Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers.

The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.

Nuclear F-actin Cytology in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

Oral cavity cancer has a low 5-y survival rate, but outcomes improve when the disease is detected early. Cytology is a less invasive method to assess oral potentially malignant disorders relative to the gold-standard scalpel biopsy and histopathology. In this report, we aimed to determine the utility of cytological signatures, including nuclear F-actin cell phenotypes, for classifying the entire spectrum of oral epithelial dysplasia and oral squamous cell carcinoma. We enrolled subjects with oral potentially malignant disorders, subjects with previously diagnosed malignant lesions, and healthy volunteers without lesions and obtained brush cytology specimens and matched scalpel biopsies from 486 subjects. Histopathological assessment of the scalpel biopsy specimens classified lesions into 6 categories. Brush cytology specimens were analyzed by machine learning classifiers trained to identify relevant cytological features. Multimodal diagnostic models were developed using cytology results, lesion characteristics, and risk factors. Squamous cells with nuclear F-actin staining were associated with early disease (i.e., lower proportions in benign lesions than in more severe lesions), whereas small round parabasal-like cells and leukocytes were associated with late disease (i.e., higher proportions in severe dysplasia and carcinoma than in less severe lesions). Lesions with the impression of oral lichen planus were unlikely to be either dysplastic or malignant. Cytological features substantially improved upon lesion appearance and risk factors in predicting squamous cell carcinoma. Diagnostic models accurately discriminated early and late disease with AUCs (95% CI) of 0.82 (0.77 to 0.87) and 0.93 (0.88 to 0.97), respectively. The cytological features identified here have the potential to improve screening and surveillance of the entire spectrum of oral potentially malignant disorders in multiple care settings.

Global feedforward active noise control in vibro-acoustic cavities without increasing structural vibrations.

Interior noise in vibro-acoustic cavities may be generated due to acoustic and structural disturbances. Earlier studies have shown that for global control, the maximum reduction in acoustic potential energy can be realised by using an optimum combination of acoustic and structural actuators. However, it is observed that this reduction in interior noise may also be accompanied with an increase in kinetic energy of the cavity structure. This paper presents the development of a feedforward technique for active noise control in vibro-acoustic cavities ensuring that the noise reduction does not lead to an increase in kinetic energy. The problem is formulated as a constrained minimisation problem to minimise the acoustic potential energy subject to a constraint that the kinetic energy does not increase. Through a numerical study, it is shown that the optimum solution of the above problem indeed is favourable in terms of reduction in acoustic potential energy in the cavity and kinetic energy of the structure. The paper further proposes a method for solution of this constrained minimisation problem using a penalty function method and solution of sequential unconstrained problems. The proposed method is validated through a numerical study on a car-like cavity for single- and multi-tonal noise.

Structural sensing of interior sound for active control of noise in structural-acoustic cavities.

This paper proposes a method for structural sensing of acoustic potential energy for active control of noise in a structural-acoustic cavity. The sensing strategy aims at global control and works with a fewer number of sensors. It is based on the established concept of radiation modes and hence does not add too many states to the order of the system. Acoustic potential energy is sensed using a combination of a Kalman filter and a frequency weighting filter with the structural response measurements as the inputs. The use of Kalman filter also makes the system robust against measurement noise. The formulation of the strategy is presented using finite element models of the system including that of sensors and actuators so that it can be easily applied to practical systems. The sensing strategy is numerically evaluated in the framework of Linear Quadratic Gaussian based feedback control of interior noise in a rectangular box cavity with a flexible plate with single and multiple pairs of piezoelectric sensor-actuator patches when broadband disturbances act on the plate. The performance is compared with an "acoustic filter" that models the complete transfer function from the structure to the acoustic domain. The sensing performance is also compared with a direct estimation strategy.

Periodic dynamics of fermionic superfluids in the BCS regime.

We study the zero temperature non-equilibrium dynamics of a fermionic superfluid in the BCS limit and in the presence of a drive leading to a time-dependent chemical potential µ(t). We choose a periodic driving protocol characterized by a frequency ω and compute the fermion density, the wavefunction overlap, and the residual energy of the system at the end of N periods of the drive. We demonstrate that the BCS self-consistency condition is crucial in shaping the long time behaviour of the fermions subjected to the drive and provide an analytical understanding of the behaviour of the fermion density nkF (where kF is the Fermi momentum vector) after a drive period and for large ω. We also show that the momentum distribution of the excitations generated due to such a drive bears the signature of the pairing symmetry and can be used, for example, to distinguish between s- and d-wave superfluids. We propose experiments to test our theory.

5-day/5-drug myeloablative outpatient regimen for resistant neuroblastoma.

5-day/5-drug (5D/5D) is a novel high-dose regimen administered with autologous hematopoietic SCT (HSCT). It was designed to maximize cytoreduction via high dosing of synergistically interacting agents, while minimizing morbidity in patients with resistant neuroblastoma (NB) and ineligible for clinical trials due to myelosuppression from previous therapy. 5D/5D comprises carboplatin 500 mg/m(2)/day on days 1-2, irinotecan 50 mg/m(2)/day on days 1-3, temozolomide 250 mg/m(2)/day on days 1-3, etoposide 200 mg/m(2)/day on days 3-5 and cyclophosphamide 70 mg/kg/day on days 4-5. HSCT is on day 8. Sixteen patients received 21 courses. Treatment was in the outpatient clinic. Responses were noted against progressive disease (PD) that had developed while patients were off, or receiving only low-dose, chemotherapy but not against PD that emerged despite high-dose chemotherapy. Responses were also seen in patients with PD or stable disease after (131)I-metaiodobenzylguanidine therapy. Grade 3 toxicities were limited to transient elevations in liver enzymes (three courses) and hyponatremia (one course). Bacteremia occurred in 2/21 (10%) courses. Hematological recovery allowed patients to be enrolled on clinical trials. In conclusion, 5D/5D (including HSCT) spares vital organs, entails modest morbidity, shows activity against resistant NB and helps patients meet eligibility requirements for formal clinical trials.

In vivo efficacy of an alcohol-based surgical hand disinfectant containing a synergistic combination of ethylhexylglycerin and preservatives.

Alcohol-based surgical hand disinfectants are widely available in healthcare settings. Some currently marketed alcohol-based products use active concentrations of antimicrobials to achieve the required efficacy, raising the risk for exposure to potentially irritating levels of antimicrobials. This study compares the in vitro and in vivo efficacy of an alcohol-based surgical hand preparation containing 70% ethanol and preservative levels of chlorhexidine gluconate (CHG) and benzalkonium chloride (BZK) in synergistic combination with ethylhexylglycerin (Surgicept) with a surgical hand disinfectant containing 61% ethanol and 1% CHG (Avagard). The in vivo efficacy of Surgicept and Avagard was evaluated in volunteers using the Tentative Final Monograph method of the Food and Drug Administration (FDA), and their prolonged effect against transient pathogens was compared using a pig skin model. Surgicept exceeded the FDA requirements for surgical hand antiseptic with mean log(10) reductions of 2.36, 3.3 and 3.54 in resident flora 1 min after initial application, and showed a persistent effect with mean log(10) reductions of 2.23, 2.73 and 3.3, 6h post application on days 1, 2 and 5, respectively. Surgicept showed a superior prolonged effect against transient bacteria compared with Avagard. Surgicept (70% alcohol and preservative levels of CHG and BZK) may provide similar in vivo efficacy as Avagard (61% ethanol and 1% CHG).

Topotecan, thiotepa, and carboplatin for neuroblastoma: failure to prevent relapse in the central nervous system.

We report on a three-drug myeloablative regimen designed to consolidate remission and to prevent central nervous system (CNS) relapse of high-risk neuroblastoma (NB). Sixty-six NB patients received topotecan 2 mg/m2/day, x 4 days; thiotepa 300 mg/m2/day, x 3 days; and carboplatin approximately 500 mg/m2/day, x 3 days. Post-SCT treatments included radiotherapy, immunotherapy, 13-cis-retinoic acid, +/-oral etoposide. Significant nonhematologic toxicities were mucositis and skin-related in all patients, convulsions in three patients, and cardiac failure and venocclusive disease of liver in one patient each. Grade 2 hepatotoxicity led to truncating cytoreduction in two patients; both later relapsed in brain. Among 46 patients transplanted in first complete/very good partial remission (CR/VGPR), event-free survival is 54% (s.e.+/-8%) at 36 months post-SCT; notable events were three non-NB-related deaths (adenovirus on day +9, bowel necrosis at 5 months, multiorgan failure at seven months) and four relapses in brain. Of 12 patients transplanted with evidence of NB, two became long-term event-free survivors and two relapsed in the brain. Of eight patients transplanted in second or greater CR/VGPR, one became a long-term event-free survivor and seven relapsed though not in the CNS. This regimen has manageable toxicity but does not prevent CNS relapse.

An alcohol hand rub containing a synergistic combination of an emollient and preservatives: prolonged activity against transient pathogens.

A new alcohol-based hand antiseptic (Octoxy hand rub) containing a synergistic combination of an emollient (Octoxyglycerine) and preservatives was developed and evaluated for immediate and prolonged activity against transient bacteria. The in vitro and in vivo antimicrobial efficacy was compared with other alcohol hand rubs containing preservative/antimicrobial (Prevacare and Avagard). In vitro evaluation was carried out using a tube-dilution method and a pig-skin model. Rapid and prolonged efficacy in vivo was evaluated against Staphylococcus epidermidis on the hands of volunteers. Octoxy hand rub was 100% effective in rapidly killing pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium in vitro. In volunteers, all three hand rubs gave a significant reduction in microbial count within 15s. Octoxy hand rub showed significantly higher efficacy against S. aureus and Escherichia coli than Avagard and Prevacare 15 min after application to the pig-skin model, and against S. epidermidis in both the pig-skin model and in volunteers. Use of Octoxy hand rub with broad-spectrum immediate and prolonged antimicrobial activity may be a very effective way of improving hand hygiene without exposing the hands to higher concentrations of antimicrobials.

Antiseptic impregnated endotracheal tubes for the prevention of bacterial colonization.

The effect of endotracheal tubes (ETTs) impregnated with chlorhexidine (CHX) and silver carbonate (antiseptic ETTs) against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter aerogenes [organisms associated with ventilator-associated pneumonia (VAP)], was evaluated in a laboratory airway model. Antiseptic ETTs and control ETTs (unimpregnated) were inserted in culture tubes half-filled with agar media (airway model) previously contaminated at the surface with 10(8) cfu/mL of the selected test organism. After five days of incubation, bacterial colony counts on all ETT segments were determined. Swabs of proximal and distal ends of the agar tract in antiseptic and control models were subcultured. The initial and residual CHX levels, (five days post-implantation in the model) were determined. Cultures of antiseptic ETTs revealed colonization by the tested pathogens ranging from 1-100 cfu/tube, compared with approximately 10(6) cfu/tube for the control ETTs (P < 0.001). Subcultures from proximal and distal ends of the agar tract showed minimal or no growth in the antiseptic ETTs compared with the control ETTs (P < 0.001). The amount of CHX retained in the antiseptic ETTs after five days of implantation was an average of 45% of the initial level. Antiseptic ETTs prevented bacterial colonization in the airway model and also retained significant amounts of the antiseptic. These results indicate that the effectiveness of antiseptic-impregnated ETTs in preventing the growth of bacterial pathogens associated with VAP may vary with different organisms.

Molecular phylogeny in 3-D.

Molecular phylogenetic trees are constructed in three dimensions relative to the distribution of MW and pl classes and immunocrossreactivity against polyclonal antibodies to lens crystallins, as well as multiple sequence alignment between amino acid sequences, coding nucleotide sequences and the gene nucleotide sequences for beta-globin. Euclidian distances are estimated to position species in x, y, z space by multidimensional scaling and merged with bootstrap-tested branching pattern of Fitch & Margoliash plots to obtain 3-D phylogenetic tree. Compared to single attributes, phylogenetic trees based on multiple parameters allow significant repositioning of rodents, chiroptera and primates.

Expression of nerve growth factor during the development of nervous system in early chick embryo.

In the chick gastrula, nerve growth factor (NGF) is localized to the endoblast mesoblast presumptive head ectoderm but not in the presumptive neuroectoblast. During early morphogenesis the dorsal body ectoderm presumptive neural crest cells exhibit strong NGF positive cell surface reaction. NGF appears to be a marker of cells participating in morphogenetic movements but not early neural differentiation. NGF is localized where neural folds fuse and cells die allowing detachment of the neural tube from head ectoderm as well as in dead cells in the neurocoele. NGF reactivity in cells lining the evaginated extremities of the optic vesicle the floor of the neural tube the splanchnopleure heart primordia the inner outer surfaces of somites is suggestive of the role of NGF in primitive organ shaping.

The neural inductive response of competent chick ectoblast decreases away from the host axis and correlates with an increased proliferative activity.

We have assessed the quality and quantity of the neural inductive response of the chick gastrula ectoblast located at increasing distancefrom the host axis. In a stage 4 gastrula, entire ectoblast exhibits neural competence. The quality of induced neural tissue shifts from deuterencephalic type in the area pellucida to archencephalic type in the area opaca and primitive medullary or palisade type atthe margin of overgrowth with a concomitant reduction in the number of induced neural cells. In contrast, the mitotic and 3H-TdR labelling frequencies in the competent ectoblast increase with increasing distance from the host axis and in a proportion inverse to the amount of induced neural tissue. It is suggested that the strong neural inductive response is correlated with low proliferative activity, or longer cell cycle time, of the competent ectoblast.

The neural inductive signal is transferred to ectoblast in 1-2 h but a continued contact with mesoblast for 2-3 h is essential for neuralization in the chick area pellucida.

In the area pellucida of the chick gastrula, the Hensen's node (HN) graft must contact the competent ectoblast for at least 4 h to promote neural induction. When we removed the grafted HN after 1 to 3 h and replaced it by a non-inducing post nodal (PN) fragment, a 1-2 h contact with HN was found to be sufficient to promote neural induction. When HN graft was removed after 3 or 4 h and replaced by PN, the neural inductive response was substantially improved towards formation of archencephalic structures. Thus, our results indicate that neural induction takes place in two steps. In the first step, a contact with HN for 1-2 h is sufficient to transferthe inductive signal which is stabilized through a second step involving continued cell-cell contact with even non-inducing PN mesoblast.

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding.

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors.

Tumor-restricted surface antigens may be targets for diagnosis and immune-based therapies. Monoclonal antibody 8H9 is a murine IgG1 hybridoma derived from the fusion of mouse myeloma SP2/0 cells and splenic lymphocytes from BALB/c mice immunized with human neuroblastoma. By immunohistochemistry, 8H9 was highly reactive with human brain tumors, childhood sarcomas, and neuroblastomas, and less so with adenocarcinomas. Among primary brain tumors, 15 of 17 glioblastomas, 3 of 4 mixed gliomas, 4 of 11 oligodendrogliomas, 6 of 8 astrocytomas, 2 of 2 meningiomas, 3 of 3 schwannomas, 2 of 2 medulloblastomas, 1 of 1 neurofibroma, 1 of 2 neuronoglial tumors, 2 of 3 ependymomas, and 1 of 1 pineoblastoma tested positive. Among sarcomas, 21 of 21 Ewing's/primitive neuroectodermal tumor, 28 of 29 rhabdomyosarcomas, 28 of 29 osteosarcomas, 35 of 37 desmoplastic small round cell tumors, 2 of 3 synovial sarcomas, 4 of 4 leiomyosarcomas, 1 of 1 malignant fibrous histiocytoma, and 2 of 2 undifferentiated sarcomas tested positive with 8H9. Eighty-seven of 90 neuroblastomas, 12 of 16 melanomas, 3 of 4 hepatoblastomas, 7 of 8 Wilms' tumors, 3 of 3 rhabdoid tumors, and 12 of 27 adenocarcinomas also tested positive. In contrast, 8H9 was nonreactive with normal human tissues including bone marrow, colon, stomach, heart, lung, muscle, thyroid, testes, pancreas, and human brain (frontal lobe, cerebellum, pons, and spinal cord). Reactivity with normal cynomolgus monkey tissue was restricted similarly. Indirect immunofluorescence localized the antigen recognized by 8H9 to the cell membrane. The antigen is proteinase sensitive and is not easily modulated off the cell surface. 8H9 immunoprecipitated a M(r) 58,000 band after N-glycanase treatment, most likely a protein with a heterogeneous degree of glycosylation. This novel antibody-antigen system may have potential for tumor targeting.

In vitro evaluation of the risk of developing bacterial resistance to antiseptics and antibiotics used in medical devices.

The risk of development of resistance in Staphylococcus epidermidis to the antibiotics and antiseptics impregnated in central venous catheters was evaluated. The culture was passaged 10-20 times through subinhibitory concentrations of different antimicrobials, singly and in combination, and the MIC of each antimicrobial before and after passage was compared. There was a 10- to 16-fold increase in the MIC of the combination of minocycline and rifampicin, while no significant increase in the MIC of minocycline alone was seen. The MIC of rifampicin was 25,000-fold higher against strains passaged through rifampicin alone (as compared with that for the original strain), while the increase was only 80-fold when it was combined with minocycline for passage. There was no substantial change in susceptibility to the antiseptic chlorhexidine when used alone or in combination with either silver sulphadiazine or triclosan (the MIC of triclosan alone increased eight-fold). In time-kill studies, synergy was observed between chlorhexidine and both triclosan and silver sulphadiazine. Zone of inhibition tests of catheters impregnated with minocycline and rifampicin showed that their activity against rifampicin-resistant strains was lower than that against the susceptible strain. On the other hand, the activity of the antiseptic (chlorhexidine and silver sulphadiazine) catheters against the rifampicin-resistant and -susceptible strains was similar. Although this study indicates that antibiotic catheters may be at a higher risk of being colonized by antibiotic-resistant bacteria, the implications of these results in clinical settings need to be determined.

In vitro and in vivo efficacy of catheters impregnated with antiseptics or antibiotics: evaluation of the risk of bacterial resistance to the antimicrobials in the catheters.

OBJECTIVE: To compare the efficacy of a new antiseptic catheter containing silver sulfadiazine and chlorhexidine on the external surface and chlorhexidine in the lumens to an antibiotic catheter impregnated with minocycline and rifampin on its external and luminal surfaces. DESIGN: Experimental trial. METHODS: Antimicrobial spectrum of catheters was determined by zones of inhibition. Resistance to luminal colonization was tested in vitro by locking catheter lumens with Staphylococcus epidermidis or Staphylococcus aureus culture after 7 days of perfusion. In vitro development of resistance to the antiseptic or antibiotic combination used in catheters was investigated. In vivo efficacy was tested (rat subcutaneous model) by challenge with sensitive or antibiotic-resistant bacteria. RESULTS: Antiseptic and antibiotic catheters exhibited broad-spectrum action. However, antibiotic catheters were not effective against Candida species and Pseudomonas aeruginosa. Both catheters prevented luminal colonization. Compared to controls, both test catheters resisted colonization when challenged with S aureus 7 and 14 days' postimplant (P<.05). Repeated in vitro exposure of S epidermidis culture to the antibiotic and antiseptic combinations led to small increases in the minimum inhibitory concentration (15 times and 2 times, respectively). Unlike the antibiotic catheter, the in vitro and in vivo activity of the antiseptic catheter was unaffected by the resistance profile of the test organism. Antiseptic catheters were more effective than antibiotic catheters in preventing colonization by rifampin-resistant S epidermidis in vivo (P<.05). CONCLUSIONS: Antiseptic and antibiotic catheters exhibit similar efficacy; however, when challenged with a rifampin-resistant strain, the antibiotic catheter appeared to be more susceptible to colonization than the antiseptic device.

Safety and efficacy of an improved antiseptic catheter impregnated intraluminally with chlorhexidine.

The safety and efficacy of a second-generation improved antiseptic catheter impregnated with silver sulfadiazine and increased levels of chlorhexidine on its outer surface and chlorhexidine alone on its luminal surfaces was compared in vitro and in vivo to standard antiseptic catheters impregnated with these antimicrobials on their outer surfaces only. In rat and pig intravenous models, the improved antiseptic catheter was significantly more effective in resisting both outer surface and luminal colonization compared with the standard antiseptic or control catheters. There was no evidence of tissue toxicity in any group.