Chimeric antigen receptor signaling: Functional consequences and design implications.

Chimeric antigen receptor (CAR) T cell therapy has transformed the care of refractory B cell malignancies and holds tremendous promise for many aggressive tumors. Despite overwhelming scientific, clinical, and public interest in this rapidly expanding field, fundamental inquiries into CAR T cell mechanistic functioning are still in their infancy. Because CAR T cells are manufactured from donor T lymphocytes, and because CARs incorporate well-characterized T cell signaling components, it has largely been assumed that CARs signal analogously to canonical T cell receptors (TCRs). However, recent studies demonstrate that many aspects of CAR signaling are unique, distinct from endogenous TCR signaling, and potentially even distinct among various CAR constructs. Thus, rigorous and comprehensive proteomic investigations are required for rational engineering of improved CARs. Here, we review what is known about proximal CAR signaling in T cells, compare it to conventional TCR signaling, and outline unmet challenges to improving CAR T cell therapy.

Cognitive behavioural therapy for eating disorders: how do clinician characteristics impact on treatment fidelity?

BACKGROUND: Clinicians routinely report not practising evidence-based treatments with eating disorders. There has been limited research investigating the impact of adaptable clinician characteristics such as self-efficacy and therapeutic optimism in this area. This study evaluated if there is a relationship between clinician therapeutic optimism, self-efficacy and the provision of evidence-based practice in the treatment of bulimia nervosa and binge eating disorder. METHOD: A survey developed for this study was administered to 100 psychologists who were recruited online via a range of organisations affiliated with psychology and/or eating disorders. The survey measured demographic factors, eating disorder treatment knowledge, treatment fidelity, the use of individual treatment components and a range of clinician characteristics including self-efficacy and therapeutic optimism. RESULTS: Results demonstrated that clinician self-efficacy was positively associated with and predicted treatment fidelity. Therapeutic optimism had significant low correlations with treatment fidelity but did not predict treatment fidelity. CONCLUSION: These findings would suggest that strengthening clinician self-efficacy is useful in improving evidence-based practice in the treatment of binge eating disorder and bulimia nervosa and may also have implications in the training of clinicians. The study also demonstrated that the use of a range of knowledge translation strategies are valuable in enhancing clinician adherence to evidence-based practice. Further research with direct measures of treatment fidelity is needed to clarify these findings.

Euhaplorchis californiensis Cercariae Exhibit Positive Phototaxis and Negative Geotaxis.

Parasites often use external cues to identify and move toward environments where they are likely to encounter suitable hosts. The trematode parasite Euhaplorchis californiensis produces cercariae that emerge from California horn snails ( Cerithideopsis californica [= Cerithidea californica]) to infect California killifish ( Fundulus parvipinnis) as second intermediate hosts. Based upon work on a congeneric Euhaplorchis species from Florida, and based on the ecology of its killifish host, we hypothesized that E. californiensis cercariae in southern California estuaries are positively phototactic and negatively geotactic, using both sunlight and gravity to guide their movement to the upper water column. To distinguish positive phototaxis from negative geotaxis, we first quantified E. californiensis movement in response to light along a horizontal plane and determined they were positively phototactic. In a second experiment, we quantified E. californiensis movement along a vertical plane in response to an overhead light, a light from below, or no light. We found that E. californiensis exhibit negative geotaxis in the absence of light, but will swim in the direction of gravity to move toward a light source from below. Thus, E. californiensis are both positively phototactic and negatively geotactic, but cercariae prioritize phototactic cues. These results suggest that E. californiensis cercariae aggregate in the open water, indicating that the pelagic zone represents an area of high infection risk for California killifish hosts.

Forensic source differentiation of petrogenic, pyrogenic, and biogenic hydrocarbons in Canadian oil sands environmental samples.

To facilitate monitoring efforts, a forensic chemical fingerprinting methodology has been applied to characterize and differentiate pyrogenic (combustion derived) and biogenic (organism derived) hydrocarbons from petrogenic (petroleum derived) hydrocarbons in environmental samples from the Canadian oil sands region. Between 2009 and 2012, hundreds of oil sands environmental samples including water (snowmelt water, river water, and tailings pond water) and sediments (from river beds and tailings ponds) have been analyzed. These samples were taken from sites where assessments of wild fish health, invertebrate communities, toxicology and detailed chemistry are being conducted as part of the Canada-Alberta Joint Oil Sands Monitoring Plan (JOSMP). This study describes the distribution patterns and potential sources of PAHs from these integrated JOSMP study sites, and findings will be linked to responses in laboratory bioassays and in wild organisms collected from these same sites. It was determined that hydrocarbons in Athabasca River sediments and waters were most likely from four sources: (1) petrogenic heavy oil sands bitumen; (2) biogenic compounds; (3) petrogenic hydrocarbons of other lighter fuel oils; and (4) pyrogenic PAHs. PAHs and biomarkers detected in snowmelt water samples collected near mining operations imply that these materials are derived from oil sands particulates (from open pit mines, stacks and coke piles).

Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase.

Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1 µM. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.

Fingerprinting of petroleum hydrocarbons (PHC) and other biogenic organic compounds (BOC) in oil-contaminated and background soil samples.

Total petroleum hydrocarbons (TPH) or petroleum hydrocarbons (PHC) are one of the most widespread soil contaminants in Canada, the United States and many other countries worldwide. Clean-up of PHC-contaminated soils costs the Canadian economy hundreds of millions of dollars annually. In Canada, most PHC-contaminated site evaluations are based on the methods developed by the Canadian Council of the Ministers of the Environment (CCME). However, the CCME method does not differentiate PHC from BOC (the naturally occurring biogenic organic compounds), which are co-extracted with petroleum hydrocarbons in soil samples. Consequently, this could lead to overestimation of PHC levels in soil samples. In some cases, biogenic interferences can even exceed regulatory levels (300 µg g(-1) for coarse soils and 1300 µg g(-1) for fine soils for Fraction 3, C(16)-C(34) range, in the CCME Soil Quality Level). Resulting false exceedances can trigger unnecessary and costly cleanup or remediation measures. Therefore, it is critically important to develop new protocols to characterize and quantitatively differentiate PHC and BOC in contaminated soils. The ultimate objective of this PERD (Program of Energy Research and Development) project is to correct the misconception that all detectable hydrocarbons should be regulated as toxic petroleum hydrocarbons. During 2009-2010, soil and plant samples were collected from over forty oil-contaminated and paired background sites in various provinces. The silica gel column cleanup procedure was applied to effectively remove all target BOC from the oil-contaminated sample extracts. Furthermore, a reliable GC-MS method in combination with the derivatization technique, developed in this laboratory, was used for identification and characterization of various biogenic sterols and other major biogenic compounds in these oil-contaminated samples. Both PHC and BOC in these samples were quantitatively determined. This paper reports the characterization results of this set of 21 samples. In general, the presence of petroleum-characteristic alkylated PAH homologues and biomarkers can be used as unambiguous indicators of the contamination of oil and petroleum product hydrocarbons; while the absence of petroleum-characteristic alkylated PAH homologues and biomarkers and the presence of abundant BOC can be used as unambiguous indicators of the predominance of natural organic compounds in soil samples.

Forensic differentiation of biogenic organic compounds from petroleum hydrocarbons in biogenic and petrogenic compounds cross-contaminated soils and sediments.

"Total petroleum hydrocarbons" (TPHs) or "petroleum hydrocarbons" (PHCs) are one of the most widespread soil pollutants in Canada, North America, and worldwide. Clean-up of PHC-contaminated soils and sediments costs the Canadian economy hundreds of million of dollars annually. Much of this activity is driven by the need to meet regulated levels of PHC in soil. These PHC values are legally required to be assessed using standard methods. The method most commonly used in Canada, specified by the Canadian Council of Ministers of the Environment (CCME), measures the total hydrocarbon concentrations in a soil by carbon range (Fraction 1: C(6)-C(10); Fraction 2: C(10)-C(16), Fraction 3: C(16)-C(34): and Fraction 4: C(34)+). Using the CCME method, all of the materials extractible by a mixture of 1:1 hexane:acetone are considered to be petroleum hydrocarbon contaminants. Many hydrocarbon compounds and other extractible materials in soil, however, may originate from non-petroleum sources. Biogenic organic compounds (BOCs) is a general term used to describe a mixture of organic compounds, including alkanes, sterols and sterones, fatty acids and fatty alcohols, and waxes and wax esters, biosynthesized by living organisms. BOCs are also produced during the early stages of diagenesis in recent aquatic sediments. BOC sources could include vascular plants, algae, bacteria and animals. Plants and algae produce BOCs as protective wax coating that are released back into the sediment at the end of their life cycle. BOCs are natural components of thriving plant communities. Many solvent-extraction methods for assessing soil hydrocarbons, however, such as the CCME method, do not differentiate PHCs from BOCs. The naturally occurring organics present in soils and wet sediments can be easily misidentified and quantified as regulated PHCs during analysis using such methods. In some cases, biogenic interferences can exceed regulatory levels, resulting in remediation of petroleum impacts that are not actually present. Consequently, reliance on these methods can trigger unnecessary and costly remediation, while also wasting valuable landfill space. Therefore, it is critically important to develop new protocols to characterize and differentiate PHCs and BOCs in contaminated sediments. In this study, a new reliable gas chromatography-mass spectrometry (GC-MS) method, in combination with a derivatization technique, for characterization of various biogenic compounds (including biogenic alkanes, sterols, fatty acids and fatty alcohols) and PHCs in the same sample has been developed. A multi-criteria approach has been developed to positively identify the presence of biogenic compounds in soil and sediment samples. More than thirty sediment samples were collected from city stormwater management (SWM) ponds and wetlands across Canada. In these wet sediment samples, abundant biogenic n-alkanes, thirteen biogenic sterols, nineteen fatty carboxylic acids, and fourteen fatty alcohols in a wide carbon range have been positively identified. Both PHCs and BOCs in these samples were quantitatively determined. The quantitation data will be used for assessment of the contamination sites and toxicity risks associated with the CCME Fraction 3 hydrocarbons.

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor.

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (alpha v beta6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an alpha v beta6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-zeta. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin alpha v beta6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.

Zincergic innervation of the forebrain distinguishes epilepsy-prone from epilepsy-resistant rat strains.

Zinc is released from a subset of cerebral cortical neurons whereupon it exerts a powerful modulatory influence on excitatory and inhibitory neurotransmission. A number of studies have suggested that alterations in the regulation of zinc may contribute to the genesis of epilepsy. Here, we tested this hypothesis by examining the distribution of zinc-containing axon terminals in rats selectively bred for an innate susceptibility (FAST) or resistance (SLOW) to the development of kindling-induced seizures. Zinc was stained histochemically and levels of staining were quantitatively assessed. We found that the levels of synaptic zinc were significantly lower in the SLOW rats throughout the telencephalon. This relative reduction was most pronounced in limbic cortices where levels were less than 30% of FAST rats. These results suggest that innate differences in the homeostatic regulation of synaptic zinc, particularly in limbic cortices, may underlie differences in epileptogenicity.

A prospective, randomized clinical trial comparing two hyperbaric treatment protocols for carbon monoxide poisoning.

INTRODUCTION: The optimal hyperbaric oxygen (HBO2) treatment protocol for acute carbon monoxide (CO) poisoning is unknown. This is indicated by one study that found 18 different protocols to treat CO poisoning by North American multiplace hyperbaric facilities. A pilot study was conducted to evaluate the feasibility of randomizing patients to different protocols and to determine whether any large differences in clinical outcome were present between the two most common protocols. METHODS: Adult patients with accidental CO poisoning resulting in transient loss of consciousness, presentation to the emergency department within 12 hours, primary language English, high school education, and residence within 100 miles of the hyperbaric facility were recruited. Enrolled patients were randomized to one HBO2 treatment at 2.4 atmospheres absolute (atm abs) pressure with 90 minutes of 100% oxygen breathing vs. treatment by the US Air Force CO protocol (3.0 atm abs maximum pressure). A neurocognitive screening test was performed immediately after hyperbaric treatment and repeated 14-21 days later. RESULTS: From 1995 to 2002, 30 patients age 21 to 88 years were randomized, 18 to treatment at 2.4 atm abs and 12 to 3.0 atm abs. Average carboxyhemoglobin level for the population was 24.8 +/- 8.8% (mean +/- SD). Delay to hyperbaric treatment averaged 313 +/- 129 minutes. Neither variable was different between treatment groups. Six patients had abnormal neurocognitive testing immediately following hyperbaric treatment, 4 in the 2.4 atm abs group (22%) and 2 in the 3.0 atm abs group (17%) (P=0.71). One patient in each group demonstrated abnormality on delayed testing (p=0.75). One in each group did not return for follow-up. CONCLUSIONS: It is feasible to randomize CO-poisoned patients to different hyperbaric treatment protocols. Determination of differences in efficacy between treatment protocols will require a large multicenter trial with the use of detailed neurocognitive testing.

Modulation of synaptic zinc in barrel cortex by whisker stimulation.

The cortical representation of the body surface is not fixed, but rather, is continuously modified by ongoing changes in sensory experience. Although the cellular and molecular mechanisms that subserve these changes are uncertain, increasing evidence suggests that synaptically-released zinc may play a role. Zinc is released from a subset of glutamatergic neurons and can modulate postsynaptic excitability by regulating the activation of glutamate and GABA receptor-gated ion channels. Previously, we have shown that whisker plucking, a manipulation commonly used to induce cortical map plasticity, results in a rapid and robust increase in staining levels for synaptic zinc in deprived regions of the barrel cortex. In the present study, we examined the effect of increased whisker activity, analogous to what may happen during tactile learning or exploratory behavior in a natural setting, on synaptic zinc levels in the adult barrel cortex. Our results indicate that stimulation of whiskers caused a selective decrease in zinc levels within layer 4 of the barrel hollow corresponding to the stimulated whisker. Quantitatively, levels of staining were significantly reduced at 3 h, and showed even greater reductions following 12 and 24 h of stimulation. However, these changes were not long-lasting, as levels of staining in the stimulated barrel returned to control values within 24 h after stimulation had ceased. These data indicate that zincergic circuits are highly sensitive to ongoing changes in sensory experience and may participate in moment-to-moment changes in the functional connectivity of the cerebral cortex.

Measuring socio-economic inequalities in the presentation of injuries to a paediatric A&E department: the importance of an epidemiological approach.

OBJECTIVES: To contrast the socio-economic pattern of childhood injuries presenting to a paediatric accident and emergency (A&E) department revealed by using both a numerator-based and a denominator-based approach to the analysis of injury surveillance data. METHODS: Injury surveillance data collected during 1997-1998 at a Glasgow children's hospital A&E department were analysed. Socio-economic status was measured using Carstairs' deprivation index. Data from West Glasgow postcode sectors only were analysed in order to optimize epidemiological validity. Socio-economic patterning of injury was investigated in two ways-numerator-based and denominator-based. RESULTS: A total of 12,762 children (0-14 years) living in West Glasgow attended the A&E department of the Royal Hospital for Sick Children over the study period. Both analytical approaches showed a clear and statistically significant excess of injury presentations in children from more deprived postcode sectors, but the variation appeared much greater in the numerator-based rather than the denominator-based approach. In regression analysis, however, only the denominator-derived rates showed a statistically significant linear trend across deprivation categories. CONCLUSION: The most appropriate and accurate means of measuring the extent of socio-economic (and other) inequalities in injury risk is to adopt a population-based rather than numerator-based perspective on the data collected by injury surveillance systems.

Detoxification of mercury in the environment.

In this study, a "green chemistry" approach was developed as an option for remediation of toxic mercury in the environment. Twenty mercury compounds were treated with an environmentally friendly agent cyclodextrin to produce stable non-toxic mercury in soil and water. The binding efficiency was determined using high performance liquid chromatography with diode-array detection. The stability of the cyclodextrin mercury complexes toward environmental microorganisms in water was estimated under OECD guidelines using gas chromatography-mass spectrometry. The toxicity of the cyclodextrin mercury compounds to terrestrial organisms was investigated by use of internationally recognized toxicity methods using mercuric acetate as a model contaminant. Key process conditions, for example pH, temperature, and amount of detoxifying agent were investigated and found to have significant effects on the toxicity of mercury. It was found that organic and inorganic mercury pollutants could be mineralized in the environment with cyclodextrins. The bound mercury compounds resisted biodegradation and were found to be non-toxic to environmental microorganisms under laboratory conditions.

Culturable bacteria in subglacial sediments and ice from two Southern Hemisphere glaciers.

Viable prokaryotes have been detected in basal sediments beneath the few Northern Hemisphere glaciers that have been sampled for microbial communities. However, parallel studies have not previously been conducted in the Southern Hemisphere, and subglacial environments in general are a new and underexplored niche for microbes. Unfrozen subglacial sediments and overlying glacier ice samples collected aseptically from the Fox Glacier and Franz Josef Glacier in the Southern Alps of New Zealand now have been shown to harbor viable microbial populations. Total direct counts of 2-7 x 10(6) cells g(-1) dry weight sediment were observed, whereas culturable aerobic heterotrophs ranged from 6-9 x 10(5) colony-forming units g(-1) dry weight. Viable counts in the glacier ice typically were 3-4 orders of magnitude smaller than in sediment. Nitrate-reducing and ferric iron-reducing bacteria were detected in sediment samples from both glaciers, but were few or below detection limits in the ice samples. Nitrogen-fixing bacteria were detected only in the Fox Glacier sediment. Restriction fragment analysis of 16S rDNA amplified from 37 pure cultures of aerobic heterotrophs capable of growth at 4 degrees C yielded 23 distinct groups, of which 11 were identified as beta-Proteobacteria. 16S rDNA sequences from representatives of these 11 groups were analyzed phylogenetically and shown to cluster with bacteria such as Polaromonas vacuolata and Rhodoferax antarcticus, or with clones obtained from permanently cold environments. Chemical analysis of sediment and ice samples revealed a dilute environment for microbial life. Nevertheless, both the sediment samples and one ice sample demonstrated substantial aerobic mineralization of 14C-acetate at 8 degrees C, indicating that sufficient nutrients and viable psychrotolerant microbes were present to support metabolism. Unfrozen subglacial sediments may represent a significant global reservoir of biological activity with the potential to influence glacier meltwater chemistry.

Hyperbaric oxygen treatment protocols for mandibular osteoradionecrosis.

To determine hyperbaric oxygen (HBO2) treatment practices for osteoradionecrosis (ORN) of the mandible in North America, we surveyed hyperbaric facilities listed in the 1998 UHMS Chamber Directory. A survey response rate of 99.7% was achieved. Among the 316 facilities listed, 280 treat or would treat mandibular ORN with HBO2. Twelve different hyperbaric treatment protocols for the condition were reported. Approximately three-quarters of facilities utilize a protocol administering 90 minutes of 100% oxygen breathing at a treatment pressure of 2.4-2.5 atmospheres absolute (atm abs). The remaining one-quarter of facilities apply alternate hyperbaric treatment protocols. In summary, mandibular ORN is commonly treated at North American hyperbaric facilities but there is a lack of uniformity with regard to the protocol utilized for hyperbaric oxygen administration. There are no clinical data to support that any one treatment protocol is superior in out come to any other.

Experience-dependent regulation of synaptic zinc is impaired in the cortex of aged mice.

Zinc plays an important role in synaptic signaling in the mammalian cerebral cortex. Zinc is sequestered into presynaptic vesicles of subpopulations of glutamatergic neurons and is released by depolarization, in a calcium-dependent manner. As the majority of mechanisms that have been suggested to participate in experience-dependent alterations in synaptic strength in the cerebral cortex implicate signaling by glutamate, it stands to reason that zincergic signaling might also be crucial. Here we show that synaptic zinc is rapidly and dynamically modulated in relation to alterations in sensory input and that this response is highly age-dependent. Juvenile, adult, and aged mice were subjected to whisker removal and levels of staining for synaptic zinc in deprived and non-deprived cortical barrels were quantitatively assessed at post-deprivation times ranging from 3 h to 21 days. In the first 12 h, zinc levels increased slightly, but significantly, in all groups. At later time points, zinc levels increased robustly (23%) in the youngest group by 24 h and remained elevated through 7 days. By contrast, deprivation-induced changes in zinc staining in aged animals, achieved their maximal levels at 12 h (approximately 10%) and steadily declined thereafter. Adult animals revealed a biphasic, intermediate change with time. In all age groups, levels of zinc staining returned to baseline by 21 days after whisker plucking. However, only in juvenile and adult mice did we observe that the level of zinc staining in deprived barrel hollows, was correlated with the length of whiskers as they regrew. Our data suggest that alterations in the regulation of synaptic zinc may be involved with decrements of synaptic plasticity that accompany senescence.

Determination of Cr3+, CrO42-, and Cr2O72- in environmental matrixes by high-performance liquid chromatography with diode-array detection (HPLC-DAD).

A high-performance liquid chromatographic method with diode array detection (HPLC-DAD), based on chelation with ammonium pyrrolidinedithiocarbamate (APDC), has been developed for the determination of chromium species. Determination of Cr3+, CrO42-, and Cr2O72- was performed for standards and synthetic environmental matrixes. This method is robust, rugged, and can be used for rapid routine determination of chromium species with high precision and reliability. Sample pretreatment is simple. The method is capable of discriminating not only between Cr(III) and Cr(VI) but also between the chemical forms of Cr(VI) - CrO42- and Cr2O72-. By analysis of numerous samples the method has been shown to be selective, sensitive, and free from matrix interference, which is crucial for the determination of chromium species in difficult-to-analyze environmental matrixes. This method has been validated by means of an interlaboratory study. Although different speciation techniques were used during this study, there was good agreement between results from the two laboratories. The method detection limits were 7 and 4 mg L(-1) for Cr3+ and Cr2O72-, respectively. Recoveries of the analytes from spiked samples were 98% and 100% for Cr3+ and Cr2O72-, respectively. Both were based on a 10-mL sample volume spiked with 0.4 mg L(-1) chromium.

The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1.

It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ~450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.

An immunohistochemical study of osteoprotegerin in the human dental pulp.

This study investigated the expression of osteoprotegerin (OPG) in healthy and inflamed dental pulps. Histological sections 7 microm thick of 47 teeth, either caries-free or affected by gross caries, were used. Sections were stained with hematoxylin-eosin, and other sections of the same specimen were subjected to the avidin-biotin peroxidase complex immunohistochemical procedure for detection of OPG. The study focused on the coronal pulp that was divided into peripheral and central regions. In the peripheral pulp healthy and inflamed specimens showed high OPG immunoreactivity of the odontoblastic layer. When no inflammation was present in the central pulp OPG immunoreactivity was light. Fibroblasts and endothelial cells showed immunoreactivity ranging from none to intense. When inflammation was present in the central pulp the chronic inflammatory cells showed intense immunoreactivity.