Advancing RAS/RASopathy therapies: An NCI‐sponsored intramural and extramural collaboration for the study of RASopathies

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1‐related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non‐NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.     

Early consolidation of instrumental learning requires protein synthesis in the nucleus accumbens

It is widely held that long-term memories are established by consolidation of newly acquired information into stable neural representations, a process that requires protein synthesis and synaptic plasticity. Plasticity within the nucleus accumbens (NAc), a major component of the ventral striatum, is thought to mediate instrumental learning processes and many aspects of drug addiction. Here we show that the inhibition of protein synthesis within the NAc disrupts consolidation of an appetitive instrumental learning task (lever-pressing for food) in rats. Post-trial infusions of anisomycin immediately after the first several training sessions prevented consolidation, whereas infusions delayed by 2 or 4 hours had no effect. However, if the rats were allowed to learn the task, the behavior was not sensitive to disruption by intra-accumbens anisomycin. Control infusions into the medial NAc shell or the dorsolateral striatum did not impair learning; in fact, an enhancement was observed in the latter case. These results show that de novo protein synthesis within the NAc is necessary for the consolidation, but not reconsolidation, of appetitive instrumental memories.     

Mechanical properties of four methylmethacrylate-based resins for provisional fixed restorations

The use of a provisional restoration is an important phase in the treatment of the dental prosthetic patient. A good provisional restoration should satisfy the following requirements: pulpal protection, positional stability, ease in cleaning, accurate margins, wear resistance, dimensional stability, and serve as a diagnostic aid in treatment assessment and esthetics. There is a tendency for discoloration, occlusal wear, and fracture that eventually leads to unnecessary repair. Heat-processed and reinforced methacrylate-based resins have been used to improve the mechanical and physical properties of provisional restorations. Among various improvements, the interpenetrating network crosslinked PMMA (IPN) has been shown to have superior mechanical properties if manufactured through a dough compression molding process at 130 degrees C. However, there have been no published data that relate with the use of this material for fixed provisional restorations.The objective of this study was to compare four methyl methacrylate-based resins for provisional crowns and bridges with varying processing cycles, including JET [self-cure], ACRALON [heat-cured], titanium dioxide filled PMMA [heat-cured], and IPN [heat-cured denture tooth resin]. Properties studied included transverse strength, toughness, rigidity, and hardness. From the results of this study the following conclusions can be made: the IPN group may have had a lower degree of conversion as demonstrated by decreased strength, toughness, and hardness data as compared with Acralon. Increasing the polymerization cycle of unmodified Acralon resin causes a significant increase in strength.     

Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins.

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.     

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.     

Frequency domain models of the EEG

The structure of the normal resting EEG crosspectrum SVV(omega) is analyzed using complex multivariate statistics. Exploratory data analysis with Principal Component Analysis (PCA) is followed by hypothesis testing and computer simulations related to possible neural generators. The SVV(omega) of 211 normal individuals (ages 5 to 97) may be decomposed into two types of processes: the xi process with spatial isotropicity reflecting diffuse, correlated cortical generators with radial symmetry, and processes that seem to be generated by more spatially concentrated, correlated sources. The latter are reflected as spectral peaks such as the process. The eigenvectors of the xi process are the Spherical Harmonic Functions which explains the recurring pattern of maps characteristic of the spatial PCA of qEEG data. A new method for estimating sources in the frequency domain which fits dipoles to the whole crosspectrum is applied to explain the characteristics of the localized sources.     

L-dopa stimulates the release of [3H]gamma-aminobutyric acid in the basal ganglia of 6-hydroxydopamine lesioned rats

L-DOPA stimulated the K(+)-induced [3H]GABA (gamma-aminobutyric acid) release from slices of substantia nigra pars reticulata, entopeduncular nucleus, globus pallidus and caudate-putamen isolated from the ipsilateral side of 6-hydroxydopamine-lesioned rats, but the release from ipsilateral subthalamic slices was not affected. In substantia nigra, L-DOPA stimulation (EC50 = 1 microM) of [3H]GABA release was dose-dependently blocked (IC50 = 0.1 microM for the stimulation caused by 10 microM L-DOPA) by the D1 antagonist SCH 23390, but was not affected by (-)-sulpiride, a D2 antagonist. SCH 23390 also blocked the stimulation in the other nuclei. The DOPA decarboxylase inhibitor NSD-1015 (500 microM) did not prevent the stimulation induced by L-DOPA in all of the studied nuclei. The results suggest that L-DOPA is able to activate D1 receptors located on the terminals of striatal projections via the dopamine formed by a decarboxylation mediated by an NSD-1015-resistant enzyme. Activation of the presynaptic D1 receptors results in stimulation of GABA release.     

Determinants of cockroach and mouse exposure and associations with asthma in families and elderly individuals living in New York City public housing.

BACKGROUND: Asthma prevalence is high in the inner city, and morbidity has been associated with cockroach and mouse allergens. OBJECTIVE: To characterize the relationships among pests, allergens, pesticides, and asthma in New York City public housing. METHODS: In 324 apartments, dust samples collected from beds and kitchens were analyzed for cockroach (Bla g 2) and mouse (mouse urinary protein [MUP]) allergens, pest populations were monitored, and residents were interviewed about home characteristics and asthma symptoms. RESULTS: Cockroaches were found in 77% of the apartments, and evidence of mice was found in 13%. Allergens and pesticide use were associated with pest infestation, and 15% of residents reported using illegal pesticides. The percentage of apartments with high allergen levels varied significantly by building (Bla g 2: P = .002; MUP: P = .03), as did the percentage of apartments with cockroaches (P = .002) and daily mouse sightings (P = .02). Thirty-seven percent of the apartments had at least 1 resident with physician-diagnosed asthma. In family buildings, apartments with high Bla g 2 levels had 1.7 times greater odds of having an asthmatic resident (95% confidence interval, 1.2-2.3). In senior citizen buildings, apartments with high MUP levels had 6.6 times greater odds of having an asthmatic resident (95% confidence interval, 1.4-31.7), controlling for smoking and other potential confounders. CONCLUSIONS: Previous studies have identified home characteristics associated with the presence of cockroaches and mice, but the present findings suggest that building-level characteristics can affect high pest exposure. Furthermore, the high asthma prevalence in residents and the use of illegal pesticides highlight the need for safe and effective building-wide pest control strategies.