Hz) with increasing amplitude. The loads and numbers of cycles to failure were recorded. Micro-CT was also used to identify the fracture modes. Statistical analysis was performed using Student's t-test. The level of significance was set at 0.05. RESULTS: The mean fracture loads for the static loading and cyclic loading groups were 769 ± 171 N and 720 ± 92 N, respectively. There was no significant difference between the two groups (P > 0.05). The proportions of longitudinal, cuspal and mixed-mode fractures under cyclic loading were 50%, 20% and 30%, respectively. Longitudinal fractures occurred with larger numbers of cycles and higher average loads per cycle compared with the other fractures. Static loading produced only cuspal fractures. CONCLUSIONS: Longitudinally cracked premolar teeth with root fillings were successfully produced using the step-stress cyclic loading method. This provides a more clinically representative methodology for studying cracked teeth in a laboratory setting.

19p13.2 microduplication causes a Sotos syndrome-like phenotype and alters gene expression.

Up to 90% of individuals affected by Sotos syndrome have a pathogenic alteration of NSD1 (encodes nuclear receptor-binding Su-var, enhancer of zeste, and trithorax domain protein 1), a histone methyltransferase that functions as both a transcriptional activator and a repressor. Genomic copy number variations may also cause a Sotos-like phenotype. We evaluated a three-generation family segregating a Sotos-like disorder characterized by typical facial features, overgrowth, learning disabilities, and advanced bone age. Affected individuals did not have a detectable NSD1 mutation, but rather were found to have a 1.9 Mb microduplication of 19p13.2 with breakpoints in two highly homologous Alu elements. Because the duplication included the DNA methyltransferase gene (DNMT1), we assessed DNA methylation of peripheral blood and buccal cell DNA and detected no alterations. We also examined peripheral blood gene expression and found evidence for increased expression of genes within the duplicated region. We conclude that microduplication of 19p13.2 is a novel genomic disorder characterized by variable neurocognitive disability, overgrowth, and facial dysmorphism similar to Sotos syndrome. Failed compensation of gene duplication at the transcriptional level, as seen in peripheral blood, supports gene dosage as the cause of this disorder.

A reproducible oral microcosm biofilm model for testing dental materials.

AIMS: Most studies of biofilm effects on dental materials use single-species biofilms, or consortia. Microcosm biofilms grown directly from saliva or plaque are much more diverse, but difficult to characterize. We used the Human Oral Microbial Identification Microarray (HOMIM) to validate a reproducible oral microcosm model. METHODS AND RESULTS: Saliva and dental plaque were collected from adults and children. Hydroxyapatite and dental composite discs were inoculated with either saliva or plaque, and microcosm biofilms were grown in a CDC biofilm reactor. In later experiments, the reactor was pulsed with sucrose. DNA from inoculums and microcosms was analysed by HOMIM for 272 species. Microcosms included about 60% of species from the original inoculum. Biofilms grown on hydroxyapatite and composites were extremely similar. Sucrose pulsing decreased diversity and pH, but increased the abundance of Streptococcus and Veillonella. Biofilms from the same donor, grown at different times, clustered together. CONCLUSIONS: This model produced reproducible microcosm biofilms that were representative of the oral microbiota. Sucrose induced changes associated with dental caries. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first use of HOMIM to validate an oral microcosm model that can be used to study the effects of complex biofilms on dental materials.

Quantifying dental biofilm growth using cross-polarization optical coherence tomography.

AIMS: Quantifying the ex vivo growth of complex multispecies dental biofilms using cross-polarization 1310-nm optical coherence tomography (CP-OCT) system was investigated. METHODS AND RESULTS: Bacterial microcosms, which were derived from plaque samples of paediatric subjects, were incubated in a biofilm reactor system containing discs of different dental materials for 72 h with daily sucrose pulsing (5×). CP-OCT analysis of biofilm mass was validated with crystal violet (CV) assays at various growth stages of these complex biofilms. CP-OCT was able to filter out the back-reflected signals of water layers in the hydrated biofilm and allowed for direct biofilm quantification. The overall depth-resolved scattering intensity of the biofilm showed very strong positive correlation with CV assay quantification (Spearman's ρ = 0.92) during the growth phase of the biofilm. CONCLUSION: CP-OCT was able to quantify the mass of the biofilm by measuring the overall depth-resolved scattering of the biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: CP-OCT has the ability to nondestructively monitor biofilm growth and elucidate the growth characteristics of these microcosms on different dental material compositions.

Accelerated Fatigue Model for Predicting Composite Restoration Failure.

An empirical method is proposed to predict the clinical performance of resin composite dental restorations by using laboratory data derived from simple specimens subjected to chemical degradation and accelerated cyclic fatigue. Three resin composites were used to fill dentin disks (2-mm inner diameter, 5-mm outer diameter, and 2 mm thick) made from bovine incisor roots. The specimens (n = 30 per group) were aged with different durations of a low-pH challenge (0, 24, and 48 h under pH 4.5) before being subjected to diametral compression with either a monotonically increasing load (fast fracture) or a cyclic load with a continuously increasing amplitude (accelerated fatigue). The data from 1 material were used to establish the relationship between laboratory time (number of cycles) and clinical time to failure (years) via the respective survival probability curves. The temporal relationship was then used to predict the clinical rates of failure for restorations made of the other 2 materials, and the predictions were compared with the clinical data to assess their accuracy. Although there were significant differences in the fast fracture strength among the groups of materials or durations of chemical challenge, fatigue testing was much better at separating the groups. Linear relationships were found between the laboratory and clinical times to failure for the first material (R2 = 0.90, 0.90, and 0.62 for the 0-, 24-, and 48-h low-pH groups, respectively). The clinical life of restorations made of the other 2 materials was best predicted with data from the 48-h low-pH groups. In conclusion, an accelerated fatigue model was successfully calibrated and applied to predict the clinical failure of resin composite restorations, and the predictions based on data obtained from chemically aged specimens provided the best agreement with clinical data.

Validation of finite element models for orthodontic aligners.

PURPOSE: Clear thermoplastic aligners have become popular in orthodontics, but the biomechanics of these devices is not well understood. Neither is the tooth movement induced by such devices. The aim of this study was to develop and validate finite element (FE) models for clear thermoplastic teeth aligners for orthodontic force prediction. METHODS AND MATERIALS: FE models were created from Micro-CT scans of an aligner and a model arch of teeth with one of the incisors tipped buccal-lingually by 2.4°. The models were uniformly meshed with 0.3-mm long elements. Linear-elastic mechanical properties provided by the material manufacturers were used. Fitting of the two components was simulated using Abaqus's interference fit, followed by frictional surface-to-surface interaction. The assembled FE model was validated by comparing its prediction for the teeth-aligner gaps and aligner surface strains with experimental data. The experimental teeth-aligner gaps were obtained from the Micro-CT scans whereas the aligner surface strains were measured using a 2-camera digital image correlation (DIC) system. RESULTS: Good agreement between prediction and measurement was obtained for both the teeth-aligner gaps and aligner surface strains. The linear regression between prediction and measurement for teeth-aligner gaps sampled at different positions had a R2 value of 0.99. The mean difference between prediction and measurement for the aligner surface strains (von Mises) over 1544 nodes on the labial side and 1929 nodes on the lingual side was 0.07% and 0.01%, respectively, both being lower than the mean background noise. CONCLUSION: A FE model for clear thermoplastic teeth aligners has been successfully developed and validated. The model can therefore be used with confidence to predict the forces and moments applied to teeth by the aligners, thus improving our understanding of the biomechanics of such devices and the tooth movement they induce.

A Review of Mechano-Biochemical Models for Testing Composite Restorations.

Due to the severe mechano-biochemical conditions in the oral cavity, many dental restorations will degrade and eventually fail. For teeth restored with resin composite, the major modes of failure are secondary caries and fracture of the tooth or restoration. While clinical studies can answer some of the more practical questions, such as the rate of failure, fundamental understanding on the failure mechanism can be obtained from laboratory studies using simplified models more effectively. Reviewed in this article are the 4 main types of models used to study the degradation of resin-composite restorations, namely, animal, human in vivo or in situ, in vitro biofilm, and in vitro chemical models. The characteristics, advantages, and disadvantages of these models are discussed and compared. The tooth-restoration interface is widely considered the weakest link in a resin composite restoration. To account for the different types of degradation that can occur (i.e., demineralization, resin hydrolysis, and collagen degradation), enzymes such as esterase and collagenase found in the oral environment are used, in addition to acids, to form biochemical models to test resin-composite restorations in conjunction with mechanical loading. Furthermore, laboratory tests are usually performed in an accelerated manner to save time. It is argued that, for an accelerated multicomponent model to be representative and predictive in terms of both the mode and the speed of degradation, the individual components must be synchronized in their rates of action and be calibrated with clinical data. The process of calibrating the in vitro models against clinical data is briefly described. To achieve representative and predictive in vitro models, more comparative studies of in vivo and in vitro models are required to calibrate the laboratory studies.

Osteomalacia: a case series of patients with atypical clinical orthopaedic presentations.

Osteomalacia is uncommon in an affluent subtropical city like Hong Kong, where sunlight exposure is adequate and nutritional support is good. We present three patients who had osteomalacia with different presentations. A 74-year-old male with oncogenic osteomalacia presented with multiple bone pain. His biochemical markers returned to normal 4 days postoperatively after resection of a second toe giant cell tumour of tendon sheath. A 62-year-old woman with a history of liver problem and proximal muscle weakness was admitted with atraumatic fracture of the left distal humerus due to osteomalacia. An 81-year-old vegetarian woman with inadequate sun exposure complained of multiple bone pains. Subsequent investigation revealed dietary- and sunlight-deficient osteomalacia with multiple bony abnormalities including marked femur bowing.

Nonlysosomal vesicles (acidosomes) are involved in phagosome acidification in Paramecium.

Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.

Retrieval of lysosomal membrane and acid phosphatase from phagolysosomes of Paramecium caudatum.

Little is known about the fate of lysosomal membrane in phagocytic cells. Because the age of the digestive vacuoles in Paramecium caudatum can be easily determined, we have been able to study the dynamic membrane events in the older vacuoles. Late in the phagolysosomal stage (DV-III) the vacuole membrane undergoes a burst of tubule formation. The tubules expand into vesicles which have characteristics resembling lysosomes in both thin sections and freeze-fracture replicas. The tubules also contain acid phosphatase activity when they arise from acid phosphatase-reactive vacuoles. We conclude that after active digestion lysosomal membrane is retrieved in whole or in part along with some membrane-associated hydrolases. A logical extension of these results is that the lysosome-like vesicles, after being recharged with hydrolases by fusing with primary lysosomes, are recycled back to DV-II for reuse.

Vesicle transport along microtubular ribbons and isolation of cytoplasmic dynein from Paramecium.

Cytoplasmic microtubule-based motility in Paramecium was investigated using video-enhanced contrast microscopy, the quick-freeze, deep-etch technique, and biochemical isolations. Three distinct vesicle populations were found to be transported unidirectionally along the cytopharyngeal microtubular ribbons. This minus-end-directed movement exhibited unique in vivo features in that the vesicle transport was nonsaltatory, rapid, and predominantly along one side of the microtubular ribbons. To identify candidate motor proteins which may participate in vesicle transport, we prepared cytosolic extracts of Paramecium and used bovine brain microtubules as an affinity matrix. These preparations were found to contain a microtubule-stimulated ATPase which supported microtubule gliding in vitro. This protein was verified as a cytoplasmic dynein based upon its relative molecular mass, sedimentation coefficient of 16S, susceptibility to vanadate photocleavage, elevated CTPase/ATPase ratio, and its typical two-headed dynein morphology. This dynein was directly compared with the axonemal dyneins from Paramecium and found to differ by five criteria: morphology, sedimentation coefficient, CTPase/ATPase ratio, vanadate cleavage patterns, and polypeptide composition. The cytoplasmic dynein is therefore not an axonemal dynein precursor, but rather it represents a candidate for supporting the microtubule-based vesicle transport which proceeds along the microtubular ribbons.

An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques.

Physical fixation by rapid freezing followed by freeze-fracture and deep-etching has provided the means for potentially seeing the three-dimensional arrangement in the native state of particles on mitochondrial inner membranes. We have used these techniques to study the tubular cristae of Paramecium in the hope of determining the arrangement of F1 complexes, their abundance, and location in the membranes. We also sought information regarding other respiratory complexes in these membranes. Our results, supported by stereo pairs, show that F1 complexes are arranged as a double row of particles spaced at 12 nm along each row as a zipper following the full length of the outer curve of the helically shaped tubular cristae. There are an average of 1,500 highly ordered F1 complexes per micrometer squared of 50-nm tubular cristae surface. The F1 complexes definitely lie outside the membranes in their native state. Other particle subsets, also nonrandomly arrayed, were seen. One such population located along the inner helical curve consisted of large 13-nm-wide particles that were spaced at 30 nm center-to-center. Such particles, because of their large size and relative abundance when compared to F1 units, resemble complex I of the respiratory complexes. Any models attempting to understand the coupling of respiratory complexes with F0F1 ATPase in Paramecium must take into account a relatively high degree of order and potential immobility of at least some of these integral membrane complexes.

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.

The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

Risk factors for clustering of tuberculosis cases: a systematic review of population-based molecular epidemiology studies.

BACKGROUND: Many molecular epidemiology studies have been conducted to identify risk factors for clustering of tuberculosis (TB) cases in the population. OBJECTIVE: To estimate the impact of commonly investigated risk factors on TB clustering. METHODS: Ten electronic databases were searched up to January 2006 along with a hand search of the International Journal of Tuberculosis and Lung Disease and bibliographies of review articles. Meta-analyses of odds ratios (ORs) for various risk factors were conducted using random effect models, stratified by TB incidence. Meta-regressions were employed to account for the heterogeneity in clustering proportions and the magnitudes of risk. FINDINGS: The TB clustering proportion varied greatly (7.0-72.3%) among 36 studies in 17 countries. In multiple meta-regression analyses, high TB incidence, mean cluster size and conventional contact tracing were significantly associated with higher clustering. The pooled ORs (95%CIs) for low and high/intermediate TB incidence studies, using a cut off of 25/100000 per year, were 3.4 (2.7- 4.2) and 1.6 (1.3-2.1) for local-born status, 1.6 (1.5-1.7) and 1.7 (1.3-2.2) for pulmonary TB and 1.2 (1.1-1.3) and 1.3 (1.1-1.7) for smear-positive cases, respectively. Male sex, local birth, alcohol abuse and injection drug use were significantly higher risks in low TB incidence studies than in the high/intermediate ones. INTERPRETATION: Meta-analyses yielded significant estimates of ORs for several risk factors across both levels of TB incidence. Alcohol abuse, injection drug use and homelessness--all characteristics of marginalized populations--were found to be consistently significant in populations of low TB incidence. More research is needed to better understand TB transmission dynamics in high-burden countries.

Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.

Membrane trafficking and processing in Paramecium.

Cellular membranes are made in a cell's biosynthetic pathway and are composed of similar biochemical constituents. Nevertheless, they become differentiated as membrane components are sorted into different membrane-limited compartments. We summarize the morphological and immunological similarities and differences seen in the membranes of the various interacting compartments in the single-celled organism, Paramecium. Besides the biosynthetic pathway, membranes of the regulated secretory pathway, endocytic pathway, and phagocytic pathway are highlighted. Paramecium is a multipolarized cell in the sense that several different pools of membrane-limited compartments are targeted for exocytosis at very specific sites at the cell surface. Thus, the method used by this cell to sort and package its membrane subunits into different compartments, the processes used to transport these compartments to specific locations at the plasma membrane and to other intracellular fusion sites, the processes of membrane retrieval, and the processes of membrane docking and fusion are reviewed. Paramecium has provided an excellent model for studying the complexities of membrane trafficking in one cell using both morphological and immunocytochemical techniques. This cell also promises to be a useful model for studying aspects of the molecular biology of membrane sorting, retrieval, transport, and fusion.

Focal choroidal excavation in patients with central serous chorioretinopathy.

PURPOSE: To evaluate the prevalence and clinical features of focal choroidal excavation (FCE) in patients presenting with central serous chorioretinopathy (CSC). METHODS: This is a retrospective consecutive case series of consecutive patients with CSC who were referred for spectral domain optical coherence tomography (SD-OCT) between January 2010 and December 2011. Medical records were reviewed and clinical features including presence of FCE in SD-OCT, fluorescence angiography (FA), and indocyanine green angiography (ICGA) were studied. RESULTS: Among the 116 CSC patients assessed, FCE was found in 11 eyes of 7 (6.0%) patients. FCE was associated with subretinal fluid in six eyes of six patients and serous pigment epithelial detachment in three eyes of two patients. The mean central subfield retinal thickness of CSC eyes with FCE was 283.7 µm, compared with 377.5 µm for CSC eyes without FCE (Mann-Whitney U-test, P=0.020). Five FCE eyes of five patients had focal leakage on FA. Choroidal hyperpermeability on ICGA was found in seven CSC eyes with FCE, with four eyes showing hypofluorescent spot corresponding to the FCE. After a mean follow-up of 16 months, visual acuity of all 11 eyes with FCE remained stable or improved at the last follow-up. CONCLUSION: FCE is not an uncommon feature in patients with CSC and might be associated with choroidal hemodynamic disturbances.

Modulation of the digestive lysosomal system in Paramecium caudatum. III. Morphological effects of cytochalasin B.

Morphological explanations for the effects of cytochalasin B (CB) on the digestive vacuole system of Paramecium caudatum were sought using electron-microscopic studies. Cytochalasin B (0.3 mM) essentially stopped vacuole release without stopping vacuole growth. Acidosome fusion with the new vacuoles was inhibited and was accompanied by the formation of discoidal vesicle-lined clumps of microfilament-like material next to these vacuoles. Vacuole membrane retrieval at the open cytoproct was inhibited and was accompanied by the loss of the microfilament network typical of this region. At 0.15 mM CB vacuole release and fusion of the acidosomes and lysosomes with the vacuoles were not stopped, but membrane retrieval at the cytoproct was affected. Cytochalasin B at the higher concentration also affected the movement of the horseradish peroxidase (HRP)-labeled vesicles away from the nascent vacuole. Membrane retrieval by the formation of tubules of 35 to 50 nm diameter from membrane of early and late vacuole stages was also affected by CB. It is conceivable that all of these effects were the result of the disruption of membrane associated actin networks. By contrast, several highly organized microfilamentous systems in the cortex and oral region of these cells appeared to be unaffected by CB or dimethyl sulfoxide (DMSO) at the concentrations used. These results show that CB-susceptible structures assume a key role in the normal functioning of the digestive vacuole system and in vacuole membrane retrieval and reutilization in Paramecium.