MOLECULAR MECHANISMS UNDERLYING THE TRANSCRIPTIONAL REGULATION OF T HELPER 17 AND REGULATORY T CELLS

CD4+ T helper (Th) lymphocytes are vital for integrating immune responses by orchestrating the function of other immune cell types. Naïve Th cells can differentiate into different effector subsets that are characterized by their cytokine profile and immune regulatory functions. These subsets include Th1, Th2, Th17, natural and inducible regulatory T cells (nTreg and iTreg respectively), among others. We focused our investigation on two Th lineages, Th17 and regulatory T cells, with opposing functions in the immune system. These subsets have been suggested to be reciprocally regulated since they both require TGF-b for their development. We investigated the role of the Treg-associated master transcription factor Foxp3, and found that Foxp3 inhibits Th17 cell generation by preventing the transcriptional activity of the two main Th17-specific transcription factors, nuclear orphan receptors RORa and RORgt. At the molecular level, we identified two different functional domains in Foxp3 required for such inhibition: the LQALL sequence in exon 2 and the TIP60/HDAC7 binding domain. These domains could be crucial to either prevent the association of the nuclear receptors to coactivators or to recruit histone deacetylases to RORa- or RORgt-target genes. Since TGF-b is a common cytokine required for the commitment towards both Th lineages, we determined the role of the TGF-b-dependent signaling pathway in the generation of each subset. By using mice with deficiencies in signaling molecules downstream of TGF-b, we found that while Smad2, Smad3 and Smad4 are required for the generation of iTreg cells, only Smad2 is indispensable for the induction of IL-17-producing cells, suggesting that TGF-b induces these T helper lineages through differential signaling pathways. Thus, our findings describe novel transcriptional regulatory mechanisms that control the generation of two T helper lineages with opposing functions. These findings could provide novel therapeutic targets to treat diseases where the balance of these T cells is dysregulated, such as in autoimmunity, chronic infectious diseases and cancer.

Three-dimensional electron beam dose calculations

The MDAH pencil-beam algorithm developed by Hogstrom et al (1981) has been widely used in clinics for electron beam dose calculations for radiotherapy treatment planning. The primary objective of this research was to address several deficiencies of that algorithm and to develop an enhanced version. Two enhancements have been incorporated into the pencil-beam algorithm; one models fluence rather than planar fluence, and the other models the bremsstrahlung dose using measured beam data. Comparisons of the resulting calculated dose distributions with measured dose distributions for several test phantoms have been made. From these results it is concluded (1) that the fluence-based algorithm is more accurate to use for the dose calculation in an inhomogeneous slab phantom, and (2) the fluence-based calculation provides only a limited improvement to the accuracy the calculated dose in the region just downstream of the lateral edge of an inhomogeneity. The source of the latter inaccuracy is believed primarily due to assumptions made in the pencil beam's modeling of the complex phantom or patient geometry.^ A pencil-beam redefinition model was developed for the calculation of electron beam dose distributions in three dimensions. The primary aim of this redefinition model was to solve the dosimetry problem presented by deep inhomogeneities, which was the major deficiency of the enhanced version of the MDAH pencil-beam algorithm. The pencil-beam redefinition model is based on the theory of electron transport by redefining the pencil beams at each layer of the medium. The unique approach of this model is that all the physical parameters of a given pencil beam are characterized for multiple energy bins. Comparisons of the calculated dose distributions with measured dose distributions for a homogeneous water phantom and for phantoms with deep inhomogeneities have been made. From these results it is concluded that the redefinition algorithm is superior to the conventional, fluence-based, pencil-beam algorithm, especially in predicting the dose distribution downstream of a local inhomogeneity. The accuracy of this algorithm appears sufficient for clinical use, and the algorithm is structured for future expansion of the physical model if required for site specific treatment planning problems. ^

Comment on “The phase relation between atmospheric carbon dioxide and global temperature” Humlum et al. [Glob. Planet. Change 100: 51–69.]: Isotopes ignored

A recent study relying purely on statistical analysis of relatively short time series suggested substantial re-thinking of the traditional view about causality explaining the detected rising trend of atmospheric CO2 (atmCO2) concentrations. If these results are well-justified then they should surely compel a fundamental scientific shift in paradigms regarding both atmospheric greenhouse warming mechanism and global carbon cycle. However, the presented work suffers from serious logical deficiencies such as, 1) what could be the sink for fossil fuel CO2 emissions, if neither the atmosphere nor the ocean – as suggested by the authors – plays a role? 2) What is the alternative explanation for ocean acidification if the ocean is a net source of CO2 to the atmosphere? Probably the most provocative point of the commented study is that anthropogenic emissions have little influence on atmCO2 concentrations. The authors have obviously ignored the reconstructed and directly measured carbon isotopic trends of atmCO2 (both δ13C, and radiocarbon dilution) and the declining O2/N2 ratio, although these parameters provide solid evidence that fossil fuel combustion is the major source of atmCO2 increase throughout the Industrial Era.

Phosphorylation in the regulation of muscle-specific transcription

The contents of this dissertation include studies on the mechanisms by which FGF and growth factor down-stream kinases inactivate myogenin; characterization of myogenin phosphorylation and its role in regulation of myogenin activity; analysis the C-terminal transcriptional activation domain of myogenin; studies on the nuclear localization of myogenin and characterization of proteins that interact with PKC.^ Activation of muscle transcription by the MyoD family requires their heterodimerization with ubiquitous bHLH proteins such as the E2A gene products E12 and E47. I have shown that dimerization with E2A products potentiates phosphorylation of myogenin at serine 43 in its amino-terminus and serine 170 in the carboxyl-terminal transcription activation domains. Mutations of these sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. Consistent with the role of phosphorylation at serine 170, analysis of the carboxyl-terminal transcriptional activation domain by deletion has revealed a stretch of residues from 157 to 170 which functions as a negative element for myogenin activity.^ In addition to inducing phosphorylation of myogenin, E12 also localizes myogenin to the nucleus. The DNA binding and dimerization mutants of myogenin show various deficiencies in nuclear localization. Cotransfection of E12 with the DNA binding mutants, but not a dimerization mutant, greatly enhances their nuclear binding. These data suggest that the nuclear localization signal is located in the DNA binding region and myogenin can also be nuclear localized by virtue of dimerizing with a nuclear protein.^ FGF is one of the most potent inhibitors of myogenesis and activates many down-stream pathways to exert its functions. One of these pathway is the MAP kinase pathway. Studies have shown that Raf-1 and Erk-1 kinase inactivate transactivation by myogenin and E proteins independent of DNA binding. The other is the PKC pathway. In transfected cells, FGF induces phosphorylation of thr-87 that maps to the previously identified PKC sites in the DNA binding domain of myogenin. Myogenin mutant T-N87 could resist the inhibition directed to the bHLH domain by FGF, suggesting that FGF inactivates myogenin by inducing phosphorylation of this site. In C2 myotubes, where FGF receptors are lost, the phosphatase inhibitor, okadaic acid, and phorbal ester PdBu, can also induce the phosphorylation of thr-87. This result supports the previous observation and suggests that in myotubes, other mechanisms, such as innervation, may inactivate myogenin through PKC induced phosphorylation.^ Many functions of PKC have been well documented, yet, little is known about the activators or effectors of PKC or proteins that mediate PKC nuclear localizations. Identification of PKC binding proteins will help to understand the molecular mechanism of PKC function. Two proteins that interact with the C kinase (PICKS) have been characterized, PICK-1 and PICK-2. PICK1 interacts with two conserved regions in the catalytic domain of PKC. It is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK2 is a novel protein with homology to the heat shock protein family. It interacts extensively with the catalytic domain of PKC and is localized in the cytoplasm in a punctate pattern. PICK1 and PICK2 may play important roles in mediating the actions of PKC. ^

A prospective cohort study evaluating the state of medical readiness in the United States Air Force and risk factors to improve the medical readiness posture

A cohort of 418 United States Air Force (USAF) personnel from over 15 different bases deployed to Morocco in 1994. This was the first study of its kind and was designed with two primary goals: to determine if the USAF was medically prepared to deploy with its changing mission in the new world order, and to evaluate factors that might improve or degrade USAF medical readiness. The mean length of deployment was 21 days. The cohort was 95% male, 86% enlisted, 65% married, and 78% white.^ This study shows major deficiencies indicating the USAF medical readiness posture has not fully responded to meet its new mission requirements. Lack of required logistical items (e.g., mosquito nets, rainboots, DEET insecticide cream, etc.) revealed a low state of preparedness. The most notable deficiency was that 82.5% (95% CI = 78.4, 85.9) did not have permethrin pretreated mosquito nets and 81.0% (95% CI = 76.8, 84.6) lacked mosquito net poles. Additionally, 18% were deficient on vaccinations and 36% had not received a tuberculin skin test. Excluding injections, the overall compliance for preventive medicine requirements had a mean frequency of only 50.6% (95% CI = 45.36, 55.90).^ Several factors had a positive impact on compliance with logistical requirements. The most prominent was "receiving a medical intelligence briefing" from the USAF Public Health. After adjustment for mobility and age, individuals who underwent a briefing were 17.2 (95% CI = 4.37, 67.99) times more likely to have received an immunoglobulin shot and 4.2 (95% CI = 1.84, 9.45) times more likely to start their antimalarial prophylaxsis at the proper time. "Personnel on mobility" had the second strongest positive effect on medical readiness. When mobility and briefing were included in models, "personnel on mobility" were 2.6 (95% CI = 1.19, 5.53) times as likely to have DEET insecticide and 2.2 (95% CI = 1.16, 4.16) times as likely to have had a TB skin test.^ Five recommendations to improve the medical readiness of the USAF were outlined: upgrade base level logistical support, improve medical intelligence messages, include medical requirements on travel orders, place more personnel on mobility or only deploy personnel on mobility, and conduct research dedicated to capitalize on the powerful effect from predeployment briefings.^ Since this is the first study of its kind, more studies should be performed in different geographic theaters to assess medical readiness and establish acceptable compliance levels for the USAF. ^

A STUDY OF ZINC CONCENTRATION IN HAIR AS AN INDICATION OF ZINC IMBALANCES

Trace metal imbalances have been implicated in several disease and nutritional states. There is mounting concern to identify the nutritional balance of the trace metals needed for growth, mental acuity and physical functioning. These two factors, diseases in which trace metals show involvement and nutritional balance, have made it necessary to be able to accurately describe the trace metal balances of an individual. Although several investigators have measured the concentration of trace metals in the hair and related those observed concentrations to various disease and nutritional states, no one has satisfactorily answered the questions of whether hair is useful to determine trace metal imbalances, whether the concentrations found in hair reflect tissue or serum concentrations of the trace metals, or whether any tissue accurately reflects body status of the trace metals.^ Male mice were used to examine several tissues, heart, liver, kidney, spleen, intestine, brain, bone, hair and serum for copper and zinc concentrations. The environment and dietary intake of the animals were carefully controlled, so that environmental and physical variables were minimized. Dietary intake of zinc was varied while copper intake was held constant. Each experimental diet group was matched with a pair fed control group.^ Of the tissues examined, only the serum was indicative of an early state of zinc imbalance. Neither hair nor the other tissues examined for copper and zinc concentrations were indicative of an acute zinc imbalance in a normal mature mouse. Zinc deficiencies or excesses may manifest themself differently in the chronic imbalance state or in the weanling, aged or traumatized mouse. The tissue response to zinc imbalance may vary in these cases. ^

A New Technique for Firn Grain-Size Measurement Using SEM Image Analysis

Firn microstructure is accurately characterized using images obtained from scanning electron microscopy (SEM). Visibly etched grain boundaries within images are used to create a skeleton outline of the microstructure. A pixel-counting utility is applied to the outline to determine grain area. Firn grain sizes calculated using the technique described here are compared to those calculated using the techniques of Cow (1969) and Gay and Weiss (1999) on samples of the same material, and are found to be substantially smaller. The differences in grain size between the techniques are attributed to sampling deficiencies (e.g. the inclusion of pore filler in the grain area) in earlier methods. The new technique offers the advantages of greater accuracy and the ability to determine individual components of the microstructure (grain and pore), which have important applications in ice-core analyses. The new method is validated by calculating activation energies of grain boundary diffusion using predicted values based on the ratio of grain-size measurements between the new and existing techniques. The resulting activation energy falls within the range of values previously reported for firn/ice.

Contribution of Starlette, Stella, and AJISAI to the SLR-derived global reference frame

The contribution of Starlette, Stella, and AJI-SAI is currently neglected when defining the International Terrestrial Reference Frame, despite a long time series of precise SLR observations and a huge amount of available data. The inferior accuracy of the orbits of low orbiting geodetic satellites is the main reason for this neglect. The Analysis Centers of the International Laser Ranging Service (ILRS ACs) do, however, consider including low orbiting geodetic satellites for deriving the standard ILRS products based on LAGEOS and Etalon satellites, instead of the sparsely observed, and thus, virtually negligible Etalons. We process ten years of SLR observations to Starlette, Stella, AJISAI, and LAGEOS and we assess the impact of these Low Earth Orbiting (LEO) SLR satellites on the SLR-derived parameters. We study different orbit parameterizations, in particular different arc lengths and the impact of pseudo-stochastic pulses and dynamical orbit parameters on the quality of the solutions. We found that the repeatability of the East and North components of station coordinates, the quality of polar coordinates, and the scale estimates of the reference are improved when combining LAGEOS with low orbiting SLR satellites. In the multi-SLR solutions, the scale and the Z component of geocenter coordinates are less affected by deficiencies in solar radiation pressure modeling than in the LAGEOS-1/2 solutions, due to substantially reduced correlations between the Z geocenter coordinate and empirical orbit parameters. Eventually, we found that the standard values of Center-of-mass corrections (CoM) for geodetic LEO satellites are not valid for the currently operating SLR systems. The variations of station-dependent differential range biases reach 52 and 25 mm for AJISAI and Starlette/Stella, respectively, which is why estimating station dependent range biases or using station-dependent CoM, instead of one value for all SLR stations, is strongly recommended.This clearly indicates that the ILRS effort to produce CoM corrections for each satellite, which are site-specific and depend on the system characteristics at the time of tracking,is very important and needs to be implemented in the SLR data analysis.

Context-Aware Adaptive Opportunistic Routing in Mobile Ad-hoc and Sensor Networks

Mobile ad-hoc networks (MANETs) and wireless sensor networks (WSNs) have been attracting increasing attention for decades due to their broad civilian and military applications. Basically, a MANET or WSN is a network of nodes connected by wireless communication links. Due to the limited transmission range of the radio, many pairs of nodes in MANETs or WSNs may not be able to communicate directly, hence they need other intermediate nodes to forward packets for them. Routing in such types of networks is an important issue and it poses great challenges due to the dynamic nature of MANETs or WSNs. On the one hand, the open-air nature of wireless environments brings many difficulties when an efficient routing solution is required. The wireless channel is unreliable due to fading and interferences, which makes it impossible to maintain a quality path from a source node to a destination node. Additionally, node mobility aggravates network dynamics, which causes frequent topology changes and brings significant overheads for maintaining and recalculating paths. Furthermore, mobile devices and sensors are usually constrained by battery capacity, computing and communication resources, which impose limitations on the functionalities of routing protocols. On the other hand, the wireless medium possesses inherent unique characteristics, which can be exploited to enhance transmission reliability and routing performance. Opportunistic routing (OR) is one promising technique that takes advantage of the spatial diversity and broadcast nature of the wireless medium to improve packet forwarding reliability in multihop wireless communication. OR combats the unreliable wireless links by involving multiple neighboring nodes (forwarding candidates) to choose packet forwarders. In opportunistic routing, a source node does not require an end-to-end path to transmit packets. The packet forwarding decision is made hop-by-hop in a fully distributed fashion. Motivated by the deficiencies of existing opportunistic routing protocols in dynamic environments such as mobile ad-hoc networks or wireless sensor networks, this thesis proposes a novel context-aware adaptive opportunistic routing scheme. Our proposal selects packet forwarders by simultaneously exploiting multiple types of cross-layer context information of nodes and environments. Our approach significantly outperforms other routing protocols that rely solely on a single metric. The adaptivity feature of our proposal enables network nodes to adjust their behaviors at run-time according to network conditions. To accommodate the strict energy constraints in WSNs, this thesis integrates adaptive duty-cycling mechanism to opportunistic routing for wireless sensor nodes. Our approach dynamically adjusts the sleeping intervals of sensor nodes according to the monitored traffic load and the estimated energy consumption rate. Through the integration of duty cycling of sensor nodes and opportunistic routing, our protocol is able to provide a satisfactory balance between good routing performance and energy efficiency for WSNs.

Systematic reviews published in higher impact clinical journals were of higher quality.

OBJECTIVES To compare the methodological quality of systematic reviews (SRs) published in high- and low-impact factor (IF) Core Clinical Journals. In addition, we aimed to record the implementation of aspects of reporting, including Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow diagram, reasons for study exclusion, and use of recommendations for interventions such as Grading of Recommendations Assessment, Development and Evaluation (GRADE). STUDY DESIGN AND SETTING We searched PubMed for systematic reviews published in Core Clinical Journals between July 1 and December 31, 2012. We evaluated the methodological quality using the Assessment of Multiple Systematic Reviews (AMSTAR) tool. RESULTS Over the 6-month period, 327 interventional systematic reviews were identified with a mean AMSTAR score of 63.3% (standard deviation, 17.1%), when converted to a percentage scale. We identified deficiencies in relation to a number of quality criteria including delineation of excluded studies and assessment of publication bias. We found that SRs published in higher impact journals were undertaken more rigorously with higher percentage AMSTAR scores (per IF unit: β = 0.68%; 95% confidence interval: 0.32, 1.04; P < 0.001), a discrepancy likely to be particularly relevant when differences in IF are large. CONCLUSION Methodological quality of SRs appears to be better in higher impact journals. The overall quality of SRs published in many Core Clinical Journals remains suboptimal.

Visual acuity of dentists in their respective clinical conditions.

OBJECTIVES This study examined the impact of age and magnification on the near visual acuity of dentists in their private practice under simulated clinical conditions. MATERIALS AND METHODS Miniaturized visual tests were fixed in posterior teeth of a dental phantom head and brought to 31 dentists in their respective private practice. The visual acuity of these dentists (n = 19, ≥40 years; n = 12, <40 years) was measured in a clinical setting under the following conditions: (a) natural visual acuity, distance of 300 mm; (b) natural visual acuity, free choice of the distance; and (c) loupe and additional light source, if available. RESULTS The visual acuity under the different clinical conditions varied widely between individuals. The older group of dentists had a lower median visual acuity value under all clinical conditions. This difference was highly significant for natural visual acuity at a free choice of distance (p < 0.0001). For younger dentists (<40 years), visual acuity could be significantly improved by reducing the eye-object distance (p = 0.001) or by using loupes (p = 0.008). For older dentists (≥40 years), visual acuity could be significantly improved by using loupes (p = 0.0005). CONCLUSIONS Visual performance decreased with increasing age under the specific clinical conditions of each dentist's private practice. Magnification aids can compensate for visual deficiencies. CLINICAL RELEVANCE The question of whether findings obtained under standardized conditions are valuable for the habitual setting of each dentist's private practice seems clinically relevant.

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Sensitive Questions in Online Surveys: An Experimental Evaluation of the Randomized Response Technique and the Crosswise Model

Self-administered online surveys provide a higher level of privacy protection to respondents than surveys administered by an interviewer. Yet, studies indicate that asking sensitive questions is problematic also in self-administered surveys. Because respondents might not be willing to reveal the truth and provide answers that are subject to social desirability bias, the validity of prevalence estimates of sensitive behaviors from online surveys can be challenged. A well-known method to overcome these problems is the Randomized Response Technique (RRT). However, convincing evidence that the RRT provides more valid estimates than direct questioning in online surveys is still lacking. A new variant of the RRT called the Crosswise Model has recently been proposed to overcome some of the deficiencies of existing RRT designs. We therefore conducted an experimental study in which different implementations of the RRT, including two implementations of the crosswise model, were tested and compared to direct questioning. Our study is a large-scale online survey (N = 6,037) on sensitive behaviors by students such as cheating in exams and plagiarism. Results indicate that the crosswise-model RRT---unlike the other variants of RRT we evaluated---yields higher prevalence estimates of sensitive behaviors than direct questioning. Whether higher estimates are a sufficient condition for more valid results, however, remains questionable.

Current role of enzyme analysis for urea cycle disorders

Urea cycle disorders (UCD) are due to defects of any of its six enzymes or two transporters. The definitive diagnosis of defects of the three mitochondrial enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase I (CPS1) and ornithine transcarbamylase (OTC) depends on either molecular mutation analysis or measurement of enzyme activity, whereas the diagnosis of deficiencies of the three cytosolic enzymes argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase I (ARG1) is usually straightforward, based on marker metabolites. Enzyme assays for all UCD have been used since their first description, for disease confirmation and in some instances even for prenatal diagnosis. The genetic bases of the UCD have only been unraveled from the 1980s; the last gene cloned being the NAGS gene in 2002. In this review we discuss the enzymatic assays for all urea cycle enzymes from a historical perspective, their potential and drawbacks, and the current role of enzymatic analysis in UCD in general.

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.