A2ML1 and otitis media: novel variants, differential expression, and relevant pathways.

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.

'Gel-Stacks' gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study.

Large DNA molecules (>20 kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.

Gel-Stacks technology provides multiple non-covalent molecular presentation modes, coupled with an unusually facile reagent delivery system designed for large-scale analytes, enhancing live imaging and manipulation. Enhanced further by modeling and software, Gel-Stacks technology becomes adaptable to a broad range of experimental applications.

The Role of Dental Practitioners in the Management of Oncology Patients: The Head and Neck Radiation Oncology Patient and the Medical Oncology Patient.

This narrative review addresses the role of a dentist in the management of oncology patients, highlighting the oral complications that arise in head and neck radiation oncology patients and medical oncology patients. The prevention and management of these complications are discussed.

Pandora's box in the dental clinic.

BACKGROUND: In June 2018, the Ministry of Health received notification from 2 hospitals about 2 patients who presented with overwhelming Enterobacter kobei sepsis that developed within 24 hours after a dental procedure. We describe the investigation of this outbreak. METHODS: The epidemiologic investigation included site visits in 2 dental clinics and interviews with all involved healthcare workers. Chart reviews were conducted for case and control subjects. Samples were taken from medications and antiseptics, environmental surfaces, dental water systems, and from the involved healthcare professionals. Isolate similarity was assessed using repetitive element sequence-based polymerase chain reaction (REP-PCR). RESULTS: The 2 procedures were conducted in different dental clinics by different surgeons and dental technicians. A single anesthesiologist administered the systemic anesthetic in both cases. Cultures from medications, fluids and healthcare workers' hands were negative, but E. kobei was detected from the anesthesiologist's portable medication cart. The 2 human isolates and the environmental isolate shared the same REP-PCR fingerprinting profile. None of the 21 patients treated by the anesthesiologist in a general hospital during the same period, using the hospital's medications, developed infection following surgery. CONCLUSIONS: An outbreak of post-dental-procedure sepsis was linked to a contaminated medication cart, emphasizing the importance of medication storage standards and strict aseptic technique when preparing intravenous drugs during anesthesia. Immediate reporting of sepsis following these outpatient procedures enabled early identification and termination of the outbreak.

A single-molecule barcoding system using nanoslits for DNA analysis : nanocoding.

Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.

A simple dialysis device for large DNA molecules.

The potential of genomic DNA is realized when new modalities are invented that manipulate large DNAs with minimal breakage or loss of sample. Here, we describe a polydimethylsiloxane-polycarbonate membrane device to remove small molecules from a sample while retaining large DNAs. Dialysis rates dramatically change as DNA size in kb (M) increases and DNA dimensions become comparable to pore size, and chain characteristics go from rod-like to Gaussian. Consequently, we describe empirical rates of dialysis, R, as a function of M as falling into two regimes: DNAs ≤ 1 kb show R(M) ∼e -t/τM (t = time, τM = time constant), while DNAs ≥1.65 kb slowly passage with R(M) ∼M -1.68; such partitioning potentiates single-molecule imaging.

Residual Roots as an Anatomical Guide for Implant Placement: Case Series With Two-Year Follow-up.

As placement of implants into immediate sites involves management of the remaining root structure, these residual roots may be used as a guide for the development of osteotomy. This aids in implant positioning and prevents drill slippage into the residual root spaces during osteotomy drilling. Following completion of the osteotomy, the remaining root structure is extracted prior to implant placement into the site. The aim of this study is to assess the success rate of implants when the residual roots were used as anatomical guides for osteotomy. One hundred implants were placed in 57 patients, and 4 different types of implants were used: 47 Bioner TOP DM implants, 20 Nobel Biocare Replace implants, 25 Biohorizons Tapered Internal implants (Birmingham, Ala), and 8 Alpha-Bio Tec SPI Implants. The implants were placed into 57 patients. Osteotomies were placed through intact residual roots, which acted as anatomical guides for implant surgical placement. Patients had a follow-up period of 2 years, and in that time none reported discomfort after implant placement. There were no signs of peri-implantitis observed in any of the patients. Of all the implants placed, the Bioner TOP DM implant showed the least amount of crestal bone loss. Placing implants through residual roots as an anatomical guide is a useful technique that shows good results over a 2-year follow-up period.

Instrumented Lumbar Corpectomy and Spinal Reconstruction Comparing rhBMP-2/Compression-Resistant Matrix, rhBMP-2/Absorbable Collagen Sponge/Ceramic Granules Mixture, and Autograft in Two Different Devices: A Study in Sheep.

STUDY DESIGN: Fusion success with rhBMP-2 and autograft in titanium or PEEK corpectomy devices was evaluated in a sheep lumbar corpectomy model. The 6 treatment groups included titanium mesh or PEEK corpectomy devices filled with rhBMP-2 on a compression-resistant matrix (CRM) carrier; rhBMP-2 in a morselized absorbable collagen sponge (ACS) carrier combined with resorbable ceramic granules; and autograft. OBJECTIVE: The aim of this study was to determine fusion rates associated with 2 different preparations of rhBMP-2 as well as autograft in an instrumented ovine lumbar corpectomy model 6 months postoperatively. SUMMARY OF BACKGROUND DATA: Vertebral reconstruction with corpectomy devices requires bone graft. Bone graft substitutes have the potential to avoid a second operation, donor site pain, and attendant morbidity associated with autograft. METHODS: Twenty-four sheep in 6 treatment groups underwent lumbar corpectomy via a retroperitoneal trans-psoas approach. Spines were reconstructed with autograft, rhBMP-2 on a CRM, or rhBMP-2 on an ACS mixed with ceramic granules. Grafting materials were placed in either a titanium mesh or PEEK conduit in spines with internal fixation. Computed tomographic (CT) scans were evaluated for fusion. Undecalcified histology was used to evaluate for fusion as well as the amount and extent of graft incorporation and graft resorption. RESULTS: Regardless of corpectomy device used, rhBMP-2/CRM or rhBMP-2/ACS with MASTERGRAFT resulted in a 100% fusion rate. The autograft group had a lower (75%) radiographic fusion rate. Using either preparation of rhBMP-2 resulted in the length of the defect filling with solid bone. Autograft fragments and ceramic granules were incorporated into the fusion masses with much of the ceramic granules being resorbed by 6 months. CONCLUSION: Both of the rhBMP-2 formulations have the potential to effect bony fusion and vertebral reconstruction within the corpectomy devices.

Application of positron emission tomography/computed tomography in radiation treatment planning for head and neck cancers.

18-fluorodeoxygluocose positron emission tomography/computed tomography ((18)FDG-PET/CT) provides significant information in multiple settings in the management of head and neck cancers (HNC). This article seeks to define the additional benefit of PET/CT as related to radiation treatment planning for squamous cell carcinomas (SCCs) of the head and neck through a review of relevant literature. By helping further define both primary and nodal volumes, radiation treatment planning can be improved using PET/CT. Special attention is paid to the independent benefit of PET/CT in targeting mucosal primaries as well as in detecting nodal metastases. The utility of PET/CT is also explored for treatment planning in the setting of SCC of unknown primary as PET/CT may help define a mucosal target volume by guiding biopsies for examination under anesthesia thus changing the treatment paradigm and limiting the extent of therapy. Implications of the use of PET/CT for proper target delineation in patients with artifact from dental procedures are discussed and the impact of dental artifact on CT-based PET attenuation correction is assessed. Finally, comment is made upon the role of PET/CT in the high-risk post-operative setting, particularly in the context of radiation dose escalation. Real case examples are used in these settings to elucidate the practical benefits of PET/CT as related to radiation treatment planning in HNCs.

Radiation therapy does not impact local complication rates after free flap reconstruction for head and neck cancer.

OBJECTIVE: To determine whether external beam radiation therapy (XRT), administered either before or after surgery, increases the rate and/or severity of local postoperative complications in patients with head and neck cancer who undergo microvascular free flap reconstruction. DESIGN: Retrospective cohort study. SETTING: University of Washington Medical Center, Seattle, a tertiary care hospital. PATIENTS: A total of 100 consecutive patients underwent fibular free flap reconstruction of the mandible. The study cohort was divided according to radiation treatment status: (1) no XRT (28 patients), (2) preoperative XRT (37 patients), and (3) postoperative XRT (35 patients). The median follow-up after surgery was 11 months (range, 1-89 months). MAIN OUTCOME MEASURES: Rate and severity of local postoperative complications. RESULTS: Fifty-four patients (54%) had at least 1 postoperative complication. There were no differences among the 3 XRT subgroups in the overall proportion of patients with complications of any severity (15 [54%] of 28 patients in the no XRT group, 24 [65%] of 37 patients in the preoperative XRT group, and 16 [46%] of 35 patients in the postoperative XRT group; P = .26, chi(2) analysis). There were also no differences seen when mild and severe complication rates were specifically examined (P = .58 and P = .10, respectively). No case of complete flap loss was observed. We noted no significant correlations between the rate of postoperative complications and the following covariates: total radiation dose, size of radiation field, disease stage, exposure to chemotherapy, presence of serious medical comorbidities, patient age, or history of tobacco use. CONCLUSIONS: Our experience suggests that XRT can be safely administered before or after surgery to patients undergoing head and neck free flap reconstruction at an experienced surgical referral center. Postoperative complication rates were not significantly affected by administration, timing, dose, or extent of XRT.

Anti-tumor necrosis factor agents reduce corticosteroid use compared with azathioprine in patients with Crohn's disease.

BACKGROUND: Corticosteroids are effective for inducing remission of Crohn's disease, but should not be used long term due to risk of adverse events. Benefits of immunosuppressants (e.g., azathioprine) and anti tumor necrosis factor (anti-TNF) agents include reduced reliance on corticosteroid-based therapies and avoidance of corticosteroid-associated adverse events. Our aim was to evaluate corticosteroid-sparing effects in patients with Crohn's disease upon being newly initiated on an anti-TNFα agent or azathioprine. METHODS: An analysis of US patient claims data from January 2008 to October 2011 was conducted using Truven Health MarketScan Research databases. Corticosteroid-sparing within 12 and 24 months after initiation of an anti-TNF agent (adalimumab, certolizumab pegol, or infliximab) or azathioprine was evaluated. RESULTS: In total, 2900 patients received a prescription for corticosteroids within the 6 month period before the initiation of an anti-TNF agent (63%) or azathioprine (37%). When certolizumab pegol, infliximab, or adalimumab were collectively compared with azathioprine, patients initiated on an anti-TNF agent avoided further prescriptions for corticosteroids at a greater rate than patients receiving azathioprine at 12 (43% vs. 27%, respectively; P < 0.0001) and 24 months (33% vs. 23%, respectively; P = 0.028). Individually, all anti-TNF agents showed higher rates of corticosteroid-sparing compared with azathioprine at 12 (P < 0.0001-0.011), but not 24 months (P = 0.0086-0.24). Key limitations of this study include lack of data regarding disease severity, response and assumptions of improvement, and compliance. CONCLUSIONS: Patients with Crohn's disease were able to avoid new prescriptions for corticosteroids at a statistically higher rate when treated with an anti-TNF agent. These results demonstrate that the anti-TNF agents are superior to azathioprine for minimizing exposure to corticosteroids.

Sleep medicine care under one roof: a proposed model for integrating dentistry and medicine.

Integrating oral appliance therapy into the delivery of care for sleeprelated breathing disorders has been a challenge for dental and medical professionals alike. We review the difficulties that have been faced and propose a multidisciplinary care delivery model that integrates dental sleep medicine and sleep medicine under the same roof with educational and research components. The model promises to offer distinct advantages to improved patient care, continuity of treatment, and the central coordination of clinical and insurance-related benefits.

How low can you go? Use of low- and standard-dose liposomal amphotericin B for treatment of invasive fungal infections.

OBJECTIVES: Recommended doses of liposomal amphotericin B (L-AMB) range from 3 to 6 mg/kg/day, but 1mg/kg/day may be equally effective and a lower cost alternative for many indications. The objective of this analysis was to assess indications and clinical outcomes of patients who received low-dose (1mg/kg/day rounded up in 50-mg increments) and standard-dose (≥2 mg/kg/day) L-AMB. METHODS: This was a retrospective analysis of adult L-AMB recipients with suspected invasive fungal infections (IFI) at a single center from 2006 to 2011. The primary outcome was clinical response at the end of treatment. Secondary outcomes included survival and toxicity. Results were analyzed using Chi-square and descriptive statistics. RESULTS: Of 89 adult L-AMB recipients included, 36 had proven or probable IFIs. Nineteen (53%) received low doses and 17 (47%) received standard doses. Median doses were 1.5 and 3.0mg/kg/day. Cryptococcus was the most common fungal pathogen in the low-dose group (37%), and Candida spp. in the standard-dose group (47%). Forty-seven percent of subjects in both groups improved clinically. Sixty-eight percent of low-dose recipients and 76% of standard-dose recipients survived to discharge. Rates of nephrotoxicity and hypokalemia were comparable. CONCLUSIONS: Comparable rates of clinical improvement, survival to discharge, and toxicity were identified among low- and standard-dose L-AMB recipients.

Nanochannel confinement: DNA stretch approaching full contour length.

Fully stretched DNA molecules are becoming a fundamental component of new systems for comprehensive genome analysis. Among a number of approaches for elongating DNA molecules, nanofluidic molecular confinement has received enormous attentions from physical and biological communities for the last several years. Here we demonstrate a well-optimized condition that a DNA molecule can stretch almost to its full contour length: the average stretch is 19.1 µm ± 1.1 µm for YOYO-1 stained λ DNA (21.8 µm contour length) in 250 nm × 400 nm channel, which is the longest stretch value ever reported in any nanochannels or nanoslits. In addition, based on Odijk's polymer physics theory, we interpret our experimental findings as a function of channel dimensions and ionic strengths. Furthermore, we develop a Monte Carlo simulation approach using a primitive model for the rigorous understanding of DNA confinement effects. Collectively, we present a more complete understanding of nanochannel confined DNA stretching via the comparisons to computer simulation results and Odijk's polymer physics theory.

A single-molecule barcoding system using nanoslits for DNA analysis.

Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro- and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.

The effect of dental artifacts, contrast media, and experience on interobserver contouring variations in head and neck anatomy.

OBJECTIVES: To investigate interobserver variability in the delineation of head-and-neck (H&N) anatomic structures on CT images, including the effects of image artifacts and observer experience. METHODS: Nine observers (7 radiation oncologists, 1 surgeon, and 1 physician assistant) with varying levels of H&N delineation experience independently contoured H&N gross tumor volumes and critical structures on radiation therapy treatment planning CT images alongside reference diagnostic CT images for 4 patients with oropharynx cancer. Image artifacts from dental fillings partially obstructed 3 images. Differences in the structure volumes, center-of-volume positions, and boundary positions (1 SD) were measured. In-house software created three-dimensional overlap distributions, including all observers. The effects of dental artifacts and observer experience on contouring precision were investigated, and the need for contrast media was assessed. RESULTS: In the absence of artifacts, all 9 participants achieved reasonable precision (1 SD < or =3 mm all boundaries). The structures obscured by dental image artifacts had larger variations when measured by the 3 metrics (1 SD = 8 mm cranial/caudal boundary). Experience improved the interobserver consistency of contouring for structures obscured by artifacts (1 SD = 2 mm cranial/caudal boundary). CONCLUSIONS: Interobserver contouring variability for anatomic H&N structures, specifically oropharyngeal gross tumor volumes and parotid glands, was acceptable in the absence of artifacts. Dental artifacts increased the contouring variability, but experienced participants achieved reasonable precision even with artifacts present. With a staging contrast CT image as a reference, delineation on a noncontrast treatment planning CT image can achieve acceptable precision.

Erythropoietic response to anemia in chronic hepatitis C patients receiving combination pegylated interferon/ribavirin.

OBJECTIVES: In hepatitis C virus (HCV)-infected patients receiving pegylated interferon (PEG-IFN)/ribavirin (RBV) combination therapy, anemia is a well-known side effect. The purpose of this study was to describe the time course and extent of hemoglobin (Hb) changes and the erythropoietic response to PEG-IFN/RBV-induced anemia. METHODS: In this multicenter, observational, 8-wk study, laboratory parameters were measured weekly for 8 wk or until early withdrawal. Primary endpoints included changes in Hb and serum erythropoietin (sEPO) from baseline to week 8; other measures were changes in reticulocytes and RBV dose. The predictive value of baseline factors for maximum Hb decline was assessed. RESULTS: In the 97 evaluable patients, mean Hb decreased from 14.4 +/- 1.4 g/dl (baseline) to 11.9 +/- 1.3 g/dl (week 8). Twenty-one percent of patients withdrew before week 8. The estimated erythropoietic response was lower than that seen in two historic control populations of iron deficiency anemia patients. Mean RBV dose decreased from 986 +/- 190 mg/day (baseline) to 913 +/- 228 mg/day (week 8). Fifty-seven out of 77 (74%) patients who completed the study maintained their initial prescribed RBV dose. Patients maintained on the initial dose of RBV who had a higher baseline Hb and viral load showed a trend toward larger Hb declines. Platelets and white blood cells (WBCs) also declined during the study. CONCLUSIONS: HCV-infected patients receiving PEG-IFN/RBV therapy have reductions in Hb, platelets, and WBCs, possibly due to bone marrow suppression. They also have diminished endogenous sEPO production for their degree of anemia.

Shear-induced migration in flowing polymer solutions: simulation of long-chain DNA in microchannels [corrected].

We simulate dilute solution dynamics of long flexible polymer molecules in pressure driven flow in channels with widths of roughly 0.1-10 times the polymer bulk radius of gyration. This is done using a self-consistent coarse-grained Langevin description of the polymer dynamics and a numerical simulation of the flow in the confined geometry that is generated by the motions of polymer segments. Results are presented for a model of DNA molecules of approximately 10-100 microm contour length in micron-scale channels. During flow, the chains migrate toward the channel centerline, in agreement with well-known experimental observations. The thickness of the resulting hydrodynamic depletion layer increases with molecular weight at constant flow strength; higher molecular weight chains therefore move with a higher average axial velocity than lower molecular weight chains. In contrast, if the hydrodynamic effects of the confining geometry are neglected, depletion of concentration is observed in the center of the channel rather than at the walls, contradicting experimental observations. The mechanisms for migration are illustrated using a simple kinetic theory dumbbell model of a confined flexible polymer. The simple theory correctly predicts the trends observed in the detailed simulations. We also examine the steady-state stretch of DNA chains as a function of channel width and flow strength. The flow strength needed to stretch a highly confined chain away from its equilibrium length is shown to increase with decreasing channel width, independent of molecular weight; this is fairly well explained using a simple blob picture.