RH) and temperature error magnitudes of at most 2% RH and 0.3 °C respectively, and similar measurement time response, would be an adequate and less expensive substitute for a dewpoint hygrometer. Furthermore, a combination capacitive humidity instrument requires no servicing compared to a dewpoint hygrometer which requires a bias adjustment and mirror cleaning each week. These findings make unattended BREB measurements of sensible heat flux and evaporation cheaper and more reliable with the system easier to assemble and service and with reduced instrument power.The possibility of reliable, reasonably accurate and relatively inexpensive estimates of sensible heat and latent energy fluxes was investigated using a commercial combination thin-film polymer capacitive relative humidity and adjacent temperature sensor instrument. Long-term and unattended water vapour pressure profile difference measurements using low-power combination instruments were compared with those from a cooled dewpoint mirror hygrometer, the latter often used with Bowen ratio energy balance (BREB) systems. An error analysis, based on instrument relative humidity and temperature errors, was applied for various capacitive humidity instrument models. The main disadvantage of a combination capacitive humidity instrument is that two measurements, relative humidity and temperature, are required for estimation of water vapour pressure as opposed to one for a dewpoint hygrometer. In a laboratory experiment using an automated procedure, water vapour pressure differences generated using a reference dewpoint generator were measured using a commercial model (Dew-10) dewpoint hygrometer and a combination capacitive humidity instrument. The laboratory measurement comparisons showed that, potentially, an inexpensive model combination capacitive humidity instrument (CS500 or HMP50), or for improved results a slightly more expensive model (HMP35C or HMP45C), could substitute for the more expensive dewpoint hygrometer. In a field study, in a mesic grassland, the water vapour pressure measurement noise for the combination capacitive humidity instruments was greater than that for the dewpoint hygrometer. The average water vapour pressure profile difference measured using a HMP45C was highly correlated with that from a dewpoint hygrometer with a slope less than unity. Water vapour pressure measurements using the capacitive humidity instruments were not as accurate, compared to those obtained using a dewpoint hygrometer, but the resolution magnitudes for the profile difference measurements were less than the minimum of 0.01 kPa required for BREB measurements when averaged over 20 min. Furthermore, the longer-term capacitive humidity measurements are more reliable and not dependent on a sensor bias adjustment as is the case for the dewpoint hygrometer. A field comparison of CS500 and HMP45C profile water vapour pressure differences yielded a slope of close to unity. However, the CS500 exhibited more variable water vapour pressure measurements mainly due to its increased variation in temperature measurements compared to the HMP45C. Comparisons between 20-min BREB sensible heat fluxes obtained using a HMP45C and a dewpoint hygrometer yielded a slope of almost unity. BREB sensible heat fluxes measured using a HMP45C were reasonably well correlated with those obtained using a surface-layer scintillometer and eddy covariance (slope of 0.9629 and 0.9198 respectively). This reasonable agreement showed that a combination capacitive humidity instrument, with similar relative humidity ( Theoretical aspects of the BREB method in relation to data exclusion have been examined –3. The mCellier and Olioso  concludede facto standard for flux measurements, instrument cost and a greater power requirement may prohibit simultaneous measurements within the same or in different catchments over different canopy types or for simultaneous flux measurements above areas subjected to different management. Furthermore, EC measurements require many corrections [While the EC method is the rections  and therrections  with difrections . The amoBREB automatic height exchange  and fixeBased on previous experiences and personal communications with other researchers, the dewpoint hygrometer of the BREB system used for long-term field measurements may after some time yield unreliable data due to the sensor's inability to maintain a stable bias. Given other practical problems such as servicing and power requirements for dewpoint hygrometer systems employed in distant locations, the objective of this work was to investigate the use of a BREB system, by substituting the dewpoint hygrometer with a combination thin-film polymer capacitive relative humidity and air temperature instrument, for long-term and unattended estimation of sensible heat and latent energy fluxes. An error analysis is presented for two different combination capacitive humidity instrument models, which are compared to a dewpoint hygrometer, in the laboratory and in a mesic grassland, for their ability to measure water vapour pressure profile differences for an extended period. Furthermore, to test the adequacy of the combination capacitive humidity instruments used in the field for flux measurements, comparisons of BREB-measured sensible heat flux against that obtained using the path-weighting surface-layer scintillometer (SLS) method, dependent on Monin-Obukhov Similarity Theory (MOST), and the standard EC method are presented. Such comparisons have not been presented previously in the literature.2.2.1.A brief description of the BREB method for measuring sensible and latent energy flux is presented. The BREB method relies on the K-theory hypothesis for which sensible heat flux, for example, is proportional to an exchange coefficient for sensible heat flux and the profile air temperature gradient.LE (W m−2) is given by:ρ is the density of air (kg m−3), pc the specific heat capacity of air at constant pressure (J kg−1 K−1), γ the psychrometric constant (approximately 0.066 kPa K−1 at sea level), wK the exchange coefficient (m2 s−1) for latent energy flux, and z2 and z1 above the soil surface respectively where Bowen , buildineen used .H is given by:hK is the exchange coefficient for sensible heat flux (m2 s−1) and z2 and z1 above the soil surface respectively where H and LE fluxes are indicative of energy losses at the surface and are negative.Similarly, the sensible heat flux β is defined as:β can be determined by measuring air temperature and water vapour pressure at two levels in the atmosphere. Ignoring photosynthesis, advection and physically and biochemically stored fluxes, the surface energy balance is given by:nR is the net irradiance and S the soil heat flux. Combining wK = hK. The equality of these two exchange coefficients, referred to as the Similarity Principle, allows the BREB method to be used without the need for atmospheric stability adjustments. A limitation of the BREB method occurs when the denominator of et al. [Following Bowen , the rativen by:Rn=−LE−H−S)/(1+β)H=−β where the % unit is in RH units and not a percentage of the measured RH (for RH between 0 and 90%) and temperature accuracy is within 0.2 °C to 20 °C , for the HMP45C is given by:T ≥ 20 °Cfor T ≤ 20 °Cand for T≥ 0 °C is given by:and that for a CS500 for The HMP45C  combinatto 20 °C . For theδT| for the CS500 varies linearly from 0.3 to 0.62 °C respectively compared to 0.3 °C for the HMP45C for the same temperature range but with a minimum for |δT| of 0.2 °C at 20 °C. Therefore, the HMP45C has a more accurate temperature sensor than that used in the CS500.These relationships are depicted in e (kPa), is calculated from the measured capacitive sensor RH and the saturation water vapour pressure se (kPa) calculated from the adjacent temperature sensor using:T (°C) is the sensor-measured airstream temperature. Hence the error magnitude in water vapour pressure, δe (kPa), is estimated from the error in RH, δRH, and error in se, sδe (kPa), using:∂e/∂RH = se/100 and ∂e/s∂e = RH/100 and where s∂e = (s∂e/∂T)·δT = Δ·δT where Δ (kPa °C−1) is the slope of the saturation water vapour pressure vs temperature relationship at temperature T:The water vapour pressure of an airstream, δe for any relative humidity and temperature combination sensor is given by:Finally then, from se and δRH, is usually much greater than the second term except when temperature is low and RH is high. Thus the error δe, mainly governed by δRH, increases with temperature. The error δe is temperature dependent since both se and Δ are temperature dependent and the information provided by the manufacturer shows that δT is also temperature dependent. For the HMP45C, combining T ≥ 20 °C or for T < 20 °CT ≥ 0 °C.In δRH = ±2% (for 0% ≤ RH ≤ 90%) and for the CS500 δRH = ±3% (for 10% ≥ RH ≥ 90%), |δe| was determined as a function of temperature between 5 and 50 °C and RH between 10 and 90 %. The calculations show : δRH = ±2 % and δT = ±0.4 °C . The RH and T errors for the CS215 are similar to those for the Campbell HMP35C combination instrument  except that the CS215 contains a single-chip element that includes both relative humidity and temperature sensors. The Campbell 155A combination instrument is based on a Vaisala model 180 Humicap capacitive humidity sensor and a platinum resistance thermometer. Measurement errors are specified in δe| values for the CS215 are marginally greater than those for the HMP45C  with an altitude of 671.3 m. The long-term annual rainfall for Pietermaritzburg is 928 mm (58 years of data) and the mean maximum and mean minimum air temperatures are 24.8 and 12.3 °C respectively (South African Weather Service). During winter, there was frequent dewfall and occasional frost. Management practices at the research site included mowing  and burning (in August when firebreaks are established and in October).3.3.BREB field measurements were conducted from January to July 2004. Water vapour pressure profile difference comparisons were made using a Dew-10 dewpoint hygrometer and two combination capacitive humidity instruments, the HMP45C and CS500. The minimum fetch distance for the site for the prevailing wind direction was 135 m for the BREB systems. The mixed grassland area to the south of the site was more exposed with a slight slope increase. The adjacent area had occasional trees. To the north of the study site, there was a residential area and occasional tall trees.−1. Air continuously sampled from each height was drawn to the respective mixing bottle housed in the instrument enclosure and then to the chamber containing either the Dew-10 or combination humidity instrument.BREB systems, modified from Campbell 023A systems  and connEach capacitive humidity instrument was sealed in a metal chamber. Unlike the dewpoint hygrometer chamber, the capacitive humidity chambers were heavily insulated using layers of mirror tape to dampen sudden changes in temperature. Tubing in the vicinity of the chamber was covered with adhesive aluminium tape in an attempt to further reduce temperature differences between the humidity and temperature sensors and that of the airstream. The Bev-a-line tubing  used for the water vapour pressure measurements was as short as possible to minimise the transit time of the air from the intake point to the instrument. Furthermore, the filter cover of the combination capacitive humidity instruments was removed to improve the time response to relative humidity. The inlet Teflon filter (1-μm pore size) at each of the two intake positions was however retained for both BREB capacitive humidity systems to prevent entry of liquid water and dirt. The voltages corresponding to the HMP45C relative humidity and air temperature were differential. The corresponding CS500 voltages were single-ended, as recommended by the manufacturer. Every 1 s, the airstream atmospheric water vapour pressure was calculated from the measured relative humidity and temperature. Averages and standard deviations were calculated every 20 min.In the first field experiment, the HMP45C and CS500 instruments were placed in individual humidity chambers. The outlet of the one chamber was connected to the inlet of the other. The inlet of the first chamber was connected to the inlet airstream from the mixing bottles and the outlet of the second bottle was connected to the inlet of the pump. The outlet of the pump was vented to the atmosphere. In this fashion, the two instruments were exposed to the same water vapour pressure.In a second experiment, measurements were made using two chambers connected in-line with the one chamber containing a HMP45C and the second chamber containing a Dew-10 dewpoint hygrometer. Most of the measurements were with the inlet air passing through the HMP45C chamber first and then entering the dewpoint hygrometer chamber and then out to the pump. This arrangement would reduce the influence of the dewpoint hygrometer measurement method on the HMP45C measurements. The dewpoint hygrometer required a weekly bias adjustment for convergence on the correct dewpoint. The capacitive humidity instruments required no attention.The air temperature differences between two levels were measured using a pair of naturally-ventilated and unshielded 75-μm type-E thermocouples. At each level, a parallel combination of 75-μm thermocouples was used. Extra insulation was used to cover the thermocouple connectors at the thermocouple join. Extra precautions were taken by covering and thermally insulating the point at which the thermocouple wires were connected to the datalogger. The thermocouples were inspected for damage, cleanliness, insects and cobwebs during each site visit and replaced or cleaned.H and LE  have been presented [H every 20 min by application of the semi-empirical MOST. A beam distance of 101 m was used. The SLS was positioned at a height of 1.68 metres above the soil surface. The SLS signal processing unit was connected to a computer via the computer serial port. Vegetation height, weighted over the beam path length [The methodology and details for the dual-beam SLS measurements  for calculating sensible heat flux . The sonic anemometer measurements were processed on-line. No coordinate rotations [w and sonicT, for determining H, was calculated using A three-dimensional sonic anemometer  was used as an EC system to measure otations  were pos4.Before the results of the flux measurements for the various field methods could be compared, a study was undertaken to investigate the possible substitution of the dewpoint hygrometer by a combination capacitive humidity instrument.4.1.de) were simulated using a LI610 dewpoint generator. The de values generated were compared against measurements in three separate experiments using a Dew-10 dewpoint hygrometer and HMP35C and CS500 combination capacitive humidity instruments. Judging by the root mean square error (RMSE) for the three measurement comparisons . Presumably, for aerodynamically rougher surfaces such as tree canopies, the magnitude of de and dT would decrease and therefore the separation distance between the upper and lower arms may need to be increased. This increase may require a greater fetch, limiting use of the BREB method if this is not possible.The measured  1.0 kPa . Measurede for the HMP45C and CS500 instruments were also good  for high evaporative demand conditions (vs 0.0009 kPa respectively) for the low evaporative demand conditions  the saturated water vapour corresponding to the sensor temperature sensorT. Therefore, assuming that sensorRH is unchanged, an increase in sensorT results in a non-linear increase in se(sensorT) causing an increase in airstreame. Similarly, a decrease in sensorT results in a decrease in se(sensorT) causing a decrease in airstreame.The variation in HMP45C-measured temperature in the chamber is shown for a 20- and 2-min period  for the The step-like variation in temperature , 7a is dThe water vapour pressure measurements of the CS500 are more variable than those obtained using the Dew-10 dewpoint hygrometer or the HMP45C (data not shown). The reason for this could be that the CS500 temperature sensor is less accurate than that used for the HMP45C . For exaWith the procedures specified and based on the data collected, it would appear that the HMP45C may be used as a substitute for the dewpoint hygrometer. Field measurements of water vapour pressure profile differences, with a resolution magnitude of at least 0.01 kPa, were possible using a capacitive humidity instrument .An advantage of the HMP45C is that it does have a switch control option to turn the instrument on, using datalogger control, just before measurements and then immediately off. The CS500 does not have a built-in switch control option and this results in an increased current drain if the instrument is continuously powered. This disadvantage for the CS500 is obviated when using a CR10X, CR23X, CR800 or CR1000 that can provide for switched 12 V control. However, the HMP50  is cheaper than the HMP45C and the Dew-10 .4.3.H obtained using capacitive humidity instruments and a Dew-10 dewpoint hygrometer is shown (A relatively long-term measurement comparison between is shown . As expeis shown . The comis shown . The HMPH measured using the BREB method using the HMP45C and the SLS (−2 and a slope value of less than 1. Of note is that the BREB estimate of H requires netR and S measurements (H. Besides measurement errors, measurement differences for the different methods are due to the differing theoretical bases for the methods, the spatial separation of the measurement systems, differing measurement heights, differing footprints and that the SLS measurements represent a path-weighted estimate [The comparison between  the SLS  and EC m the SLS  are reasurements  whereas estimate ,34 as weH for the EC and BREB methods, for days ranging from wet and overcast to cloudless conditions, also show reasonable correspondence. For some days (101 and 102 and 104 to 106, H compared to EC whereas on other days (32 and 34, H. During the night-time, usually when dew occurs, fluctuations occur in the data particularly for the BREB method (day of year 31 to 35, Temporal plots of H and LE, it is recommended that for water vapour pressure measurements, either the HMP45C be used, or a combination polymer capacitive instrument with similar or better accuracy for both RH and T. Alternatively, the CS500 or HMP50 could be used but with a more accurate temperature sensor.For BREB calculations of 5.RH and T respectively, yielded less accurate water vapour pressure differences with more measurement noise and an increased RMSE of 34.89 W m−2 in the sensible heat flux comparisons obtained using a dewpoint hygrometer compared to a RMSE of 8.06 W m−2 using the HMP45C. Modifications to the combination capacitive humidity instrument included removal of the instrument membrane filter and providing a more thermally stable environment for the combination instrument chamber and adjacent connecting hoses. This substitution makes the BREB system much cheaper with little servicing required by the capacitive humidity instrument compared to the dewpoint hygrometer used. The combination capacitive humidity instruments show more water vapour pressure noise than the dewpoint hygrometer but the 20-min average of the profile water vapour pressure differences compare well with the corresponding dewpoint hygrometer measurements. Further modifications could include increasing the equilibration time from 40 s to 60 s and using a thermocouple to measure the temperature adjacent the capacitive humidity sensor or using a single-chip relative humidity and temperature capacitive instrument placed in a chamber. Sensible heat flux obtained using a HMP45C instrument compared to either surface-layer scintillometer or eddy covariance estimates yielded a RMSE of about 38 W m−2 and a slope less than 1.Based on the laboratory and field experimental measurements, a combination polymer capacitive humidity instrument such as the HMP45C with a relative humidity magnitude accuracy of 2% or better and a temperature accuracy of at most 0.3 °C, perhaps 0.4 °C, is a reasonable substitute for the cooled mirror dewpoint hygrometer of a BREB system for measuring water vapour pressure profile differences. A less accurate combination capacitive humidity instrument, the CS500 or HMP50, with an accuracy of 3% and 0.4 °C for


Diagnostic and treatment approaches for sarcoidosis have changed dramatically over the past decade. Yet, the most recent reports of trends in hospitalizations of sarcoidosis patients are over ten years old. The objectives of this study were to determine the incidence of sarcoidosis among hospitalized patients and to analyze recent trends and seasonality of hospitalizations in sarcoidosis patients.We performed a retrospective cohort study of the Nationwide Inpatient Sample from 1998 through 2008. We identified all hospitalizations with a primary or secondary diagnosis of sarcoidosis (ICD-9-CM code 135). Incidence was modeled as a seasonal time series about a linear trend.Time series analysis of the monthly number of hospitalizations revealed a distinct positive linear trend. Over the study period, the number of hospitalized patients with sarcoidosis increased from 37,516 to 70,947 cases. Trends were most pronounced in patients older than 55 years (p < 0.0001), African Americans (p < 0.0001), females (p = 0.0289), and non-Medicaid populations (p < 0.0001). Hospitalizations are seasonal with highest incidence in January through March.Hospitalizations among sarcoidosis patients have almost doubled during the past decade, with disproportionate rate increases in African Americans, women, and older patients. The rate also increases among patients with insurance other than Medicaid. This study indicates the need for heightened surveillance of sarcoidosis patients given the unknown consequences of evolving treatment approaches. Our results point to a need for research investigating risk factors for hospitalization, including medications, co-morbidities, demographics, and socioeconomic status. Sarcoidosis is a systemic granulomatous inflammatory disease of unknown etiology which can cause significant morbidity ,2. It isPrior studies focusing on hospitalization trends among patients with sarcoidosis report conflicting results. A study of U.S. Navy personnel showed a steep decline in hospitalizations for sarcoidosis over the time period of 1975 to 2001, with the authors concluding that this was due to improvements in protection from occupational exposures that may have been causing sarcoidosis, or reflecting improvement in a ‘sarcoid-like’ disease from these exposures .Since these studies were published, diagnostic and treatment approaches have changed dramatically. More sensitive imaging technologies to detect and monitor organ involvement are now available, and increased use of immunosuppressive alternatives to prednisone  has altered clinical management over recent years -13. DespThe goal of this study was to determine the incidence of sarcoidosis among hospitalized patients and to analyze the recent trend in hospitalizations among patients with sarcoidosis. We also examined demographics, hospital mortality, length of stay, and seasonal disease patterns.th Revision, Clinical Modification (ICD-9-CM) code 135. We then aggregated all cases of sarcoidosis by month to produce a national sample of cases of sarcoidosis over time. Cases of sarcoidosis were assigned to a calendar month on the basis of the date that the patient was admitted to the hospital. The numbers of sarcoidosis cases were further converted to incidence rates to account for the variation in total overall number of all hospitalized discharges in each month.All data were extracted from the Nationwide Inpatient Sample (NIS), the largest all-payer database of national hospital discharges in the United States. The purpose of the NIS is to specifically identify, track, and analyze national trends in healthcare utilization, access, charges, quality, and outcomes. It is maintained as part of the Healthcare Cost and Utilization Project by the Agency for Healthcare Research and Quality (AHRQ) and contains data from a 20 % stratified sample of nonfederal acute hospitals . This saThe yearly series for hospital mortality rate and average length of stay were also extracted to monitor the characteristics of sarcoidosis patients over time. For each year, the hospital mortality rate is defined as the number of deaths during hospitalization over the total number of sarcoidosis cases in that year.We performed a time series analysis of the sarcoidosis rate over the study period. Specifically, we built a seasonal autoregressive integrated moving average (SARIMA) model, which accounted for the serial correlation and allowed us to test for a potential trend and for seasonality in sarcoidosis incidence. After obtaining a suitable model for the national data, we then applied the same model structure to each sub-series stratified by the dichotomous variables age (< 55 vs. > = 55), race (black vs. non-black), gender , and payer (Medicaid vs. non-Medicaid). We chose 55 years old as an age cut-off in order to capture the second bimodal peak of presentation of sarcoidosis patients in their 60’s that has been previously noted in epidemiological studies . In a siTo determine if there was a trend in hospital mortality rate, we fit a logistic regression model with the year as the independent variable. Generalized estimating equations were used for parameter estimation and an autoregressive correlation structure was incorporated to account for the temporal association in the outcome. To investigate whether there was a trend in average length of stay, we fit an analogous linear regression model, assuming normality for the distribution of the outcome.All statistical analyses were performed using R version 2.11.1  and SAS 9.2 .Over the study period, the estimated number of hospitalized patients with sarcoidosis almost doubled from 37,516 cases in 1998 to 70,947 cases in 2008. Our time series analysis of the sarcoidosis incidence rate revealed a significant upward trend  remained in the final model, but the first-order seasonal moving average (p = 0.3195) and linear time trend p = 0.7259) were no longer significant  and patients with a primary diagnosis of sarcoidosis . A significant decrease in average length of stay was also observed for general sarcoidosis population , but not for patients with a primary diagnosis of sarcoidosis .Our results show a dramatic increase in the number of overall hospitalizations of sarcoidosis patients in the U.S. between 1998 and 2008. We found the highest increase in the rate of hospitalizations among African Americans, females, older patients, and those with insurance other than Medicaid. However, for those admitted with sarcoidosis listed as the primary diagnosis, the incidence was stable during the study period with no significant differences in trends between races, gender, or insurance status. Thus, it appears that this increasing trend is driven by admissions for people with sarcoidosis, rather than because of sarcoidosis.Although sarcoidosis was first described over 100 years ago, the cause is still unknown, treatment is still controversial, and epidemiologic data are difficult to collect due to the rarity of the disease and diverse clinical presentations. Our findings are important as there are few published data on the burden of hospitalizations in this disease in the past ten years. Our results clearly demonstrate that there is an increasing burden of inpatient medical care required by sarcoidosis patients, and that this trend is greater in older patients, women, and African Americans.With respect to race, gender, and age, our results are similar to those published by Foreman et al who reported finding hospitalizations more frequently among females, older patients, and African Americans . SarcoidIn contrast to previous work, however, we found that patients with insurance other than Medicaid actually had a higher increase in the rate of hospitalization compared to Medicaid patients. We speculate that this difference may be due to differential practice patterns or access to care/travel issues between patients with Medicaid and those with alternative insurance. For instance, those with insurance may be receiving more aggressive immunosuppression with corticosteroids, potentially more use of costly immunosuppressive alternatives to prednisone (with increased risky side effects), more diagnostic procedures, or may be able to more easily access care , thereby influencing treatment and monitoring decisions by practitioners. Advancing radiographic imaging technology has also made it easier to find potential sarcoidosis cases and identify more subtle lung findings, which may have in turn led to more aggressive treatment approaches than in times of monitoring only with chest x-rays. Thus, the use of more sensitive imaging in insured populations may contribute to more aggressive immunosuppression.Our hypothesis that the increase in hospitalizations is associated with more aggressive immunosuppressive treatments is further supported by the observation that hospitalizations with a primary sarcoidosis diagnosis are not increasing; increasing admission rates due to primary diagnoses other than sarcoidosis suggest that factors other than sarcoidosis itself are driving this trend. In addition, a recent retrospective study of healthcare utilization showed that sarcoidosis patients with greater corticosteroid use had a greater number of unscheduled emergency department visits . FurtherBecause other studies have suggested a seasonal peak of diagnosis of sarcoidosis cases in the early spring, we investigated the seasonality of hospital admissions among patients with a sarcoidosis diagnosis ,26. GeneThere are several limitations to our study. First, a major limitation is that we use that we use administrative data, and do not have access to laboratory, radiographic or medication data. Second, we cannot confirm our hypothesis that immunosuppressive drugs are driving the change in epidemiology, nor can the coded diagnosis of sarcoidosis be confirmed. We also cannot report the burden of repeated hospitalizations. Due to the sampling design in the AHRQ database, there are no individual identifiers that can link patients across multiple admissions or over multiple years. Third, it is possible that medical advances are sustaining patients for longer, leading to increased number of hospitalizations that are not necessarily related to the severity or treatment of sarcoidosis. Fourth, it is also possible that subgroups with limited access to outpatient care  may be admitted more frequently. Fifth, racial data in large databases may be missing or inaccurate. Last, it is possible that the increase in hospitalizations is due to an increase in the prevalence of sarcoidosis during the study period. However, hospitalizations specifically for a primary diagnosis have remained stable, suggesting that increased incidence of sarcoidosis is not the driving factor for hospitalization. The incidence in populations in the United Kingdom and Australia (both with similar incidence data to the United States) show that the diagnosis of sarcoidosis is not increasing over time ,28.This study highlights the need for heightened surveillance of sarcoidosis patients, as the requirement for inpatient care in this group is increasing. Our observations are important given the unknown implications of evolving diagnostic and treatment approaches over this period of time. Our results also point to a need for research investigating risk factors for hospitalization, including the effects of medications, co-morbidities, demographics, geographic patterns, socioeconomic status, and differing practice patterns.AHRQ: Agency for healthcare research and quality; ACF: Autocorrelation function; ICD-9-CM: International classification of diseases, 9th revision, clinical modification; SARIMA: Seasonal autoregressive integrated moving average; U.S.: United States.The authors declare that they have no financial or non-financial competing interests.AKG, the guarantor of the manuscript, contributed to the design of the study, was responsible for data collection and analyses, and contributed to the writing and revising of the first draft of the manuscript. MY was responsible for data analyses, and contributed to the writing and revising of the manuscript. FT was responsible for data analyses, and contributed to revising the manuscript. JEC contributed to the design of the study, data analyses, and to the writing and revising of the manuscript. PMP contributed to the design of the study, data collection and analyses, and to the writing and revising of the first draft of the manuscript. All authors read and approved the final manuscript.This work was supported by the National Institutes of Health, Grant 1KL2RR024980: Institute for Clinical and Translational Science, University of Iowa.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/12/19/prepub


G93A ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders.Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1 Neurotrophins are potent regulators of survival and function of mammalian nervous system neurons Although TrkB or BDNF are not required for motoneuron survival during development, BDNF/TrkB kinase signaling is required for maintenance of the cholinergic phenotype of adult motor neurons and prevents embryonic and postnatal motoneuron cell death in a variety of experimental paradigms  mice were acquired from The Jackson Laboratory  and crossed to TrkB.T1 knockout mice backcrossed on C57/B16J background for at least 10 generations SOD1th section  was collected for analysis. Motoneurons were visualized by immunofluorescent staining for Choline acetyltransferase  and by Cresyl Violet staining and counted by the stereological modified optical fractionator method multiplying the number of counted neurons per section with the sampling fraction.Mice were anesthetized and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde. The lumbar region of the spinal cord (L1–L5) was removed and 50-µm transverse sections were cut on a cryostat. Every 10Muscle strength and coordination were assessed by an accelerating rota-rod  that rotates from 4 to 40 RPM over 5 min To monitor the progression of the disease we used two different parameters: early progression of disease and end-stage disease in the animals. Starting at 8 weeks of age, animals were weighed three times weekly. Given that animals start losing weight with disease progression, we defined the early progression of disease as the duration between the time of peak weight and the time when animals lose 10% of peak weight due to muscle atrophy. End-stage was defined as the age of actual death of the animal or the age at which paralysis rendered the animal unable to right itself within 20 seconds when placed on its side Animals were scored for disease signs and symptoms at 20 weeks based on the following scale 0 =  normal functions, no sign of disease onset.1 =  distal muscle (paw) weakness of one limb.2 =  distal muscle weakness of two limbs, slight wobble when walking.3 =  weakness of proximal muscle (leg) of one limb plus weakness in two distal muscles, clear wobble when walking.4 =  both hind limbs paralyzed with dragging of hind limbs.5 =  animal is immobile.The adenosine A2A receptor agonist CGS21680 was purchased from Tocris (USA) or custom synthesized from ChemieTek . CGS21680 was dissolved in 5% DMSO prior to use. Starting at 8 weeks of age animals received a single daily intraperitoneal injection of CGS21680 or vehicle (5% DMSO) and were sacrificed at different time points depending on the experiments. The dose of CGS21680 (5mg/kg) was determined on the basis of our pilot study and other studies GraphPad software (InStat and Prism) was used for all statistical analysis. Comparisons of motor neuron numbers and rota-rod performance between the groups at different ages was done by ANOVA followed by post-hoc Tukey-Kramer Multiple comparison test or Dunn test. Survival analysis  was subjected to Kaplan-Meier Statistics. A Log rank P value was calculated to compare the survival curves. Neurological scores were compared by Mann-Whitney test. Data are represented as Mean ± SEM.


The bone and cartilage destruction seen inrheumatoid arthritis (RA) is caused by synovial pannus formation, which is characterized by aberrant proliferation of synovial fibroblasts. Inhibition of synovial proliferation has recently been reported to be a promising therapeutic strategy for RA.However, the specific mechanism underlyingdysregulated proliferation of synovial fibroblasts remains unclear.We aimed toidentify and characterize genesthat are involved in the aberrant proliferation of synovial fibroblasts.Microarray analysiswas performed to identifythe genes that had upregulated expression inmice with collagen-induced arthritis (CIA). The effect of candidate genes on the proliferation of synovial fibroblasts was screened using antisense oligodeoxynucleotides and small interfering RNAs (siRNAs).SPACIA1/SAAL1 (synoviocyte proliferation-associated in CIA 1/serum amyloid A-like 1)that was associated with aberrant proliferation of synovial fibroblasts. Immunohistochemicalanalysis indicated that SPACIA1/SAAL1 was strongly expressed in the foot joints of mice with CIA and in the thickened synovial lining of the human RA synovium. Transfection of siRNA targeting SPACIA1/SAAL1into RA synovial fibroblastscould inhibit tumor necrosis factor (TNF)α-induced proliferation more effectively thanit could inhibit serum-induced proliferation.In addition,the antiproliferative effect of SPACIA1/SAAL1 siRNA was caused byinhibition of cell cycle progression and not by induction of apoptosis.We established transgenic (Tg) mice that overexpressed SPACIA1/SAAL1. These Tg mice did not spontaneously develop arthritis or cancer. However,inducing CIA causedgreatersynovial proliferation and worse diseasein Tg mice thanin wild-type mice.We identified a novel gene named SPACIA1/SAAL1 plays an important role in the aberrant proliferation of synovial fibroblasts under inflammatory conditions.


Tgr from 1600 to 2000°C). For all the considered growth temperatures, few layers of graphene (FLG) conformally covering the 100 to 200-nm wide terraces of the SiC surface have been observed by high-resolution cross-sectional transmission electron microscopy (HR-XTEM). Tapping mode atomic force microscopy (t-AFM) showed the formation of wrinkles with approx. 1 to 2 nm height and 10 to 20 nm width in the FLG film, as a result of the release of the compressive strain, which builds up in FLG during the sample cooling due to the thermal expansion coefficients mismatch between graphene and SiC. While for EG grown on on-axis 4H-SiC an isotropic mesh-like network of wrinkles interconnected into nodes is commonly reported, in the present case of a vicinal SiC surface, wrinkles are preferentially oriented in the direction perpendicular to the step edges of the SiC terraces. For each Tgr, the number of graphene layers was determined on very small sample areas by HR-XTEM and, with high statistics and on several sample positions, by measuring the depth of selectively etched trenches in FLG by t-AFM. Both the density of wrinkles and the number of graphene layers are found to increase almost linearly as a function of the growth temperature in the considered temperature range.In this work, we present a nanometer resolution structural characterization of epitaxial graphene (EG) layers grown on 4H-SiC (0001) 8° off-axis, by annealing in inert gas ambient (Ar) in a wide temperature range ( Graphene has attracted the interest of the scientific community due to its excellent electronic transport properties ,2, whichTgr, ramp rate, pressure in the chamber) and on the initial surface conditions of SiC wafers, e.g., the termination (Si- or C-face), the miscut angle, the defectivity. It has been shown that the rate of Si evaporation can be strongly reduced by performing the annealing in inert gas ambient at atmospheric pressure instead than in vacuum [Tgr ~1300°C in vacuum to Tgr > 1600°C at atmospheric pressure [The latter method is the strongest candidate for graphene based electronics, owing to the fact that few layers of graphene (FLG) can be grown on a semiconductor substrate without any need to transfer . Graphene such produced is known as epitaxial graphene (EG). Graphene growth during annealing of SiC relies on the interplay of two different mechanisms: (i) the preferential Si sublimation from SiC surface, which leaves an excess of C atoms in the near surface region, and (ii) the diffusion of these C atoms on the SiC surface and their reorganization in the two-dimensional graphene lattice structure. Both mechanisms depend in a complex way both on the annealing conditions . The modifications in the surface morphology after thermal treatments at the different temperatures were studied by tapping mode atomic force microscopy (t-AFM), showing the increase in the density of peculiar corrugations (wrinkles) in the EG multilayers with the growth temperature. High-resolution cross-sectional transmission electron microscopy (HRXTEM) was used to determine the number of graphene layers on very small sample areas. An alternative method to estimate the EG thickness on larger areas, based on etching out trenches selectively in graphitized SiC and measurements of the depth of those trenches by AFM, is also presented. The accuracy of this method is discussed.In this work, we present a structural characterization of EG growth on the Si face of 8° off-axis 4H-SiC  by anneaTgr from 1600 to 1700°C, or in an inductively heated reactor at Tgr = 2000°C. The substrates used for graphene growth were lowly doped (approx. 1014 cm-3) epitaxially grown SiC layers on top of highly doped, 8° off-axis 4H-SiC (0001).Graphene growth was carried out in argon (Ar) ambient in a commercial furnace by Centrotherm Thermal Solutions GmbH + Co. KG, Blaubeuren, Germany at temperatures k = 20 to 80 N/m, oscillation frequency 332 to 375 kHz in high-amplitude mode were employed.t-AFM measurements were carried out with Veeco DI3100 atomic force microscope fitted with Nanoscope V controller. Commercial silicon probes with spring constant Cross-sectional observations of the few layers of graphene grown on SiC were carried out using a JEOL JEM 2010F transmission electron microscope with a Schottky field emission gun operating at an acceleration voltage of 200 kV. Thin specimens for TEM observation were prepared by Ar-ion thinning by the following procedure: the graphene-on-SiC samples were mechanically ground to 70 μm thick, and the center of the sample was thinned by dimple grinding, followed by Ar-ion beam thinning under a low angle of 8° at 5 kV.2 plasma (25 sccm) treatment for 16 min in an Roth & Rau Microsys 400 ICP-RIE (inductively coupled plasma) equipment. The step height between etched and not etched regions was determined by t-AFM. Before each t-AFM map, we acquired amplitude-distance curves both on not-etched and etched regions and the amplitude setpoint as well as the free amplitude were chosen accordingly, in order to operate in the repulsive interaction regime [HRXTEM images allow an extremely precise determination of the number of grown layers, but on very small areas. An alternative method to determine the thickness of the grown FLG film on larger areas has been also developed. Stripes were patterned onto graphitized SiC substrates by optical lithography. Graphene was then selectively removed from these stripes with an On regime  on both The etch rates of graphene and bare SiC were calibrated independently. To this aim, graphene flakes were exfoliated from HOPG on SiC and their thickness was measured after different etching times by AFM. A patterned virgin SiC sample was etched under identical conditions and the etched depth was also measured by AFM. For the used plasma conditions, the etch rate of graphene is much higher than that of bare SiC.A morphological AFM image of the virgin 4H-SiC (0001) surface is reported in Figure A root mean square (RMS) roughness of 0.2 nm is obtained from this surface analysis.The morphological transformation of SiC surface after annealing is described in the following. AFM images of the surface morphology for the samples annealed at 1600, 1700, and 2000°C are reported in Figure From the linescan in the direction parallel to the steps (d), it can be observed that the features in the morphology map are some peculiar corrugations with 1 to 2 nm typical height. These features appear on the phase linescan as a peak surrounded by two valleys. It is worth noting that these peculiar corrugations run preferentially in direction orthogonal to the step edges of the substrate.In order to get further insight on the structural properties of the graphene films grown at the different temperature and to clarify the nature of these peculiar corrugations, HRXTEM analyses were carried out.As an example, in Figure From Figure These peculiar defects have been observed also by other authors in the case of few layers of graphene grown on the C face  or on th2) as a function of Tgr, as obtained by a statistical analysis of a large number of AFM images collected each sample, is reported in Figure Comparing the morphology and phase images in Figure 2 plasma. This plasma treatment is known to remove efficiently carbonaceous species through a chemical reaction leading to the formation of CO2. In Figure t0 ~2 nm of SiC is etched during the plasma treatment, due to the physical action of the plasma. This depth must be subtracted while evaluating the number of layers on graphitized SiC. Hence the number of layers can be estimated according to the relation n = (D - D0)/Dgr, being Dgr the interlayer separation between two stacked graphene layers (Dgr ≈ 0.35 nm). The average number of grown layers as a function of Tgr is reported in Figure In the following, we will study the increase in the average number of graphene layers as a function of the growth temperature. HRXTEM images on the samples annealed at 1600, 1700 and 2000°C are reported in Figure Few-layers of graphene grown on 4H-SiC (0001) 8° off-axis, by annealing in inert gas ambient (Ar) in a wide temperature range (from 1600 to 2000°C) have been structurally characterized by atomic force microscopy and HR-XTEM. For all the considered growth temperatures, FLG conformally covering the 100 to 200-nm wide terraces of the SiC surface have been observed. The formation of wrinkles (approx. 1 to 2 nm high and 10 to 20 nm wide) preferentially oriented in the direction perpendicular to the step edges of the SiC terraces has been observed in the FLG film, as a result of the release of the compressive strain accumulated in FLG during the sample cooling due to the thermal expansion coefficients mismatch between graphene and SiC. This parallel orientation of wrinkles is peculiar of EG grown a vicinal SiC surface, whereas for EG grown on on-axis 4H-SiC an isotropic mesh-like network of wrinkles interconnected into nodes is commonly reported. The observed phenomenon deserves further investigations. Both the density of wrinkles and the number of graphene layers are found to increase almost linearly as a function of the growth temperature in the considered temperature range.CVD: chemical vapor deposition; EG: epitaxial graphene; FLG: few layers of graphene; HR-TEM: high-resolution cross-sectional transmission electron microscopy; HOPG: highly oriented pyrolithic graphite; RMS: root mean square; t-AFM: tapping mode atomic force microscopyThe authors declare that they have no competing interests.FG and VR conceived the study. FG coordinated the experiment and participated to the analysis of the data. FG, MR and RY worked on graphene growth. CV and SS carried out the t-AFM measurements and the analysis of the data. CV prepared the sample cross-sections for HR-TEM analyses and CB carried out the HR-TEM measurements. FG and CV wrote the article. All the authors read and approved the manuscript.


We conclude that prolonged hypoxia down-regulates BDNF-TrkB signaling leading to an increase in TNF-α in the cortex, while DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α levels in the hypoxic cortex.We investigated whether δ-opioid receptor (DOR)-induced neuroprotection involves the brain-derived neurotrophic factor (BDNF) pathway. We studied the effect of DOR activation on the expression of BDNF and other proteins in the cortex of C57BL/6 mice exposed to hypoxia (10% of oxygen) for 1–10 days. The results showed that: (1) 1-day hypoxia had no appreciable effect on BDNF expression, while 3- and 10-day hypoxia progressively decreased BDNF expression, resulting in 37.3% reduction ( Evidenctrindole .Based on the facts from above mentioned studies, we speculate that the mechanism of DOR neuroprotection against hypoxic injury could involve regulation of BDNF-TrkB signaling pathway and its interaction with the regulation of TNF-α. As an initial step towards testing this possibility, we have performed this investigation to determine if DOR activation affects the expression of BDNF, TrkB and TNF-α in the brain following hypoxic exposure for varying durations.p > 0.05) (p < 0.05) (p > 0.05) (p > 0.05) (We first examined the expression of BDNF and DOR in mouse brain. As shown in  > 0.05) , and fur < 0.05) . In cont > 0.05) , suggest > 0.05) . The DOR > 0.05)  did not p < 0.05) in the hippocampus after 1-day hypoxic exposure as shown in p > 0.05) observed after 3 and 10 days of hypoxic exposure   .vs. 100% of the control level, p > 0.05)  , suggestSimilarly as in the cortex, the DOR expression  showed np > 0.05) after 1, 3 and 10 days of hypoxic exposure   . The DOR > 0.05) .p < 0.05)) (p > 0.05) suggesting that UFP-512 attenuated the hypoxia-induced reduction of 140 KDa TrkB expression after 3 days of hypoxic exposure. Although UFP-512 did not significantly affect the hypoxia-induced reduction in 140 kDa TrkB expression after 10 days of hypoxic exposure, the expression of 140 kDa TrkB in UFP-512  was higher compared to that without hypoxic exposure  (We examined the expression of both full-length and truncated TrkB receptors. As shown in < 0.05)) . In cont < 0.05) .p < 0.05) (p > 0.05) suggesting that UFP-512 attenuated the hypoxia-induced truncation of 140 kDa TrkB.The production of truncated isoform of TrkB (90 kDa) receptors is induced in response to an injury . Our res < 0.05) . In contp > 0.05) (p > 0.05), and was significantly upregulated after 10 days of hypoxic exposure  (p < 0.05)  , but the < 0.05) . In cont < 0.05)  suggestip > 0.05), but gradually decreased after 3 days , and 10 days of hypoxic exposure  (p < 0.05)  . In cont < 0.05)  suggestip > 0.05; pCREB/CREB: 106.3% ± 3.7%, 99.1% ± 2.8% and 97.7% ± 2.8% of the control level respectively, p > 0.05). DOR activation also had no appreciable effect on the expression of these proteins (p > 0.05)  .We report three major observations in the cortex exposed to hypoxia: (1) Hypoxia-induced damage is dependent on hypoxic duration, since prolonged (3- and 10-day) hypoxia caused a significant reduction in BDNF expression in the cortex when compared to short-term (1 day) hypoxia, and this reduction was partially reversed following DOR activation; (2) DOR activation partially reversed the hypoxia-induced reduction in functional TrkB (140-kDa) expression and attenuated the hypoxic increase in truncated TrkB (90-kDa) in the cortex exposed to 3- or 10-day hypoxia; (3) prolonged hypoxia (10 days) significantly upregulates the expression of TNF-α in the cortex but down-regulates the CD11b expression that was completely reversed following DOR activation. In addition, we observed that the levels of pCREB and pATF-1 were not significantly altered in the hypoxia-exposed cortex. These results support our hypothesis that the DOR-induced neuroprotection is mediated by an underlying BDNF-TrkB signaling pathway. Prolonged hypoxia causes a down-regulation of BDNF-TrkB signals leading to an increase in TNF-α in the cortex, while DOR activation upregulates BDNF-TrkB signals thereby decreasing the level of TNF-α in the hypoxic cortex.This is the first study that demonstrates partial reversal of hypoxia-induced reduction of BDNF and functional TrkB expression as well as hypoxia-induced increase in truncated TrkB (90-kDa) and TNF-α expression in the cortex following DOR activation without any change in DOR density. DOR expression plays an important role in protection against cerebral hypoxia. Our previous studies have demonstrated that cortical DOR activation is protective against hypoxia and glutamate excitotoxicity in neurons ,24. DOR in vitro and in vivo studies. A blockade of endogenous BDNF activity exacerbates the effects of cerebral ischemia [BDNF is an important neuroprotective factor that has been proven to increase neuronal tolerance against hypoxia/ischemia in both ischemia . Furtherischemia . BDNF biischemia . TrkB haischemia . The proischemia . Our stuBrain injury is influenced by multiple factors, including stress duration and neuronal age ,28. The In a sharp contrast, the subcortical regions and cerebellum did not show any significant alteration in BDNF expression under hypoxic exposure for various periods . This is partially because the subcortical and cerebellar regions may be relatively less sensitive to hypoxia and their BDNF can be compensated by transportation along cortical, nigral and brainstem neurons during hypoxia –32. In fAlthough neurons are the chief source of BDNF, it may also come from the microglia. Microglia are immune cells present in the central nervous system and activated in response to injury and diseases and are known to produce neurotrophic factors such as BDNF, glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which provide trophic support to neurons during injury . PioneerHowever, activated microglia exerts both beneficial and deleterious effects on neurons. For example, activated microglia following cerebral ischemia express a variety of proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which induce neuroinflammation and neurotoxicity . The avaThe BDNF gene is one of the targets of CREB pathway . The DORTo summarize, we propose a potential neuroprotective mechnism involving interactions among DOR, BDNF-TrkB and TNF-α that is schematically illustrated in ad libitum. All animal procedures were approved by the University of Texas medical school at Houston Animal Care and Use Committee  and conformed to the National Institutes of Health Guide for the Care and Use of Animals in Research.Male C57BL/6 mice  at the age of 30-day old (P30) were maintained under a 12 h light/12 h dark cycle and 22 °C with standard rodent chow n = 15); (2) Hypoxia with saline (n = 15); (3) DOR activation with UFP-512 treatment under normoxic conditions (n = 15); and (4) DOR activation with UFP-512 treatment under hypoxic conditions (n = 15). Saline or UFP-512 (1 mg/kg) was daily administrated by intraperitoneal injection. In the hypoxic mice, the first injection was administered at 30-min prior to hypoxia induction. The numbers of the animals used for 3 time points  were 4, 5, and 6 respectively in each group under 4 experimental conditions .In total, 60 mice were randomly divided into 4 groups: (1) Normoxia control with saline (2 levels and humidity in the chamber were kept constant as the ambient levels. O2 levels in the chamber were strictly kept at (10 ± 0.5)% by constantly flushing with nitrogen that was automatically controlled by a ProOx P110 Oxygen Controller with E702 Oxygen Sensor . The ProOx O2 controller has an extremely wide range from 0.1% O2 all the way up to 99.9% O2 with the variation of 0.1%, which provides a very important tool for our research in terms of accurate control of O2 level. The mice were exposed to hypoxia for 1, 3 or 10 days. For both the saline control and UFP-512 control groups, mice were treated in the similar fashion as the hypoxic exposure mice, but under normoxia (21% of O2 level) instead of hypoxia.To expose mice to hypoxic stress, 2 cages of mice (2 or 3/cage) were placed in a plexiglass chamber (30″W x 20″D x 20″H) . The chamber was connected with the outside environment via holes in the wall of the chamber; therefore, COThe chamber used was custom designed, based on the experimental demands and was fitted with two eight-inch iris ports to access the animals during a prolonged hypoxic exposure in order to keep exposure changes to a minimum. Our daily saline or UFP-512 administration was carried out in the hypoxic chamber by accessing the animals with hands stretching through the iris ports in the front wall of the chamber. Therefore, all the procedures were uniform and strictly controlled for hypoxic exposure.UFP-512 (H-Dmt-Tic-NH-CH(CH2–COOH)-Bid), a specific and potent DOR agonist –46, was 133) were obtained from Millpore . Rabbit monoclonal anti-CREB and TrkB was obtained from Cell Signaling Technology . Mouse monoclonal anti-TNF alpha and rabbit polyclonal anti-CD11b was obtained from Abcam Inc . Rabbit polyclonal anti-BDNF was purchased from Alomone Labs Ltd (Cat: ANT-010). Mouse monoclonal anti-β-actin and RIPA buffer were obtained from Sigma-Aldrich . Horseradish peroxidase conjugated goat anti-rabbit/mouse IgG (H + L) were purchased from Invitrogen . Laemmli sample buffer, Precision Plus Protein™ Dual Color Standards and mini-protean precast gels were obtained from Bio-rad .Rabbit polyclonal anti-DOR and anti-phospho-CREB . The supernatants of tissue homogenates  of the cortex, subcortex, cerebellum and hippocampus from each mouse were boiled at 100 °C in laemmli sample buffer (Bio-Rad) for 5 min. After, the samples were electrophoresed on 4%–15% mini-protean precast gels (Bio-Rad), and transferred to 0.20 μm nitrocellulose membrane (Whatman). Membranes were blocked with 5% (m/v) nonfat dry milk in 0.1% Tween 20  for 1 h at room temperature and subsequently incubated overnight at 4 °C in the blocked buffer with the p-CREB (1:1000), CREB(1:2000), CD11b(1:800), TrkB (1:1000), TNF alpha (1:1000) and BDNF (1:200) antibodies, respectively. The membranes were washed with 0.1% Tween 20, and then treated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG  for 1 h at room temperature. Peroxidase activity was visualized with a super signal West Pico Chemiluminescent Substrate . Stripping filters and reprobing for β-actin were carried out for normalization. Controls for nonspecific binding were determined by omission of the primary antibody. The proteins were quantified by measuring optical densities of immunostained bands using an image analysis system .Mice were sacrificed at day 1, 3 or 10, and the brain was removed quickly. The cortical, subcortical , cerebellar and hippocampal tissues were dissected on ice, frozen immediately on dry ice, and then stored at −80 °C until use. The tissues were ultrasonically homogenized in a RIPA buffer and protease inhibitor cocktail, and the homogenates were centrifuged at 12,000× p values < 0.05.Two independent and blinded investigators examined the end point assessments. Data was expressed as mean ± SEM. One-way ANOVA with the post hoc test of Least Significant Difference (LSD) was used to analyze the difference between various groups. Statistical significance was determined based on versus the downregulation of the pro-inflammatory cytokine, TNF-α, suggests their role in the mechanism of DOR-mediated neuroprotection against hypoxia. Targeting the DOR-BDNF-TrkB pathway provides a potential therapy for neuroprotection against hypoxic/ischemia encephalonpathy. However, further studies are needed to ascertain the mechanisms of DOR-mediated regulation of BDNF-TrkB and TNF-α under hypoxic conditions.Our data suggest that the cortical neurons and microglia lose their ability to maintain normal levels of BDNF and functional TrkB after a prolonged exposure to hypoxia. Therefore, both BDNF and functional TrkB protein levels decrease, along with a significant increase in the level of TNF-α expression following a severe hypoxic injury. DOR activation can partially reverse the hypoxia-induced decrease in the BDNF and functional TrkB levels and protect the brain from hypoxic/ishcmic injuury. The upregulation of BDNF along with its TrkB receptor and CD11b


Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.Long interspersed (L1) and  Alu elements accounting for more than one fourth of the human genome, are still actively amplifying. It is widely believed that retroelements insert randomly in the genome. Retroelements newly inserted in the germ line may cause genetic disease, if a functional gene is disrupted. Up to now, only ∼65 well-characterized pathogenic retroelement insertions in 31 different human genes have been reported. Therefore, retrotransposition is suspected to be underdiagnosed as disease-causing mutation mechanism. Reporting 18 novel insertions in the NF1 gene, all identified by a comprehensive RNA–based mutation analysis protocol, we show that L1 and Alu insertions represent 0.4% of all NF1 mutations. Strikingly, we found three integration sites within this 280-kb gene that were used twice independently to insert a retroelement. One of these sites was located in a 1.5-kb “hotspot” region where four additional integration sites clustered. These findings, together with three additional integration sites used multiple times independently to insert retroelements in other genes, indicate that some genomic sites may be especially prone to host newly retrotransposed elements. As some of these sites are embedded in “hotspot” regions, larger flanking sequences may play a role in making these sites particularly vulnerable.Repetitive retrotransposable elements, including LINE1 and  Alu sequences belonging to the family of short interspersed nuclear elements (SINEs) still actively amplify in the human genome, by a process called retrotransposition. L1 elements comprise ∼17% of the human genome sequence Alu elements comprising more than 10% of the human genome can retrotranspose in a non-autonomous process, using proteins encoded by L1 elements to mediate their mobility Alu elements are named master or source Alu elements Alu elements. Both elements are transcribed from an internal promoter Alu transcripts may be encoded directly from the genomic site of transcription Alu transcripts are then terminated at the 3′ end with a short run of U's 3′-AA/TTTT-5′ (and derivates thereof) Alu elements are reverse transcribed by the same process called “target primed reverse transcription” (TPRT), but they need to “borrow” the factors for TPRT from L1 elements Alu element including the poly(A) tail flanked by short direct repeats of the duplicated target site. For a detailed description of the mechanisms of autonomous and non-autonomous retrotransposition see Long interspersed nuclear elements (LINE-1 or L1 elements) and Alu elements dimorphic with respect to presence/absence at a given site contribute significantly to structural variation of the human genome. Furthermore, these studies show that current activity of mobile elements is likely to be higher than previously appreciated.Although retrotransposable elements have no immediate function in the cell, their motility can be important for genome plasticity and the creation of genetic variation Alu elements are believed to insert randomly into the human genome. Hence, de novo transposition of an element may occasionally cause an inherited disease when the element is inserted into (or at proximity of) the coding region of a functional gene de novo L1 EN-mediated insertions of retrotransposable elements causing genetic diseases have been reported Alu, 15 L1, four SINE/VNTR/Alu (SVA) composite element insertions as well as three simple poly(A) insertions de novo insertions were found in 31 different genes including one insertion in the NF1 gene NF1 gene: one poly(T), three L1 and 14 Alu insertions were all identified using an RNA-based core assay as the starting point for comprehensive NF1-gene analysis illustrating the strength of this approach in identifying also this complex type of mutations. Of note, six of the integration sites were located within a relatively small genomic region of 1500 bp between NF1 exons 21 (16) and 23 (18) . Even more striking, three of the integration sites within the NF1 gene were used twice. Our results indicate and confirm that some genomic locations may be especially prone to L1 EN mediated retrotransposition.L1 and NF1 mutation analysis in the two centers (UAB and MUI) uncovered heterozygous (∼50% of the transcripts affected) splicing alterations in 18 NF1 index patients (Alu elements inserted within the exonic sequences. In addition to the approximately 280-bp long Alu elements a 60–120-bp long poly(A) tail -or a poly(T) tail- was inserted depending on whether the Alu element was inserted in the sense or antisense orientation with respect to the NF1 reading frame . We reasoned that the substantial increase in size of the mutant exons containing the Alu/L1 insertions, compared to the wild type exons caused allelic drop-out under standard PCR conditions. We increased the extension time of the PCR reactions and/or increased the size of the PCR amplicons to enhance the amplification of the larger mutant alleles and, hence, facilitate the detection of possible de novo Alu insertions. Amplification of all exons with so far unexplained splicing alterations using PCR conditions optimized to amplify larger PCR products , 23 (18) and 9 (7) did not allow resolution of the cause of their mis-splicing in the patients UAB-R01429, UAB-R316001 and UAB-R91409, respectively. Use of a Taq polymerase enabling amplification of extremely long PCR products (up to >6 kb) was needed to amplify the mutant exons which had 10- and 17-times increased in size compared to their wild type normal allele in patients UAB-R316001 and UAB-R01429, respectively. A 6-kb full-length L1 element inserted in sense orientation into exon 39 (30) was identified in patient UAB-R01429 . Full seAluY element into intron 10 (8) cannot be determined due to a 71-bp deletion associated with this insertion that causes also lack of a TSD. Nevertheless, the most likely insertion site matching a L1 EN cleavage site is tentatively given in AluY and AluYb8 element inserted into the splice acceptor site of intron 14 (10c). The same L1 EN cleavage site was used once at the anti-sense strand to insert the AluY element in sense orientation with respect to the coding sequence of the NF1 gene (integration site according to HGVS nomenclature c.1642-1_1642) and in an unrelated patient once at the sense strand to insert the AluYb8 element in anti-sense orientation (integration site according HGVS nomenclature c.1642-12_1642-11). Furthermore, we found in two non-related patients . Both Alu elements were inserted in sense orientation and flanked by the same sized TSD . The length of TSDs flanking the two insertions differed between both patients (de novo insertions were distributed over the entire NF1 gene from exon 6 (4c) to intron 48 (39).Alignment of the sequences at the integration sites  shows thized TSD . Finallyized TSD  -one of patients  confirmiAlu elements , two insertions at different integration sites in exon 22 (17) and one insertion in exon 6 (4c) , 30 nucleotides downstream of the intron-exon 21 (16) border, resulted in the use of a cryptic 3′ splice site downstream of the integration site and loss of the first 229 bp of the exon 21 (16) (mutation UAB-R39428 in AluYa5 element into exon 47 (38) resulted in at least two different splicing effects, i.e. skipping of the exon and use of an exonic cryptic 5′ splice site upstream of the integration site  also resulted in a more complex splicing defect. This element lacks the first 39 bp at its 5′ end , leading to transcripts containing the first 61 nucleotides of this Alu element but lacking the 3′ half of exon 12 (10a), downstream of the Alu integration site (type 5 effect in Four out of 14 Insertion of the 5′-truncated L1 element into exon 23 (18) also caused simple exon skipping in the transcripts of the patient UAB-R316001 (type 2 splicing effect in Alu and L1 elements as well as the insertion of a poly(T) element lead to altered splicing of the transcripts from the mutant allele. None of the inserted Alu or L1 elements was fully included into the transcripts.Taken together, all NF1 gene. This is the largest number of disease causing mutations of this type found in a single human gene. Sixteen of these insertions were found in a cohort of 4300 unrelated NF1 mutation-positive patients analyzed at UAB and two in a cohort of 200 unrelated NF1 mutation-positive patients analyzed at MUI. Taken together, this suggests that retrotransposon insertions account for ∼0.4% (18/4500) of NF1 mutations identified. This frequency is two to four times higher than previously anticipated Here we report the identification of 18 pathogenic L1 EN-mediated insertions into the Alu, L1 and poly(T) stretch insertions, it was necessary in most of the cases to modify the PCR conditions to prevent allelic drop-out of the substantially larger mutant allele. Hence, most of these mutant alleles would be missed by an exon by exon-sequencing approach. Similar findings were reported for the BRCA1 and BRCA2 genes Firstly, mutations due to retrotransposition have long been thought to be underestimated, since they may be missed by mutation detection methods relying on PCR amplification of small amplicons, i.e. exons, from genomic DNA NF1 gene is particularly susceptible to retrotransposon integration. It is striking that three specific sites within the NF1 exons 21 (16) and 33 (25) and the splice acceptor site of intron 14 (10c) hosted each two independently retrotransposed elements. Moreover, one of these sites is located in a small region of approximately 1.5 kb within the ∼280-kb NF1 gene where six of the 18 insertions cluster. Together, these finding strongly support the notion of an non-random de novo insertion of retrotransposons in the human genome F8 and F9 mutated in hemophilia B, may have hotspot regions for retrotransposon integration APC, F9 and BTK, have been reported to be used twice independently for L1 EN-mediated retrotransposon insertions NF1 gene, a tenth (6/66) of the well characterized sites that harbor disease causing retrotransposon insertions have been used multiple times with half of them identified in the current study. It remains to be elucidated which features make these sites particularly vulnerable to L1 EN-mediated insertions. The fact that some of these sites are embedded in a larger sequence context that appears to be a hotspot region for insertions may indicate that flanking sequences and possibly also the chromatin structure in these regions may play a role.Secondly, it is possible that the Alu and L1 elements have a similar retrotransposition efficiency via-à-vis the molecular retrotransposition machinery NF1 gene also in previously published disease causing retrotranspositional insertions a preponderance of Alu over L1 insertions can be observed. As a possible explanation Dewannieux et al. trans mobilization of Alu elements even if they are unable to promote their own transposition. In other words, there appear to be more potential “drivers” for Alu retrotransposition than there are for L1 retrotransposition Alu over L1 insertions It has been suggested that Alu, L1 and simple poly(T) insertions. Hence, our data confirm and extend several observations deduced from 48 previously analyzed pathological insertions NF1 coding sequence. All Alu insertions belong to the evolutionary youngest “Y” Alu family  sequence inserted into the NF1 exon 25 (19b) may represent a severely truncated Group III Alu insertion AluY element inserted into intron 10 (8) was accompanied by a 71-bp deletion and lacked a TSD. Different possible mechanisms for the loss of genomic sequences in association with EN-dependent retroelement insertions have been discussed AluY insertion into NF1 intron 10 (8). In both models the first-strand L1 EN-mediated nick would have occurred at position c.1186-85_1186-86 in intron 10 (8)  element F8 gene in a hemophilia A patient NF1 gene switched and continued from the anti-sense strand. The presence of a 2-bp micro-homology that may have promoted strand switching due to secondary binding between the RNA-template and the 3′-end of the 5′-overhang at the antisense strand supports the model of twin priming According to Kojima Alu insertions identified here directly affect splice sites. Nevertheless, all L1 EN-mediated insertions in this study affected NF1 splicing in the patients. The predominant effect of the 13 exonic Alu, L1 and poly(T) insertions was exon skipping (6/13 cases) or use of a cryptic splice site -either a cryptic 5′ splice site upstream (3/13 cases) or a cryptic 3′ splice site downstream of the integration site (1/13 cases). In one of 13 cases both of these splice effects were observed in the same patient. In only two cases part of the Alu or L1 element were contained in the mRNA transcripts, inserted into exon 12 (10a) and 39 (30) respectively. Overall, we do not believe that ascertainment bias can explain why exon skipping and/or use of cryptic splice site are the main effects of the de novo Alu and L1 insertions in exonic sequences. Firstly, because we have no evidence that the long-range RT-PCR reactions used in the applied assays would miss Alu insertions in exons or introns that lead to full or partial exonisation of the inserted sequences in the mRNA transcripts. On the contrary, duplications of single or multiple exons which lead to a similar size increase of the RT-PCR products were readily detected by our assays Alu and L1 insertion is weakening of the exon definition resulting in altered splicing of the affected exon. Currently it is not fully evaluated by which mechanisms the inserted sequences reduce exon recognition by the splicing machinery. It has been proposed that inserted sequences may disrupt specific cis-acting exonic splice elements, such as exonic splicing enhancers (ESEs)  weakens exon definition simply by increasing the size of the affected exon and/or by disrupting the exonic structure of cis acting elements in a more general sense, e.g. by increasing the distance of exonic cis regulatory elements and the splice sites of the exon. In this respect it is of note that the use of cryptic 5′- and 3′- splice sites in exons 21 (16) and 25 (19b) is also observed in transcripts from mutant NF1 alleles that carry single nucleotide alterations destroying the respective natural 5′- and 3′- splice sites of these exons  caused a complex splicing effect, i.e. insertion of a cryptic exon embedded in the inserted L1 element and skipping of the preceding exon 9 (7). A similar splicing defect was described in a chronic granulomatous disease patient who carried a truncated L1 element in intron 5 of the Alu insertions. In addition, the RNA-based comprehensive NF1 mutation detection approaches unambiguously identify and functionally characterize other classes of mutations usually missed by DNA-based mutation detection strategies, such as intronic alterations outside the canonical splice site dinucleotides (GT-AG) (including deep intronic mutations), as well as silent and missense mutations with an effect on splicing De novo retrotransposon insertions within the 3′- or 5′-UTR can lead to reduced expression by the disruption of gene regulatory elements NF1 mutation analysis assay.Our results clearly show that RNA-based mutation analysis strategies have the potential to detect disease-causing L1 and NF1 testing to two centers, i.e. the Medical Genomics Laboratory at the University of Alabama in Birmingham (UAB) and the Division of Human Genetics, Medical University Innsbruck (MUI). Informed consent was obtained from all patients. This study was approved by the ethical committee from both institutions.All mutations reported were uncovered in samples from unrelated index patients sent for clinical NF1 mutation analysis is a direct cDNA sequencing approach that is based on the amplification of the entire NF1 coding region in three  or five (Innsbruck) overlapping RT-PCR fragments and subsequent sequencing of the entire PCR products with 18 (20) internal primers. Details on the RT-PCR reactions and primers can be found in In both laboratories the primary assay for comprehensive NF1 nucleotide numbering is based on GenBank reference sequence NM_000267.3 with the A of the ATG start codon being nucleotide position c.1. The integration sites are given according to the HGVS nomenclature after the first duplicated sequence regardless of whether the first nick occurred at the sense or the anti-sense strand and the thereof resulting orientation of the insertion with respect to the NF1 coding sequence. Exons are numbered according to the reference sequence with the widely known legacy numbering given in parenthesis.Alu/L1 insertion within or in close vicinity of the exon  or Taq DNA Polymerase . To amplify the mutant alleles containing a truncated 1753-bp and a full-length 6-kb L1 element, respectively, Expand Long PCR Taq  was used. To determine the precise sequence of the inserted Alu element as well as the duplicated sequence at the insertion site the PCR products showing a band of increased size were cloned according to the manufacturer's instructions into the TOPO-TA cloning vector pCR 4-TOPO  and individually sequenced. Alternatively or in addition, primers were designed to specifically amplify only the mutant alleles containing the Alu insertions. These primers contained at their 5′-end the exonic sequences immediately upstream/downstream of the insertion site and at the 3′-end a few nucleotides of either the Alu or the poly(A/T) stretch. All primers are listed in Alu sequences with the consensus sequences of the different Alu families  with the current SALSA-MLPA-kit P081-B1 and/or P082-B1 . When MLPA results were negative for a genomic deletion of the respective exon and no alteration was identified explaining the observed splicing defect, sequences were meticulously reanalyzed to uncover a possible exon see . To dimiFigure S1AluYa5 insertion in exon 47 (38). The sequence generated with a reverse primer from a RT-PCR product from the patient shows the border of NF1 exons 47 (38) and 48 (39). In addition to the wild type transcript two aberrantly spliced transcripts can be deduced from the sequence. One aberrant transcript lacks the entire exon 47 (38) and the other the last 62 nucleotides of exon 47 (38) due to the use of an exonic cryptic 5′-splice site upstream of the integration site.Detection of two aberrant splice products due to an (TIF)Click here for additional data file.Figure S2AluYa5 insertion in NF1 exon 47 (38). A) Agarose gel showing PCR products generated from gDNA from a control individual (C) and the patient (P) harboring an AluYa5 insertion in NF1 exon 47 (38), . PCR product of the patient shows a faint extra band of larger size (arrow) that is not present in the control. B) Sequences of the PCR products from the control individual and the patient. The sequence of the patient shows a faint background sequence (a poly(T) stretch) starting at nucleotide c.6952 . This indicates the insertion of a retrotransposon, in this case an AluYa5 element, in anti-sense direction with respect to the NF1 coding sequence at this site.Detection of an (TIF)Click here for additional data file.Figure S3AluYa5 insertion in NF1 exon 47 (38). A) Scheme showing the strategy to amplify the AluYa5 insertion. Exons are shown as boxes and introns as lines. The Alu-insertion is shown as an arrow in anti-sense orientation. In order to specifically amplify the mutant allele containing the Alu sequence two Alu insertion specific primers (Alu_47f and Alu_47r) spanning the exon 47 (38)-Alu insertion border were designed. These primers were used together with their respective regular exon primer (47r or 47f) at the opposite site of the exon resulting in fragments of 600 bp and 1000 bp, respectively, each containing the Alu insertion. C) Agarose gel showing the result of the Alu-insertion specific PCRs derived from the patient (P) and a control individual (C). B) Sequence analysis of the specific Alu-insertion product generated by the Alu insertion-specific reversed primer and the respective regular forward primer, shows the anti-sense orientation of the Alu insertion.Specific PCR for the (TIF)Click here for additional data file.Figure S4Alu sequences with the consensus sequence of the AluY, AluYa5 and AluYb8 subfamilies. Identical nucleotides between all sequences are indicated by capital letters. In order to maximize the alignment gaps were introduced (dashed). The reference sequences of the Alu subfamilies were taken from Repbase Giri Sequence alignment of the 14 (TIF)Click here for additional data file.Figure S5NF1 exon 39 (30) of a patient (R01429). The sequence of the hot L1 element is taken from Alignment of the full length L1 sequence with the consensus sequence of the hot L1 element. The element was found inserted in sense orientation into the (TIF)Click here for additional data file.Figure S6NF1 coding sequence at least 1088 bp from the center of the L1 ORF2 and ends with the 3167th nucleotide of the 3825-bp L1 ORF2 suggesting that during the process of retrotransposition the orientation of the reverse transcription from the L1-RNA template that started at the sense strand of the NF1 gene switched and continued from the anti-sense strand. The duplicated nucleotides of the TSD are underlined. The 130-bp cryptic exon embedded in the L1 element is indicated in a darker shade of green. The sequence used to design L1 insertion-specific primers is underlined. The splices site scores of the cryptic exon as calculated by Splice Site Prediction by Neural Net (http://www.fruitfly.org/seq_tools/splice.html) is 0.62 for the 5′ splice site and 0.86 for the 3′ splice site.Truncated L1 element inserted into intron 9 (7). Genomic sequence of intron (IVS) 9 (7) (black letters) with truncated L1 element (green letters) inserted at position c.1062+195_1062+196 and the flanking exons 9 (7) and 10 (8) (red letters). The inserted sequence contains at the 5′-end a poly(T) stretch indicating that the poly(A) tail of the L1 transcript had annealed to the sense strand where reverse transcription from the template started. However, the 3′-end of the inserted sequence contains in sense orientation with regard to the (TIF)Click here for additional data file.Table S1NF1 exons, Alu and L1 sequences. (B) List of PCR programs.(A) List of primers for amplification of (PDF)Click here for additional data file.