Use of plasma induced modification of biomolecules (PLIMB) to evaluate hemin dissociation from fish and bovine methemoglobins.

Hemin dissociation occurs much faster from fish methemoglobin (metHb) compared to mammalian metHb yet the mechanism remains poorly understood. This may involve enhanced solvent access to His(E7) of fish metHbs by a protonation mechanism. Plasma induced modification of biomolecules (PLIMB) produces free radicals that covalently modify solvent accessible residues of proteins, and so can provide insight regarding accessibility of hydronium ions to protonate His(E7). PLIMB-induced modifications to heme crevice sites of trout IV and bovine metHb were determined using tandem mass spectrometry after generating peptides with Trypsin/Lys-C. αHis(CE3) was more modified in trout attributable to the more dynamic nature of bovine αHis(CE3) from available crystal structures. Although His(E7) was not found to be more modified in trout, aspects of trout peptides containing His(E7) hampered modification determinations. An existing computational structure-based approach was also used to estimate protonation tendencies, suggesting His(E7) of metHbs with low hemin affinity are more protonatable.

Memristive Behavior Enabled by Amorphous-Crystalline 2D Oxide Heterostructure.

The emergence of memristive behavior in amorphous-crystalline 2D oxide heterostructures, which are synthesized by atomic layer deposition (ALD) of a few-nanometer amorphous Al2 O3 layers onto atomically thin single-crystalline ZnO nanosheets, is demonstrated. The conduction mechanism is identified based on classic oxygen vacancy conductive channels. ZnO nanosheets provide a 2D host for oxygen vacancies, while the amorphous Al2 O3 facilitates the generation and stabilization of the oxygen vacancies. The conduction mechanism in the high-resistance state follows Poole-Frenkel emission, and in the the low-resistance state is fitted by the Mott-Gurney law. From the slope of the fitting curve, the mobility in the low-resistance state is estimated to be ≈2400 cm2 V-1 s-1 , which is the highest value reported in semiconductor oxides. When annealed at high temperature to eliminate oxygen vacancies, Al is doped into the ZnO nanosheet, and the memristive behavior disappears, further confirming the oxygen vacancies as being responsible for the memristive behavior. The 2D heterointerface offers opportunities for new design of high-performance memristor devices.

A physiologically relevant 3D collagen-based scaffold-neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models.

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

Plasma-Generated OH Radical Production for Analyzing Three-Dimensional Structure in Protein Therapeutics.

Protein three-dimensional structure dynamically changes in solution depending on the presence of ligands and interacting proteins. Methods for detecting these changes in protein conformation include 'protein footprinting,' using mass spectrometry. We describe herein a new technique, PLIMB (Plasma Induced Modification of Biomolecules), that generates µs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. PLIMB was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution.

NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis.

Dual specificity protein phosphatase 26 (DUSP26) is overexpressed in high-risk neuroblastoma (NB) and contributes to chemoresistance by inhibiting p53 function. In vitro, DUSP26 has also been shown to effectively inhibit p38 MAP kinase. We hypothesize that inhibiting DUSP26 will result in decreased NB cell growth in a p53 and/or p38-mediated manner. NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a novel DUSP26 small molecule inhibitor, shows effective growth inhibition and induction of apoptosis in NB cell lines. NB cell lines treated with small hairpin RNA (shRNA) targeting DUSP26 also exhibit a proliferation defect both in vitro and in vivo. Treatment of NB cell lines with NSC-87877 results in increased p53 phosphorylation (Ser37 and Ser46) and activation, increased activation of downstream p38 effector proteins (heat shock protein 27 (HSP27) and MAP kinase-activated protein kinase 2 (MAPKAPK2)) and poly ADP ribose polymerase/caspase-3 cleavage. The cytotoxicity resulting from DUSP26 inhibition is partially reversed by knocking down p53 expression with shRNA and also by inhibiting p38 activity with SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine). In an intrarenal mouse model of NB, NSC-87877 treatment results in decreased tumor growth and increased p53 and p38 activity. Together, these results suggest that DUSP26 inhibition with NSC-87877 is an effective strategy to induce NB cell cytotoxicity in vitro and in vivo through activation of the p53 and p38 mitogen-activated protein kinase (MAPK) tumor-suppressor pathways.

The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma.

Neuroblastoma is an aggressive pediatric malignancy which is >98% p53 wild-type at diagnosis. As a primary repressor of p53 activity and part of a p53-activated negative feedback loop, targeting of mouse double minute 2 homolog (MDM2) is an attractive therapeutic approach to reactivation of p53. Since development of the first selective MDM2 inhibitor, Nutlin-3a, newer compounds have been developed for increased potency and improved bioavailability. Herein, we sought to determine the efficacy and specificity of a second-generation MDM2 inhibitor, RG7388, in neuroblastoma cell lines and xenografts and examine its effect on the p53-independent pathway of hypoxia-inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF). Cell viability and apoptosis studies were performed on the neuroblastoma cell lines, NGP, SH-SY5Y, LAN-5, LAN-5 si-p53 (p53 silenced), and SK-N-AS (p53 null). RG7388 potently decreased cell proliferation and activated p53-dependent apoptosis. Tumor-bearing mice treated with RG7388 demonstrated significant tumor inhibition by 59% in NGP (P = 0.003), 67% in SH-SY5Y (P = 0.006), and 75% in LAN-5 (P = 0.0019) p53 wild-type xenograft tumors, but no inhibitory effect on LAN-5 si-p53 or SK-N-AS p53-silenced/null xenograft tumors. Moreover, RG7388 was found to inhibit the p53-independent pathway of HIF-1α/VEGF with decreased gene expression and alteration of angiogenesis. Our study supports the further evaluation of RG7388 as a novel treatment option in p53 wild-type neuroblastoma at diagnosis and relapse.

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis.

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation.

Embryonal cancer can arise from postnatally persistent embryonal remnant or rest cells, which are uniquely characterized by the absence of p53 mutations. Perinatal overexpression of the MycN oncoprotein in embryonal cancer precursor cells causes postnatal rests, and later tumor formation through unknown mechanisms. However, overexpression of Myc in adult tissues normally activates apoptosis and/or senescence signals as an organismal defense mechanism against cancer. Here, we show that perinatal neuroblastoma precursor cells exhibited a transiently diminished p53 response to MycN oncoprotein stress and resistance to trophic factor withdrawal, compared with their adult counterpart cells from the TH-MYCN(+/+) transgenic mouse model of neuroblastoma. The adult stem cell maintenance factor and Polycomb group protein, Bmi1 (B-cell-specific Moloney murine leukemia virus integration site), had a critical role at neuroblastoma initiation in the model, by repressing p53 responses in precursor cells. We further show in neuroblastoma tumor cells that Bmi1 could directly bind p53 in a complex with other Polycomb complex proteins, Ring1A or Ring1B, leading to increased p53 ubiquitination and degradation. Repressed p53 signal responses were also seen in precursor cells for other embryonal cancer types, medulloblastoma and acute lymphoblastic leukemia. Collectively, these date indicate a general mechanism for p53 inactivation in some embryonal cell types and consequent susceptibility to MycN oncogenesis at the point of embryonal tumor initiation.

Dual-specificity phosphatase 26 is a novel p53 phosphatase and inhibits p53 tumor suppressor functions in human neuroblastoma.

Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy.

An inner medullary concentrating process actuated by renal pelvic/calyceal muscle contractions: assessment and hypothesis.

We propose a mechanism of an inner medullary concentrating process in which water extraction is accomplished by a colloid osmotic mechanism and hydrostatic pressure. There are 3 essential features of the proposal: 1. the fluid compartmental structure of the inner medullary interstitium: owing to molecular exclusion, negatively charged macromolecules, i.e. hyaluronan and extravasated plasma albumin form separate compartments (the HA and the EPA compartments); the resulting Gibbs-Donnan effect governs the movements of both ions and water. 2. NaCl, in high concentration in the inner medulla conditioned by the outer medullary countercurrent processes, significantly reduces the equilibrium colloid osmotic pressure between these compartments. 3. Urea, also accumulated here by special transport mechanisms, increases the mobility of water molecules and the flexibility of the HA fibrils by loosening hydrogen bonds. These features suggest that rhythmic, small pressure increases of the pelvic/calyceal muscles squeeze dilute fluid out of the HA compartment and, at the same time, accelerate the outflow of fluid and albumin into the ascending vasa recta from the EPA compartment. Further, they suggest a mechanism for the phenomenon that living organisms utilize hydrostatic pressure generated by muscle contractions in water economy namely, concentrating and diluting body fluids.

The MYCN oncogene is a direct target of miR-34a.

Loss of 1p36 heterozygosity commonly occurs with MYCN amplification in neuroblastoma tumors, and both are associated with an aggressive phenotype. Database searches identified five microRNAs that map to the commonly deleted region of 1p36 and we hypothesized that the loss of one or more of these microRNAs contributes to the malignant phenotype of MYCN-amplified tumors. By bioinformatic analysis, we identified that three out of the five microRNAs target MYCN and of these miR-34a caused the most significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines with MYCN amplification. Quantitative RT-PCR showed that neuroblastoma tumors with 1p36 loss expressed lower level of miR-34a than those with normal copies of 1p36. Furthermore, we demonstrated that MYCN is a direct target of miR-34a. Finally, using a series of mRNA expression profiling experiments, we identified other potential direct targets of miR-34a, and pathway analysis demonstrated that miR-34a suppresses cell-cycle genes and induces several neural-related genes. This study demonstrates one important regulatory role of miR-34a in cell growth and MYCN suppression in neuroblastoma.

Two fluid compartments in the renal inner medulla: a view through the keyhole of the concentrating process.

Approximately four decades ago, the countercurrent theory became influential in studies on the concentrating process in the mammalian kidney. The theory successfully represented the concentrating process in the outer medulla, but the problem of the concentrating mechanism in the inner medulla, as defined by Homer Smith has remained essentially intractable. In a recent comprehensive review by Knepper and coworkers of various theories and models, attention was refocused on the possible role of hyaluronate (HA) in the inner medullary concentrating process. The authors proposed a hypothesis that HA can convert hydrostatic pressure to concentrating work.Here, we briefly survey the earlier ideas on the role imputed to HA and present a new hypothesis which is different from that of Knepper and coworkers. We estimate that the hydrostatic pressures available in the inner medulla can account only for a very small fraction of the concentrating work. We hypothesize that the role of HA is tied up with extravasated plasma albumin and suggest that owing to the property of HA solutions to exclude other macromolecules, extravasated plasma albumin and HA constitute two fluid compartments in the interstitium in the inner medulla. In this proposed two-compartment model, the Gibbs-Donnan distribution influences the movement of ions and water between the HA and the extravasated albumin compartment. To relate the hypothetical role of HA to the concentrating process, we briefly describe new results obtained by other investigators on the accumulation of urea in the inner medulla. This subject has been critically reviewed recently by Yang & Bankir.Many processes have been identified as contributing to the concentrating process in the mammalian inner medulla. We speculate that among these many processes, the primary responsibility for the final concentration of the excreted urine may be portioned out differently in different mammalian species.

Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis.

Minichromosomal maintenance protein 7 (MCM7) is an essential component of the replication helicase complex (MCM2-7) required for DNA replication. Although this function is highly conserved among eukaryotes, additional functions for the MCM molecules continue to be described. Minichromosomal maintenance protein 7 is a marker for proliferation and is upregulated in a variety of tumors including neuroblastoma, prostate, cervical and hypopharyngeal carcinomas. To further investigate the general role of MCM7 in tumorigenesis, we generated a mouse model with deregulated MCM7 expression targeted to the basal layer of the epidermis using the keratin 14 (K14) promoter (K14.MCM7). When subjected to a two-stage chemical carcinogenesis protocol (dimethylbenz[alpha]anthracene (DMBA) initiation with 12-ortho-tetradecanoylphorbol-13-acetate promotion), K14.MCM7 mice showed significantly increased incidence and prevalence of tumor development relative to controls. Furthermore, within 40 weeks of treatment over 45% K14.MCM7 mice exhibited tumors that had converted to squamous cell carcinomas versus none in the control group. As predicted from previous skin carcinogenesis studies using DMBA as the initiating agent, Ras mutations where found in more than 90% of tumors isolated from K14.MCM7 mice. Whereas previous studies have shown that MCM7 is useful as a proliferation marker, our data suggest that deregulated MCM7 expression actively contributes to tumor formation, progression and malignant conversion.

Mode locking in reversed-field pinch experiments.

The MHD mode trajectory in the Madison Symmetric Torus reversed-field pinch has been found to obey the sine-Gordon equation. Corresponding to experiment, a perturbation analysis predicts the locations of mode locking to be at the vacuum chamber poloidal and/or toroidal gaps. The mode's energy dissipates when it locks, as shown by a decaying spiral phase-plane trajectory. Unlocked modes travel around the torus without an abrupt energy loss. By varying key machine parameters obtained by statistical analysis, the probability of locking in accordance with the experimental results can be predicted.

Neurotologic follow-up after radiation of posterior fossa tumors.

OBJECTIVE: Stereotactic radiation treatment, also known as gamma knife surgery or radiosurgery, has come into acceptance as a treatment alternative to surgical removal for posterior fossa tumors. The purpose of this article is to describe the role of the neurotologist in the optimal management of neurotologic complications after stereotactic radiation, as illustrated by five patients. STUDY DESIGN: Retrospective chart review. PATIENTS: Five patients who underwent stereotactic radiation of posterior fossa tumors. MAIN OUTCOME MEASURES: Presence or absence of neurotologic complications (tumor growth, hearing loss, imbalance/ataxia, vertigo, and facial paralysis) or neurosurgical complaints (facial numbness, motor weakness, headache, hydrocephalus, and subarachnoid cysts). RESULTS: Postradiation neurotologic complaints included vertigo, imbalance/ataxia, and progressive hearing loss in four of the five patients. Continued tumor growth occurred in two patients; two patients had no growth; in one patient the tumor became smaller. The complications of facial nerve paralysis, facial numbness, motor weakness, headache, hydrocephalus, cerebellar edema, and posterior fossa arachnoid cyst formation occurred less frequently. CONCLUSIONS: Stereotactic radiation of posterior fossa tumors can produce significant neurotologic problems. It is imperative that neurotologists remain involved in the follow-up care of patients with posterior fossa tumors to offer optimal treatment alternatives for the neurotologic disorders.

Malignant glomus tumors.

OBJECTIVES/HYPOTHESIS: To ascertain the incidence of malignancy in a large glomus and skull base series and to compare the clinical course of such patients with those who do not have malignant skull base lesions. STUDY DESIGN: Retrospective review of all skull base surgery cases treated at the Otology Group between 1970 and 1995. RESULTS: In a series of 175 jugulotympanic glomus tumors, 9 cases (5.1%) were identified. The surgery required for their extirpation is more extensive than nonmalignant glomus tumors. Attendant deficits and mortality from these procedures are accordingly greater. Five-year survival in this limited population was 72%. Prolonged periods of survival are possible with distant metastases. CONCLUSIONS: This rate of malignancy should advocate against a watchful, waiting approach. Radiation therapy is not advocated as a primary modality for this type of tumor, as it may lead to recurrence/persistence with either subsequent malignant degeneration and metastases or local recurrence.

Which culprit is causing your patient's otorrhea?

Otorrhea varies in appearance and can be accompanied by a multitude of symptoms. Recognition of what various combinations indicate--whether otitis externa, chronic otitis media, Langerhans' cell histiocytosis, or basal or squamous cell carcinoma--is essential. Physicians must also be familiar with the tests appropriate for confirming otorrhea's cause. Once proper diagnosis is made, effective treatment can follow.

Hearing loss: the invisible disability.

Loss of hearing is a national health problem with significant physical and psychological repercussions. Although there is no cure for certain forms of hearing loss, many patients can be helped, especially when the problem is recognized early. The authors discuss the important role of primary care physicians in early identification, management, and counseling of hearing-impaired patients.

Symptoms of patients with myasthenia gravis receiving treatment.

The symptoms of myasthenia gravis (MG) are often simply classified as excessive fatigue rather than evaluated as different signs of disease progression. The purpose of this study was to evaluate the medical symptoms of patients with MG who had been under treatment for many years. Patients diagnosed with MG were compared to healthy controls. A survey questionnaire was used and differences were evaluated using non-parametric statistics. Health care givers should be aware of these differences in order to facilitate early appropriate treatment, to decrease disability, and to increase the quality of life.