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Phlebotomine sandflies (diptera: psychodidae) are widespread in the tropics and subtropics, and they transmit leishmania, protozoan parasites which cause visceral leishmaniasis (vl) and various forms of cutaneous leishmaniasis (cl) infecting more than 350 million people in more than 80 countries . They have a wide distribution, though mainly in the tropics and subtropics (lane 1993). Approximately 700 species of phlebotomine sand flies distributed among 6 genera but only two of them, phlebotomus in the old world and lutzomyia in the new world are introduced medically importance (lewis 1982, lane 1987, lane 1993, sharma and singh 2008), and only 10% of them act as the disease vector . Further, about 30 species of these are important from public health standpoint (who 1990, desjeux 2000, sharma and singh 2008). The blood - feeding females of phlebotomine sand flies (diptera: psychodidae, phlebotomine) include natural vectors of protozoa of the genus leishmania (kinetoplastida: trypanosomatidae), which are the parasitic causative agents of mammalian leishmaniasis (killick - kendrick 1990). Cutaneous leishmaniasis are reported from more than 50% of the 31 provinces in iran (yaghoobi - ershadi et al . 2007, rassi et al 2007, rassi et al . 2011). In the middle east, phlebotomine sand fly populations occasionally occur in great numbers, resulting in biting pressure of 1,000 bites per person per night (coleman et al . Although the impact on adult sand fly biting activity was not determined (blow et al . 2007), these preliminary results suggested that commercial mosquito traps might be good candidates for inclusion into integrated sand fly control or suppression programs in desert settings (hoel et al . 2000). In this study, sampling of sand flies were carried with disney trap, malaise, sticky traps, cdc light trap, co2 light trap, shannon traps and animal - baited trap . The purpose of the current study was to assess which of these traps could catch a larger number or show the most diversity of sand flies in the interval times . Some of them are currently used in routine sand fly surveillance programs (alexander 2000). Previous studies aiming at determining the species composition and distribution patterns of sand flies in kashan revealed eleven phlebotomus spp . And six sergentomyia spp . Some ideas for precise sampling in order to determine species composition, nocturnal activity and biodiversity of different sand fly was species in cl focus at different topographic condition e.g. Mountain / plain . Furthermore the effects of some environmental factors such as temperature, relative humidity and height above sea level were studied in relation to fluctuation considered in pattern of nocturnal activity as well as sand fly biodiversity . The studywas carriedoutin both plain region about 5 km far from northeast and mountainous region about 40 km far from southwest of kashan district, isfahan province, in central iran (fig . The geographical coordinates is 51 29 54.6 e and 33 58 52.9 n for plain region and 51 13 54.6 e and 33 58 52.9 n mountainous region with at an altitude of 951 m and 1823 m respectively . The maximum and minimum temperatures were 38 c and 19 c in plain region and were 45 c and 15 c in mountainous region during the study period . Kashan district, isfahan province, central iran seven traps were employed for sampling of sand flies . The sampling was repeated three times during the peak activity of sand flies in both plain and mountainous regions . Turning of sand flies sampling were 5 times per night starting 8:00pm and ending 6:00 am at outdoors using sticky traps (4 papers for 2 hours, totally 20 papers per day), disney trap, malaise, cdc light trap, co2 light trap (0.5 kg dry ice), shannon traps (both black and white nets) and animal - baited trap (fig . 2). Sticky traps were changed every two hours and as the traps were changed, the new ones were replaced in the same location . Other collection methods were also performed over the same 10 hours period using mouth aspirator to collect the sand flies . All sand flies at these traps were captured during 1015min in every two hours . Collected sand flies for sticky traps (2030 cm papers coated with castor oil) and disney trap were removed from sticky papers using entomological needles or fine brushes, preserved in 70% ethanol, and kept in microtubes before identification . At other traps, sand flies were also collected at interval of 2 hours by aspirator starting 8:00 pm ending 6:00 am . The sand flies were anesthetized with chloroform and transferred to microtube containing 70% ethanol . Then specimens transported to laboratory research center for mounting and identification . Different methods used for sand fly sampling after recording the sampling data and locations, some sand fly specimens which collected with sticky traps were washed in acetone and rest of them transferred to 70% ethanol . At appropriate time the identification keys of theodor and mesghali 1964, lewis 1982, rassi et al . The permanent mounted, labelled and identified microscope slides were deposited in medical entomology and zoology museums, school of public health, tums under code no . Gc22st5 - 92 . To determine the relationship between temperature and relative humidity (rh) and the abundance or activity of sand flies in both study regions (plain and mountain) associated with traps some meteorological data were recorded data were analyzed using spss 20 . In order to ensure normality and homogeneity of variances before subjecting to statistical analysis, data on the number of flies collected were square - root transformed . The significance difference of effect of the traps and time was analyzed in relation to sand flies species caught in two regions using univariate analysis of variance (anova) and chi - square tests . Total sand fly species caught constituted the dependent variable, while sex and traps and time were chosen as the fixed factor . Basic correlation matrices were used to determine the existence of a correlation between the abundance of the sand flies, average hourly temperature and relative humidity in the study area . The studywas carriedoutin both plain region about 5 km far from northeast and mountainous region about 40 km far from southwest of kashan district, isfahan province, in central iran (fig . The geographical coordinates is 51 29 54.6 e and 33 58 52.9 n for plain region and 51 13 54.6 e and 33 58 52.9 n mountainous region with at an altitude of 951 m and 1823 m respectively . The maximum and minimum temperatures were 38 c and 19 c in plain region and were 45 c and 15 c in mountainous region during the study period . The sampling was repeated three times during the peak activity of sand flies in both plain and mountainous regions . Turning of sand flies sampling were 5 times per night starting 8:00pm and ending 6:00 am at outdoors using sticky traps (4 papers for 2 hours, totally 20 papers per day), disney trap, malaise, cdc light trap, co2 light trap (0.5 kg dry ice), shannon traps (both black and white nets) and animal - baited trap (fig . Sticky traps were changed every two hours and as the traps were changed, the new ones were replaced in the same location . Other collection methods were also performed over the same 10 hours period using mouth aspirator to collect the sand flies . All sand flies at these traps were captured during 1015min in every two hours . Collected sand flies for sticky traps (2030 cm papers coated with castor oil) and disney trap were removed from sticky papers using entomological needles or fine brushes, preserved in 70% ethanol, and kept in microtubes before identification . At other traps, sand flies were also collected at interval of 2 hours by aspirator starting 8:00 pm ending 6:00 am . The sand flies were anesthetized with chloroform and transferred to microtube containing 70% ethanol . Then specimens transported to laboratory research center for mounting and identification . After recording the sampling data and locations, some sand fly specimens which collected with sticky traps were washed in acetone and rest of them transferred to 70% ethanol . At appropriate time, the sand flies were mounted in puri s medium . The identification keys of theodor and mesghali 1964, lewis 1982, rassi et al . The permanent mounted, labelled and identified microscope slides were deposited in medical entomology and zoology museums, school of public health, tums under code no . To determine the relationship between temperature and relative humidity (rh) and the abundance or activity of sand flies in both study regions (plain and mountain) associated with traps some meteorological data were recorded . Data were analyzed using spss 20 . In order to ensure normality and homogeneity of variances before subjecting to statistical analysis, data on the number of flies collected were square - root transformed . The significance difference of effect of the traps and time was analyzed in relation to sand flies species caught in two regions using univariate analysis of variance (anova) and chi - square tests . Total sand fly species caught constituted the dependent variable, while sex and traps and time were chosen as the fixed factor . Basic correlation matrices were used to determine the existence of a correlation between the abundance of the sand flies, average hourly temperature and relative humidity in the study area . A total of 1445 sand flies belonging to 15 species of the genus phlebotomus and 5 of the genus sergentomyia were collected . Of the collected total, s. sintoni was found to be the most prevalent (37.86%) species while ph . Papatasi the proven vector of l. major, l. turanica and l. gerbili in isfahan accounted for 31.76% of the sand flies that were identified . Dentata (0.3%), s.(ser .) Antennata (0.2%), s.(ran .) Pawlowskyi (0.3%), s.(par .) Palestinensis (0.1%), ph. (syn .) Ansarii (0.1%), ph. (para .) Kazeruni (1.7%), ph. (par .) Caucasicus group (0.4%), ph. (par .) Alexandri (1.1%), ph. (lar .) Wenyoni (0.6%), ph. (lar .) Tobbi (1%), ph. (lar .) Major (3.5%), ph. (lar .) Keshishiani (0.2%), ph. (lar .) Kandelaki (0.1%), ph. (adl .) Longiductus (0.1%), ph. (adl .) Halepensis (0.1%), ph. (adl .) The population of sand flies was found to be the lowest in cdc light trap and co2 light trap . Population size was raised with two highest peaks in disney trap and sticky trap at 22:0024:00 pm . The average monthly temperature and relative humidity values ranged between 21.8328.33 c and 24.8336.33% respectively . During the night, when the maximum number of sand flies (29.25%) was collected at 22:0024:00 pm, the average temperature and rh were found to be 26.92 c and 26.5%, respectively, while the minimum number of sand flies was sampled at 02:0004:00 am (11.62%) with an average temperature of 23.08 c and 33.5% relative humidity (fig . Correlation of two environmental conditions with nocturnal activity of sand flies the number of sand flies collected using different types of traps during the study period is presented in table 1 . 24.2% and 10.5% of sand flies were collected using sticky traps and shannon trap (black net), respectively . Collections using animal baited trap and shannon trap (white net) constituted 8.5% and 7.3% of the total collection . Neither co2 light trap (5.0%) nor cdc light trap (3.3%) and malaise trap (0.1%) showed a high efficiency during the study . A significant interaction was revealed between the collection methods and the number of individuals collected using of univariate analysis of variance (anova) (p <0.05), and there was significant interaction between the trapping methods and the proportion of males and females collected (p <0.05). When significant effects of traps were established (p <0.05), differences among traps were exposed that co2 and standard cdc light traps, shannon trap (black and white net) and animal baited trap displayed some similar efficiency, whereas sticky papers and disney trap differed from the others (p <0.05). Prevalent sand flies collected by different traps, kashan, isfahan province, 2011 results with respect to the nocturnal activity indicated that even though the number sand flies declined rapidly between 02:00 and 04:00h . There was no significant difference between hourly pattern either in the species prevalence or in the activity of the species in different traps (fig . 4). Correlation between collection methods and density of abundance sand flies species in the study area in kashan city this study is the first detailed research in terms of species composition, density and nocturnal activity of sand flies using different methods of capturing in an endemic focus of cutaneous leishmaniasis in kashan district, isfahan province, iran . Trapping over the summer season during the peak of sand flies activity revealed that s. sintoni (37.9%), p. papatasi (31.8%) and p. sergenti (20.1%) are the most abundant and prevalent species in these regions respectively (doroudgar et al . Both the previous and recent findings showed that p. papatasi the main vector of l. major, l. turanica and l. gerbili in isfahan province, p. sergenti the proven vector of l. tropica and l. gerbili in this region, also s. sintoni were represented with high populations in the study areas (seyedi rashti and nadim 1992, doroudgar et al . Although sand fly abundance was not strongly and significantly influenced by the variations in average monthly temperature and relative humidity during the six months of survey in cukurova plain, south anatolia, turkey, the maximum number of sand flies was recorded in the hottest and driest seasons between june and september when the average temperature was comparatively high, the relative humidity was low and the rainy days ranged between 1.5 to 3.5 days . The role of climatic factors on the seasonal distribution of sand flies in the arid areas of india exhibited that the majority of species preferred comparatively higher temperatures and low rh%, a prerequisite for survival in arid and semi - arid conditions (singh 1990). Our study results are concordance with the other survey in relation to effects of temperature and relative humidity on abundance of sand flies . It seems that special range of both high temperature and low relative humidity caused the high abundance of sand fly species in our study regions . The results showed that before midnight (between 22:0024:00 pm) sand flies were very active . Although cdc and co2 light traps are used extensively in the field studies of sand flies (alexander 2000), in cukurova plain in turkey sticky traps have no known attractiveness and have generally been used for determining species composition of an area as they randomly sample the species where they are set (kasap et al . 2009), whereas our findings showed that disney trap and sticky traps have been attractiveness more than other traps . Our findings in terms of the efficiency of the sampling methods, showed these two trapping methods appeared to be the most productive for both estimating the number of sand flies and the species composition in the study area, in agreement with some previous studies . It seems that the sticky traps provide more realistic results than cdc light traps that could attract additional phototropic sand flies . The effective range of cdc light traps was less than 5 m (wheeler et al . Study in northern italy showed that when compared to sticky traps, co2 traps were more effective in collecting sand flies and addition of a light source improved the catches (veronesi et al . The nocturnal activity patterns of sand flies have been reported for old world species (roberts 1994, guernaoui et al . According to our results, sand flies activity was not significantly different over time even though total counts decreased between 04.0006.00h during late summer . It seems to be the most important factor affecting sand fly nocturnal activity in similar studies was low humidity, followed by low wind velocity and high temperature (roberts 1994, guernaoui et al . Therefore, more detailed studies with respect to the seasonal variations and the effects of abiotic conditions, other than temperature and relative humidity, such as cloud cover, wind velocity and lunar cycle on the nocturnal activity of sand flies may improve knowledge of the behavior of sand flies for important epidemiologically surveys.
Peritoneal drainage of cerebrospinal fluid (csf) is widely used by neurosurgeons to treat hydrocephalus . The first uses of laparoscopy to aid in the abdominal placement of ventriculoperitoneal (vp) and lumboperitoneal (lp) shunts were reported in 1993 and 1999, respectively . These early series suggested that laparoscopy was safe and effective and provided direct visual confirmation of shunt position and function . Comparative studies of minimally invasive shunt placement versus traditional open techniques have suggested laparoscopy is not only a reliable technique, but it has fewer complications as well . Shunt dysfunction still occurs in a substantial number of cases, with reported estimates of abdominal complications ranging from 5% to 47% . However, experience with laparoscopy in the diagnosis and treatment of shunt malfunction has been limited . This series reviews our experience with laparoscopy in the management of vp and lp shunt malfunction in adult patients, as well as diagnostic laparoscopy in patients with indwelling shunts who presented with abdominal pain . We conducted a retrospective review of all adult patients undergoing laparoscopic revision of vp and lp shunts or diagnostic laparoscopy for assessment of abdominal complaints or shunt function in patients with a vp or lp shunt . Institutional review board approval was obtained from the university of maryland human subjects research committee for this study . Data collected include age, sex, diagnoses, indications for surgery, operative details, and short - term results . For initial assessment, pneumoperitoneum was established to a pressure of 10 mm hg to 15 mm hg by using the veress needle technique . Placement of the trocar was dictated by the side of the shunt, and the patient's previous surgical incisions . An additional 5-mm port was placed under direct vision in a location that provided easy manipulation of the tip of the shunt catheter . In any case where a new shunt was placed, we used a technique previously described by the senior investigator in this study (jsr) with a percutaneous peel - away sheath serving as an introducer for the shunt catheter . An additional 5-mm port may be placed as necessary to facilitate the conduct of the operation and allow for the 2-handed technique . Patients with vp shunts were placed in a supine position, whereas patients with lumboperitoneal shunts were necessarily placed in a lateral decubitus position . For initial assessment, pneumoperitoneum was established to a pressure of 10 mm hg to 15 mm hg by using the veress needle technique . Placement of the trocar was dictated by the side of the shunt, and the patient's previous surgical incisions . An additional 5-mm port was placed under direct vision in a location that provided easy manipulation of the tip of the shunt catheter . In any case where a new shunt was placed, we used a technique previously described by the senior investigator in this study (jsr) with a percutaneous peel - away sheath serving as an introducer for the shunt catheter . An additional 5-mm port may be placed as necessary to facilitate the conduct of the operation and allow for the 2-handed technique . Patients with vp shunts were placed in a supine position, whereas patients with lumboperitoneal shunts were necessarily placed in a lateral decubitus position . Ten patients underwent 13 laparoscopic procedures for shunt - related complications of a ventriculoperitoneal or lumboperitoneal shunt . Bmi = body mass index; ptc = pseudotumor cerebri; nph = normal pressure hydrocephalus; sah = subarachnoid hemorrhage; csf = cerebrospinal flui . All patients in our series were female, with an average age of 42.4 years (range, 29 to 74). Most were also morbidly obese, with an average body mass index (bmi) of 40.7 . The most common condition requiring initial shunt placement was pseudotumor cerebri (60% of patients), but other diagnoses included normal pressure hydrocephalus, intracranial hemorrhage, syringomyelia, and spina bifida . Indications for the laparoscopic procedure included suspected shunt failure (n=10), shunt migration (n=1), and abdominal pain (n=2). Shunt replacement or revision was ultimately successful in all patients as documented by the laparoscopic visualization of csf draining from the end of the shunt tubing at the time of the operation (figure 1). Eighty percent of patients undergoing revisional surgery were treated successfully with a single surgical procedure . In one case, the abdominal portion of the shunt had become detached and was retrieved laparoscopically before insertion of a new shunt catheter (figure 2). Complications developed in 3 patients: 1 patient developed a leak from the lumbar wound that required surgical oversewing, and 2 patients required multiple abdominal procedures to establish proper shunt function . One of these patients (patient 3 in table 1) had migration of her original shunt catheter into the extraperitoneal space (figure 3). A large extraperitoneal collection of csf was noted in the right lower quadrant . During the initial procedure, the collection was drained laparoscopically, and the shunt catheter was positioned into the peritoneal cavity . On the second postoperative day, the patient developed recurrent headaches that prompted a computerized tomography scan . The catheter had again migrated into the extraperitoneal space . In the subsequent laparoscopic procedure, the peritoneal portion of the catheter was relocated to a position in the right upper quadrant . (a) computerized tomography image depicting extraabdominal migration of catheter tip with corresponding cerebrospinal fluid collection . (b) laparoscopic view, fluid collection drained intraabdominally . (c / d) catheter positioned into the peritoneal cavity as the extraabdominal fluid collection is marsupialized . In the second case of postoperative shunt dysfunction (patient 6 in table 1), the patient had had a total of 34 prior shunt placements and revisions (figure 4). During the year of this study, she required initial revision, followed by a valve replacement and ultimately a lumboperitoneal shunt placement . Laparoscopic lysis of adhesions of patient with history of both lumboperitoneal and ventriculoperitoneal shunting . In this series, 2 patients underwent laparoscopy for abdominal pain presumably related to the shunt catheter . In 1 patient, however, she also had a small incisional hernia at the shunt insertion site, which was repaired laparoscopically . Postoperatively, the patient had complete resolution of her abdominal pain . In the second patient, the diagnostic laparoscopy was unremarkable, but the shunt catheter was noted to be extraperitoneal . This shunt was not revised, as the patient no longer had the neurologic symptoms that resulted in the placement of the original shunt . Shunt malfunction may be caused by ventricular catheter obstruction, valve problems, distal catheter obstruction, pressure mismatch, or component disconnection . Common presenting features of shunt dysfunction include headache, mental status changes or drowsiness, and vomiting . Computerized tomography or magnetic resonance imaging may not show a change in ventricular size, but a comparison to a previous examination must be performed . To evaluate the patency of a ventriculoperitoneal shunt, a this involves injection of a small quantity of nonionic contrast material into the valve of a ventricular shunt system . Serial films are obtained to document forward flow of contrast material and csf.8 patency can also be evaluated via nuclear medicine techniques or manometric evaluations; however, significant variations can occur in shunt flow rates and volumes during the course of the day . As a result, the traditional open technique for placement of the shunt involves creating a limited laparotomy and blindly introducing the catheter into the abdominal cavity . The use of laparoscopy facilitates this procedure by allowing for placement of the catheter under direct vision and confirming shunt function by visualization of drainage of csf from the shunt tubing . However, even the laparoscopic approach is not a guarantee against shunt - related complications . The use of a peel - away introducer sheath to insert the tubing through the abdominal wall greatly facilitates the procedure . However, the addition of a second trocar allows the operator to grasp the catheter and direct it toward an area of the abdomen free of adhesions . Several authors have advocated the use of mini - laparoscopy in the placement of shunts . In our patients, the median number of prior procedures was 3, and laparoscopy was ultimately successful in all cases . Abdominal access in this patient population was readily obtained utilizing a veress needle and a direct access trocar without abdominal injury or complications . Other authors have reported similar success with the placement of vp and lp shunts in patients with prior surgery . In this series, 20% of patients required a second surgical procedure to correct shunt dysfunction . However, each of these difficulties was addressed by subsequent laparoscopy, leading to an ultimate success rate of 100% . Complications may be anticipated, given the typical history and status of the patient population . In addition to the multiple previous shunt procedures, the mean american society of anesthesiologists classification status for this patient population was 3, indicating significant systemic disturbance . A 2-mm to 5-mm incision may be used for access to the peritoneal cavity, rather than the long incision required in open placement for patients with a thick abdominal wall . As mentioned earlier, although only basic laparoscopic skills are required for vp shunt placement, laparoscopic lp shunt placement requires slightly more advanced skills due to the increased difficulty in abdominal access in the lateral position . Numerous case reports have described techniques for laparoscopic drainage of csf pseudocysts, the removal of disconnected distal catheters, and the repositioning of shunts into more favorable locations . In our series, laparoscopy was additionally utilized as a diagnostic tool for abdominal pain in 2 patients . In both patients, one patient was found to have a small incisional hernia that was repaired laparoscopically . In the second patient, other authors have similarly utilized laparoscopy in the diagnosis and management of abdominal pain following shunt placement . In all shunt revisions, the laparoscopic technique provided a minimally invasive approach to the successful manipulation or replacement of the shunt documented by visualization of csf drainage . The safety and efficacy of this technique has been shown in children, the obese, and the multiply operated on abdomen . This report represents the largest published series on the laparoscopic management of complications of vp and lp shunting in adults . Ventriculoperitoneal shunting is commonly performed by neurosurgeons without the assistance of an abdominal surgeon . In this series, all procedures were performed under the direction of both a neurosurgeon and a general surgeon . Although basic laparoscopic skills may be taught to neurosurgeons, widespread adoption of the laparoscopic abdominal technique may not be expected . Although it may not be feasible in all centers, it is a safe, reliable, and effective technique at our institution . Laparoscopy is a safe and effective technique for the management of complications following shunt placement, assessment of shunt function, and diagnosis of abdominal complaints . Our series represents the largest published series of laparoscopic management of shunt complications in adults . Future prospective trials will more clearly delineate the roles of laparoscopy in this patient population.
Previous studies have implicated that endothelial dysfunction plays an important role in the onset and progression of atherosclerosis . Tes, a novel tumor suppressor gene, is located in a common fragile site on human chromosome 7q31.2, designated as fra7 g . It is predicted to encode a highly conserved protein of 421 amino acids named testin . Testin has been found to play an important role in focal adhesion and is present in the cytoplasm . Recently, it has been reported that the expression of tes was decreased in the left anterior descending aortic tissue from patients with coronary artery disease (cad) compared with non - cad subjects . In this report, we aimed to determine the cellular origin of tes expression in aorta and examine whether its expression is affected in the aortic tissue during the pathogenesis of atherosclerosis . Thirty - two healthy purebred new zealand rabbits were used at the age of 34 months with body weights from 1.5 to 2.0 kg . All experimental protocols were approved by the institutional animal care and use committee at tianjin medical university . The rabbits were randomly divided into two groups as the following: (1) the normal diet (nd) group (n = 12) were fed on 100 g / d of nd; (2) high fat diet (hfd) group (n = 20) were fed on the mixture of 100 g / d of 1% cholesterol, 5% lard, 10% egg yolk powder and 84% nd . Body weight and serum lipid levels were measured at 0, 4 and 12 weeks after enrollment . Thoracic aortas were rapidly removed, cleaned of adventitia, cut into 5-mm ring segments . The specimens were fixed in 4% formaldehyde and sectioned for routine hematoxylin and eosin staining . Tissue structure and pathological features of atherosclerosis after fixation, the tissue sections were rehydrated twice for 5 min in phosphate buffer saline (pbs), blocked for 1 h in pbs with 10% normal goat serum followed by incubation in a humid chamber with 50 l of anti - testin primary antibody (1:100 dilution; abcam, ab78499, usa) and anti - cd31 primary antibody (1:50 dilution; abcam, ab9498, usa) in 10% serum overnight at 4c . The slides were rinsed twice for 5 min in pbs before being incubated for 2 h in a humid chamber with 50 l of anti - rabbit immunoglobulin g conjugated with fluorescein isothiocyanate (1:100 dilution; sigma, usa) solution to visualize testin . Stained sections were rinsed twice for 5 min in pbs, stained for 10 min with 50 l of 1 mg / ml 4,6-diamidino-2-phenylindole (dapi) in 200 ml pbs, then rinsed again twice for 5 min in pbs, and mounted with an aqueous mounting medium before being examined under confocal microscope (olympus, japan). Total rna was isolated from aortic tissue using the trizol solution (invitrogen, usa). Total of 1 g rna was used for first - strand complementary dna synthesis using random primer (invitrogen, usa) and superscript ii transcriptase (invitrogen, usa) according to manufacturer's instruction . Real - time polymerase chain reaction (pcr) was performed using iq sybr green supermix (bio - rad, usa) with cfx-96 real - time pcr detection system (bio - rad, usa). The primers used for tes and glyceraldehyde-3-phosphate dehydrogenase (gapdh) as an internal control in the reaction were: 5-cctgtccagaaccaggcatt - 3/5 - ttctttcggtactgtgcccc-3 (tes gene, 98 bps), and 5-tgagaatctgcccctcttcac - 3/5-cgttgctgtcgagactttattga-3 (gapdh, 110 bps). Total protein extract was prepared from aortic tissue by homogenize tissue in radio immunoprecipitation assay lysis buffer (sigma, r0278, usa). Proteins were separated on nupage sds - page gel (invitrogen, usa) and transferred to a nitrocellulose transfer membrane (whatman, uk) according to the manuscript's instruction . The primary antibodies used for protein detection included anti - testin (1:500 dilution; abcam, ab78499, usa) and gapdh (1:1000 dilution; santa cruz, usa). Membranes were then incubated with horseradish peroxidase - conjugated secondary antibodies (abcam, usa). Proteins were visualized by enhanced chemiluminescence system (western blot detection kit, ge healthcare). Developed signals were digitally recorded and quantified with melanie two - dimensional gel software analysis (sib, switzerland). Statistical analysis was performed by one - way analysis of variance among groups . For comparison of data before and after dietary intervention thirty - two healthy purebred new zealand rabbits were used at the age of 34 months with body weights from 1.5 to 2.0 kg . All experimental protocols were approved by the institutional animal care and use committee at tianjin medical university . The rabbits were randomly divided into two groups as the following: (1) the normal diet (nd) group (n = 12) were fed on 100 g / d of nd; (2) high fat diet (hfd) group (n = 20) were fed on the mixture of 100 g / d of 1% cholesterol, 5% lard, 10% egg yolk powder and 84% nd . Body weight and serum lipid levels were measured at 0, 4 and 12 weeks after enrollment . Thoracic aortas were rapidly removed, cleaned of adventitia, cut into 5-mm ring segments . The specimens were fixed in 4% formaldehyde and sectioned for routine hematoxylin and eosin staining . Tissue structure and pathological features of atherosclerosis the detection of testin on tissue samples was inspected via immunohistochemical and immunofluorescence staining . After fixation, the tissue sections were rehydrated twice for 5 min in phosphate buffer saline (pbs), blocked for 1 h in pbs with 10% normal goat serum followed by incubation in a humid chamber with 50 l of anti - testin primary antibody (1:100 dilution; abcam, ab78499, usa) and anti - cd31 primary antibody (1:50 dilution; abcam, ab9498, usa) in 10% serum overnight at 4c . The slides were rinsed twice for 5 min in pbs before being incubated for 2 h in a humid chamber with 50 l of anti - rabbit immunoglobulin g conjugated with fluorescein isothiocyanate (1:100 dilution; sigma, usa) solution to visualize testin . Stained sections were rinsed twice for 5 min in pbs, stained for 10 min with 50 l of 1 mg / ml 4,6-diamidino-2-phenylindole (dapi) in 200 ml pbs, then rinsed again twice for 5 min in pbs, and mounted with an aqueous mounting medium before being examined under confocal microscope (olympus, japan). Total rna was isolated from aortic tissue using the trizol solution (invitrogen, usa). Total of 1 g rna was used for first - strand complementary dna synthesis using random primer (invitrogen, usa) and superscript ii transcriptase (invitrogen, usa) according to manufacturer's instruction . Real - time polymerase chain reaction (pcr) was performed using iq sybr green supermix (bio - rad, usa) with cfx-96 real - time pcr detection system (bio - rad, usa). The primers used for tes and glyceraldehyde-3-phosphate dehydrogenase (gapdh) as an internal control in the reaction were: 5-cctgtccagaaccaggcatt - 3/5 - ttctttcggtactgtgcccc-3 (tes gene, 98 bps), and 5-tgagaatctgcccctcttcac - 3/5-cgttgctgtcgagactttattga-3 (gapdh, 110 bps). Total protein extract was prepared from aortic tissue by homogenize tissue in radio immunoprecipitation assay lysis buffer (sigma, r0278, usa). Proteins were separated on nupage sds - page gel (invitrogen, usa) and transferred to a nitrocellulose transfer membrane (whatman, uk) according to the manuscript's instruction . The primary antibodies used for protein detection included anti - testin (1:500 dilution; abcam, ab78499, usa) and gapdh (1:1000 dilution; santa cruz, usa). Membranes were then incubated with horseradish peroxidase - conjugated secondary antibodies (abcam, usa). Proteins were visualized by enhanced chemiluminescence system (western blot detection kit, ge healthcare). Developed signals were digitally recorded and quantified with melanie two - dimensional gel software analysis (sib, switzerland). Statistical analysis was performed by one - way analysis of variance among groups . For comparison of data before and after dietary intervention we examined the extent of atherosclerosis in rabbits fed with hfd and nd for 12 weeks . As expected, all of the rabbits in hfd group had a significantly higher level of serum lipids [table 1] and presence of atherosclerotic plaque in the thoracic aorta compared with nd group [figure 1]. Levels of serum lipids in two groups at different periods (mmol / l, mean sd) * compared with nd group, p <0.05 . Tg: triglyceride; tc: total cholesterol; ldl - c: low - density lipoprotein cholesterol; hdl - c: high- density lipoprotein cholesterol; sd: standard deviation; nd: normal diet; hfd: high - fat diet . High incidence of atherosclerotic plaque in high - fat diet (hfd) group when compared with natural diet (nd) group (hematoxylin and eosin staining). Representative sections from aortic tissues in hfd group (b and c) showed atherosclerotic plaque, whereas nd group (a) was normal . By immunostaining in aortic tissues with anti - testin antibody and confocal microscopy, we observed testin signal was predominantly detected in the endothelium as identified with cd31 staining . A relatively weaker signal was also detected in the subendothelial areas [figure 2a2c]. Moreover, the majority of atherosclerotic plaque displayed testin immunoreactivity on segments bearing the plaque but also on those distal from the plaque [figure 2d2 g]. Significant differences in testin signal intensities were observed among different segments, suggesting that testin expression may correlate and contribute to vascular phenotypic heterogeneity and its possible role in atherosclerosis expression of testin protein in the normal and atherosclerotic aortic tissues . (a c, 10) the signal for testin co - localized with the cd31 signal, indicating that testin was expressed in endothelial cells (arrow); (d and e, 20; f and g, 10) immunohistochemical and immunofluorescence staining analysis of the normal aorta and plaque with an anti - testin antibody . It showed that testin was expressed predominantly in the endothelium of normal aorta and plaque (arrows). We assessed the expression levels of the tes gene in the aortic tissues obtained from hfd and nd groups . Real - time pcr analysis demonstrated that tes mrna was markedly reduced by approximately 10-fold in the hfd group compared with the nd group (p = 0.015) [figure 3a]. Using western blot analysis, on the same experimental subjects showed testin expression was decreased at protein level in the hfd group in comparison with atherosclerotic - free nd group (p = 0.01) [figure 3b and 3c]. These results suggested that reduced tes / testin expression was associated with hfd induced atherosclerosis . Decreased tes / testin expression in atherosclerotic aorta compared with nonatherosclerotic samples . (a) tes mrna expression from 12 nonatherosclerotic aorta compared with 20 atherosclerotic aorta arteries by real - time reverse transcription - polymerase chain reaction . The mrna expression level of tes in atherosclerotic aorta was significantly lower than in nonatherosclerotic aorta arteries; (b) testin protein expression by western blot analysis with 12 nonatherosclerotic (n) aorta and 20 atherosclerotic aorta arteries (a) showed that testin expression was lower in atherosclerotic aorta arteries . Glyceraldehyde-3-phosphate dehydrogenase was used as loading control; (c) western blot images in (b) was scanned, quantified, and plotted, which showed testin expression was significantly decreased in atherosclerotic aorta . We examined the extent of atherosclerosis in rabbits fed with hfd and nd for 12 weeks . As expected, all of the rabbits in hfd group had a significantly higher level of serum lipids [table 1] and presence of atherosclerotic plaque in the thoracic aorta compared with nd group [figure 1]. Levels of serum lipids in two groups at different periods (mmol / l, mean sd) * compared with nd group, p <0.05 . Tg: triglyceride; tc: total cholesterol; ldl - c: low - density lipoprotein cholesterol; hdl - c: high- density lipoprotein cholesterol; sd: standard deviation; nd: normal diet; hfd: high - fat diet . High incidence of atherosclerotic plaque in high - fat diet (hfd) group when compared with natural diet (nd) group (hematoxylin and eosin staining). Representative sections from aortic tissues in hfd group (b and c) showed atherosclerotic plaque, whereas nd group (a) was normal . By immunostaining in aortic tissues with anti - testin antibody and confocal microscopy, we observed testin signal was predominantly detected in the endothelium as identified with cd31 staining . A relatively weaker signal was also detected in the subendothelial areas [figure 2a2c]. Moreover, the majority of atherosclerotic plaque displayed testin immunoreactivity on segments bearing the plaque but also on those distal from the plaque [figure 2d2 g]. Significant differences in testin signal intensities were observed among different segments, suggesting that testin expression may correlate and contribute to vascular phenotypic heterogeneity and its possible role in atherosclerosis expression of testin protein in the normal and atherosclerotic aortic tissues . (a c, 10) the signal for testin co - localized with the cd31 signal, indicating that testin was expressed in endothelial cells (arrow); (d and e, 20; f and g, 10) immunohistochemical and immunofluorescence staining analysis of the normal aorta and plaque with an anti - testin antibody . It showed that testin was expressed predominantly in the endothelium of normal aorta and plaque (arrows). We assessed the expression levels of the tes gene in the aortic tissues obtained from hfd and nd groups . Real - time pcr analysis demonstrated that tes mrna was markedly reduced by approximately 10-fold in the hfd group compared with the nd group (p = 0.015) [figure 3a]. Using western blot analysis, on the same experimental subjects showed testin expression was decreased at protein level in the hfd group in comparison with atherosclerotic - free nd group (p = 0.01) [figure 3b and 3c]. These results suggested that reduced tes / testin expression was associated with hfd induced atherosclerosis . Decreased tes / testin expression in atherosclerotic aorta compared with nonatherosclerotic samples . (a) tes mrna expression from 12 nonatherosclerotic aorta compared with 20 atherosclerotic aorta arteries by real - time reverse transcription - polymerase chain reaction . The mrna expression level of tes in atherosclerotic aorta was significantly lower than in nonatherosclerotic aorta arteries; (b) testin protein expression by western blot analysis with 12 nonatherosclerotic (n) aorta and 20 atherosclerotic aorta arteries (a) showed that testin expression was lower in atherosclerotic aorta arteries . Glyceraldehyde-3-phosphate dehydrogenase was used as loading control; (c) western blot images in (b) was scanned, quantified, and plotted, which showed testin expression was significantly decreased in atherosclerotic aorta . In this study, we characterized the expression change of tes / testin in aortic tissues during hfd induced atherosclerosis using a well - established rabbit model . Our data showed that tes / testin levels were significantly decreased in the hfd treated aortic tissues at both mrna and protein levels . Using confocal analysis, we detected that testin was mainly expressed in cd31 luminal endothelium while a weaker expression was also detected in the subendothelium area . In this study, tes / testin was expressed in endothelium, and was down - regulated in atherosclerotic tissues, which suggested a possible role for testin in the pathogenesis of atherosclerosis; however, the underlying mechanism is not clear and needs to be further investigated . First, the cellular composition of the aortic tissues in atherosclerotic subjects may be altered from basal status . In fact, the loss of endothelial cells (ecs) is a hallmark pathological in atherosclerotic tissue, which can lead to reduced expression of testin . Second, endothelium dysfunction due to pro - atherosclerotic and pro - inflammatory stimulation in the aortic tissue following hfd can lead to down - regulation of testin expression by genetic and epigenetic regulatory machinery . Indeed, it has been demonstrated that 5 cpg island methylation of the tes gene can silence its expression in gastric cancer, and a similar mechanism may have occurred in atherosclerotic endothelium . Third, tes gene expression can also be regulated by mirnas, which may be induced in the process of atherosclerosis . Finally, single nucleotide polymorphisms (snps) may contribute to regulating the expression of tes gene . These possible mechanisms should be the subject of future investigations . As a component of focal adhesions, testin interacts with the cytoskeletal protein such as zyxin, talin, vasodilator - stimulated phosphoprotein, mena, extractable nucler antigen (evl), alphall - spectrin, actin and actin - related proteins 7a . However, the functional role of testin in the cardiovascular system is not fully understood . Reported that overexpression of tes decreased monocyte adhesion to ecs or the migration of monocytes across the ec layer, while knockdown increased these activities . This result indicated that tes may play a potentially important role in the homing and local infiltration of pro - inflammatory cells at atherosclerotic plaques, thus contributes to the initiation and progression of the pathology . In addition, zhu et al . Have demonstrated that mir-29b decreased tes mrna expression, whereas matrix metalloproteinases-2 (mmp-2) increased tes expression, respectively, and it is well - established that mmp-2 is associated with atherosclerosis by regulating vascularization and inflammatory response . Moreover, magno et al . Identified testin can also interact with calcium - sensing receptor (car), and influence cytoskeletal function via enhancing the car - mediated rho signaling pathway . All these results suggested that testin may play a protective role in endothelium, and its loss of expression contributes to the initiation and progression of atherosclerosis through multiple mechanisms . In short, we characterized the expression of tes gene in the aortic tissue of atherosclerotic rabbits . We showed that tes / testin expression was significantly decreased in aortic tissues with atherosclerotic lesions compared with normal vessels . We further demonstrated that testin was strongly expressed in cd31 luminal lining with a weaker expression in the subendothelium area . Future studies are needed to fully uncover the underlying mechanisms of tes in the pathogenesis of cardiovascular diseases and to explore the possibility of targeting tes as a potential therapy.
In recent years, there is increasing evidence to suggest that prokaryotes adopt primitive organelle - like structures called bacterial microcompartments or nanocompartments, depending on their size . Such assemblies localize and compartmentalize multiple enzymes and substrates involved in specific metabolic pathways . The ability to mimic and understand enzymatic activity in confinement would provide ground - breaking insight into these assemblies and in organelles in general . One of the main challenges is to controllably package and coencapsulate different enzymes noncovalently within the same compartment as exemplified by nature . There has been some success using coiled - coil helices, peptide tags, and protein protein fusion constructs to direct enzymatic cargo encapsulation into protein cages . However, such approaches often lead to covalently connected protein cargo or inefficient loading (i.e., formation of empty cage assemblies). To circumvent these problems, we seek a versatile approach that would promote a noncovalent co - encapsulation of enzymes within a single protein cage in vitro . The cowpea chlorotic mottle virus (ccmv) is an ideal candidate to mimic bacterial nanocompartments, owing to its size and biocompatibility . The ccmv capsid is 28 nm in diameter and is based on a t = 3 lattice (t, triangulation number), with 12 pentamers and 20 hexamers of identical monomers of capsid protein (cp) organized as 90 dimers . Similar to bacterial compartments, it has multiple pores in the capsid shell (around 2 nm), which allows molecules and substrates to diffuse in and out . The cp n - terminal region is enriched in positively charged residues, termed the arginine - rich motif (arm), that face the capsid inner surface . After removal of native single - strand (ss) rna cargo, the arm can trigger reassembly of capsid protein dimers in the presence of an appropriate negatively charged template, resulting in the formation of monodisperse virus - like particles (vlp). Encapsulation of enzymes in vlps has been shown to stabilize and protect the enzymes, and its ease of modification enables new applications . In this contribution, single- and complementary - stranded dna tags are chemically attached to the exterior of chosen enzymes, resulting in negatively charged complexes that induce the co - encapsulation inside ccmv capsids . Unlike covalent interactions, electrostatic interactions between the dna tags and the interior of the capsid provide a tunable system, enabled, for instance, by changing the salt concentration or by varying the length of the dna chains . Hence, this makes our system a model for natural bacterial compartments (e.g., the encapsulins) where the confined enzymes are not covalently bound but rather included in the protein cage by noncovalent, multivalent interactions . Noncovalent encapsulation mediated by nucleic acid tags has been reported with the use of genetic engineering and/or only focused on a single enzyme . Using this strategy, we were able to confine two separate cascade systems in vitro, for which the glucose oxidase (gox), a 160 kda dimeric enzyme, is chosen as the primary enzyme for both encapsulated cascades . Gox catalyzes the oxidation of glucose into gluconolactone (which undergoes spontaneous hydrolysis into gluconic acid) and produces hydrogen peroxide as the side product . In the first cascade system, hydrogen peroxide produced by gox is consumed by the so - called dnazyme, a peroxidase - mimic formed in situ by a specific sequence of ssdna in the presence of hemin (figure 1a). In the second cascade system, in the presence of atp and nadp, gluconic acid produced by gox is consumed by a secondary enzyme, gluconokinase (gck) that is coencapsulated inside the ccmv - like particles, followed by a nonencapsulated tertiary enzyme, 6-phosphogluconate dehydrogenase (6-pgdh), to form ribulose-5-phosphate and nadph, the latter of which can be monitored spectroscopically (figure 1b). Therefore, the nucleic acid tags in this work are useful both as a secondary biocatalyst (in cascade system i) and as negatively charged tags to trigger the encapsulation of the enzyme(s) (in cascade systems i and ii). Schematic representation of the enzyme pathways (encapsulated processes shown in gray boxes). Gox oxidizes glucose to gluconic acid and produces h2o2, which dnazyme uses for subsequent reaction with abts inside ccmv capsid . The conversion of glucose to d - gluconate-6-p occurs at the interior of the ccmv capsids, whereas the conversion of d - gluconate-6-p into ribulose-5-p occurs at the exterior of the ccmv capsid catalyzed by tertiary enzyme, 6-pgdh . We anticipated that functionalization and subsequent hybridization of gox and gck with (complementary) single - stranded dna strands should promote their co - encapsulation into ccmv capsids (figure 2). We suspect that the size of the enzyme cargo as well as the number and spatial distribution of negative charges anchored to the enzyme surface might play a role in determining the efficiency of the encapsulation . To modify and hybridize the relevant enzymes, the lysine residues of gox and gck were functionalized with a heterobifunctional linker using sulfo - nhs coupling followed by maleimide thiol chemistry on the single stranded dna (ssdna) or its complementary sequence (csdna), respectively (see figures s1 and s2). All hybridized complexes were purified initially by spin - filtration to remove excess dna . Furthermore, size - exclusion chromatography (sec) was used for the gox gck dual - enzyme complex to remove nonhybridized gck and gox prior to encapsulation (figure s3). Encapsulation of different enzyme dna hybrids inside ccmv capsids (gray) at ph 7.5 . Encapsulation of (a) ssdna in yellow, (b) gox, in blue, functionalized with ssdna, (c) gck, in green, functionalized with the complementary ssdna in red, (d) gox conjugated to gck . The specific sequence of ssdna is catalytically active in the presence of hemin . Encapsulation of gox ssdna, gck csdna, or hybridized gox gck in ccmv at ph 7.5 led to the formation of stable capsid - like assemblies, which were purified by sec (figure 3a d). The elution volume (12 ml), together with the relative absorbance ratio (260 nm/280 nm)> 1, are characteristic features of intact ccmv capsids containing dna - based cargo . Control experiments with no dna tags (i.e., nonfunctionalized gox and/or gck) confirmed that enzymes lacking ssdna or csdna cannot be encapsulated (figure s4). As anticipated, the dna strands provide the required negative charges for reassembly into virus - like particles . This further ensures that no empty particles are obtained with this strategy and that formed particles always contain a negatively charged cargo . Size - exclusion chromatograms for ccmv containing (a) ssdna, (b) gox ssdna, (c) gck csdna, and (d) gox gck, with monitoring at = 260 (red), 280 (blue), and 450 nm (black), for dna, ccmv, and flavin (gox), respectively . Negatively stained transmission electron microscopy of (e) ssdna, (f) gox ssdna, (g) gck csdna, and (h) gox gck encapsulated ccmv assemblies . The assembled ccmv - like particles were characterized by negative staining transmission electron microscopy (tem) (figure 3e h) and dynamic light scattering (dls, figure s5), showing spherical structures of around 20 nm in diameter . The size of around 20 nm indicates the formation of t = 1 icosahedral symmetry that is composed of 60 identical capsid subunits . Furthermore, co - encapsulation of gox and gck in a single particle was confirmed with sds - page and western blot analyses (figures s6 and s7). Additionally, their concentrations and relative ratios were estimated by gel densitometry, which suggested a gox / gck / capsid protein ratio of approximately 1:1.4:60 . Since the capsid protein is composed of 60 identical subunits, we estimate that only a single gox ssdna is confined inside ccmv - like particles for cascade system i and a hybrid of 1 gox dna and 1 or 2 gck csdna is confined for cascade system ii . To confirm the assembly of ccmv - like particles with t = 1 icosahedral symmetry, we analyzed the gox ssdna - loaded ccmv - like particles with cryo - electron microscopy (cryo - em) to calculate their native three - dimensional reconstruction (3dr) (figure 4a). The sample contained particles with spherical and elongated profiles as well as irregular assemblies (figure 4a, inset). Two - dimensional classification followed by a three - dimensional classification using relion software resulted in two sizes of icosahedral capsids with t = 1 architecture . Whereas class i capsids were 214 in diameter (figure 4b), class ii capsids were 226 (figure 4c). Three - dimensional cryo - em reconstructions of gox ssdna - loaded ccmv capsids . (a) cryo - electron micrograph of gox ssdna - loaded ccmv capsids . Two - dimensional class averages derived from the final 15481 particle data set (inset). (b) surface - shaded representation of the outer surface of the class i t = 1 capsid (diameter 21.4 nm) viewed along a 2-, 3-, and 5-fold axis of icosahedral symmetry (top to bottom). Models of the class i t = 1 capsid, with the front half of the cargo and protein shell removed (right). (c) surface - shaded representations of the outer surface of the class ii t = 1 capsid (diameter 22.6 nm) (as in b). Capsids i and ii made up 50% of the total particles in the sample (70% class i, 30% class ii). The cryo - em images analyzed for processing are in fact snapshots of the dynamic states of the sample; the ratio observed could be due to displacement of dynamic equilibrium toward class i t = 1 capsids (70% class i, 30% class ii). The two particle sizes might be related to the reported dynamic swelling of the t = 3 ccmv native capsid, as the size difference of 5% involves a 7 outward radial expansion and widening of the pores, hinting at possible structural breathing . In both t = 1 capsids, the pentamer bases were strongly connected to the underlying gox ssdna cargo, although the capsid surface pores were distinct . Whereas class i capsid pentamers barely left any space between their lateral contacts, those of class ii capsids were clearly separated and left large pores, especially at the icosahedral 2-fold axes (figure 4b, c, arrows). Docking of ccmv capsid protein (cp) dimer into the cryo - em density maps of gox - ssdna - loaded t = 1 vlp showed major structural differences of the two classes (figure 5a, b). Connecting densities between pentamers and cargo were mediated by residues 4250 of the cp n - terminal region (figure 5c, dark blue), although the preceding region (residues 2741) could also be involved (figure 5c, pink). The cp c - terminal ends were responsible for cp dimer assembly in class i ccmv t = 1 capsids (figure 5a, arrows; figure 5d, red). The hinge angle formed between cp dimers in gox ssdna - loaded t = 1 class i capsids was 60 (figure 5d); it resembles that found at the quasi-2-fold axes of the swollen t = 3 ccmv capsid and in other cp dimers such as the phthalocyanine - loaded t = 1 vlp . Ccmv cp dimers are the building blocks of native t = 3 virion capsids as well as of in vitro assembled structures such as tubes and icosahedral capsids with t = 1 (containing 30 cp dimers), t = 2 (60 dimers), and t = 3 (90 dimers) architecture . The outward expansion of the class ii capsid pentamer entailed the disappearance of or a great reduction in dimeric contacts (figure 5b, arrows) and indicated that these interactions contribute much less to class ii capsid stability than to that of class i capsids . Assuming the same building block is involved, the class ii capsids are based on pentamers bound weakly by the cp c - terminal ends (figure 5e, red), which adhere strongly to the polyanionic cargo . Pseudoatomic model of gox ssdna - loaded ccmv capsids . (a) t = 1 class i capsid viewed down a 3-fold axis from outside, with docked ccmv cp atomic coordinates . (b) class ii t = 1 capsid viewed down a 3-fold axis from outside (as in b). (c) pentamer contacts with the cargo mediated by residues 4250 in the n - terminal region (dark blue, bottom view). The n - terminal region residues 2741 might also contribute to cargo side view (top), top view (bottom). The hinge dihedral angle is indicated . Cp monomers in the class ii dimer are 6.5 further apart than class i dimers (n - terminal 2732 region is omitted). After imposing icosahedral symmetry in class i and ii capsids, we observed the packed cargo as a hollow sphere (9.6 10 and 1.2 10, respectively), with numerous connections to the t = 1 capsid inner surface . Based on the atomic model of gox (pdb 1gal), several copies of gox could be encapsulated in the capsid, although our biochemical analyses indicated the presence of a gox dimer only . This discrepancy is probably due to the chemical modification of accessible lys residues of gox that are covalently bound to ssdna . Capsid connections observed in the 3d cryo - em maps probably represent the interaction of the arm region with negatively charged dna strands . Gox ssdna packaging resulted in a slightly disordered icosahedral capsid (also reflected in a limited map resolution), but this cargo enabled structural polymorphism with weak cp interactions in the dimer . Both t = 1 capsids coexist in dynamic equilibrium, probably enabled because the cp ssdna interactions are more flexible (or less well - defined) than the cp ssrna interactions . To our knowledge, this is the first demonstration of the formation of a t = 1 ccmv - like structure templated by a biological soft material that also displays an extreme capsid swelling . Following structural characterization of enzyme dna complexes inside t = 1 ccmv - like particles, we proceeded to monitor the enzymatic activity of both cascade systems to examine whether the encapsulated complexes were still catalytically active . For cascade system i (figure 1a and figure 6a), we deliberately chose the ssdna sequence coupled to the gox to be that of a hemin - binding dna quadruplex, the so - called dnazyme . In the presence of hemin, the ssdna spontaneously forms a scaffold that mimics the catalytic properties of horseradish peroxidase (hrp). Dnazyme was monitored via the production of abts at = 410 nm upon addition of glucose to the system . The activity plot obtained for encapsulated gox dnazyme shows that both gox and dnazyme remained catalytically active after encapsulation (figure 6b and figure s8a). An increase in both km values (2.2-fold) and kcat values (1.7-fold) is observed when the system is encapsulated (summarized in table s1). (a) schematic representation of cascade system i in the presence of a competing enzyme, catalase (encapsulated processes shown in gray boxes). (b) kinetic measurements of cascade system i; the production of abts was monitored at = 410 nm at different glucose concentrations . (c) kinetic measurements of cascade system ii; the production of nadph was monitored at = 340 nm at different glucose concentrations . (d) kinetic measurements for the production of abts in the presence of and after ph inactivation of the competing enzyme, catalase . For cascade system ii consisting of gox, gck, and 6-pgdh (figure 1b), we monitored the formation of the end product, nadph at = 340 nm, upon addition of glucose to the system . Both enzymes (gox and gck) are therefore required for the reaction and its visualization at 340 nm . The activity profile in figure 6c and figure s8b confirms that both enzymes were present in the system and still active upon hybridization and subsequent encapsulation . While the km values remain similar for both systems, the kcat values show a 2-fold increase for the encapsulated system (summarized in table s1). Based on the recurring trends, a slightly higher turnover number (kcat) upon pathway encapsulation is estimated, although the protein concentration determination by gel densitometry is expected to have a large deviation and consequently also the kcat . An eventual increase might be the result of a local enhancement in effective molarity due to confinement or of the channeling effect when multiple enzymes in a cascade pathway are brought to a close proximity inside a confined system . The enzymatic activities observed for both cascade pathways indicated that the substrate glucose was able to diffuse into the capsid shell . We further investigated whether the intermediate of the cascade could also diffuse freely or was trapped inside the cagelike structure during the reaction . In order to confirm the state of the intermediate, h2o2 is broken down to water and oxygen by the enzyme catalase, and it can therefore act as an external competitor with the dnazyme . In contrast, if the h2o2 intermediate is trapped inside ccmv (as proposed for the bacterial microcompartments), the kinetics of abts production should remain unaltered . Instead, we observed almost complete suppression of abts production in the presence of catalase (figure 6d). Only upon lowering the buffer to ph 4 and hence inactivating catalase (t = 60 min) could the h2o2 conversion by dnazyme be restored while maintaining the stability of the particles, as confirmed by sec and tem analyses in figure s9 . Taken together, in agreement with a previous report, we also observed that the ccmv capsid shell is permeable to small molecules such as h2o2, which can diffuse out of the capsid shell and react with the competing enzyme . Nevertheless, it should be noted that the dnazyme is likely to exhibit lower catalytic efficiency and lower affinity to h2o2 compared to catalase, which could also lead to the diffusion of h2o2 out of the ccmv capsid . We have presented a highly effective strategy of using single - stranded dna for the controlled noncovalent packing of enzyme cascades in a single protein capsid assembly . To demonstrate the versatility of this strategy, two different cascade systems based on glucose oxidase were assembled inside the protein shell of ccmv at ph 7.5 and this encapsulation strategy resulted in icosahedral structures of approximately 20 nm, which were further analyzed with 3d cryo - em . The resulting 3d reconstruction provides the first - time demonstration of t = 1 structured assemblies of ccmv around a biological soft matter template . In addition, an extra - swelling phenomenon was indicated on the basis of the coexistence of two differently sized particles of similar structure and origin . The method presented for assembling virus - like particles can provide a structural and functional basis to analyze bacterial protein organelles and will further improve our understanding of their containment properties and biochemical function . The ssdna (5-hs-(ch2)6-gggtagggcgggttgggtttt-3) and csdna (5-hs-(ch2)6-aaaacccaacccgccctaccc-3) oligonucleotide sequences were synthesized by eurofins mwg operon . For the coupling of dna to enzymes, the bifunctional cross - linker sulfo - emcs (n-[-maleimidocaproyloxy] sulfosuccinimide ester) was purchased from pierce . D - gluconate / d - glucono--lactone assay kit was purchased from megazyme and used as provided . All other reagents were purchased from sigma - aldrich or fluka unless stated otherwise and were used without further purification . A stock solution of hemin (5 mm) was prepared in dmso and stored in the dark at 4 c . Hcl, 500 mm nacl, 50 mm mgcl2, 1 mm dtt, ph 7.5) to obtain ccmv dimer coat proteins (ccmv - cp) (500 m). Gck, and gox gck were buffer exchanged against milli - q water using amicon ultra centrifugal filters (30 kda or 10 kda mwco). Gck and ccmv cp (in assembly buffer) were mixed in a 4:1 (v / v) ratio and incubated for 2 h at 4 c before purification by size - exclusion chromatography (sec) using a superose 6 10/100 gl column, eluting with 50 mm tris hcl, 100 mm nacl, 10 mm mgcl2, 0.5 mm dtt at ph 7.5 . Protein fractions were collected and analyzed by sds page, agarose gel, and western blot analysis . For the complete procedure, samples (5 l) were applied onto formvar carbon - coated grids . Uranyl acetate (5 l, 1% w / v) was added and the excess liquid was drained after 20 s and dried for 30 min at room temperature . The samples were examined on a feg - tem (phillips cm 30) operated at 300 kv acceleration voltages . Ssdna - loaded vlp (5 l) were applied to one side of quantifoil r 2/2 holey grids, blotted, and plunged into liquid ethane in a leica em cpc cryofixation unit . The grids were analyzed in a tecnai g2 electron microscope equipped with a field emission gun operating at 200 kv, and images were recorded under low - dose conditions with a fei eagle ccd at a detector magnification of 69,444x (2.16 / pixel sampling rate). Image processing operations were performed using xmipp and relion, and graphic representations were produced with ucsf chimera . The xmipp automatic picking routine was used to select 15481 particles, and defocus was determined with ctffind . Images were 2d - classified using the appropriate relion routine and 7932 isometric particles were selected . The structure of phthalocyanine - loaded ccmv t = 1 capsid was filtered out to 30, and the cargo density was masked . This map was used as an initial model for 3d classification of spherical particles, using relion to select 5572 (class i) and 2318 (class ii) particles; these data sets were used to obtain the final 3drs using the relion autorefinement routine . Resolution was assessed by gold standard fsc between two independently processed half - data sets . Applying a correlation limit of 0.5 (0.3), the resolution for class i and ii 3d maps was 22.7 (22.2) and 25.6 (21.3), respectively . The chimera fitting tool was used to dock the atomic structure of a whole pentamer from the x - ray structure of ccmv (pdb entry 1cwp) into the cryoem maps . Substrate solutions containing various glucose concentrations (01 m, 180 l) were prepared . An enzyme solution containing either (1) free gox gck (60 l) or (2) encapsulated gox 6-pgdh (55 u / ml, 2 l) and 16.1 mm nadp+ containing 69.4 mm atp were added to each reaction mixture at ph 7.5, according to the manufacturer s instructions (megazyme kit). The reaction was started upon addition of glucose (120 l) to enzyme (82 l), and formation of reduced nadph was monitored at = 340 nm in 100 s time intervals over 2 h at 27 c . Stock solutions containing both substrates glucose (ranging from 01 m) and 4 mm abts were freshly prepared at room temperature . Dnazyme (30 l) and hemin (30 l, 5 m) or (2) encapsulated gox dnazyme (30 l) and hemin (30 l, 5 m) were prepared and incubated at rt for 2 h. to each enzyme containing solution (60 l) was added the substrate solution containing both glucose and abts (120 l), and the reaction was monitored immediately at = 410 nm for the conversion of abts to abts at 27 c in 100 s time intervals over 2 h. control experiments containing hemin, glucose, and abts were performed under the same reaction conditions . Experimental data were corrected for background absorbance (using the control experiment as a reference). The concentration of abts or nadph was determined using the lambert beer law, assuming extinction coefficients of abts (410 nm = 36000 m cm) or nadph (340 nm = 6300 m cm) before plotting concentration (m) vs time (min) curves, from which the velocity (v) was determined (m / min). A dilution factor (df) relative to the enzyme (gox) and a proportionality factor of abts to substrate consumption (p = 1/2) were used to correct the velocity values as described in eq 1.1
Castleman s disease, giant lymph node hyperplasia, is an autoimmune, lymphoproliferative disease that shows itself with the enlargement of lymph nodes and varied clinical presentations . Castleman s disease commonly involves mediastinum and hence it is thoracic in most of the reported cases . It is a rare disease that presents itself by hyperplasia of lymph nodes with no malignant origin . This disease is known by other names such as angiofollicular lymph node hyperplasia or giant lymph node hyperplasia . Simply, the pathology of this disease can be attributed to the hypervascular of lymph nodes and hyalinization of vessels . From a pathological perspective, three variants of this type of tumor are recognized: hyaline vascular cd type, plasma cell type, and mixed type . From a clinical standpoint, this tumor is found in two types of unicentric and multi - centric, in which the unicentric type is much more common . Previous studies have presented it as an unusual finding in retroperitoneal ct imaging . In this study, we aim at introducing one patient with castleman s disease who had referred with clinical picture of occasional abdominal pain . A 34-year - old woman complaining of occasional abdominal pain referred to the surgery clinic . There was no remarkable point in physical examination and the patient did not have any other clinical symptoms . Laboratory findings only reported microcytic anemia (mch: 18.5, mcv: 63, hemoglobin 10.2 g / di). Imaging results were chest and abdominal x - ray without any remarkable point . In abdominal ultrasonography, a solid and firm tumor with 12.25.36.6 cm was reported in patient s retropritoneum . For more evaluation, the tumor was not attached to the walls of the intestines and it did not cause a blockage . With coordinates obtained by ct scan and ultrasonography (us), a big tumor was found in the retropritoneum that was solid and firm but was not attached to the walls of the intestine or to the lymph nodes of that area . In the operation area, the tumor was completely removed in order to treat patent and more reviews . From the macroscopic point of view, the tumor was shaped like an egg, covered by a fibrous thick capsule, with no bizarre appearance and without visible bleeding on its surface (figure 1). In cutting points, its surface was homogeneous, chocolaty and in terms of consistency, hard . Although it was similar to lipoma in early studies, but the initial pathologic study reported its lymphoid origin that rejected the possibility of malignancy . Macroscopic view of the resected castleman s tumor (the tumor was shaped like an egg, covered by fibrous thick capsule, with no bizarre appearance and bleeding). More histopathological investigation of tumor was an extensive lymphatic tissue containing hyperplastic follicles that was placed in the frame of a lymphoid tumor . Mantle zones of follicles had spread and small germinal centers were seen (figure 2). Microscopic view; expanded mantle zone and the small germinal centers of the lymphoid follicles have shown apparently . Hyaline vascular type revealed castleman s disease (h&e, x400). Among these germinal centers, interfollicular stroma as hyperplasia is defined as post - capillaries venules, in which combination of plasma cells and eosinophils are seen . Histopathology of these samples gave a definitive diagnosis of angiofollicular lymph node hyperplasia, which in this case was hyaline vascular type . A chest ct scan was performed at the end of treatment and its review implied the absence of a similar tumor elsewhere . The patient did not have any particular postoperative problems and was discharged on day-7 after surgery . Patient s follow up was done until 9 months after discharge for symptoms of recurrence or any kind of clinical abnormalities . Our study was approved by the medical ethics committee, according to the helsinki declaration . Castleman s disease is a kind of rare pathology, usually benign, with unclear etiology and prevalence . It is reported in childhood and adolescence periods in much lower numbers . In most of the reported cases, the disease arises in the chest and especially in the mediastinum . From other involved areas, we can mention mesentery, armpits, neck and in very rare cases in retroperitoneum . Despite the fact that the etiology of this disease remained unknown, we can cite autoimmune disease, and some viral diseases as predisposing factors for this disease, abnormality of test results and clinical examinations . None of the above diseases was seen in this reported case, even until the end of the follow - up period . Therefore, these immunity factors are discussed as predisposing factors and not as a main etiological factor . As mentioned, the castleman s disease is clinically divided into two groups of unicentric and multi - centric . In unicentric type, that is more common, the possibility of invasion is lower and is seen with hyaline vascular type . Therefore, it is difficult and sometimes impossible to diagnose only based on disease symptoms before the operation and pathology study, especially in such diagnosis . Among clinical tests, it is possible that complete blood to be along with anemia with small changes, similar to what was observed in our patient, while this finding is not specific . In some case reports, lack of result from these cannot be a reason to reject castleman s disease . Some studies started their diagnostic procedures with endoscopic ultrasound - guided fine - needle aspiration (eus - fna). In this study, we used ultrasonography after abdominal x - ray leading to tumor detection and locating its exact location by using an abdominal ct scan . Of course, since no study with a high sample size has been conducted in this field, sensitivity and specificity of none of these diagnostic methods were exactly identified . Histopathology of tumor tissue after surgery is the only way for tumor (and its type) diagnosis . Most of the castleman s tumor is seen as homogeneous and hypoechoic tumor in the view of the sonography . In color doppler view, it can be concluded that the radiological view is not specific enough to detect this disease and the observed views could be mistaken with each type of benign or malignant lymphomatous tumor . In this study, similar to most other published case reports, the tumor was of the unicentric type while multi - centric type is less common and should be noted with a broader therapeutic approach . It should be noted that in this disease, the localized type often responds to surgical treatment alone . Complete removal of tumor and its margin will suffice with the treatment of laparotomy and even laparoscopy . These kinds of tumors do not have invasive behavior and are completely benign . On the other hand, castleman s disease is a kind of rare vascular hyperplasia that has often benign and with noninvasive behavior . There is no reliable diagnostic method and its definitive diagnosis is based on histopathology report . It is often found in the chest, especially in the mediastinum and is asymptomatic in most cases.
The paternal contribution to an embryo plays an important role in understanding early developmental processes and their effect on the health of a child . Sperm cells are highly differentiated, polarized and specialized, containing only the constituents required during and after fertilization (early embryonic development). However, the contribution of the male gamete to embryogenesis has not been well investigated . For quite some time, sperm have been considered mere vectors that carry the paternal genetic component to the oocyte . However, the contribution of sperm to the embryo has recently been better elucidated, with accumulating evidence suggesting that various spermatozoal components actively participate in early human development (1 - 4). During fertilization, the sperm transmits not only nuclear dna but also oocyte activation factor (oaf) (critical for fertilization), centrosomes (critical for cell division) (5), and a population of messenger rna (mrna) that are of critical developmental importance (1,6). Studies investigating the epigenetic modifications in the developing sperm cell have provided new insights that may establish a more critical role for the sperm epigenome in the developing embryo . These non - genetic modifications include dna methylation, histone tail modifications, targeted histone retention and protamine incorporation into the chromatin, all of which have a significant influence on the developing sperm cell . Sperm require these changes not only to shield the dna throughout spermatogenesis but apparently, they also require these changes to contribute to the developmental program of the future embryo . Damage to genetic constituents and perturbations in the maintenance of these epigenetic changes have been demonstrated to affect fertilization potential and the early development of the embryo (7). This chapter will focus on the possible genetic and epigenetic contributions of sperm to the development of the human embryo . The maturation of spermatozoa consists primarily of three important phases: the initial proliferative phase, the meiotic division of the chromosomes, and the final maturation step (spermiogenesis). Of the 28010 human sperm normally ejaculated into the vagina, only 200 reach the ampullary region of the oviduct where fertilization takes place (8). Fewer than 1 in 10,000 sperm get close enough to the egg to complete the process of fertilization . However, even these highly competent sperm are frequently not sufficient for sustaining the later development of the embryo . Sperm cells are highly specialized vehicles for transporting chromatin cargo, which consists of dna and its associated proteins . The chromatin in mammalian sperm can broadly be divided into three major structural domains: (1) the vast majority of sperm dna is coiled into toroids by protamines, (2) a much smaller percent (5 - 15%) remains bound to histones and thus retains its nucleosomal structure, and (3) dna that is attached to the sperm nuclear matrix at mars (matrix attachment regions) at intermediate intervals of approximately 50 kb throughout the genome (9). Sperm chromatin is essential for sperm function and subsequent embryonic development; defects in sperm chromatin have been linked to natural reproductive malfunctions, such as spontaneous abortion and assisted reproductive failure (10 - 12). These defects can include disrupted dna integrity, which may affect fecundity and embryo growth, leading to embryo loss (13 - 15). Furthermore, the alteration of chromatin - associated proteins contributes to decreased fertility and poor embryonic growth (16). Most of the techniques used to detect sperm chromatin defects only detect gross defects in dna integrity (17), while the roles of the associated proteins remain a mystery . Here, we will first discuss the role of sperm dna and its associated proteins during embryonic growth . Dna damage (also referred to as dna denaturation or fragmentation) is a common feature of human spermatozoa that affects dna quality . Sperm dna damage is thought to be induced by several mechanisms: (1) apoptosis during the process of spermatogenesis; (2) dna breaks generated during the remodeling of sperm chromatin during the process of spermiogenesis; (3) post - testicular dna fragmentation, which is induced by oxygen radicals, including the hydroxyl radical and nitric oxide, during sperm transport through the seminiferous tubules and the epididymis; (4) dna fragmentation induced by endogenous caspases and endonucleases; (5) radiotherapy and chemotherapy; and (6) environmental toxicants and xenobiotics (18,19). Much concern has been expressed regarding the influence of sperm dna integrity on abnormal reproductive outcomes (20,21). However, one of the well - established and extensively studied causes of dna damage is oxidative stress, which causes the oxidation of the dna bases and leads to the formation of various types of dna adducts . These dna adducts alter the function of the sperm genome, ultimately influencing in vivo or in vitro conception and the subsequent events that occur during early development (22). Dna fragmentation is more frequent in caudal epididymal and ejaculated sperm than testicular sperm (23 - 25). The persistence of high rates of dna damage in ejaculated sperm is obvious, as mature spermatozoa possess a limited capacity to repair oxidative dna lesions (26). It has been suggested that mammalian spermatozoa contain a mechanism by which they can digest their own dna upon exposure to a stressful environment (27); however, the question of whether this mechanism is sufficient to cope with extensive damage arises . The answer lies in several studies postulating that the presence of a critical level of unrepaired dna damage in embryos generated in vivo / in vitro explains the block in embryo development (28). The authors of these studies have arrived at the conclusion that a late paternal effect is responsible for blocking embryo development, which is evident from the outcomes of various assisted reproductive techniques (arts). The fragmentation of sperm dna affects post - implantation embryonic development in icsi procedures; specifically, high levels of sperm dna fragmentation can compromise the viability of an embryo, resulting in pregnancy loss (21,29). Sperm carrying damaged dna can complete the initial process of fertilization; however, the developmentally necessary genes in the damaged sperm dna may hinder embryonic development upon activation of the embryonic genome at the 3-cell stage . Furthermore, sperm dna damage has been linked to delayed chromosomal instability in blastocysts and post - implantation developmental abnormalities (30,31). However, very little is currently known regarding the nature of germline dna damage and the extent to which germ cells are capable of eliminating damaged dna and completing the process of dna repair . Oocytes have been shown to possess different repair pathways to handle a certain level of dna damage in sperm (32). There is little information regarding the fidelity and nature of this oocyte repair mechanism, but these mechanisms are negatively impacted by age . The majority of couples with reduced reproductive fitness may be of advanced age, and women above 35 - 40 years of age may have a defective dna repair system that may not be able to repair extensive dna damage . Oocyte repair mechanisms may also be inhibited by the accumulation of oxidized bases, such as ethenonucleosides, in the sperm genome . The accumulation of such dna adducts is mutagenic and, if not corrected, may increase the mutagenic load in early embryos . The analysis of sperm dna fragmentation is a potentially valuable method for explaining the paternal origin of some unexplained and repeated icsi fertilization and implantation failures (29). Such analysis may even help to determine the most efficient art procedure (33) to reduce the probability of negative paternal contribution . A recent systematic review and meta - analysis of 2,969 couples showed a significant association between the level of dna damage and pregnancy loss in both art and spontaneous conceptions (34). These data affirm the clinical indication for the evaluation of sperm dna damage prior to infertility treatment and the need to investigate the association between sperm dna damage and recurrent pregnancy loss (10,35). The intact structural framework of chromatin, which consists of molecular regulatory factors, is also required for proper embryonic development (9). Among the three types of sperm chromatin structures discussed earlier, histone - bound sperm dna and mars are inherited by the embryo and are most likely required for proper development . At least 2 - 15% of mammalian sperm chromatin is bound to histones rather than protamines (36 - 38). Histones are interspersed throughout the genome, primarily located at gene promoters (39). Another independent study also concluded that entire gene families important for embryo development are preferentially associated with histones in human spermatozoa (38). However, it will be interesting to know whether sperm histones are transmitted to the developing embryo . (40,41) demonstrated that histones with specific modifications in the sperm cell are present in the paternal pronucleus, suggesting that these histones were never replaced . While the protamines in sperm chromatin are replaced with histones supplied by the oocyte after fertilization (42,43), this may not be necessary in regions where histones are already present in the sperm dna . In the sperm nucleus, the chromatin is organized into loop domains that are attached to a proteinaceous structure, termed the nuclear matrix, every 20 - 120 kb . This organizes the chromatin into functional loops of dna that help regulate dna replication and gene transcription (9). Several studies have demonstrated a functional role for the sperm nuclear matrix during early embryogenesis . These data suggest two roles for the sperm nuclear matrix (44 - 47): to facilitate the proper association of dna with the nuclear matrix (required for paternal pronuclear dna replication in the one - cell embryo) and to serve as a checkpoint for sperm dna integrity after fertilization . The maturation of spermatozoa consists primarily of three important phases: the initial proliferative phase, the meiotic division of the chromosomes, and the final maturation step (spermiogenesis). Of the 28010 human sperm normally ejaculated into the vagina, only 200 reach the ampullary region of the oviduct where fertilization takes place (8). Fewer than 1 in 10,000 sperm get close enough to the egg to complete the process of fertilization . However, even these highly competent sperm are frequently not sufficient for sustaining the later development of the embryo . Sperm cells are highly specialized vehicles for transporting chromatin cargo, which consists of dna and its associated proteins . The chromatin in mammalian sperm can broadly be divided into three major structural domains: (1) the vast majority of sperm dna is coiled into toroids by protamines, (2) a much smaller percent (5 - 15%) remains bound to histones and thus retains its nucleosomal structure, and (3) dna that is attached to the sperm nuclear matrix at mars (matrix attachment regions) at intermediate intervals of approximately 50 kb throughout the genome (9). Sperm chromatin is essential for sperm function and subsequent embryonic development; defects in sperm chromatin have been linked to natural reproductive malfunctions, such as spontaneous abortion and assisted reproductive failure (10 - 12). These defects can include disrupted dna integrity, which may affect fecundity and embryo growth, leading to embryo loss (13 - 15). Furthermore, the alteration of chromatin - associated proteins contributes to decreased fertility and poor embryonic growth (16). Most of the techniques used to detect sperm chromatin defects only detect gross defects in dna integrity (17), while the roles of the associated proteins remain a mystery . Here, we will first discuss the role of sperm dna and its associated proteins during embryonic growth . Dna damage (also referred to as dna denaturation or fragmentation) is a common feature of human spermatozoa that affects dna quality . Sperm dna damage is thought to be induced by several mechanisms: (1) apoptosis during the process of spermatogenesis; (2) dna breaks generated during the remodeling of sperm chromatin during the process of spermiogenesis; (3) post - testicular dna fragmentation, which is induced by oxygen radicals, including the hydroxyl radical and nitric oxide, during sperm transport through the seminiferous tubules and the epididymis; (4) dna fragmentation induced by endogenous caspases and endonucleases; (5) radiotherapy and chemotherapy; and (6) environmental toxicants and xenobiotics (18,19). Much concern has been expressed regarding the influence of sperm dna integrity on abnormal reproductive outcomes (20,21). However, one of the well - established and extensively studied causes of dna damage is oxidative stress, which causes the oxidation of the dna bases and leads to the formation of various types of dna adducts . These dna adducts alter the function of the sperm genome, ultimately influencing in vivo or in vitro conception and the subsequent events that occur during early development (22). Dna fragmentation is more frequent in caudal epididymal and ejaculated sperm than testicular sperm (23 - 25). The persistence of high rates of dna damage in ejaculated sperm is obvious, as mature spermatozoa possess a limited capacity to repair oxidative dna lesions (26). It has been suggested that mammalian spermatozoa contain a mechanism by which they can digest their own dna upon exposure to a stressful environment (27); however, the question of whether this mechanism is sufficient to cope with extensive damage arises . The answer lies in several studies postulating that the presence of a critical level of unrepaired dna damage in embryos generated in vivo / in vitro explains the block in embryo development (28). The authors of these studies have arrived at the conclusion that a late paternal effect is responsible for blocking embryo development, which is evident from the outcomes of various assisted reproductive techniques (arts). The fragmentation of sperm dna affects post - implantation embryonic development in icsi procedures; specifically, high levels of sperm dna fragmentation can compromise the viability of an embryo, resulting in pregnancy loss (21,29). Sperm carrying damaged dna can complete the initial process of fertilization; however, the developmentally necessary genes in the damaged sperm dna may hinder embryonic development upon activation of the embryonic genome at the 3-cell stage . Furthermore, sperm dna damage has been linked to delayed chromosomal instability in blastocysts and post - implantation developmental abnormalities (30,31). However, very little is currently known regarding the nature of germline dna damage and the extent to which germ cells are capable of eliminating damaged dna and completing the process of dna repair . Oocytes have been shown to possess different repair pathways to handle a certain level of dna damage in sperm (32). There is little information regarding the fidelity and nature of this oocyte repair mechanism, but these mechanisms are negatively impacted by age . The majority of couples with reduced reproductive fitness may be of advanced age, and women above 35 - 40 years of age may have a defective dna repair system that may not be able to repair extensive dna damage . Oocyte repair mechanisms may also be inhibited by the accumulation of oxidized bases, such as ethenonucleosides, in the sperm genome . The accumulation of such dna adducts is mutagenic and, if not corrected, may increase the mutagenic load in early embryos . The analysis of sperm dna fragmentation is a potentially valuable method for explaining the paternal origin of some unexplained and repeated icsi fertilization and implantation failures (29). Such analysis may even help to determine the most efficient art procedure (33) to reduce the probability of negative paternal contribution . A recent systematic review and meta - analysis of 2,969 couples showed a significant association between the level of dna damage and pregnancy loss in both art and spontaneous conceptions (34). These data affirm the clinical indication for the evaluation of sperm dna damage prior to infertility treatment and the need to investigate the association between sperm dna damage and recurrent pregnancy loss (10,35). The intact structural framework of chromatin, which consists of molecular regulatory factors, is also required for proper embryonic development (9). Among the three types of sperm chromatin structures discussed earlier, histone - bound sperm dna and mars are inherited by the embryo and are most likely required for proper development . At least 2 - 15% of mammalian sperm chromatin is bound to histones rather than protamines (36 - 38). Histones are interspersed throughout the genome, primarily located at gene promoters (39). Another independent study also concluded that entire gene families important for embryo development are preferentially associated with histones in human spermatozoa (38). However, it will be interesting to know whether sperm histones are transmitted to the developing embryo . (40,41) demonstrated that histones with specific modifications in the sperm cell are present in the paternal pronucleus, suggesting that these histones were never replaced . While the protamines in sperm chromatin are replaced with histones supplied by the oocyte after fertilization (42,43), this may not be necessary in regions where histones are already present in the sperm dna . In the sperm nucleus, the chromatin is organized into loop domains that are attached to a proteinaceous structure, termed the nuclear matrix, every 20 - 120 kb . This organizes the chromatin into functional loops of dna that help regulate dna replication and gene transcription (9). Several studies have demonstrated a functional role for the sperm nuclear matrix during early embryogenesis . These data suggest two roles for the sperm nuclear matrix (44 - 47): to facilitate the proper association of dna with the nuclear matrix (required for paternal pronuclear dna replication in the one - cell embryo) and to serve as a checkpoint for sperm dna integrity after fertilization . In the previous section, the importance and contributions of sperm chromatin were discussed . In this section, we will further elaborate the role of sperm rnas and their indispensability to the spermatozoa and embryo development . In the late 1950s and early 1960s, controversy regarding the role of sperm rnas arose, and the presence of these molecules was questioned . Various landmark studies in the 1970s concluded that bovine spermatozoa were transcriptionally active but this activity was localized to the mitochondria (48 - 50). Contributing to these controversies, pessot et al . Demonstrated the presence of nuclear rna in rat and human sperm . The rna was extracted from sperm and analyzed by electrophoresis on a 10% polyacrylamide gel and 7 m urea . The electrophoretic profile revealed a complex set of bands ranging in size from trna to high - molecular - weight components . On average, an rna content of approximately 0.1 pg per rat or human sperm was found (51). The repackaging of dna into a nucleotorroidal conformation, which is approximately 20 times more condensed, enables the complete shutdown of the spermatid nucleus (52,53). The gradual shutdown of rna transcription begins during meiosis, when the paired sex chromosomes are accommodated in the male germ cell, leading to the repression of gene expression on the x and y chromosomes (54,55). The shutdown of transcriptional activity in human spermatozoa has been confirmed (56), strengthening the previous observations . The retention of mrnas in spermatozoa begins to occur during the early stages of spermatogenesis . In the late 1990s, various studies documented the presence of different types of rnas in human spermatozoa . First documented the presence of c - myc mrna in the mid - piece and tail region of human spermatozoa (57). Mrnas coding for the following molecules have been found in human ejaculate spermatozoa: hla (58), integrins (59), cyclic nucleotide phosphodiesterases (60), the l - type calcium channel and n - cadherin (61,62), estrogen and progestin - like receptors (63,64), nitric oxide synthase (nos) (65), and, surprisingly, insulin (66), among others (67). Complex rna populations have also been reported in the sperm of cows (68,69) and human spermatozoa (1,70,71). These mrnas were assumed to be residues left over from spermatogenesis, but there is evidence that the spermatozoa deliver a unique set of mrnas to the oocyte . The first group of mrnas has a specific function during spermatogenesis but does not exhibit an obvious function post - fertilization . The presence of this set of remnant mrnas could serve as a diagnostic tool with which to follow the fidelity of the later phases of spermatogenesis (71). A second group of rnas (e.g., mrna coding for plc - z) also originates from the testicular germ cells and may have an additional role in the fertilized oocyte . A third not yet extensively studied group of sperm mrnas (e.g., mrna coding for clusterin) may originate from a non - testicular source and, after incorporation into the sperm, could be introduced into the oocyte during fertilization . It is of considerable interest that foreign rna constructs can be introduced into the sperm cell to be expressed by the oocyte after fertilization . These non - testicular rna - containing sperm can take up dna and rna in vitro; these molecules are not only delivered to but also expressed in the fertilized oocyte (72,73). This in vitro experiment showed that, in theory, foreign sperm rna could play a role in early embryogenesis . Furthermore, the fact that some of these spermatozoal mrnas are also found in zygotes indicates that these transcripts may be functionally important (74). (75) were the first to prove that sperm rna can influence embryo development by studying the kit gene - derived heritable effect, which appeared to be affected in heterozygous mice carrying a wild - type kit allele and a silent kit allele . This work was followed by a study showing that the pathological overexpression of cdk9, a key regulator of cardiac growth in the mouse, could be induced and heritably transmitted following the intraooplasmic injection of rnas targeting the gene, including the sequence - related expression of mir-1 mirna . Mir-1 is among a number of small inhibitory rnas that are present in sperm (76). A recent report also demonstrated that some mrnas can be translated de novo, supporting the hypothesis that a population of mrnas may have a function during or beyond the process of fertilization (77). As there is no evidence that the proteins encoded by the majority of mrnas found in mature spermatozoa are also present in sperm (67), these mrnas may be viewed as potential contributors to early embryogenesis . In addition to mrna, spermatozoa are enriched in antisense rnas and micrornas (mirnas). These mirnas have been detected in human spermatozoa, raising the possibility that these molecules may play a role in early fertilization events (6) and embryo development by regulating the expression of various genes . For example, sperm - borne microrna-34c is required for the first cleavage division in mice (78). A recent survey of small rnas in sperm also revealed a complex population of male - derived sncrnas (small, non - coding rnas) that are available for delivery upon fertilization (79). In addition to finding the mirna class previously detected in mature mouse, porcine, and human spermatozoa (6,80,81), this survey also identified pirnas for the first time (79). Any alteration in the amount or composition of sperm mrnas may indicate abnormalities in spermatogenesis, which may later affect embryo development . The mrna fingerprints of normozoospermic and teratozoospermic men have been shown to differ (82). Additionally, variations in the expression of two sperm rnas coding for ldhc transcript variant 1 and tpx1 have been reported in men with poor sperm motility (83). The expression profiling of human spermatozoa by serial analysis of gene expression (sage) revealed 389 clustered genes, with a highly selective grouping among the most abundant sage tags (85). Approximately 25% of these tags (96) were related to dna - dependent transcription or transcriptional regulation . In addition, a comparison of the spermatozoa used in homologous intrauterine insemination showed a difference in the transcripts of the two groups (patients achieving pregnancy versus those who did not; both fresh and frozen spermatozoa were used) (86). The authors found 741 exclusive transcripts that were expressed only in the pregnant group and 976 transcripts that were expressed only in the non - pregnant group . Several studies have reported that certain sperm transcripts do have an important role in early embryogenesis . The presence of mrna transcripts encoding psg-1 and hla - e in human spermatozoa has previously been confirmed (84,85). Conversely, microarray analysis of the transcriptome of the human oocyte did not demonstrate the presence of psg-1 mrna and showed that hla - e mrna was down - regulated 87 . An investigation of these transcripts showed significantly higher levels of psg1 and hla - e mrna in the fertile group than the infertile group (88). In accordance with this study, a preliminary examination of the psg1 gene also showed a lower level of psg1 expression in the male partners of couples experiencing recurrent pregnancy loss than in fertile men (in press). It has been speculated that the psg1 protein may play a crucial role in supporting early gestation and protecting the fetus from the maternal immune system . This hypothesis is supported by the fact that psg1 is able to modulate monocyte / macrophage metabolism to regulate t - cell activation and proliferation . In addition, psg1 induces the secretion of anti - inflammatory cytokines by monocytes (89). We recently investigated the expression of a few important genes (wnt5a, hsp90, and prm2) that have been postulated to have a critical role in early development (unpublished). The prm2 and hsp90 expression patterns were significantly altered in the male partners of couples with idiopathic recurrent pregnancy loss, while no association with recurrent pregnancy loss was found for wnt5a . Scientists investigating the heat - shock response (hsr) have focused on developmental processes because of the remarkably unusual characteristics of heat shock protein (hsp) expression in pre - implantation embryos and gametogenesis . A striking hsp expression pattern is exhibited in embryos during gametogenesis and in stem cell and differentiation models, and the expression of these proteins was shown to be stage - specific in both tissue models and male germ cells, the latter of which exhibited impaired abilities to mount a classical hsr . In addition, spermatogenesis and pre - implantation embryos showed extreme sensitivity to heat stress . The basal levels of hsfs and, even more interestingly, the ratios between different hsfs, which could vary from one individual to another, could contribute to reproductive success versus infertility or developmental success versus failure in humans . Our study revealed significantly higher levels of hsp90 and significantly lower prm2 levels in the male partners of couples with idiopathic recurrent pregnancy loss . This result may be due to higher levels of free radicals causing oxidative stress, which is compensated for by increased hsp90 expression . High free radical levels in spermatozoa may cause a pronuclear block, impair cleavage and lead to blastomere fragmentation and poor - quality blastocysts . The glutathione system (gs) is an oxidative stress defense system in sperm that is specifically controlled by gpx family members and has been correlated with embryo morphology on day 3 . The results of this study indicated that sperm - derived mrna may condition the human embryo and persist to the cleavage stage (90). As explained earlier, protamines play a crucial role in the condensation of sperm chromatin and the protection of the paternal genome from internal and external environmental insults . Altered prm2 expression is reported among men with poor fertilizing capacity (91), and a lack of prm2 leads to sperm dna damage and embryo death in mice (92). While high - throughput technologies have provided a glance at the mrna population contained in spermatozoa, future studies should focus on the functional aspects of these rnas in the growing embryo . The results from such studies will further strengthen the correlation between the mrna fingerprint of sperm and embryogenesis . Telomeres are evolutionarily conserved tandem hexameric repeats at the ends of chromosomes that maintain genomic integrity and chromosome stability . Telomeres serve as a biological clock and undergo attrition at a rate of 50 - 200 bp per cell division . The telomeres in germ cells are 10 - 20 kb in length, compared with 5 - 10 kb in somatic cells . The inheritance of telomere length in the embryo is a complex trait codetermined by the length of the telomeres in the sperm and ovum, the age of the father, free radical levels and gender . Tandem telomere repeats are rich in guanine, the nucleotide with the lowest oxidative potential and, thus, the most susceptibility to oxidative damage . We have previously shown high levels of free radicals to be associated with dna damage in the semen and sperm, as well as shorter telomeres in the male partner of infertile couples and couples experiencing idiopathic recurrent pregnancy loss . As telomeres are histone - bound and located in the periphery of the sperm nucleus, telomeres are highly susceptible to oxidative damage . This induces gc to ta transitions, single- and double - strand breaks and accelerated telomere shortening . Inheriting shortened telomeres from the father may thus result in impaired cleavage and embryonic development . Reported that sperm telomeres are the first structures to respond to the oocyte signal for pronucleus formation (93), and rodriguez and colleagues (94) later reported that shortened sperm telomeres are associated with sperm dna fragmentation and abnormal embryonic development . Concordance in telomere length is required for synapsis, homologous recombination, and normal chromosome segregation . Sperm with shortened telomeres show segregation abnormalities and nondisjunction, thus giving rise to aneuploid sperm after meiosis . Shortened telomere can increase the incidence of offspring with major or minor congenital malformations, childhood cancers, perinatal morbidity, developmental delay, and failure to thrive . In an ongoing study in our laboratory, we have found significantly shortened telomeres in the male partners of couples experiencing idiopathic recurrent pregnancy loss . We did not find an association with high levels of reactive oxygen species in this pilot study; however, this was a very clinically significant finding, and studies are still ongoing to further validate this result . Genomic imprinting is a parental, origin - specific, gene - marking phenomenon that is crucial for normal mammalian development . Imprinted genes are characterized by epigenetic modifications (95,96), including dna methylation, and are associated with differentially methylated regions (dmrs) that are methylated on either the paternal or maternal allele . Epigenetics refers to phenotypic changes that are caused by mechanisms other than changes in the dna sequence (thus the name epi- (above or over) genetics). The methylation of primary dmrs is presumed to be maintained throughout embryonic development, including the pre - implantation stages, during which extensive demethylation of the genome takes place . Male germ cells undergo unique and extensive chromatin and epigenetic remodeling soon after their specification (determination to become a spermatocyte) and during the differentiation process to become a mature spermatozoon (97). The epigenetic program of sperm is unique and tailored to meet the needs of this highly specialized cell . The unique nuclear protein landscape in sperm creates a chromatin structure that is between six and 20 times more dense than nucleosome - bound dna, ultimately resulting in a tightly condensed nucleus (53,98). Although the mechanisms regulating and orchestrating specification and spermiogenesis remain poorly understood, some progress has been made in elucidating the molecular changes associated with these complex cellular changes . Recent studies analyzing sperm dna methylation, histone modifications and the rna transcripts in spermatozoa have further established the role of the sperm epigenetic program in the developing embryo . The importance of dna methylation has been demonstrated globally, regionally, and at the single locus level in both humans and animal models . Multiple targeted studies have been performed in animal models to establish a clear role of dna methylation in sperm and embryos . El hajj et al . (99) suggested that the improper methylation of repetitive elements may be linked to recurrent pregnancy loss . Methylation abnormalities in the crem promoter were observed in a subset of patients with protamine ratio abnormalities, as well as in patients presenting with various forms of male factor infertility (100). Recent data demonstrate that the aberrant methylation of promoters for specific genes (e.g., dazl and mthfr) and general gene classes, such as imprinted loci, is strongly associated with various forms of infertility and sperm defects in men (101 - 103). Twin studies have played an essential role in enabling the estimation of phenotypic heritability, and these studies now offer an opportunity to study epigenetic variation as a dynamic quantitative trait . Monozygotic (mz) twin studies have proven to be very effective in answering key questions ranging from the genetics of social behavior and the nature versus nurture question to the heritability of phenotypic variation and disorders (104,105). Several studies have examined dna methylation patterns in twins, and a recent study found that mz twins exhibit a considerable degree of variability in dna methylation patterns, which may impact the variability of gene expression and possible differences in disease susceptibility . Along with the current interest in cpg methylation, recent data have suggested that the intermediates formed during dna demethylation may be important epigenetic regulators . Most prominent among these intermediates is 5-hydroxymethylcytosine (5-hmc). A recent study by pastor et al . Revealed a pattern of 5-hmc enrichment in transcriptionally poised genes in stem cells (106). This unique localization suggests that 5-hmc has a role in embryonic stem cells and possibly the epigenome of multiple other cell types . The timing of the establishment and removal of methylation is critical to normal spermatogenesis . During cell division, the dna in male germ cells is packaged in nucleosomes comprised of histone 2a (h2a), histone 2b (h2b), histone 3 (h3) and histone 4 (h4), all of which are susceptible to covalent modifications . Histone modifications, such as acetylation, methylation, ubiquitylation and phosphorylation, have emerged as the main players regulating epigenetic modifications . Each of these chemical modifications to histones can influence gene repression and/or activation . In post - implantation mammalian embryos, germ cells undergo several changes in their epigenetic profile during the different stages of meiosis . Pgcs enter mitotic arrest in males, whereas pgcs are arrested in prophase of meiosis i in females . Global nuclear remodeling occurs in haploid round spermatids, although some histone marks, such as h3k9me2, on the inactive x chromosome are retained (107,108). The testis - specific linker histone variant h1t2 appears at this stage and plays a crucial role in chromatin condensation during spermiogenesis (108). Later, the linker histone variant hils1 (histone-1-like protein in spermatids 1) is expressed in elongated spermatids . In the histone - protamine exchange process, nuclear histones become hyperacetylated during spermiogenesis and disassemble shortly thereafter, replaced by transition proteins (tp1 and tp2). In the final stage of spermiogenesis, the transition proteins are removed and replaced by protamines (109). The incorporation of protamines into sperm chromatin induces dna compaction, which is important for the formation of spermatozoa and for providing a safe environment for the genome . The presence of somatic - like chromatin in the sperm nucleus could transmit different epigenetic information to offspring . (110) suggested that the genome - wide dna methylation pattern observed during spermiogenesis changes little after the pachytene spermatocyte stage . Many epigenetic modifiers, including dna methyltransferases, histone modification enzymes and their regulatory proteins, play essential roles in germ cell development . Some of these modifiers are specifically expressed in germ cells, whereas others are more widely expressed . The crucial roles of germ - cell - specific genes, such as dnmt3l and prdm9, were revealed by conventional knockout studies (111 - 113). A recent report showed numerous intra- and inter - individual differences in the dna methylation of human sperm samples, which could contribute to phenotypic differences in offspring . Furthermore, it has been reported that sperm samples from oligospermic patients often contain dna methylation defects at imprinted loci (114,115). Kobayashi and his associates reported methylation defects in 17 of 78 embryos conceived by art . He found that seven of these 17 embryos had inherited these altered methylation patterns paternally, while the rest were believed to have resulted from the art process . The altered hormonal milieu associated with art and the retrieval of epigenetically immature oocytes may result in an increase in epigenetic / imprinting defects in children conceived through art . Imprinting errors in the developing fetus have been identified and shown to cause severe pathologies . Evidence suggests that prader - willi syndrome (pws) and angelman syndrome (as) arise from the functional loss of several paternally expressed genes . The prader - willi / angelman imprinted domain on human chromosome 15q11-q13 imprinting defects affecting the pws / as region can arise from the failure to demethylate the pws - sro in the male germ line, the failure to methylate the maternal pws - sro, or the failure to maintain pws - sro methylation after fertilization (116). Some studies have also suggested that the use of art increases the risk of imprinting diseases, as the germ cells of infertile couples are prone to epigenetic instability . A pattern of decreased genome - wide methylation in sperm has also been associated with poor embryo quality in rats and decreased ivf pregnancy rates in humans (117). (117) used 5-methyl - cytosine immunostaining as an indicator of the genome - wide methylation pattern in sperm . He showed that decreased global methylation in semen samples from normozoospermic men was related to a poor pregnancy outcome from ivf (118), suggesting that global methylation status independently affects embryogenesis . Both the complex path of sperm production and the delicate balance of epigenetic and genetic factors during sperm maturation contribute to the formation of a mature sperm with the ability to fertilize an oocyte and contribute to the developing embryo . It has been proposed that the level of dna methylation in human sperm could be linked to their ability to initiate a pregnancy in an assisted reproduction procedure (118). The epigenome cycles through a series of precisely timed methylation changes during development, making the epigenome vulnerable to interference from environmental exposure (119). As the embryo grows, these parent - of - origin imprints are maintained in somatic tissues but erased in primordial germ cells so that imprints can be re - established in a sex - specific manner during gametogenesis . Histone modifications are thought to play a role in this sex - specific mark establishment, as the extensive loss of histone methylation and acetylation occurs along with the loss of dna methylation (120). The methylation marks are then sustained throughout the individual's lifetime until methylation marks are erased and re - established following fertilization of the next generation (120). Epigenetic programming plays an important role in an organism's response to environmental stress during critical developmental periods (121). Epigenetic changes are not only heritable in somatic cells but can also be maintained during meiosis . Transgenerational epigenetic inheritance is governed by chromatin remodeling in drosophila melanogaster (122), and the inheritance of coat color in successive generations of the agouti viable yellow mouse is controlled by epigenetic mechanisms associated with the agouti allele (123). The agouti viable yellow (a) and axin - fused (axin) mice are unique animal models that carry the a and axin metastable epialleles, respectively . Researchers have used these mice to show that several nutritional and environmental exposures can alter epigenetic programming during gestation . For instance, exposure to methyl donors, such as folic acid, can hypermethylate the a and axin alleles, leading to offspring mice that are brown (pseudoagouti) and to mice with straightened tails, respectively (124,125). The sperm cell has a highly differentiated and specialized morphology, and the epigenome of human sperm is unique, elegant and essential to embryogenesis . Understanding the epigenetics of sperm and spermatogonia may be key in understanding the mechanisms of pluripotency, which has broad implications for potential therapies . Efforts should be aimed at identifying select candidate alleles that are key factors in embryogenesis or that are representative of the genome at large and are predictive of abnormal epigenetic modifications . Future studies will likely focus on the epigenetics of both gametes, as well as on the changes observed throughout embryogenesis . Thus, investigating the effects of genetic and epigenetic alterations in sperm and how these modifications arise and affect embryonic genes will be important in the prevention, diagnosis and treatment of disease . Sperm are highly polarized cells that are both transcriptionally and translationally silent . At the time of fertilization, sperm transfer not only nuclear dna but also oocyte activation factor, centrosomes, long - lived mrnas, and small non - coding rnas . It is believed that these mrnas are transcripts for key developmental genes and that the small non - coding rnas modify post - fertilization events . Due to its high polyunsaturated fatty acid content and the loss of the majority of cytosolic antioxidants, mitochondria are the first site of free radical production and free radical - induced dna damage . Thus, the sperm mtdna is reduced to disposable elements at the time of fertilization . However, mtdna accumulates sequence variations that produce high levels of free radicals, which damage both the mitochondrial and nuclear genomes . The mtdna copy number in healthy sperm is 1 - 1.4, compared with approximately 5 - 10-fold more in sperm with morphological abnormalities and impaired motility . This high copy number of mtdna when aging oocytes with a defective genetic filter are fertilized . The mtdna copy number has a profound impact on the methylation pattern of nuclear genes . In a previous study from our laboratory, we established sperm dna fragmentation indices of 30 and 26% in infertile males and the male partners of couple experiencing idiopathic recurrent pregnancy loss, respectively . The sperm genome is partitioned into a peripheral compartment that has histone - bound dna, retains the nucleosomal structure and thus maintains imprints . This region of the genome contains promoters for developmentally important genes, transcription factors, signaling factors and micrornas . The retention of histones is not random but is significantly enriched in many developmentally important loci . Recent studies have also shown that the mtdna copy number has a significant impact on the nuclear methylation pattern . Thus, one cannot study the nuclear genome in isolation because of the cross - talk between the mitochondrial and nuclear genomes of sperm . Telomeres are tandemly repeating hexameric units that cap chromosomal ends and are vital for genomic integrity and chromosomal stability . Concordance in telomere length in germ cells is necessary for synapsis, recombination and chromosome segregation . Rapid telomere attrition due to oxidative stress may result in meiotic recombination defects and segregation errors . This results in meiotic arrest and the generation of gametes with increased disjunction errors and aneuploidy . The inheritance of short telomeres from sperm may also impair cleavage, as optimal telomere length is a prerequisite for cell division . This may be one of the key factors affecting pre- and post - implantation losses and the birth of offspring with major and minor congenital malformations and childhood cancers . Thus, the sperm genome is highly vulnerable to oxidative stress - induced dna damage . Various lifestyle modifications (such as the increased intake of fruits and vegetables, exercise, meditation, yoga, cessation of smoking, and reduction in alcohol intake) can improve the health of the sperm genome and result in normal embryonic development and the birth of healthy offspring . The integrity of the sperm genome and epigenome is critical for the birth of healthy offspring . These compartments include a central compact toroid, in which dna is protamine - bound . The peripheral compartment contains histone - bound dna (5 - 15%) that retains the nucleosomal structure . The histone - bound dna is highly susceptible to environmental insults, especially oxidative damage . Telomeres, the nucleoprotein structures that constitute the biological molecular clock, cap the chromosomal ends and maintain chromosomal and genomic integrity . These guanine - rich repeats are highly susceptible to free radical - induced dna damage . We believe that the rapid alteration of telomeres in the sperm genome underlies the etiology of infertility, which occurs as a result of accelerated telomere aging . Because the inheritance of telomere length is a complex trait, a short telomere length adversely affects cleavage and results in the generation of blastocysts with poor morphology . Sperm not only transfer the nuclear genome to oocytes but also transfer a stable population of developmentally important mrnas . The sperm epigenome is maintained through the retention of histones, the compaction of major portions of the genome by protamines, dna methylation, and covalent histone modifications . Because sperm lose the majority of cytosolic antioxidants at the time of spermiogenesis, sperm cells are highly vulnerable to free radical - induced dna damage . Lower levels of key dna repair enzymes have also been found in sperm (unpublished study from our laboratory). The fertilization of oocytes by such sperm, either spontaneously or through assisted reproductive techniques, may help us to understand the pathogenesis of congenital malformations, childhood cancers and perinatal morbidity.
Breast cancer is a leading cause of death and disability among women, especially young women, in low- and middle - income countries . Though incidence and overall mortality rates continue to be lower than in most high - income countries, case fatality rates from breast cancer are very high . These high case fatality rates are likely due to a lack of awareness of the benefits of detection and treatment and a scarcity of adequate facilities for detection and diagnosis, as well as poor access to primary treatment . Remarkable improvements have been achieved in the probability of survival for women diagnosed with breast cancer in the usa as compared to 60 years ago . Early detection through the use of mammography, high - quality surgery, and adjuvant therapies including chemotherapy and targeted therapies, such as hormonal therapy and, more recently the her2-directed agent trastuzumab, can be credited for much of the recent improvement in outcome for women with breast cancer in the usa . However, even prior to the routine use of mammography or adjuvant therapy, significant improvements were made in breast cancer survival, and these can be traced to relatively low - cost interventions that are still in use in high - income countries . Understanding which healthcare interventions were available and how they resulted in improvements in the probability of survival could be important, especially for designing programs in resource - constrained settings where breast cancer case fatality is high and many of the most costly and technology - intensive diagnostic and therapeutic options are not available . In many developing countries, the incidence of breast cancer is now rising sharply due to changes in reproductive factors, lifestyle, and increased life expectancy . Today, more than half of incident cases occur in the developing world [14, 15]. Combined with still high case - fatality rates, this means that mortality from breast cancer is a leading cause of death among adult women in developing countries, as well as in the developed world . In mexico, for example, breast cancer is now the second leading cause of death among women aged 30 to 54 and the leading cause of tumor - related death among adult women of all ages . The high probability of dying from breast cancer the case fatality rate, which is approximated by the ratio of mortality to income across the developing world further reflects the inequities in early detection and access to treatment [1, 17]. The number of deaths as a percentage of incident cases in 2008 was 48% in low - income, 40% in low - middle - income, and 38% in high - middle - income countries, while it was 24% in high - income countries according to the most recent globocan / iarc data . Available evidence on stage at diagnosis, though scarce, indicate that a very high proportion of cases in the developing world are detected in late stages [1, 3]. (table 1) in many underserved populations, a majority of women present with advanced disease; the figure is as high as 78% in black women in south africa . In contrast, in the united states the majority of cases are detected in localized stages of the disease (stages i and ii), a third is regionally advanced (stage iii), and only 5% are distant - stage metastatic (stage iv). Many reasons are given for the advanced stage at presentation and resultant poor survival rates in low- and middle - income countries: the stigma of breast cancer and the associated societal implications of its treatments (especially mastectomy) discourage women from seeking care early on; lack of knowledge about breast health; scant options for early detection due to limited access to routine care and examinations; and lack of access to mammography and to affordable, high - quality treatment options . In the short term, mammography and other expensive and technologically complicated resources and therapies will not likely be available to many of the world's women . Though we must continue to work at all levels to bring diagnostics and therapeutics with a proven impact on outcomes to these women as soon as possible, there are ways closer at hand to improve the immediate outlook for women in these settings . Figure 1 shows the incidence and mortality rates for breast cancer in the usa between 1940 and 2000 . From the late 1940s, thus mortality - to - incidence ratios decreased dramatically, even before the generalized use of mammography or adjuvant chemotherapy and antiestrogen therapy that commenced in the mid- to late 1970s . Table 2 presents the ratio of mortality over incidence, as an approximation of the case - fatality rate, in 5-year increments between 1950 and 1975 . Between 1950 and 1975 incidence nearly doubled, increasing from 66.6/100,000 women to 119.2/100,000, while mortality remained relatively constant, 28/100,000 and 31.6/100,000, respectively . Thus, during this time period, the ratio of mortality over incidence (an approximation of the case - fatality rate) fell from 0.42 to 0.27 representing a 36% decline: this suggests that more women were surviving their cancers in 1975 as compared to 1950 and is true for both whites and blacks . Further, the reduction in case - fatality rates is at least as large as the improvement evidenced since the introduction of mammography and adjuvant therapy . These findings suggest considerable room for reducing the high mortality - to - incidence ratio found in many developing countries even without mammography or adjuvant therapy . The increases in incidence and survival for breast cancer in the usa between 1950 and 1975 cannot be attributed only to detection of in situ cancers that would not have progressed . The proportion of in situ cases in known - stage cases in the connecticut tumor registry in that period was very small and increased from only 0.3% in 19501954 to 1.9% in 19701974, and it was largely unaffected by improved reporting and a reduction in unknown - stage tumors . Thus, the reduction in the mortality - to - incidence ratio must largely reflect outcomes for patients with invasive cancers . From 1940 to 1970, regional and advanced stages fell from 58% to 54% between 19401944 and 19501954, and to 45% in 19701974 . The period from 1940 to 1974 was a time in the usa when evidence - based medicine became more widespread, and healthcare became more generally available, including increased use of routine gynecologic and general physical examination . For example, the american cancer society began promoting self - examination for breast cancer in 1950 and routine screening by cervical cytology starting in 1952 . Further, the era of oral contraceptives in the 1960s contributed to greater interactions between healthy women and their healthcare providers . Authors who analyzed data prior to 1974 assign the improvements in survival to more effective breast education programs, increased breast cancer awareness, detection of tumors palpable with self or breast - clinical examination, and better diagnostics [19, 20]. Thus, the increase in survival rates in the usa prior to 1975 strongly suggests potential to improve breast cancer outcomes in developing countries more quickly than we will be able to make routine mammography and adjuvant therapy available . Recent studies, showing breast physical examination and breast self - examination to be unhelpful in reducing stage at diagnosis [2326], have considered only developed countries or urbanized areas of developing countries where routine healthcare is generally available, breast cancer awareness and education are high, and mammography is more routinely accessible . These data, and hence the findings, are likely to be less applicable to a population where breast cancer education and awareness are low, access to the healthcare system severely restricted, and the vast majority of patients present with advanced disease . While reducing the incidence of breast cancer is an ideal goal, the options for achieving this are limited and longer term, particularly for the developing world . Healthy lifestyle, including limiting alcohol consumption, maintenance of ideal body weight, regular physical activity, and avoidance of postmenopausal hormone replacement therapy, can have an important impact on breast cancer incidence [15, 27]. Every effort should be made to limit these risk factors and thus breast cancer risk . Yet, even with strong efforts aimed at prevention, the incidence of breast cancer is likely to increase in most developing countries due to changes in reproductive patterns including later first pregnancies, reductions in parity, and shorter duration of lactation; as well as, declines in physical activity and increased life expectancy . Earlier detection and timely, adequate surgery would likely result in substantial improvements in survival in much of the developing world . Education about breast cancer, advocacy around curability, and increased coverage of basic healthcare including skilled breast physical examinations could produce improvement in survival rates as occurred in the usa between 1950 and 1975 . Education efforts need to address the reality that many women, particularly those with less income and education, may not seek care when they feel a breast mass, because they are unaware of what it represents, are concerned about the stigma of cancer and being rejected by their community and their partners, fear the potential loss of the breast, or believe there are no effective therapies for the disease especially if all the women they have known with breast cancer died . Hiv a stigma - laden disease, that if untreated is universally fatal by contrast, it has been demonstrated that by combining education, with better and more accessible healthcare facilities, trained medical personnel, and effective therapy, patients do seek and comply with treatment and benefit from it [2830]. The ability to provide adequate affordable access to physical exams by healthcare workers is not a trivial obstacle . An essential first element is the existence of a functioning primary care system staffed by providers trusted by their community . While many countries continue to battle with a weak primary infrastructure, examples, such as the oportunidades program and seguro popular in mexico and partners in health in rural africa and haiti, provide important lessons for strengthening primary healthcare including, and often especially, interventions to improve the health of women [16, 31, 32]. These interventions are essential parts of overall health system strengthening and can help with the prevention and treatment of many diseases in addition to breast cancer . Clinical breast exams do not need to be performed by physicians or nurses . In settings where community healthcare workers have learned to care for patients with diseases as complex as hiv, multidrug resistant tuberculosis, and malaria, they could be trained to effectively perform breast exams . Large - bore core needle biopsy is a reliable method to obtain tissue for diagnosis and can be performed by trained personal in relatively simple ambulatory settings . Pathology services must be available to process the specimens but can be located regionally or outsourced globally . In many developing countries, surgery is available in regional centers, although additional training of surgeons in appropriate techniques may be needed, and women will require financial support and transportation . Where radiation therapy is not available, as is the case in many low - income countries given the high proportion of hormone receptor positive cancers, tamoxifen can be effectively combined with surgery . Unlike many treatments for breast cancer, generic tamoxifen is low cost, taken orally, and in the vast majority of patients is well tolerated and does not generate unmanageable side effects or require additional medications or care to control symptoms . Options exist to greatly expand low - cost alternatives for earlier detection and treatment of breast cancer in developing countries . Guidelines have been developed and have been stratified according to the resources available in specific countries and health systems [3336]. Many of the basic interventions focus on education, awareness building, the health of women, and expanding capacity at the primary and community healthcare levels, and thus and also contribute to overall health system strengthening . Education to improve breast health awareness, breast self - examination, and clinical breast exam are relatively inexpensive and can be incorporated into existing primary health infrastructures . Surgery and hormone therapy based on tamoxifen are cost effective, especially with early detection, and implementable in poor - resource settings . Focusing on providing these interventions in locations where they do not currently exist could dramatically improve survival . In no way does this abrogate the responsibility to eventually provide resources such as mammography, adjuvant chemotherapy, and advanced targeted therapies such as trastuzumab in these settings . However, great benefit can emerge from basic breast cancer education and awareness, integrating breast exams into primary healthcare infrastructure, and adequate surgery combined with tamoxifen . Implementation of these interventions should proceed as quickly as possible, while the more complex and costly interventions, such as mammography, are being made more available . The provision of better primary healthcare, education, and better medical outcomes will provide a solid foundation for reducing stigma and fear that will make more effective the introduction of complex technologies, such as mammography or adjuvant therapy . There is no reason not to immediately strive for the implementation of basic interventions for breast cancer care and control in all settings . All authors have completed the unified competing interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare that all authors had: (1) no financial support for the submitted work from anyone other than their employer, (2) no financial relationships with commercial entities that might have an interest in the submitted work, (3) no spouses, partners, or children with relationships with commercial entities that might have an interest in the submitted work, (4) no nonfinancial interests that may be relevant to the submitted work.
The consumption of high levels of fructose in humans and animals causes insulin resistance, lipid abnormalities, obesity, hypertension, and renal changes.1 - 5 the combination of these metabolic and cardiovascular alterations observed in fructose - fed subjects is collectively known as metabolic syndrome (ms). To model the development of ms experimentally, long - term fructose overload in rats has been used.1,2 statins (or hmg - coa reductase inhibitors) have been shown to lower arterial pressure (ap) in borderline hypertensive dyslipidemic humans . This favorable effect of statins may be a result of both lipid - based mechanisms and non - lipid - based mechanisms affecting endothelial vasoregulation and the sympathovagal balance in the disease state.6 experimental studies have shown that simvastatin improves baroreflex sensitivity (brs).7 findings from several studies have strongly suggested that simvastatin normalizes the autonomic function in individuals with heart failure, inhibiting the central mechanisms of angiotensin ii and, consequently, the superoxide production pathway.8 moreover, simvastatin may improve left ventricular function8 and reduce vascular dysfunction in mice with dyslipidemia.9 despite these positive results, the effects of statin therapy on autonomic function have not been established to date, particularly in females with ms . It is important to emphasize that significant advances in the management of cardiovascular disease and ms have been made in recent years.1,2,10,11 however, cardiovascular diseases remain the leading cause of death among women in the most developed areas of the world,12 exceeding the number of deaths in men and the combined number of deaths due to the next seven causes in women.10 because autonomic dysfunction leads to cardiometabolic disorders11 and because statins have demonstrated neuroprotective effects,12 - 15 we hypothesized that chronic simvastatin administration in female rats submitted to long - term fructose overload (18 weeks) would improve cardiac autonomic control and reduce the cardiometabolic risk . Therefore, the aim of the present study was to investigate the effects of simvastatin on the metabolic, cardiovascular and autonomic changes induced by fructose overload in female rats . Experiments were performed using 24 female wistar rats (70 days old, approximately 50 g) that were obtained from the animal shelter of sao judas tadeu university in sao paulo, brazil . The rats received standard laboratory chow (nuvital, colombo, brazil) and water ad libitum . The animals were housed in individual cages in a temperature - controlled room (22c) with a 12-h dark - light cycle . All surgical procedures and protocols were in accordance with the ethical care guidelines for experimental animals and the international animal care and use committee and were approved by the sao judas tadeu university ethical committee (protocol number 058/2007). Three experimental groups were used in this study: control (c; n = 8), fructose (f; n = 8), and fructose+simvastatin (fs, n = 8). Fructose overload was induced via dilution of d - fructose in the drinking water (100 simvastatin (5 mg / kg / day) treatment was performed by gavage for the last two weeks of fructose overload . After 18 weeks of fructose overload, the blood glucose and triglyceride concentrations were measured using a roche device (accutrend gct, roche, sao paulo, brazil) after four hours of fasting at the end of the protocol . For the insulin tolerance test (itt), the rats were fasted for two hours and then anesthetized with thiopental (40 mg / kg body weight, ip). A drop of blood was collected from the tail to measure the blood glucose concentration using the accucheck system (roche, sao paulo, brazil) before and 4, 8, 12, and 16 minutes after insulin injection (0.75 u / kg). The constant rate of decrease of the blood glucose concentration (kitt) was calculated using the 0.693/t/2 formula . The t/2 for blood glucose was calculated from the slope of the least squares analysis of the blood glucose concentrations during the linear phase of decline.16,17 after metabolic measurements, two catheters filled with 0.06 ml of saline were implanted in anesthetized rats (ketamine 80 mg / kg+xylazine 12 mg / kg) into the carotid artery and jugular vein (pe-10) for direct measurements of the ap and for drug administration, respectively . One day after the catheter placement, the rats were conscious and allowed to move freely during the experiments . The arterial cannula was connected to a strain - gauge transducer (blood pressure xdcr, kent scientific, litchfield, ct, usa), and ap signals were recorded over a 30-min period by a microcomputer equipped with an analog - to - digital converter board (codas, 2-khz sampling frequency, dataq instruments, inc ., akron, oh, usa). The recorded data were analyzed on a beat - to - beat basis to quantify the changes in the mean ap (map) and the heart rate (hr).16,18 the vagal and sympathetic effects were studied by injecting methylatropine (3 mg / kg iv, sigma - aldrich, st . Louis, mo, usa) and propranolol (4 mg / kg iv, sigma - aldrich) in a volume of 0.1 ml/100 g of body weight . The resting hr was recorded while the rats were in their cages in an unrestrained state . Methylatropine was injected immediately after the recording . Because the hr response to these drugs reaches its peak within 10 to 15 minutes, this time interval was allowed to elapse before the hr measurement was taken . On the next day, the sequence of the injections was inverted, and propranolol was injected before methylatropine.16 the sympathetic effect was determined by calculating the difference between the basal hr and the lowest hr after the administration of propranolol . The vagal effect was obtained based on the difference between the maximum hr after methylatropine injection and the basal hr . The data were expressed as the meanssem and were compared using one - way analysis of variance (anova) or repeated one - way anova followed by the student newman - keuls test . The body weight was not different between the studied groups at the beginning (c: 486 g, f: 435 g, and fs: 451 g, p>0.05) and at the end of the protocol (c: 28510 g, f: 2949 g, and fs: 2888 g, p>0.05). The fasting glucose levels were increased in f (922 mg / dl) and fs (932 mg / dl) rats compared with that in c rats (822 mg / dl, p<0.05). The blood triglyceride concentration was also higher in the f and fs groups (14212 and 18722 mg / dl, respectively) compared with that in the c group (1015 mg / dl, p<0.05). The constant rate of the plasma glucose disappearance (kitt) was reduced in the f group (3.40.32%/min) compared with that in the c group (4.40.29%/min, p<0.05) during itt, which is indicative of the insulin - resistant state in the fructose - fed rats . The simvastatin treatment increased the kitt (fs: 5.40.66%/min, p<0.05) in f rats . The f group exhibited increases in the systolic, diastolic, and mean arterial pressures (p<0.05). Simvastatin treatment did not change the ap in the f rats (p>0.05) (table 1). The resting hr showed similar values among groups (p>0.05) (table 1). The cardiac vagal effect was similar between the c (499 bpm) and f groups (465 bpm, p>0.05). Simvastatin treatment increased the vagal effect in fructose - overloaded animals (fs: 847 bpm) compared with that in c and f animals (p<0.05) (figure 1a). The sympathetic effect was enhanced in the f group (737 bpm) compared with that in the c group (487 bpm, p<0.05). This effect was normalized by simvastatin treatment (fs: 318 bpm, p<0.05) (figure 1b). The aim of this study was to determine the effects of simvastatin treatment on the metabolic, cardiovascular, and autonomic modulation in an experimental model of ms that was induced by long - term fructose overload (18 weeks) in female rats . Although simvastatin treatment did not change the blood metabolic parameters or the ap, this pharmacologic approach improved insulin resistance, reduced the exacerbated cardiac sympathetic effect and increased the vagal effect to the heart . Additionally, our findings in female rats corroborated previous data that have been obtained in male animals submitted to fructose overload; these male rats exhibited enhanced blood glucose and triglyceride levels, insulin resistance, increased ap and sympathetic activation . We should emphasize that the protocol used in our research differed from those of previous studies . Because fructose overload was performed in animals starting from their 70 day of life through their adult phase previous investigations have covered a shorter time - span and gathered data from the acute (2 - 24 h) or mid - term (1 - 9 weeks) phases of fructose administration.2 - 5,16, the rationale behind our design choice lies in the fact that metabolic and cardiovascular disorders take years to manifest as clinical alterations due to the stepwise compensatory behaviors, physiological adaptations, and new equilibrium levels . The fructose drinking model in rats resembles a state of early insulin resistance in humans, which is associated with mild hypertension.16,23 previous studies have demonstrated that increased body weight is not common in male rat or in fructose - rich chow - fed mouse models . However, fructose - rich chow - fed mouse models can develop hypertriglyceridemia, increased blood glucose concentrations, glucose intolerance or insulin resistance and hyperinsulinemia.3,5,16, in our study, the fructose - overloaded female rats displayed increased blood glucose and triglyceride concentrations and insulin resistance . Although simvastatin treatment did not alter the fasting blood glucose and triglyceride levels in our study, it increased kitt in fructose - fed rats . Most studies investigating the effects of high fructose consumption on the basal ap in male or female rats have used tail - cuff plethysmography, which is an indirect measurement of ap that can only measure systolic ap.19 - 22 increased fructose consumption leads to increases in the ap in male20,21 and female rats16 and in male mice during the dark period.5 farah et al.5 have demonstrated an increase in the low - frequency component of systolic ap in fructose - overloaded (eight weeks) male mice . These results show that increased ap is associated with sympathetic modulation in the circulation that is limited to the dark (active) period . In the present study, we observed an increase in the ap and in the sympathetic effect in the heart in fructose - fed female rats after 18 weeks of fructose consumption . The ap change in fructose - fed animals is mediated by activation of the sympathetic nervous system,5,24 impairment of the cardiac parasympathetic tonus16 and of endothelium - dependent relaxation25 and dysfunction in the angiotensin - renin system.5 our results show that the simvastatin treatment normalizes the cardiac sympathetic effect and insulin resistance . In addition, simvastatin treatment increased the cardiac vagal effect in fructose - fed female rats . However, simvastatin treatment did not change the basal ap or the blood triglyceride level . These results suggest that these statin - induced improvements might result from a pleiotropic effect that is independent of the drug's classical effect on lipids . Pliquett et al.26 have also demonstrated improvements in the brs in rabbits with heart failure after statin treatment without changes in the total plasma or high - density cholesterol levels . Additionally, if decreased ap was observed in the present study in simvastatin - treated fructose - fed rats, the autonomic improvement observed in these animals may be attributed, at least in part, to this change . Previous studies have reported lower aps in borderline hypertensive dyslipidemic humans who were treated with statins and have attributed this favorable alteration to both lipid - based mechanisms and non - lipid - based mechanisms affecting endothelial vasoregulation and the sympathovagal balance in the disease state.6 based on these findings, we hypothesize that the role of simvastatin in the autonomic nervous system is vast and includes enhancing no synthesis in the endothelium27,28 and reducing angiotensin ii induced injury, at1 receptor expression,29,30 and eta receptor expression.31 these functions indicate a potential role for statins in regulating sympathetic and vagal outflow in the central nervous system and improving the afferent or efferent arms of the cardiovascular autonomic reflexes . These autonomic pleiotropic effects of statins may account for patient outcomes and require further characterization . The results of the present study demonstrate that fructose overload in female rats induces increases in the ap and the cardiac sympathetic response, which are associated with insulin resistance . These findings reinforce the role of autonomic dysfunction in the development of early cardiometabolic disorders that are induced by a high fructose diet in female rats . Importantly, we demonstrated that a short - term simvastatin treatment may improve insulin sensitivity and cardiac autonomic control in an experimental model of ms in female rats . These effects were independent of improvements in the classical plasma lipid profile and of reductions in the ap, reinforcing the hypothesis that statins reduce the cardiometabolic risk in females with ms . However, additional studies are needed to confirm the pleiotropic effects of long - term statin treatment on autonomic dysfunction and on the outcome of women with ms . This study was supported by the conselho nacional de desenvolvimento cientfico e tecnolgico (pibic - usjt) (107977/2006 - 0), the fundao de amparo pesquisa do estado de so paulo (05/60827 - 0, 05/60828 - 6, 07/57595 - 5, 11/11267 - 2) and capes (capes- prosup).
Oral melanoma is an extremely rare tumor arising from uncontrolled growth of melanocytes found in the basal layer of oral mucous membrane . It occurs between 30 and 90 years of age, with a higher incidence in the 6th decade with a mean age of 56 years . It is having a higher prevalence in yellows, blacks, japanese, and indians of asia due to more frequent finding of melanin pigmentation in oral mucosa of these races . Green et al . Described criteria for diagnosis of primary oral melanoma which includes demonstration of melanoma in the oral mucosa, presence of junctional activity, inability to demonstrate extraoral primary melanoma . A total of 80% to 90% of oral malignant melanoma arises in the mucosa of maxillary jaw with a majority occurring on the keratinized mucosa of hard palate and gingiva . Clinically, it is easy to diagnose them as these are pigmented ones and have irregular shape and outline . These are mostly asymptomatic and detected only when there is ulceration or hemorrhage of the overlying epithelium . The delayed detection may be the cause for the poor prognosis with a 5-year survival being between 15% and 38% . The purpose of this article is to present a case of oral malignant melanoma, as well as to emphasize the necessity for early recognition and treatment of this lesion . A 48-year - old male patient reported to the department of oral medicine and diagnosis with chief complaint of pain and swelling in the upper right gums . The clinical examination revealed a large mass of 8 3 cm in dimension on buccal aspect of right maxillary alveolus involving marginal, attached, and interdental gingiva [figure 1]. Anteriorly, it extends from the gingiva of mesial surface of 22, to the gingiva in relation to 17 posteriorly . 13 was missing and 12 was displaced laterally; while, 11, 12, and 21 exhibited mobility . Primary oral malignant melanoma extending from 22 to distal aspect of 17 the palpatory findings revealed a firm consistency of lesion with mild pain . A complete examination of the lesion was done and no other primary site of the lesion was found . Correlating all clinical features, diagnosis of primary malignant melanoma of oral cavity was made and the patient was referred for further investigations . A computed tomography examination of neck, chest, abdomen, and bone scanning and ultrasounds of liver and kidney were normal excluding any diagnosis of distant metastasis . An incisional biopsy was done for the lesion under local anesthesia and the specimen was sent for histopathologic examination . The gross examination of tissue revealed a mass of 2 mm 3 mm 1 mm in size, which was black in color and firm in consistency . The hematoxylin and eosin - stained section showed a melanin - producing tumor, consisting of atypical irregularly elongated spindle and oval - shaped melanocytes, exhibiting uniformly dark, enlarged and irregular nuclei [figure 2]. In the superficial layers of the tissue, a junctional nevus with pigmentation the diagnosis of an invasive melanoma arising most likely from a pre - existing junctional nevus was made and the patient was referred to the oral and maxillofacial surgery clinic for required therapy . As per the traditional approach, partial maxillectomy of the right side was performed . To reduce the defect and to reconstruct alveolus, microvascular fibula flap was used . The orbital floor near to maxilla was reconstructed with the help of premolded titanium mesh . The histologic examination of the specimen confirmed the initial diagnosis of an invasive melanoma of the oral mucosa . The patient has been followed - up with no evidence of recurrence or metastasis either clinically or radiographically, 11 months after the tumor's resection . The hematoxylin and eosin stained section shows melanoma with invasive pattern showing large cells with pleomorphic vesicular nucleus and brown pigment (40) the hematoxylin and eosin stained section shows stratified squamous keratinized epithelium with in situmelanotic pigment growth (10) it has no known predisposing factors and is difficult to diagnose and manage . Differentiating it from a metastatic melanoma is often challenging . The first symptoms of oral melanoma described by berthelsen were those of asymptomatic swelling and occasional bleeding, where he found that only 2 (14%) patients had a pain . Because most of the melanomas are painless in their early stages, the diagnosis is often unfortunately delayed until symptoms resulting from ulceration, growth, or bleeding are noted . The pain may be the later manifestation in melanoma as in our case that again could cause delay in seeking treatment . On gross appearance, the tumors on the palate are usually flat, with a varying degree of thickness . Microscopically, the tumor cells present themselves as densely packed, large epithelioid cells with eosinophilic cytoplasm . The melanin pigment, which is located intra or extracellularly, may be abundant, but may be sparse or even absent at the light microscopic level . The prognostic value of various levels of invasion, as established in the clark's classification, does not apply for mucosal melanoma because of the absence of histologic landmarks, which are analogous to the papillary and reticular dermis . The staging systems that are applied to cutaneous melanoma are not applicable to mucosal melanomas . The american joint committee on cancer the generally followed guideline is a clinical classification stage i clinically localized disease, stage ii regional lymph node disease, and stage iii - distant disease . The tumor thickness is a reliable prognostic indicator for survival . In this case, the patient was in stage i. westbury describes a clinical classification as follows: 1only primary tumor present, and 2metastasis present, 2a adjacent skin involved, 2b adjacent lymph nodes involved, and 2ab adjacent skin and lymph nodes involved . Thus, according to these systems specification, our patient was in stage i. the etiology of malignant melanoma is essentially unknown . Tobacco use and chronic irritation from ill - fitting dentures have been considered as possible risk factors, but the evidence is weak . The cause could also be related to our patient as he was having the history of smoking from last 15 years . But most of the malignant melanomas arise de novo, from apparently normal mucosa, and about 30% are preceded by oral pigmentations for several months or even years . Some melanoma - associated antigens become expressed during transformation process from a benign melanocytic nevus to melanoma; the majorities of these are related to the melanin production process and most are hla restricted . A recent study demonstrates that the loss of heterozygosity at 12p13 and p27kip1 protein expression contributes to melanoma progression . Cytogenetic analysis and evaluation of melanocyte - specific gene-1 (msg-1) appears to be very helpful for understanding the pathogenesis of oral malignant melanoma . The current guidelines for the surgical management of primary cutaneous melanoma recommend a diagnostic excisional biopsy of the lesion followed by a wide local excision where the diagnosis is proved . However, in oral cavity, the size of the lesion or anatomic limitations, particularly the presence of teeth, may preclude the taking of excisional biopsy . Younes et al . Proposed to take an excisional biopsy with a 12 mm margin for small lesions in amenable locations, but incisional biopsy, through the thickest or the most suspicious part of the tumor, in case of a large lesion or a location in sites where an excisional procedure would involve extensive and militating surgery . Usually oral malignant melanoma can be diagnosed with confidence on hematoxylin and eosin stained sections . If pigment is completely absent (amelanotic melanoma), immunohistochemical stains are of significant help . Useful markers include s-100 protein, gp 100 (hmb-45), and mart-1 (melan - a). It has always been suggested that cutting into malignant neoplasm during incisional biopsy could result in accidental dissemination of malignant cells within adjacent tissues or blood or lymphatic stream with subsequent risk of local recurrence or regional or distant metastasis . Did find a somewhat reduced survival rate in patients with melanoma who had incisional biopsies but against the studies done by lederman and sober where they found no correlation in patient's prognosis with incisional and excisional biopsies . Distant metastasis to the lungs, brain, liver, and bones are frequently observed . The treatment of oral malignant melanoma is still controversial and there is no census regarding the best therapeutic approach . Data from several studies indicate radical resection of the primary as the treatment of choice . Regression in melanoma is a well - recognized phenomenon and may account for many of the cases of metastatic melanoma with occult primaries . Partial regression of melanoma is relatively common, but complete regression is quite rare and relatively few cases are well documented . A further feature of regression is its association with poor rather than a good prognosis . The rather nonspecific features of regressed melanoma (apparently inflammatory nodules, depigmented patches, and flat or slightly depressed scars) are easily missed or discounted unless the patient had noticed the regression . From the prognostic point of view, clinical stage at presentation is probably the most important factor in determining the outcome . It has been found by liu et al . That thickness of the tumor, cervical lymph node metastasis, presence or absence of ulceration, and the anatomic sites are all independent risk factors . It has been calculated that nodal metastasis reduces the mean survival time from 46 to 18 months . Furthermore, a tumor thickness greater than 5 mm, presence of vascular invasion, necrosis, polymorphous tumor cell morphology, and inability to properly resect the lesion with negative margins have been associated with poor survival in patients with primary melanomas of head and neck region . Despite the improvement of surgical techniques and the introduction of new chemotherapeutic agents, prognosis of this malignancy remains poor . The generally advanced stage of the tumor at initial diagnosis leads to a poorer survival of patients with mucosal melanomas as compared with patients with cutaneous melanomas and presence of vertical growth phase are associated with median survival rate . Analysis of published cases and recognition of new ones may be helpful in establishing definite classification and proposing clinical features that would facilitate its early diagnosis as a prerequisite for timely treatment and better prognosis of this rare pathology.
In physical therapy for patients of cerebrovascular, cardiovascular, respiratory or musculoskeletal diseases, assessments of muscle strength and the movement of joints are essential for treatment planning and also for lifestyle guidance . In order to evaluate patient performance, the range of motion test (rom - t) is used to assess movement impairment of the joints1, and the manual muscle test (mmt) is used to assess muscle impairment2 . Both of these test methods are fundamental skills required of every physical therapist, which they have to master in training schools . The mmt is a manual maneuver to evaluate the strength of the prime mover muscles for a given motion of a joint . The procedures employed in the mmt were determined mainly based on our present knowledge of kinesiology and electromyography (emg). Not only in diagnostic tests, the mmt has also been used for muscle training and maintaining muscle strength2 . The muscles involved in finger function are one of the most important tasks for humans, since the function of the hands is strongly related with the quality of life . The mmt of the forearm muscles are also based on emg . However, it is quite difficult to employ emg for muscles located deeply beneath the skin . It is also difficult to discriminate the actual source of the emg detected by surface electrodes3,4,5,6 . Recently, magnetic resonance imaging (mri) has been used to monitor muscle activities by using the t1, t2, and water content values of skeletal muscles that increased after muscle exercise7,8,9 . Intensive studies have been conducted on the determination of agonist muscles using the increase in t2 values10, 11, and takamori detected increases in the t2 weighted mr (t2w - mr) image intensity of the extensor digiti minimi muscle after extension exercise of the mp joint of the digitus imnimus12 . In this study, among the mmt for the forearm muscles, we selected 3 mmt for the primary mover muscles confirmed by the emg . In order to confirm the reliability of the mr images, 1) the increase in the t2w - mr image intensity was detected after the mmt of the supinator muscle or the pronator teres muscle13, 14, and 2) we also aimed to detect the mmt of a smaller muscle of the forearm, the extensor indicis muscle15 . Three healthy male volunteers participated in this study . The age, height, and weight of the subjects averaged 36.4 12.3 years, 172.3 6.9 cm, and 72.3 17.0 kg (means sd), respectively . The left forearms of the subjects were studied, and the mmt was applied by physical therapists with more than 10 years of experience . The procedures, purpose, and risks associated with the study were explained to all of the subjects, and written consent was obtained prior to the commencement of the study . The study was approved by the human research review board at the dokkyo medical university school of medicine (no . Mr images of each forearm were obtained with a 0.2 t compact mri system (mrtechnology, tsukuba, japan) equipped with an oval h solenoidal radiofrequency probe12 . The transverse t2w - mr images were measured using multi - slice spin - echo mr image sequences with a 200 200 mm field - of - view, a 128 128 data matrix, a 39 ms echo - time with a 2,000 ms repetition time, 11 slices with an interval 10 mm, a 9.5 mm slice thickness, 1 accumulation and a total image acquisition time of 4 min 16 s. each left forearm was fixed by a shell - type holder of the forearm12, and a t2w - mr image in the resting condition was measured at first . Then, manipulative isometric contraction exercise (5 sec duration) was applied to the supinator muscle, the pronator teres muscle or the extensor indicis muscle using borg s rating of perceived exertion (rpe) scale of 151716 . Immediately after the exercise, the arm position was restored to the original position, and another t2w - mr image was measured . The increase in the t2w - mr image intensity of muscle was evaluated by 3 physical therapists (m.t ., s.a . And k.y .) With 45 years of experiences with 0.2 t mri . The epimysium of the muscle was traced by hand, and used for the same trace for the t2w - mr image of the resting muscle . The image intensity of a circular region of interest (roi) with 25 pixels was measured by imagej software 1.46r (nih, bethesda, usa). In each slice, 3 rois were set in the muscle without overlapping each other, and the average image intensity (cm) was obtained . In the same slice, an roi was set in the background and sd (sdair) was obtained . The image intensity of the muscle was represented by the signal - to - noise ratio (snr): i = cm / sdair17 . An image intensity outside of the 99.9% of confidence interval (ir 3.3) was considered as a significant increase (cl=99.9%), and an image intensity within 99.9% of the confidence interval was considered not significant (n.s . ), where ir was the snr for resting muscle . T2w - mr images of the supinator muscle (a), the pronator teres muscle (b) and the extensor indicis muscle (c) of the subject #1 . (i) t2w - mr images before the exercise, (ii) t2w - mr images after the exercise, (iii) the trace around muscle (). Represents a typical result of the mmt of the supinator muscle sliced at one - third of the length of the ulna from the olecranon . The subjects were sitting, arm at the side, with the elbow flexed to 90, and the forearm in full pronation to neutral . Then, the examiner supported the elbow, and applied resistance with the heel of the hand over the dorsal (extensor) surface at the wrist (fig . The subjects #1, #2 and #3 did the exercise 82, 40 and 40 times until they reached borg s rpe scale of 1517, respectively . The image intensity of the t2w - mr image increased more than 1.7 times compared with the resting muscle, showing a significant increase (cl=99.9%) (table 1atable 1.t2w - mr image intensity before and after exercisesubject#1#2#3a) supinator musclebefore exercise19.813.516.8after exercise33.530.343.5b)pronator teres musclebefore exercise14.416.725.4after exercise30.930.646.3c)extensor indicis musclebefore exercise15.514.920after exercise29.230.236.6image intensity was represented by the signal - to - noise ratio . No significant changes were shown in the image intensities for the rest of the muscles . Transverse t2w - mr images of the supinator muscle (a), the pronator teres muscle (b) and the extensor indicis muscle (c) of the subject #1 . (i) t2w - mr images before the exercise, (ii) t2w - mr images after the exercise, (iii) the trace around muscle (). Figure 1b represents a typical result of the mmt of the pronator teres muscle sliced at one - third of the length of the ulna from the olecranon . The function of the pronator teres muscle is to pronate the forearm with the pronator quadratus muscle . The subjects were siting, arm at the side with the elbow flexed to 90 and the forearm in supination . Then, the examiner supported the elbow, apply resistance with hypothenar eminence over radius on the volar (flexor) surface of the forearm at the wrist (fig . The subjects #1, #2 and #3 did the exercise 45, 40 and 50 times until they reached borg s rpe scale of 1517, respectively . Image intensity of the t2w - mr image increased more than 1.8 times compared with the resting muscle, and represented significant increase (cl=99.9%) (table 1b). No significant changes were shown in the image intensity of the rest of the muscles . Figure 1c represents a typical result of the mmt of the extensor indicis muscle sliced at two - third of the length of the ulna from the olecranon . The extensor indicis muscle is a deep - layer, narrow skeletal muscle and its function is the extension of the index finger . The subjects were sitting with the forearm in pronation, the wrist in neutral . And the mp and ip joints in a relaxed flexion posture . Then, the examiner stabilized the wrist in neutral, and placed the index finger of the resistance hand cross the dorsum of all proximal phalanges just distal to the mp joints, and applied resistance in the direction of the flexion (fig . The subjects #1, #2 and #3 did the exercise 40, 40 and 40 times until they reached borg s rpe scale of 1517, respectively . The image intensity of the t2w - mr image increased more than 1.8 times compared with the resting muscle, showing a significant increase (cl=99.9%) (table 1c). No significant changes were shown in the image intensity of the rest of the muscles . In this study, the t2w - mr image intensity of the supinator muscle, the pronator teres muscle and the extensor indicis muscle increased (cl=99.9%) with the mmt for these muscles with borg s rep scale of 1517 . We did not detect any increases in the image intensity of the rest of the muscles . Since the first report by lovett in 191518, the mmt has been applied to evaluate the musculoskeletal and nervous systems, and the reliability and validity of the mmt is well established19 . The mmt depends on manual procedures for joint motion, such as flexion of the mp joint . Although the prime movers of a movement can be identified, secondary or accessory movers may be equally important, but definitive studies remain incomplete2 . Compared with emg, mri can image all of the muscles in a single fov in the arm . Moreover, we can observe 11 slices simultaneously along the longitudinal axis of the forearm using a multi - slice spin - echo mr sequence . Thus, mri can detect secondary or accessory movers located even distant from the primary movers in the same fov . Therefore, even with a low number of subjects, the 3 in this study, we can conclude that muscle activity was not detected in other than the primary mover muscles of the 3 mmt used in this study . It can be considered that the strength of the manual resistance of the mmt is important . In a previous report12, the mmt for the extensor digiti minimi muscle was applied until the subjects were unable to continue the extension of the digitus minimus . As a result, we detected other muscle activity in the extensor carpi ulnaris muscle that acts to extend and adduct at the carpus, in addition to the extension of the digitus minimus . Therefore, in this study, we adjusted the strength of the exercise to borg s rpe scale of 1517, as that might be suitable for the mmt of the extensor indicis muscle . The results of this study suggest that mr imaging could be useful in training physical therapists in using the mmt, because muscle activity can be visualized by mr images . We plan to study using the mmt for these muscles, such as the flexor carpi radialis muscle and the flexor carpi ulnaris muscle . In these muscles, we can expect activity due to not only the primary mover muscles, but also accessory muscles . It can be considered that mri technique will be useful to analyze mmt results, since we need not predict specific candidate muscles . Thus, mri techniques should be useful to increase the reliability of the mmt.
Bis - quinolizidine alkaloids produced by lupine species have generated much interest because of their valuable pharmacological properties . Both pharmacological and toxicological properties of these alkaloids are well known [1, 2]. Sparteine appears to offer protection to plants from leguminosae family against insects and grazing mammals [3, 4]. Several bis - quinolizidine alkaloids (sparteine, lupanine, 17-oxosparteine, 13-hydroxylupanine, angustifoline, etc .) Show antihypertensive, antipyretic, anti - inflammatory, antiarrhythmic, diuretic, hypoglicemic, hypotensive, antidiabetic, respiratory depressant and stimulant, and uterotonic properties [5, 6]. The mass spectrometry study of bis - quinolizidine alkaloids has been stimulated by the evidence of the method's ability to distinguish their stereoisomers, metamers, and positional isomers [714]. The main characteristic of the so - called hard electron - impact induced ionization (ei) of mass fragmentation of bis - quinolizidine alkaloid molecular ions is the dependence of the fragmentation pathway of the bis - quinolizidine skeleton on the stereochemistry of the a / b and c / d ring junctions . The stereochemical effects that are encountered with dissociations of stereoisomers incorporating saturated heterocycles rings are due to the ability of chemical bonds to be broken or formed . Mass spectrometry includes a broad range of techniques that have allowed us to prove the detailed structures of organic compounds in a variety of ways . Fast atom bombardment ionization (fab) is classified as a soft ionization technique in mass spectrometry and is well suited to organic compounds which contain a basic functional group . Those compounds tend to run well in positive ion mode . In the positive fab technique a high velocity, rare gas atom molecular beam was produced in the ionization source, and directed onto the sample which was in solution (in the matrix) on a target, thus causing desorption of protonated molecular ions from the sample . Generally, positive fab produces protonated molecular ions m+h with a little fragmentation, and there are some limitations because the presence of matrix gives rise to matrix - related ions . If, by chance, the sample give rise to ions at anyone of the m / z values of matrix, than the matrix should be changed . There are many references in the literature for different matrices where their molecular formulae and masses, their most frequently encountered m / z ions, and their uses have been summarized . As a continuation of our previous study it seems reasonable to extend investigation over fab mass fragmentation of isomeric oxo - substituted sparteine derivatives . Fab - ms gives useful information for the chemical characterization of different types of alkaloids [16, 17]. Fast atom bombardment (fab) mass spectral behaviour of oxo - substituted sparteine derivatives has not been reported in the literature . In a previous work we described the fragmentation routes of seven bis - quinolizidine alkaloids under fast atom bombardment conditions . We have shown that positional isomers of sparteine derivatives can be differentiated on the basis of their fab mass spectra . The aim of this study was to explain the fab mass fragmentation of isomeric sparteine lactams, for example, 2-oxosparteine (lupanine) 1, 15-oxosparteine 2, 17-oxosparteine 3, 2,17-dioxosparteine (17-oxolupanine) 4, 2,13-dioxosparteine (13-oxolupanine) 5, 2-oxo-13-hydroxysparteine (13-hydroxylupanine) 6, and 2-oxo-17-hydroxysparteine (17-hydroxylupanine) 7 (figure 1). These compounds consist of four rings, two of which (a / b) form a sofa / chair system of trans quinolizidine, the second trans system (c / d) form a boat / chair conformations . On the basis of the analysis of the fragmentation processes of 17 in the fab conditions we wished to establish whether it would be possible to distinguish the positional isomers (13; 4,5; 6,7) (figure 1). Compounds 17 were obtained in the form of free basis according to literature methods [1824]. Their spectral characteristics were consistent with the literature data [1824]. The fab spectra were produced using 3-nitrobenzyl alcohol (m - nba) as matrix . These spectra were recorded in positive mode on an amd - intectra gmbh harpstedt d-27243 model 604 two - sector mass spectrometer . For the collision - induced dissociation (cid) experiments, helium was used as a collision gas in the first field - free region (1ffr) at a pressure corresponding to 50% attenuation of the precursor ion signal . The mass spectrometric behaviour of isomeric 17 was investigated in details by the positive fab mass spectrometry combined with cid . The relative abundances of characteristic peaks of even - electron as well as matrix - derived ions are presented in table 1 . On the basis of fab and fab / cid mass spectra of 17, the fab mass fragmentations of these compounds are shown in scheme 1 . In the fab spectra of 17 apart from the expected protonated [m+h] a ions, there are also fragment ions . Fragmentation of the cyclic m+h a of 1, 2, and 3 (scheme 1, table 1) proceeds by the cleavages of two bonds of ring b and c of the sparteine skeleton . The cleavages of c6-c7 and c9-c10 bonds of ring b (for 1) or c9-c11 and c7-c17 ones of ring d (for 2 and 3) lead to the even - electron fragment ions d [c9h15n+h], at m / z 138 . The cleavages of c7-c17 and n16-c17 bonds of ring c (for 3) lead to the even - electron fragment ion b [c14h24n2+h] [m+h - co], at m / z 221 . It should be pointed out that the origin of the even - electron fragment ions b and d has been confirmed by the fab / cid mass spectra of 13 . The even - electron fragment ions a [m+h] gives the base peaks for the spectra of 2 and 3 . In the fab mass spectrum of 1 fragmentation of the cyclic protonated molecule m+h of 3 (table 1) proceeds also by the cleavages of n16-c17 and c17-c7 bonds of ring c with elimination of the neutral molecule of carbon monoxide and leads to the even - electron fragment ion b [c14h24n2+h] [m+h - co] at m / z 221 . It ought to be pointed out that in the molecules 2 and 3 in the bis - quinolizidine skeleton a and b rings form a trans double - chair system that is relatively resistant (for thermodynamic reasons) to conformational - configurational changes than a / b trans - fused rings with sofa / chair conformation of the molecule of 1 . This suggests that the structure of 2 and 3 increases the stability of m+h ions of these compounds in comparison with that of m+h ion of 1 . On the other hand the localization of oxo groups in the different position in bis - quinolizidine skeleton, that is, at c2 (ring a; 1), at c15 (ring d; 2) and c17 (ring c; 3) influences clearly on the elimination of the neutral molecule of carbon monoxide from m+h ion of 3 . Such ejection of co has been seen previously in the ei mass fragmentation of the molecular ion of 17-oxosparteine . The differences in the relative abundances of ions a in the spectra of 13 depend clearly on the differences in the conformations of bis - quinolizidine skeleton of these compounds . In the light of these data isomeric compounds 1, 2, and 3 can be distinguished from each other on the basis of the differences in the relative abundances of ions a and d (table 1, scheme 1) as well as the presence of the even - electron fragment ion b [m+h - co] in the fab mass spectrum of 3 . The common characteristic features of the fab fragmentation of the protonated molecules [m+h] a of 4 and 5 are the cleavages of bonds of rings b and c (c6-c7 and c9-c10 as well as c7-c17 and c9-c11, resp . ). The fragment ions d [c9h12no+h] at m / z 151 were obtained in this way by fab fragmentation (table 1, scheme 1). It should be pointed out that the even - electron ions [m+h] are the base peaks in the fab spectra of 4 and 5 . In the fab fragmentation of 5 (table 1, scheme 1) the elimination of a neutral molecule of water from the enol - tautomeric form of the [m+h] molecule of 5, that is, the cleavage of c13-o bond of ring d leads to the even - electron fragment ion c. isomers 4 and 5 can be distinguished by the presence of even - electron fragment ion [m+h - h2o] c [c15h20n2o+h] at m / z 245 in the fab spectrum of 5 . The absence of the elimination of water in the fab mass fragmentation of m+h a ion of 4 is probably causes by neighbouring heteroatom participation in the elimination of this small molecule . Protonated 4 shows no fragmentation by loss of water than does protonated 5 . In 4 the proton forms a hydrogen - bridge between oxygen a nitrogen atom of enol - tautomeric form of 4 thus stabilizing the ion . No such stabilization is possible in enol - tautomeric forms of 5 which undergoes fast elimination of water (figure 2). The common characteristic features of the fab fragmentation of 6 and 7 are the cleavages of c7-c17 and c9-c11 bonds of ring c of the lupanine skeleton . The fragment ions d [c9h12no+h] at m / z 151 are obtained in this way of fab fragmentation . In the fab mass spectra of 6 and 7 there are also even - electron fragment ions c [c15h22n2o][m+h - h2o] at m / z 247 . In the fab mass spectrum of 6 the base peak is ion [m+h] a and in the fab mass spectrum of 7 the base peak is ion c. the differences in the relative abundances of ions a, c, and d in the fab spectra 6 and 7 allow differentiation of these positional isomers . It ought to be pointed out that the water loss is much more favourable in the fab mass fragmentation of 7 than 6 because after this elimination in the even - electron fragment ion c of 7 the charge is probably situated on the annular nitrogen atom n16, and in the case of 6 the charge is probably situated on the carbon atom c13 . Identification and structural characterisation of isomeric bis - quinolizidine alkaloids is an important problem in their analysis . Mass spectrometry is a powerful tool for unambiguous determination of the structure of these compounds . In the literature that is no information about the fab mass fragmentation of bis - quinolizidine alkaloids . The present study has demonstrated that fab mass fragmentation of isomers of lactams of sparteine sparteine 17 (13; 4, 5; 6, 7) could be expressed as follows . (1) the fab mass fragmentation of the protonated molecules m+h of 17 proceeds mainly by the cleavages of bonds in b (c6-c7 and n1-c10) ring and c (c9-c11, c7-c17) ring of the bis - quinolizidine skeleton (table 1, scheme 1). (2) the protonated molecules m+h of investigated isomers 17 follow the common fragmentation pathways, but with differences in the relative abundances of fragment ions (table 1). (3) the differences in the relative abundances of fragment ions depend mainly on the location of the carbonyl function in the bis - quinolizidine skeleton . (4) the differences in the relative abundances of fragmentation ions depend also on the stereochemistry of a / b and c / d ring junctions of investigated 17 . (5) the main fab fragmentation of 3 involves also elimination of the neutral molecule of carbon monoxide [(m+h - co)] m / z 221 (table 1). (6) the main fab mass fragmentation of 6 and 7 involves also elimination of the h2o neutral molecule, yielding ions at m / z 245 [m+h - h2o] (5) and m / z 247 [m+h - h2o] (6, 7), respectively (table 1). (7) the differences in relative abundances of even - electron ions a and d (table 1) in the fab mass spectra of 1, 2, and 3 and the presence of even - electron ion b [m+h - co] in the fab mass spectrum of 3 allow differentiation of positional isomers 1, 2, and 3 . (8) the presence of even - electron fragment ion [m+h - oh] c allow differentiation of positional isomers 4 and 5 (table 1). (9) the differences in the relative abundances of ions a, c, and d in the fab mass spectra of 6 and 7 allow differentiation of these positional isomers.
Throughout life, we are constantly challenged by pathogens, and re - exposure to previously encountered viruses or bacteria happens frequently . Homologous re - infections are often effectively controlled by neutralizing antibodies, but pathogens that express altered serological epitopes (heterosubtypic re - infection) can bypass antibody - mediated immunity and cause a secondary infection (corti and lanzavecchia, 2013). Right at the beginning of such a re - infection, the t cell repertoire may already contain large numbers of pathogen - specific t cells, and some of these can immediately exert effector function (zhang and bevan, 2011). Moreover, preexisting pathogen - specific but insufficiently neutralizing antibodies could strongly alter infection and replication kinetics (beura et al ., 2016) of a pathogen, and this may significantly alter tissue tropism (rothman, 2011). It has been shown that cells of the innate arm of the immune system can respond more vigorously (sun et al ., 2009), and memory cd8 t cells can even contribute to controlling pathogen load in the early phase of infection in a non - cognate, innate - like fashion (chu et al ., 2013). Thus, pathogen spreading, antigen presentation, t cell activation kinetics, and levels of inflammation and tissue destruction can significantly vary between primary and secondary infections . A particular feature of heterosubtypic re - infections is that many antigens are shared in the primary and secondary infection, but the second pathogen expresses epitopes, which are new to the immune system . Such a situation of preexisting partial immunity to a pathogen can also occur in a primary infection when a pathogen happens to share an epitope with a previously encountered unrelated pathogen a phenomenon known as heterologous immunity (welsh et al ., 2010, che et al ., 2015). Though highly relevant for understanding immunity in humans, interferences between past and present infections (or vaccinations) are rarely investigated under well - defined conditions . Thus, we still have very limited insight into how existing immunity impacts t cell activation, expansion, and conversion into effector and memory t cells . Similarly, pre - existing partial immunity can significantly alter the outcome of vaccination (frahm et al ., 2012). It is also particularly important to consider that pre - existing immunity does not inevitably result in better immune protection . Instead, certain re - infections are known to be accompanied by enhanced pathology, as occurs when a dengue - virus - immune individual is exposed to a different virus serovar (rothman, 2011). Similarly, certain vaccination trials are suspected to have augmented the severity of subsequent infections (blanco et al ., 2010, moore et al ., 2008). The mechanisms underlying such disease enhancement are not clearly resolved, and it is not known how frequently this may occur . All of these points strongly underline the need to investigate immune responses in immune or partially immune individuals . Here, we utilize well - controlled experimental systems to dissect and characterize how different levels of pre - existing immunity influence t cell responses . We observed that t cell activation thresholds differ substantially between primary and secondary infections . We report that only the highest - affinity ligands induce t cell expansion in heterosubtypic re - infections, whereas minor reductions in the levels of tcr stimulation fail to induce t cell expansion . Importantly, we show that neither a shortened inflammatory response nor a globally altered antigen presentation pattern are responsible for raising the threshold . Instead, we found that even a single shared epitope recognized by previously formed memory cd8 t cells is sufficient to increase the t cell activation threshold of naive t cells . We noted that this elevated threshold is mediated by a non - transferrable endogenous memory t cell population . Together our data provide relevant insights into t cell differentiation mechanisms in secondary heterosubtypic infections and for the design of vaccines that are given multiple times . We previously reported that low - affinity t cells differentiate into effector and memory cells in primary infections (zehn et al ., 2009). We established this using recombinant listeria strains, which express full - length ovalbumin [ova] that contains either the original high - affinity, h-2k - restricted ot-1 ligand siinfekl (ova357364) or different altered peptide ligands (apls) a2> y3> q4> t4 apl (listed in decreasing ot-1 stimulatory potency). We then became interested in addressing the stimulation requirements in secondary heterosubtypic re - infections . To do so, we infected naive or wild - type listeria (lm - wt)-experienced mice with lm - ova- or apl - expressing listeria (see figure 1a). With this experimental setup, we mimic a frequently occurring situation where an individual is immune to some but not all antigens in a secondary infection . We observed that the high - affinity ot-1 ligand n4 induces similar expansion in mice with or without a previous lm - wt immunization . In contrast, ot-1 expansion in response to listeria expressing the slightly weaker sainfekl (a2) or siynfekl (y3) apl was decreased in lm - wt immune mice (figure 1b) and significantly impaired in response to q4 or t4 . Similar exclusions of low - affinity t cells were observed when we transferred ot-1 t cells before the primary lm - wt infection (figure s1a) and when memory ot-1 t cells were used instead of naive ot-1 t cells (figure s1b). Notably, both n4 and t4 induce similar carboxyfluorescein succinimidyl ester (cfse) dilution in primary infections, but only n4 induces robust proliferation in immune mice (figure 1c). Some ot-1 t cells proliferate in response to t4 in listeria - immune mice, but this did not increase total ot-1 t cells (data not shown). We therefore conclude that low - affinity stimulation leads to an abortive t cell response in heterosubtypic re - infections . To illustrate the relevance of our observation, we transferred low - affinity h-2k / ova - specific ot-3 tcr transgenic t cells (enouz et al ., 2012) into naive or lm - wt immune rip - mova mice . The lm - wt immune mice were then infected with the same challenge as in figure 1a, whereas the naive rip - mova mice received a priming dose . Using this setup, only naive mice showed diabetic blood glucose levels above 300 mg / dl (figure 1d). Next, we wanted to define the difference in the affinity range of t cells responding in primary versus heterosubtypic infections . We previously showed that the low - affinity v4 ligand activates ot-1 t cells (zehn et al ., 2009), even though it has a 1,000-fold lower ec50 than the n4 ligand (figure 2a). Interestingly, the d4 apl has an ec50 value that is 10,000-fold below n4 . To test the ot-1 t cell response to this very low - affinity ligand we confirmed that the lm - d4 strain is infectious and comparable to lm - n4 (figure s2a). Unexpectedly, even lm - d4 induced rapid initial ot-1 proliferation (figure 2b). D4 activated ot-1 t cells secrete tnf and ifn (figure 2c) and produce granzyme b (figure 2d), and they differentiate into memory t cells that can undergo secondary expansion (figure 2e). We illustrated this by transferring ot-1 cells into mice that were either infected with lm - d4 or with lm - wt . Four weeks later, we challenged the mice with vesicular stomatits virus expressing ova (vsv - ova). We noticed higher numbers of ot-1 cells in mice primed by d4 in comparison to lm - wt - primed mice that do not stimulate ot-1 t cells (figure 2e). T cells indicated that the signal 1 (tcr stimulation) activation requirement in primary infections is very low . In contrast, the difference between the normal n4 and the a2 or y3 apl is rather small (figure 2a). Nonetheless, this level already strongly impairs t cell expansion in secondary or cross - reactive infections . We therefore conclude that there is a substantial stimulation threshold difference between primary and secondary infections . To further illustrate the significance of this difference, we marked the known boundaries for ligands which induce positive and negative selection of ot-1 t cells in figure 2a, whereby t4 is the threshold ligand between positive and negative selection (figure 2a) (daniels et al . G4 is known to induce positive selection in fetal thymic organ cultures (yachi et al ., 2006). Given that d4 with a potency much below g4 supports effector t cell differentiation, we wondered whether even the presumed endogenous positive selecting ligands for ot-1, isfkfdhl from the f - actin capping protein (cp1) and rtytyekl from -catenin (hogquist et al . 2002) would drive ot-1 expansion when expressed by listeria, but this was not the case (figure 2f). We confirmed that the lack of an ot-1 response is not due to a lack of bacterial replication and that an lm-cat infection is, in this respect, comparable to an lm - n4 infection (figure s2a). E1 ova (eiinfekl) is the only known apl that supports ot-1-positive selection in vivo (stefanski et al ., 2001). The lack of a peripheral response to e1 (figure 2f) suggests that it more closely resembles the quality of ligands (cp1 and -catenin) that are thought to naturally support positive selection . Given the activation threshold differences in primary versus heterosubtypic infections, we sought to define the responsible mechanism . Depletion of cd4 t cells and nk cells from lm - wt primed mice did not restore low - affinity t cell activation (data not shown). Taking a possible role of listeria - specific memory cd8 t cells into consideration, we set up a system that allowed us to control the size of the antigen - specific memory population by sharing only one cd8 t cell epitope between two consecutive infections . We constructed listeria strains that express n4 or t4 plus the lymphocytic choriomeningitis virus (lcmv)-derived gp33 - 41 epitope . We found that lm - gp33-n4 and lm - gp33-t4 have similar in vivo growth rates (figure s2a) and induce similarly sized populations of endogenous gp33-specific t cells (data not shown and figures s2b and s2c). T cells are primed by these listeria strains in naive or lcmv immune hosts (figure 3a), we observed a 2-fold reduction in ot-1 expansion to n4 and an essentially non - detectable response to t4 in lcmv immune mice (figure 3b). This indicates that pre - existing antigen - specific memory cd8 t cells effectively raise the t cell activation threshold and that only one epitope (or two if one also considers the h-2k - restricted gp34 epitope) (hudrisier et al ., 1997) we considered that rapid listeria clearance in immune hosts might shorten the duration of antigen - presentation and lower the magnitude of concomitant inflammation (zehn et al ., 2014, prlic et al ., 2006). To address this point, we transferred cfse - labeled ot-1 t cells into naive or lm - wt - primed mice and challenged them with lm - n4 . We observed similar cfse dilution in naive and immune hosts excluding major differences in antigen presentation kinetics in the two hosts (figures 3c and 3d). Also anti - cd40-mediated dc maturation or administration of recombinant ifn to increase inflammation left the t4 response unchanged (data not shown). Next, we established comparable levels of listeria induced inflammation and tissue destruction in lcmv immune and naive mice by co - injecting lm - wt and lm - gp33-t4 into lcmv immune mice . Here, only lm - gp33-t4 are recognized by memory t cells, whereas the lm - wt infection propagates normally . This resulted in similar absolute listeria titers in lcmv immune and naive mice, but the response to t4 remained unchanged in immune mice (figure 3e). To control for an impact of the different listeria challenge doses in naive or immune mice (figures 1, 3a, and 3b), we used acta - deficient listeria . These strains cannot spread from cell to cell and are rapidly cleared by the host, allowing c57bl/6 mice to be challenged with a high listeria dose . This creates a setup in which antigen levels are primarily dependent on the amount of the initial bacterial challenge dose, but not the in vivo expansion, of injected bacteria . We observed a lack of low - affinity t cell expansion in immune mice challenged with acta - deficient lm (figure 3 g). This excludes a principle shortage in antigen presentation and a lower inflammatory response as causes of the observed effect . To determine whether the presence of memory t cells is sufficient to raise the activation threshold, we transferred gp33-specific memory t cells into new c57bl/6 hosts along with cd45.1/2 congenic ot-1 t cells . Contrasting our prior observation, the transferred memory p14 t cells reduced, but did not completely suppress, the ot-1 response to lm - gp33-t4 . In an lm - gp33-n4 infection, the same p14 dose caused only a minor impact on the ot-1 response (figure 4a). Similar outcomes were observed when p14 memory t cells were transferred 28 or 80 days after the lcmv infection (figure s3a). We considered the possibility that the number of transferred p14 t cells was too low to suppress the t4 response effectively and therefore increased the number of transferred p14 to 10 cells . Again, the t4 response was incompletely blocked, but surprisingly, this p14 dose already caused a strong decline in the high - affinity ot-1 response (figure 4b). These data suggest that transferring memory t cells is not sufficient to recapitulate the selective and complete exclusion of low - affinity t cells, as observed when endogenous memory t cells respond to a pathogen expressing a shared antigen (figure 1). To exclude a sole ot-1-dependent phenomenon, we reversed the strategy and used the recently described gp33c6 low - affinity apl for p14 t cells (utzschneider et al ., 2016). We transferred ot-1 memory and naive p14 t cells into naive b6 mice and infected them with listeria expressing ova and wild - type (lm - gp33-n4) or the low - affinity apl gp33c6 (lm - gp33c6-n4). Again, the memory ot-1 had a minor impact on the p14 response to high - affinity ligands and a much stronger impact on the lower - affinity gp33c6 response (figure s3b). These data also show that a weak or strong response of de novo primed naive ot-1 or p14 t cells does not impact the response of memory p14 (figure s3a) or ot-1 t cells (figure s3b). We also examined the phenotype of p14 t cells before (endogenous situation) and after the transfer and noticed a larger population of klrg1 t cells among endogenous compared to transferred memory t cells and a higher proportion of cd43cd27 cells (figures 4c and 4d). Both populations are thought to have immediate effector function (olson et al ., 2013). Moreover, listeria monocytogenes enters the spleen via the bloodstream and in a dendritic cell dependent fashion (neuenhahn et al ., 2006). At this stage, they will likely be in contact with effector / memory t cells that are positioned in the spleen in close proximity to the bloodstream . By injecting fluorescently labeled anti - cd8 antibody into mice, we marked the fraction of p14 t cells that are in close contact with blood (galkina et al ., 2005). Interestingly, but in line with an earlier publication (olson et al ., 2013), we saw that the klrg1 or cd43cd27 t cells (figure 4e), which show a low engraftment efficacy in our memory transfers, were enriched among the p14 cells with access to the blood stream . Therefore, we favor the conclusion that a form of tissue - resident t cell population is more effective than transferred t cells in raising the activation threshold in heterosubtypic infections . We show a clear difference in the tcr stimulation threshold between primary and secondary infections . Only very high - affinity ligands activate t cells in immune mice, whereas ligands such as a2 and y3, whose ec50 for inducing a half - maximum ifn response differed only 2- to 4-fold from the wild - type n4 peptide (figure 2a), trigger a much weaker response in immune compared to naive mice . This is in contrast to the situation in naive mice in which even ligands with 10,000-fold lower ec50 activate ot-1 t cells (figure 2a). Our observations help to understand t cell responses in heterosubtypic re - infections caused for instance by different influenza strains or dengue virus serotypes . Serial infections with the distantly related lymphocytic choriomeningitis virus and pichinde virus were shown to induce a narrowed pathogen - specific t cell repertoire because of antigens that are shared between these infections (cornberg et al ., 2006, welsh et al ., 2010). Our data indicate that this exclusion is caused by a memory t - cell - mediated elevation of the stimulation threshold and that even minimum cross - reactivity is sufficient to cause this effect . The exclusion of low - affinity t cells by previously established memory t cells has consequences for designing vaccine strategies that rely on a prime - boost regimen and intend to elicit a broad t cell repertoire (such as hiv vaccine trials). Several examples underline that optimum protection against pathogens is mediated by highly diverse t cell populations . This ensures cross - reactivity to pathogens expressing mutated epitopes, and it enables individuals to better handle a heterosubtypic infection with a related but non - identical pathogen . It also prevents the selection of escape variants (price et al ., 2004, meyer - olson et al ., 2004, t cell receptor diversity in the population of antigen - specific t cells is largely facilitated by expanding t cells, which suboptimally respond to antigen (low - affinity t cells) (zehn et al ., 2009). The focusing of the t cell repertoire as we observed it may be beneficial for the ongoing secondary infection, but the narrow repertoire may severely diminish the ability to subsequently cross - react to related antigens . Similarly, tumor immune responses rely on recruiting t cells of intermediate or low affinity given that high - affinity t cells are often eliminated by tolerance mechanisms (enouz et al ., 2012). Our data imply that vaccination strategies, which involve repetitive injections, will perform suboptimally if the vaccine vector contains previously experienced epitopes . In addition, pre - existing immunity to vaccine vectors were shown to strongly diminish the efficacy of inducing t cell immunity . Specifically, adenovirus - seropositive vaccine recipients had lower hiv - specific responses to an adenovirus 5-vectored hiv vaccine in comparison to adenovirus - seronegative recipients (frahm et al ., 2012). In line with this previous study, our data show that a minimal epitope overlap is sufficient to impact not only the quantity but also the breadth of the recruited t cell repertoire . In contrast, memory t cells do not seem to impact the clonal - like expansion of antigen - specific nk cells (johnson et al ., 2016). Our data suggest that populations of memory t cells, which are difficult to transfer, are more effective than transferrable memory populations in raising the stimulation threshold . These cells may impair the translocation of antigen - loaded dcs from the marginal zone into the t cell zone, and this barrier function may lead to altered antigen presentation and consequently a higher stimulation threshold . This is in line with a recent report that highlights the presence of a tissue - resident t cell population in the spleen (schenkel et al ., 2014). Nonetheless, it remains difficult to precisely distinguish between qualitative and quantitative causes for the observed differences between transferred and endogenous memory t cells . What supports the notion of a qualitative difference is that the transfer of a large number of memory p14 t cells, which already diminished a high - affinity response, did not resemble the effect seen with endogenous memory t cells . C57bl/6 mice were obtained from charles river, and ot-1, rip - mova, and cd45.1 congenic c57bl/6 mice were obtained from jackson laboratories . Low - affinity ot-3 tcr transgenic mice were described previously (enouz et al ., 2012). Mice were bred in specific pathogen - free (spf) facilities and infected in spf or conventional facilities at the university of lausanne . Mice that were at least 6 weeks old were used for experiments in compliance with the university of lausanne institutional regulations, and the experiments were approved by the veterinarian authorities of the swiss canton vaud . Single - cell suspensions were obtained by mashing spleens through a 100 m nylon cell strainer (bd falcon). Red blood cells were lysed with hypotonic ammonium - chloride - potassium (ack) lysis buffer . If cells were harvested less than 5 days after the infection, then the spleens were before mashing digested with 150 g / ml dnase i (roche) and 200 g / ml liberase tl (roche) for 40 min at 37c . The mouse cd8 t cell enrichment kit (miltenyi biotech) was used for cd8 t cell enrichment . Memory p14 cells were isolated by using fluorescein isothiocyanate (fitc), phycoerythrin (pe), or biotin - conjugated cd45.1 antibodies followed by anti - fitc, anti - pe, or anti - biotin microbeads (miltenyi biotech) according to the manufacturer s instructions . Cells were labeled in serum - free medium with 5 m cfse at 37c for 10 min . Mice were infected intraperitoneally (i.p .) With 2 10 plaque - forming units (pfu) of lymphocytic choriomeningitis virus (lcmv strain 53b, armstrong) grown and titrated on vero cells (utzschneider et al ., 2013). Vesicular stomatitis virus expressing siinfekl (n4) (kim et al ., 1998) was grown and titrated on bhk cells . Recombinant listeria monocytogenes strains expressing ovalbumin that contain k / ova - derived apl were previously described (zehn et al ., 2010). 2002), which contains native ovalbumin (aa134387) or ovalbumin with the siitfekl variant (zehn et al ., 2009), were digested with sapi . Oligonucleotides encoding kavynfatc (gp33) or kavyncatc (gp33c6) plus an alanine on both sides were ligated into the sapi restriction site . The constructs were inserted into wild - type or acta - deficient 10403s listeria by e. coli conjugation as previously described (lauer et al . Listeria were grown in brain heart infusion broth (oxoid, thermo fisher) to mid - log phase . Then, bacterial numbers were determined by measuring the od at 600 nm, and diluted stocks were i.v . Naive mice received 1,0003,000 colony - forming units (cfu), mice previously infected with listeria received 5 10 cfu, mice previously infected with lcmv received 2 10 cfu, and, for infection with acta - listeria, 10 cfu were injected . Bacterial loads were determined by lysing spleens in pbs supplemented with 0.1% tergitol np-40 (sigma - aldrich). Serial dilutions were spread out on brain - heart infusion plates containing streptomycin (200 g / ml), and colonies were enumerated . A large pool of siinfekl - derived synthetic altered peptide ligands were tested for h-2 kb stabilization in rma - s cells and recognition by ot-1 t cells as previously described (zehn et al ., 2009). The d4 peptide shows similar surface stabilization as the n4 peptide, but it is a weak agonist for ot-1 t cells (figure 2a). Ot-1 cells were stimulated in rpmi (10% fcs, 100 iu / ml penicillin, 100 iu / ml streptomycin, 5 m 2-me, 5 mm hepes; invitrogen) with anti - cd3/cd28-coated beads (dynabeads, invitrogen) and cultured with 50 u / ml human il-2 (chiron) in 7% co2 . 6 days after activation, 2 10 ot-1 cells and 1 10 rma cells were mixed in 96-well plates, and titrated concentrations of siinfekl or apl peptides (emc microcollections) were added . After 30 min, 7 m brefeldin a (sigma - aldrich) was added, and cultures were incubated for another 3.5 hr . Up to 4 10 cells were plated in 96-well plates in pbs supplemented with 2% fcs and 0.01% azide . Cells were surface stained for 20 min at 4c with fluorescently labeled anti - cd8 (536.7), cd127 (a7r34), klrg1 (2f1), cd27 (lg7.f9), cd43 glyco (1b11), cd4 (gk1.5), cd45.1 (a20), and cd45.2 (104) antibodies and fixed in pbs supplemented with 1% formaldehyde, 2% glucose, and 0.03% azide . For intracellular cytokine staining, cells were fixed in pbs 2% formaldehyde and permeabilized in pbs with 0.25% saponin and 0.25% bsa (perm buffer). Cells were stained in perm buffer for ifn (xmg1.2), tnf (mp6-xt22), or granzyme b (gb12). Fluorescently labeled mhc - i - gp33 multimers were used for detecting gp33-specific t cells (tc - metrix). For in vivo staining experiments, we injected mice i.v . With fluorescently labeled anti - cd8 antibodies . Mice were sacrificed 3 min later, and harvested organs were processed as indicated above . Flow cytometry data were obtained on a bd facs lsrii machine and analyzed using flowjo software (tree star). Graphpad prism was used for graphic representation of data, calculation of ec50 values, and statistical calculations . P values 0.05 were considered significant (p <0.05; p <0.01; p <0.001), and p values> 0.05 were considered non - significant (ns).
Uveal melanoma (um) is a rare intraocular cancer affecting the choroid, ciliary body, or iris . At diagnosis, almost all patients will present without evidence of metastatic disease . Despite high rates of primary tumor control, metastatic lesions, predominantly to the liver, occur in approximately 2050% of um patients . Primary tumor gene expression profile (gep) testing has shown that patients can be accurately and reliably classified into low - risk class 1 and high - risk class 2 with significantly different metastatic potentials [25]. These patient groups should be managed based upon their relative risk versus the overall population risk . For clinical care purposes, a prognostic tool should be highly accurate in its risk prediction (clinical validity), and its results should be implemented to inform patients' subsequent management plans (clinical utility). Clinical factors including age, extraocular extension, tumor size, and ciliary body involvement have been linked to higher risk for metastasis of um tumors [7, 8]. However, none of these have offered the prognostic accuracy or reproducibility required for clinical implementation . Genetic analysis has led to the identification of metastasis - associated cytogenetic abnormalities on chromosomes 1, 3, 6, and 8 [914]. In particular, the presence of monosomy 3 in primary tumor cells is a significant factor for predicting metastatic risk [10, 15, 16], and fluorescence in situ hybridization (fish), comparative genomic hybridization (cgh), multiplex ligation - dependent probe amplification (mlpa), and loss of heterozygosity (loh) have been developed to identify chromosome abnormalities [1720]. However, to our knowledge, these molecular tests to detect gains and losses of chromosomes 1, 3, 6, and 8, including the commercially available mlpa platform [17, 21], have not been clinically validated in prospective, multicenter studies and published clinical utility is limited to high - risk patients . The decisiondx - um gep test is an accurate prospectively validated molecular classifier, and its results are highly correlative to metastatic potential [2, 5], as reported by the collaborative ocular oncology group (coog). Notably, because decisiondx - um has been extensively validated exclusively on pretreatment um specimens and the training set, to which the machine - learning algorithm compares each patient sample to generate a class 1 or 2 result, consists of only such samples, nonmelanoma samples are inappropriate for prognostication with the gep panel, consistent with the exclusion criteria of the coog study . Similarly, given the lack of validation on posttreatment tumors and the potential for radiobiological effects on tumor's genomics, irradiated samples are also ineligible [23, 24]. Decisiondx - um's clinical utility has also been reported, indicating that the majority of ophthalmologists who order the test use the results to guide risk - appropriate treatment and management strategies for um patients . Herein are the first results from an interim analysis of the ongoing, prospective, multicenter clinical application of decisiondx - um gene expression assay results (clear) registry . This study tracks decision impact (surveillance regimens and treatment referral patterns), as well as clinical outcomes for decisiondx - um patients . These results add additional prospective, multicenter evidence underscoring decisiondx - um as a highly accurate and clinically actionable assay for determining risk associated with primary um tumors . After irb approval of the study (number nct02376920; clinicaltrials.gov) at participating centers (mayo clinic, rochester mn; university of virginia, charlottesville, va; retinal consultants, sacramento, ca; retina specialists of michigan, grand rapids, mi), patient consent was obtained . Data entry was performed at receipt of test results and semiannually (censor date: june 2015). Physician - obtained fnab or ffpe um tumor specimens were submitted to a centralized cap - accredited, clia - certified laboratory for gep . Frozen specimens, mostly fnab, were dispersed into rnase - free stabilization buffer immediately following biopsy, and all samples were shipped to the recipient lab on dry ice . Rna isolation was performed with the picopure rna isolation kit (molecular devices, sunnyvale, ca). All ffpe samples were prepared from enucleated globes, and tumor sections on microscope slides were shipped at room temperature to the recipient lab . Tumor tissue was macrodissected from slides using a sterile, disposable scalpel, deparaffinized in xylene, and processed for rna isolation with the ambion recoverall total nucleic acid isolation kit (life technologies corporation, grand island, ny). All rna was assessed for quantity and quality using the nanodrop 1000 system (life technologies corporation) and the agilent bioanalyzer 2100 and then converted to cdna (applied biosystems high capacity cdna reverse transcription kit; life technologies corporation). Each cdna sample underwent a 14-cycle preamplification step and was then diluted 20-fold in tris - edta buffer . Fifty microliters of each diluted sample was mixed with 50 l of 2x taqman gene expression master mix (life technologies corporation) and loaded onto a custom high - throughput microfluidics gene card containing primers specific for 12 class - discriminating genes and three endogenous control genes . Each sample was run in triplicate on an applied biosystems ht7900 instrument (life technologies corporation). Delta ct values were calculated by subtracting the mean ct of each discriminating gene triplicate from the geometric mean of the three endogenous control genes' mean ct values . Molecular class assignments were determined by comparing the 12 discriminating gene ct values from each sample to a well - characterized, proprietary um training set of low - risk class 1 and high - risk class 2 geps using a support vector machine- (svm-) learning algorithm . The predicted confidence is indicated by the discriminant score, which is inversely proportional to the proximity of the sample to the hyperplane established between class 1 and class 2 training set samples . Surveillance regimens were not prespecified but instead were independently decided upon by each participating physician utilizing the decisiondx - um result and documented as part of the registry data entry . The intensity of surveillance for each patient was categorized based upon a previously reported study and determined by the frequency of imaging (ultrasound, pet / ct, or mri) and liver function tests (lfts) that a patient received in addition to their regular eye examination follow - up . A high - intensity schedule was characterized by imaging and/or lfts every 36 months, whereas a low - intensity schedule was characterized by annual imaging and/or lfts . The prospective, multicenter clear study was designed to assess (a) the management of patients according to their decisiondx - um results (clinical utility) and (b) their documented development of metastatic disease (clinical validity). Seventy patients have been enrolled from four centers across the us (table 1). Thirty - seven (53%) were class 1 and 33 (47%) were class 2 . None of the patients who consented to be in the registry study had technical failures with gep testing . Of the class 1 patients, 30 (81%) were class 1a, while 7 (19%) were class 1b . Consistent with their high - risk gep, 12 (36%) class 2 patients experienced a metastasis, whereas only 2 (5%) class 1 patients experienced a metastasis (p = 0.002 by fisher's exact test) with a median follow - up of 2.38 years . The median time to metastasis for class 2 patients was 1.4 years (table 2). Class 2 patients had a significantly worse 3-year metastasis - free survival (mfs) rate of 63% (95% confidence interval = 43%83%) compared to 100% in class 1 patients (log rank test p = 0.003) (figure 1). The median rate of metastasis was 3.78 years for class 2 patients, while the median was not reached for the class 1 patients . The majority of the metastases were localized in the liver, but metastases were also found in the lungs, brain, and bone (table 2). Of note, one of the class 1 patients who experienced metastasis had a large tumor, while the other patient did not undergo treatment of their primary tumor . Nine out of the 12 class 2 patients who ultimately had metastases were treated by enucleation, while 2 were treated with plaque radiotherapy, and one was treated by transpupillary thermotherapy (ttt) (table 2). Tumor nodal metastasis (tnm) staging by ajcc includes tumor diameter, thickness, ciliary body involvement, and extraocular extension of the tumor . In this cohort, neither largest basal diameter nor ciliary body involvement performed as statistically significant prognostic markers (figure 2). While tumor thickness did provide significant stratification of the patients, gep showed stronger prognostic significance in kaplan - meier and multivariate analysis (table 3), similar to previous studies [2, 28]. One primary objective of clear was to document clinical management differences that are implemented for class 1 compared to class 2 patients . Of the 37 class 1 patients, the majority (n = 30) were treated with low - intensity follow - up (imaging and/or lfts every year), while all 33 class 2 patients were treated with high - intensity follow - up (imaging and/or lfts every 36 months) (figure 3). Two of the class 1 patients who received high - intensity surveillance had intermediate risk class 1b results . Only 4 out of the 37 (11%) class 1 patients were referred to medical oncology . Of note, two of these referred class 1 patients had ciliary body involvement, one of whom also had a large (22 mm diameter) tumor and displayed loss of chromosome 3 . Therefore, it is possible that the presence of these clinical features contributed to their medical oncology referrals . Due to their high - risk disease, 6 out of 33 (18%) of class 2 patients were referred to medical oncology and 8 class 2 patients (24%) were referred to adjuvant clinical trials . Importantly, four class 2 patients went on to receive systemic adjuvant therapy; three patients received combinatorial chemotherapy (tamoxifen, sunitinib, and cisplatin) within a clinical trial, while one received ivig immunotherapy . Three of the four patients remained metastasis - free at last follow - up (mean follow - up: 4.48 years; median follow - up: 4.02 years). No class 1 patients were referred to clinical trials or had systemic adjuvant therapy . Taken together, these results indicate that class 2 patients that have imaging and lfts are managed by medical oncology and are offered clinical trial participation significantly more often than class 1 patients (fisher's exact test for intensity of surveillance p <0.0001; for medical oncology / clinical trial referral p = 0.04; table 1; figure 3). The national comprehensive cancer network (nccn) has emphasized the critical importance that all participants in the medical management of cancer patients, including the patients themselves, are provided with timely, reliable, and actionable education regarding molecular testing for diagnosis and treatment . As molecular biomarker tests are increasingly available for diagnostic, prognostic, and therapeutic purposes, there is a growing need for transparency in reporting clinical validity and utility to ensure confidence in the accuracy and clinical impact of their results [6, 29]. Previous studies have reported the validity and utility of decisiondx - um to provide highly accurate prognostic information to guide individualized management of um patients [2, 5, 30]. Herein, we report the initial results of the clear prospective registry study that confirms the clinical utility of decisiondx - um, since it became clinically available . As this trial is ongoing, a final analysis of 5-year survival rates the nccn recommends that the clinical utility of a biomarker test should be determined in a prospective clinical trial . The clear results represent a second independent, prospective, multicenter study making the test unique, not only in um, but in comparison to the majority of high - complexity advanced diagnostic tests . The multicenter coog study validated decisiondx - um's ability to accurately predict patient outcomes, reporting that metastatic events were observed in only 1% of class 1 cases versus 26% of class 2 cases after 50 months of follow - up time (median follow - up was 17.4 months). Similarly, interim results from our prospective registry study to track decisiondx - um patients indicated a low - risk of metastasis for class 1 patients compared to class 2 patients (5% versus 36%, resp . ; median follow - up of 27.3 months). Taken together, the results from the clear registry add to the compelling evidence - based clinical validity of decisiondx - um . As has been demonstrated in other cancers for which prognostic and predictive testing are employed, tailoring a treatment strategy according to patient's individual tumor biology offers the potential for improved quality - of - life and more efficient utilization of healthcare resources [3134]. The clinical utility of decisiondx - um was initially reported following a review of medicare medical records that indicated the test significantly impacted patient management plans, resulting in less aggressive management of class 1 versus class 2 patients, and similar management impact results were observed from blinded surveys of ocular oncologists . The clear results also demonstrate that the clinical management of class 2 patients is associated with significantly higher surveillance intensity, including more frequent imaging, lfts, and referral to medical oncology compared to class 1 patients . The higher - intensity surveillance for class 2 patients is consistent with the goal of potentially identifying metastases earlier, thus permitting intervention, while the patient is asymptomatic and likely more amenable to treatment(s). Conversely, unnecessary surveillance can potentially be avoided for patients in whom extraocular recurrence of disease is unlikely . It should be noted that it is possible, based on a class 1 result, for a patient who ultimately experiences metastasis to receive lower frequency management, thus potentially delaying the identification of metastatic disease by a few months (i.e., at 12 months versus 36 months with annual versus more frequent surveillance, resp . ). However, given the consistency of the mfs rates between the coog study and our data presented here (table 4), we expect metastasis would occur in a small minority of class 1 patients . Furthermore, the small percentage of class 1 patients who experienced metastasis (5%) that we report here is in line with previously reported metastasis rates reported for um patients who are identified as low - risk [21, 35, 36] and for breast and colon cancer patients classified as low - risk by other tests [37, 38]. The data reported in this study are important for um because numerous clinical, pathological, and genetic characteristics of the primary tumor have been proposed as being significantly prognostic for um metastasis, yet these methods have achieved neither adequate nccn level of evidence for clinical utility nor clinical validity . Based upon multiple prospective and retrospective studies published to date, given its robust ability to identify high - risk patients, rational intervention trials to identify effective adjuvant therapies have been initiated [3942]. Four class 2 patients within this cohort pursued adjuvant treatment for their high - risk disease; three of whom were metastasis - free at last follow - up, underscoring the importance of making more clinical trials accessible for high - risk um patients . The decision to enroll class 2 patients in clinical trials is directly related to the level of evidence for metastatic propensity that has been reported for the test . The continued clinical performance and utility demonstrated in this study contributes to the high level of evidence regarding the clinical validity and utility of the decisiondx - um test . This study demonstrates that the 15-gene expression assay decisiondx - um continues to accurately predict metastatic risk for um patients, thus enhancing the molecular test's established clinical validity . Furthermore, this is the first prospective analysis of clinical utility of the assay, and this study demonstrates that the test results are being used to guide decision - making for physicians and patients in the clinic.
There have been numerous studies evaluating the anatomical distribution of vertebral fractures and consistently showing two prevalent peaks of vertebral fractures: the first one in the mid - thoracic spine region (t7/t8) and another one in the thoraco - lumbar junction (tlj)1,3,8,17,23,27). In these previous studies, approximately 4% of thoraco - lumbar vertebral compression fractures occur at l4 level1,3,8,17,23,27). Furthermore, fractures of the 5 lumbar vertebra are quite uncommon, representing only 1.2% of overall spine fractures and 2.2% of thoraco - lumbar fractures9). On the other hand, according to the previous studies on percutaneous vertebroplasty (pvp) or kyphoplasty, the incidence of pvp at l3, l4 and l5 is about 9 - 13%, 5 - 9% and 2 - 5% respectively of all their thoraco - lumbar pvp or kyphoplasty procedures5,18,21,28,29). Therefore, the approximate incidence of pvp at l3 is near to the sum of incidence of pvp at l4 or l5 . As a result of performing over 200 pvp cases for compression fracture at a single institute annually, the authors identified distinct characteristics, clinical presentations and surgical outcomes of patients with lower lumbar compression fractures (l4 or l5). As the pioneers, deramond and galibert reported their first seven procedures in 198710), and pvp has been used to manage vertebral compression fractures15). There also have been studies on the factor, fracture level in thoraco - lumbar spine, that influences the result of pvp with regard to back motion pain2,11,14,25,28). However, most previous authors did not separate the clinical features and surgical outcomes of patients with l3 compression fractures from those with l4 or l5 compression fractures2,11,13,14,18,23,24,28). If the clinical feartures and the surgical outcomes of l3 fracture were significantly different from those of l4 or l5 fractures despite classifying l3, l4 and l5 as the lower lumbar spine, those of l3 level fracture, almost half proportion of the lower lumbar spine fractures, could make some confusion and ambiguousness in analysing those of l4 and/or l5 level fractures . The authors retrospectively investigated our patients data to elucidate whether those patients treated for l4 or l5 compression fractures by pvp were similar, in terms of demography, clinical features and surgical outcomes, to those treated for l3 fractures . We then reviewed the literatures to compare the surgical outcomes of patients with l4 or l5 compression fractures with those of patients with tlj level fractures . Therefore, the purpose of this present study is to provide accurate understanding of clinical presentations and surgical outcomes as well as to identify the unique characteristics of lower lumbar osteoporotic compression fracture (ocf), which would enable physicians and patients to make more informed decisions about whether to perform the procedure and to develop more precise expectations of prognosis . Between october 2008 and july 2012, a total of 762 patients with 948 symptomatic ocfs were consecutively treated with pvp at our single institution . All procedures were performed by the same team, who obtained a detailed and standardized history . Preoperative clinical data were collected retrospectively from the medical records and assembled in database by one of the authors (sh). There were 105 l3 (11% of all procedures), 75/l4 (8%) and 46/l5 (4.8%) ocf patients respectively . Of these cases, the patients with multiple fractures were excluded for the level homogeneity . Hence, a total of 120 patients (17 male and 103 female) were investigated, in which there were 57 patients with l3, 40 patients with l4 and 23 patients with l5 ocfs respectively and they were all treated by pvp (no kyphoplasty). All patients demonstrated acute agonizing or chronic severe focal back motion pain, who did not respond to bed rest or analgesics at least for 2 weeks . Preoperative data included the presence or absence of previous history of pvp or lumbar decompressive surgery, recent trauma history prior to occurrence of acute compression fracture, leg radiating symptoms and value of bone mineral density (bmd). Postoperative data included some information about the presence or absence of cement leakage, subsequent fracture and additional lumbar decompressive surgery . On post - pvp plain images and magnetic resonance imaging (mri) after pvp) vertebral body compression ratio by calculating the height of anterior - posterior (ap) vertebral wall ratio on the lateral radiography19). Therefore, a smaller ap ratio implies a greater degree of compression or wedge deformity . Initially, the data from l5 ocf patients were compared with those from l4 ocf patients by use of chi - square statistics and students t - test (p<0.05). Thereafter, the patients with l4 or l5 ocfs were grouped (group 1) and they were compared with those with l3 ocfs (group 2) for the insignificant variables from initial comparisons . The out - patients follow - up interview and examination were performed at 1 month after pvp . The postoperative back motion pains and leg symptoms were graded by the operating surgeons as improved, unchanged or worse . In addition, long - term follow - up data were obtained via telephone interview in 105 (87.5%) of 120 patients at a median value of 22 months (range, 2 - 47 mo .) After pvp . Telephone interviews were performed by a third party, who was blinded and not involved in the treatment . The patients' preoperative economic and functional statuses were compared with their current statuses using prolo economic and functional grading scale22)(table 1). Patients were determined to have an improved score if either their economic or functional status, such as house - working or daily living activities improved after pvp or to have a worse score if either status was worse after pvp . Furthermore, an additional long - term outcome of leg pain was graded by patients as improved, unchanged or worse . There was no statistically significant difference in incidence of multiple fractures between l3, l4 and l5, which were 46%, 47%, and 50% respectively (p>0.05). The characteristics of 63 patients with single level l4 or l5 ocf are listed in table 2 . In the previous pvp histories, there was statistically significant difference between l4 and l5 (p<0.05) as well as l3 and l5 ocfs (p<0.05). In analysis of the medical chart review and follow up interview on the telephone, there was no statistical difference in postoperative back motion pain, leg radiating pain and functional or economic status between l4 and l5 ocf patients (p>0.05). 56% (13/23) of l5 ocfs and 30%(12/40) of l4 had not anterior wedge deformity but biconcave or uni - concave deformity at middle vertebral body portion . The characteristics of group 1 were compared with those of group 2 except the sole significant variable (previous pvp) from initial comparison at table 3 . The causes of leg radiating pain were mostly combined with intra - canalicular stenosis, disc herniation or partly the encroachment of corresponding intervertebral foramen by height loss due to compression fractures . Not all the cases with leg radiating pain required further lumbar decompressive surgery . In group 1, 64%(29/45) of cases with leg radiating symptoms needed further decompressive lumbar surgery, whereas 38%(9/24) of cases did in group 2 . There were 5 patients who required the foraminal decompression surgery by paraspinal approach or fusion surgery during or after pvp in group 1 (2 patients with l4 ocf and 3 patients with l5 ocf), in which functional or economic status of 4 patients (80%) was improved and 3 patients (60%) showed leg radiating pain improvements on the long - term follow up interviews . However, all further lumbar surgeries were performed for the combined intracalicular pathologies, such as lumbar disc herniation or lateral recess stenosis in group 2 . There were trends toward the higher frequency of cement leakage and needs for further pvp due to subsequent fractures in group 1 compared with those in the group 2 (41% versus 28%, 22% versus 11% respectively), however these did not reach statistical significance (p>0.05). 2 showed clinical success rates using prolo scale and there was statistically significantly worse long - term outcome in group 1 (p<0.05). Only 56% patients showed improvement in economic or functional statuses in group 1, but 72% in group 2 . There was no change of statuses in 40% of group 1 patients after pvp, on the contrary to those in 19% of group 2 . The additional long - term follow up results for leg pain were illustrated in fig . 3 . Meanwhile, the long - term follow up results of patients with leg radiating symptom in group 1 showed leg pain improvements after further decompressive lumbar surgery in 50%(13/26) patients, whereas 27%(3/11) patients were improved without additional decompressive surgery . In group 2, 56%(5/9) patients with leg pain were improved after further decompressive lumbar surgery, on the other hand, 38%(5/13) patients showed improvement without additional decompressive lumbar surgery . However, there were no statistically significant differences between the two groups (p>0.05). Osteoporotic vertebral compression fractures are not uncommon disease in the elderly people and can manifest as severe pain, functional deterioration and limited mobility . Conventional treatments, such as bed rest, bracing, and physical therapy, can result in frequent adverse effects . Furthermore, osteoporotic vertebral compression fractures can cause serious complications during surgery as well as after surgery, such as vertebral reconstruction or fusion operation under general anesthesia . Therefore, pvp is considered the treatments of choice to relieve pain and to stabilize vertebrae . Although it has been reported that pvp has a lot of advantage, the patient population that is most likely to benefit from this procedure is still uncertain1,13,20), and there have also been investigations on unfavorable outcomes and the levels of vertebral fractures as influencing factors2,11,14,25,28). However, most of these studies included l3 level as lower lumbar spine2,11,14,28). Ryu and park25) divided the fracture levels into four groups (upper and lower parts in thoracic and lumbar vertebrae respectively), and categorized l4 or l5 into lower lumbar vertebrae . Furthermore, their cases with lower lumbar fractures might involve many of cases with multiple fractures . Most of accompanying fractures would be at tlj, and the excellent effects of pvp at tlj were well known and self - evident2,5,19,25,28,29,31). Therefore, the inclusion of cases with multiple fractures can confuse the authentic outcomes of pvp for lower lumbar levels in spite of multivariate polytomous logistic regression analysis . Recently, the most notable determining factor during pvp is the concept of cemented vertebral body fraction (cvbf)13,20). To be achievable volume of intravertebral cement is becoming progressively larger as increasing the volume of vertebral body in lower lumbar spine20). Hence, the possibility to obtain an unfavorable outcome would also increase if an amount of cement was not decided in terms of the volume of fractured vertebra body and treated level . However, the ideal needle placement of our procedure was at the anterior third of the vertebral body, and the end point of cement injection during pvp was when the cement reached the posterior quarter of the vertebral body or when significant leakage occurred as described by jensen et al.12). Therefore, cvbf alone cannot explain our poor result of pvp for l4 or l5 compression fractures . The lower lumbar ocfs are unique in a number of ways: they are distinctly uncommon compared with fractures at tlj levels and their symptomatology frequently occurs without any traumatic events, which is combined with leg radiating pain, and outcome after pvp is less satisfactory than that of tlj fracture cases . There have been few studies in which the features of lower lumbar spine fractures have been divided into 2 parts, (1) l3 fracture and (2) l4 or l5 fractures, and the characteristics of their clinical presentations and surgical outcomes have been compared each other . Our data indicate that l4 and l5 vertebral ocfs are different from those at the l3 level, and fractures at the l3 level are more similar to those occurring at tlj . Therefore, the results of previously published reports on lower lumbar ocfs including a preponderance of l3 cases may mask the true features of l4 or l5 vertebral fractures . The origin of these differences is unclear, but those patients with l4 or l5 fractures may have their own spinal biomechanics which are different from those with tlj fractures . In orther words, l4 - 5 and l5-s1 segments have been reported to bear the highest loads and to undergo the most motion in the sagittal plane30), which means that it is resonable to seperate l3 from lower lumbar spine levels in this study . It is also supported by our findings that even though there were no statistical differences in demographic data and bmd between group 1 and group 2, the group 1 had the significantly higher incidence of non - traumatic fractures than that of group 2 . This different biomechanics may prevent these patients from achieving the typical excellent outcome with regard to back motion pain experienced in pvp for tlj fractures . Further clue to this unfavorable result of pvp for l4 or l5 compression fracture can be found in the previous literatures in which almehed et al.1) and el maghraoui et al.8) demonstrated and illustrated that compression severity was highest in the lower lumbar spine . Also, alvarez et al.2) insisted that favorable result of pvp could be expected in patients with the vertebral height loss less than 70% . Their study revealed that pvp for patients with more than 70% vertebral height loss was technically difficult to place the needle safely into the vertebral body with a high incidence of cement leakage into disc space, and the complete relief was limited to less than 30% of patients2). At last, our data demonstrated that group 1 had the higher incidence of cement leakage and rate of subsequent fracture than group 2, which did not reach statistical significance . However this relatively higher rate of subsequent fracture at l4 or l5 level also may contribute to the unfavorable long - term outcome of pvp, which is also suggested by our data that the long - term outcome via telephone interview of group 1 patients could be obtained in 11 patients of 14 patients who experienced the subsequent fracture, in which only 3 patients (27%) had improvement of their functional or economic status . Thermal7), and chemical26) mechanisms have been proposed, however increasing strength and stiffness may be the key elements of its principle mechanism considering previous reports4,16). Chung et al.6) used pvp for the treatment of severe leg radiating pain caused by lower lumbar ocf . They showed the excellent results in which all seven patients had experienced dramatic relief of leg radiating pain after cement injection into lower lumbar fractured body through the pedicle on the symptomatic side . Therefore, they concluded that pvp may be an effective modality of ameliorating leg radiating pain caused by ocf combined with foraminal stenosis through local stiffness mechanism . However, they did not show the overall incidence or outcome of this unique symptom by lower lumbar ocf . Furthermore, the outcome of pvp for leg radiating pain was disappointing level (27 - 56% of amelioration rate) irrespective of further decompressive lumbar surgery in our data . They suggested the clinical characteristics of those patients and described that objective evidence of radiculopathy was identified upon electromyography (emg) or neurological examination . However, the radiculopathy caused by foraminal pathology is not confirmed by clinical symptom, emg and neurological examination, but suspicious . Thus, the definite, widely acceptable and uniform criteria are necessary to confirm this unique symptom of lower lumbar ocf . The limitations of this study were that there was no detailed investigation on functional outcomes such as short form-36 or oswestry disability index, which should be necessary for making this study more valuable . However, the retrospective nature of this study was limited thorough investigation on their functional outcomes . Thus, we only investigated short term and long term postoperative prolo scale as clinical outcome . It is not possible to fully devaluate the usefulness of pvp for l4 and l5 ocf according to the long - term follow up results of this study reporting over 50% improvement, which is not quite good results to compare with those of previous studies2,5,19,25,28,29,31). Therefore, the prospective randomized controlled clinical studies are needed to demonstrate whether pvp is effective at l4 or l5 level ocf and whether those level ocf may be a marker for progressive back pain, radiating pain and overall worse outcomes . The patients with l5 ocf patients treated by pvp were more likely to have had previous pvp, and the patients with l4 or l5 ocf were more likely to be non - traumatic, presenting leg radiating symptoms and requiring an additional decompressive surgery more often than l3 ocf group . The pvp outcome in terms of postoperative back motion pain is worse for compression fractures at l4 or l5 compared with those treated at l3 level.
Renal cell carcinoma (rcc) comprises 23% of all adult malignancies and 85% of malignant renal tumors . The highest incidence is found in individuals in the sixth and seventh decades of life, 66 being the median age at diagnosis [1, 2, 3, 4, 5]. Rcc has a strong propensity to metastasize; 25% of patients initially present with distant metastasis and another 50% develop metastasis during follow - up [1, 6, 7]. It is the third most frequent neoplasm to metastasize to the head and neck region, preceded only by breast and lung cancer [1, 3, 4, 5, 8, 9, 10]. Although infrequently reported, head and neck regional metastases may be linked to rcc in up to 815% of cases [1, 2, 4, 6, 7, 8, 9]. The most affected regions of the head and neck include the paranasal sinuses, larynx, jaws, temporal bones, thyroid gland, and parotid glands . Rcc metastases to the nose and paranasal sinuses are the most frequently affected areas, followed by the tongue [1, 3, 9, 11, 12]. The most common presenting symptoms to the head and neck region include an enlarging neck mass, epistaxis, anosmia, facial pain, nasal obstruction, and diplopia . Rcc is comprised of hypervascular tumors associated with multiple arteriovenous shunts due to the release of vascular endothelial growth factor as well as other angiogenic factors . Given the fact that the kidneys receive 25% of the circulating blood volume, rcc has a high spreading potential via the blood [12, 13]. Rcc has 5 distinct histologic presentations: clear cell / conventional (75%), papillary (15%), chromophobe (5%), collecting duct (2%), and unclassified (3%). Histopathologically, clear cell rcc typically shows a compact alveolar or solid architecture with varying degrees of cystic changes . Rcc characteristically tends to exhibit numerous capillaries and thin - walled blood vessels in the supporting stroma . The cytoplasm is rich in lipids and glycogen; the latter 2 elements dissolve during processing to provide the characteristic clear cytoplasm [2, 7]. Immunohistochemical staining helps in this distinction, exhibiting focal cytokeratin positivity (vs. minor salivary gland cancers that show diffuse positivity) and a strong reaction for vimentin . Radiologic diagnosis is based on the vascular nature of the tumor, which shows moderate to marked signal enhancement on contrast ct . If contrast enhancement indicates destruction and lack of tumor calcification, metastatic rcc should be part of the differential diagnosis . Excision is usually performed to control pain and to manage any potential complications from space - occupying masses in the head and neck region, including the brain [1, 10]. Rcc does not respond well to radiation therapy, and while chemotherapy (interleukin-2, interferon-, and 5-fluorouracil) may be useful in cases of residual disease after resection, a positive response is experienced in less than 25% of patients . Radiotherapy can only improve symptomatic relief and increase quality of life for perhaps a few months . The 5-year survival rate for rcc after nephrectomy is 6075% . Excision of solitary metastatic lesions of rcc following nephrectomy results in a survival rate of 41% at 2 years and 13% at 5 years . The prognosis for patients with multiple rcc metastases is poor, with a 5-year survival rate of 07% [2, 4, 9, 14]. We report on a 62-year - old male who presented to the ophthalmology service with vi cranial nerve bilateral paresis, absence of pharyngeal reflex, dysarthria, right tongue deviation, and paralysis to the right side of the face . Ct and mri were performed, with ct showing a large expansive process at the cranial base with clivus and right petrous apex osteolysis (fig . An mri t1 sequence showed a solid isointense expansive process with bilateral internal carotid artery involvement (fig . 3). An mri t2 sequence revealed small hyperintense areas indicative of being cystic in nature or suggestive of central necrosis (fig . Ipsilateral side secretions coming from the eustachian tube indicated the mastoid region had been compromised . An additional mri t1 sequence following administration of gadolinium contrast material showed moderate enhancement with clear involvement of bilateral internal carotid arteries (fig . 5) and the cavernous sinus, posterior ethmoidal sinus, clivus and both petrous apex infiltration . The head and neck cancer committee recommended ultrasonography, which subsequently revealed a heterogeneous echogenic multilobular mass on the right kidney, with irregular contours (fig . Pelvic and abdominal ct was performed, revealing a large mass on the right kidney with irregular contours and poor definition . Further investigations performed following contrast injection revealed a heterogeneous density with a hypodense central area and some calcifications in thickness (hyperdense deposits in the body of the primary tumor: right kidney) (fig . Radiotherapy was recommended, but due to the advanced stage of the disease the patient was enrolled in a palliative care and pain control program . Metastases of malignances in the head and neck are rare . When present, they tend to be very aggressive due to the advance stage of the disease . Multidisciplinary management, clinical experience and high - resolution imaging support are crucial for determining tumor identification, primary tumor localization, and the extent of tumor involvement.
Parasites and chicks: the niah strain of e. tenella, which is virulent and maintained at the laboratory of parasitic diseases, national institute of animal health (tsukuba, japan), was used throughout this study . The parasites were maintained by passage in 2- to 3-week - old chicks (nisseiken, tokyo, japan). The chicks were housed in wire - floored cages in coccidian - free rooms and with free access to feed and water that contained no anticoccidial drugs or antibiotics . The animals were treated in accordance with protocols approved by the animal care and use committee, niah (approval nos . They were orally inoculated with 2 10 oocysts, and feces were collected after 68 days . Preparation of developmental zoites: the purified e. tenella oocysts were incubated for various time periods (0 hr, 48 hr and 144 hr) at 28c in 2.5% potassium dichromate (wako, osaka, japan), treated with sodium hypochlorite (nacalai tesque, kyoto, japan) for 20 min at 4c and then washed with phosphate - buffered saline (pbs). This chemical treatment was repeated twice, followed by washing with pbs 5 times, and processed for further analysis as described below . For the purification of sporozoites, the sporulated oocysts were broken by vortexing with glass beads for 12 min to release sporocysts . The sporocysts were treated in excystation medium, 0.25% (w / v) trypsin (merck, darmstadt, germany) and 1% (w / v) taurodeoxycholic acid (sigma, st . Louis, mo, u.s.a .) In hanks balanced salt solution (sigma), ph 7.4, at 41c in a 5% co2 incubator for 90100 min . The sporozoites were obtained by purification involving two steps, namely centrifugation at 450 g for 2 min according to a modification of a previously described method and a filtration protocol using 595 filter paper circles . Merozoites and schizonts of the second generation were purified by centrifugation using percoll (ge healthcare life sciences, piscataway, nj, u.s.a .) As reported previously . Briefly, the chicks were orally inoculated with 2 10 oocysts, and the infected ceca were removed at 96 to 108 hr post - inoculation after euthanasia of animals according to protocols approved by the animal care and use committee, niah (approval nos . After removal of the contents and washing with pbs, the ceca were cut into less than 5 mm pieces and filtered by a wire mesh . The filtered homogenate was mixed with 100% percoll to make a 30% percoll - pbs homogenate and layered over 50% percoll - pbs, which were finally layered over pbs . A total of three layers was prepared and centrifuged at 1,300 g for 15 min . After centrifugation, a high population of schizonts was seen floating on the 30% percoll - pbs; merozoites were concentrated on the 50% percoll - pbs, while erythrocytes were collected as a red pellet at the bottom . Briefly, 2-week - old chicks in groups of three were inoculated with 2 10 oocysts, and the infected ceca were removed at 72 hr, 96 hr, 120 hr, 144 hr and 168 hr post - inoculation and then fixed . Cloning and sequencing of eimeripain: the gene coding for eimeripain was partially identified from the e. tenella gene index database (sequence no . 5 and 3 rapid amplification of cdna ends (race) was performed using the smarter race cdna amplification kit (takara, otsu, japan) according to the manufacturer s protocol using these primers: etcathep - f1; caa ctt cga cca cgt gcc cat ttc tct t for the 5end and etcathep - f14; tac tgg cta gct gtg aac agc tgg for the 3end . The putative signal sequence of eimeripain was analyzed using the prediction server signalp v4.1 . By sequence analysis of race products, 1,536 bp of eimeripain this molecule also contained approximately 410 bp and 1,180 bp non - coding regions at the 5 and 3 ends, respectively, consisting of a 512 amino acid protein, with a signal peptide of 21 hydrophobic amino acids, a predicted molecular weight of 54.68 kda and a pi of 5.53 (excluding signal peptide) using peptidemass . Expression of eimeripain: the open reading frame (orf) of eimeripain was amplified by pcr from a cdna of sporulated oocysts using a set of primers [forward: reetcathep - f1 (cgg ggt acc ccg atg ccc tcc gat gat ttg ggc), reverse: reetcathep - f2 (ggg gta ccc ctc ata ggt cct gcg ctg acg g)] containing kpn i restriction sites . Pcr was performed for 5 min at 94c followed by 30 cycles of 30 sec at 94c, 30 sec at 63.4c and 2 min at 72c and a final elongation at 72c for 10 min . The pcr product and the vector ptrchisb (invitrogen, carlsbad, ca, u.s.a .) Were digested by kpn i restriction enzymes . The purified pcr product was inserted into the kpn i sites of the vector ptrchisb using dna ligation kit (takara). The resultant plasmid was transformed into competent cells of escherichia coli top10f strain (invitrogen) following the conventional method . The expression of eimeripain in e. coli with a polyhistidine - tag was performed according to the procedure described by tsuji et al . . Briefly, the transfected cells were allowed to grow in sob medium (bd, franklin lakes, nj, u.s.a .) Containing 50 mg ampicillin / ml at 37c . To induce recombinant protein expression, isopropyl - b - d - thiogalactopyranoside (iptg) was added at 1 mm concentration, and the culture was grown for an additional 4 hr at 37c . The culture was then centrifuged at 10,000 g for 20 min and 4c, and then, the pellet was resuspended in lysis buffer (20 mm sodium phosphate and 500 mm sodium chloride, ph 7.8) with egg white lysozyme (100 g / ml). The suspension was sonicated on ice with an ultrasonic processor (taitec, saitama, japan) followed by freezing and thawing . After three cycles of this treatment, the e. coli lysate was centrifuged at 23,900 g for 30 min at 4c, and the supernatant was collected . As a result, eimeripain was successfully expressed in e. coli, harvested as a soluble protein from bacterial cultures, separated on 12.5% sds page gel and detected as an approximately 60 kda fusion protein by western blotting using an anti - his monoclonal antibody (nacalai tesque) as described below . The recombinant protein was purified using probond resin (invitrogen) as described by the manufacturer . Page gel and detected as a single band by staining with 0.2% coomassi brilliant blue r-250 (wako) (fig . Protein concentration was calculated using the bca protein assay kit (pierce, rockford, il, u.s.a . ), and protein was stored at 20c until further use . Sds - page of fusion - protein recombinant eimeripain after purification of probond resin . Reverse transcription polymerase chain reaction (rt - pcr): for rt - pcr, six samples of unsporulated oocysts, sporulated oocysts incubated for 48 hr and 144 hr, and purified sporozoites, merozoites and schizonts were used . They were washed in pbs and submerged in rlt lysis buffer (qiagen, hilden, germany). Total rna from each sample was extracted using the rneasy mini kit (qiagen) according to the manufacturer s protocol . Single stranded cdna for mrna was prepared using the takara rna pcr kit (amv) ver.3.0 (takara) following the manufacturer s instructions . Four hundred nanograms of total rna was used for rt before pcr . As a control, rna from chicken cells purified by the protocol for schizonts and merozoites using non - infected ceca were used, because chicken cells might contain purified schizonts or merozoites . The synthesized cdnas were used for pcr using eimeripain [forward: etcathep - f5 (att ctg cga gat gta gag aag gat att), reverse: etcathep - r5 (ctg gat ctg aat aga aag aaa ggt aag t)] (499bp) and e. tenella actin (350bp) specific oligonucleotides . As an additional control, rna samples treated with the same protocol for synthesizing cdna without transcriptase were used . Pcr was performed for 5 min at 94c followed by 40 cycles of 30 sec at 94c, 60 sec at 53c and 60 sec at 72c and finally elongation at 72c for 10 min . Polyclonal antisera against eimeripain: an antiserum against the eimeripain was raised by immunizing balb / c mice subcutaneously with 100 g of purified recombinant protein emulsified with complete freund s adjuvant (difco laboratories, detroit, mi, u.s.a . ), followed by booster immunizations of 50 g at 2 weeks apart using the same route five times . The immunized mice were sacrificed 1 week after the last booster, and serum was collected and stored at 20c for further use . The peptide cggepkvpndknas, amino acids 257 to 270 of the mature domain of eimeripain as previously reported, were synthesized and coupled to keyhole limpet hemocyanin (klh) (pierce). This synthetic peptide was used to raise polyclonal antibodies against the mature domain of eimeripain in a japanese white rabbit by subcutaneous injection of 400 g of the antigen emulsified with complete freund s adjuvant (difco laboratories). The protocol was the same as that described above, except for the booster immunizations using 200 g of antigen . Western blotting: the sporulated oocysts (48 hr incubation) and purified sporozoites, merozoites and schizonts were resuspended in pbs . They were freeze - thawed five times, sonicated in an ice bath with the ultrasonic processor (taitec) and centrifuged at 600 g for 5 min . The concentration of crude antigens was determined by bca protein assay kit (pierce). These parasite lysates (1020 g) and the purified recombinant protein of eimeripain (2 g) were separated through 12.5% sds - page gels under reducing conditions, and the proteins were transferred onto nitrocellulose membranes (ge healthcare life sciences). The membranes were blocked with 5% skim milk in tris - buffered saline (tbs) for 30 min and then incubated with polyclonal antibodies against the recombinant eimeripain or the peptide corresponding to the eimeripain mature domain (1:1,000) for 1 hr at room temperature . They were washed with tbs containing 0.05% tween 20 (tbs - t) and then incubated with alkaline phosphate - conjugated goat anti - mouse or rabbit igg (h + l) (zymed, south san francisco, ca, u.s.a .) As a secondary antibody for 1 hr . The membrane was washed again with tbs - t, and the bound antibody was developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (bcip / nbt, promega, madison, wi, u.s.a . ). Indirect immunofluorescence assay: the purified sporozoites and merozoites were applied to slide glass, air - dried and fixed by methanol . These prepared slides and sections of infected ceca were blocked with 10% goat serum (mp biomedicals, santa ana, ca, u.s.a .) For 30 min, washed three times with 0.05% tween 20 in pbs (pbst) and then incubated with polyclonal antibodies (diluted 1:1,000) against recombinant eimeripain and eimeripain mature domain peptide for 1 hr . The slides were washed three times with pbst and then incubated with green fluorescence - labeled mouse and rabbit igg secondary antibodies [alexa fluor488 goat anti-(mouse igg) (h + l) or anti-(rabbit igg) (h + l); invitrogen] for 1 hr . They were observed under a fluorescence microscope (leica, wetzlar, germany). Transcription of eimeripain: in the present study, the total rna that was first extracted from several stages of e. tenella was subjected to rt - pcr analysis for transcription profiling of eimeripain . By successful amplification of an approximately 500 bp fragment, we confirmed that eimeripain was expressed in all examined stages, namely oocyst (0 hr, 48 hr and 144 hr after sporulation), sporozoite, schizont and merozoite stages, but not in samples of chicken cells or in any of the controls (fig . Detection of eimeripain (499 bp) (a) and e. tenella actin (350 bp) (b) specific rna . L; dna marker, 1; rna from unsporulated oocysts, 2; from oocysts sporulated at 48 hr, 3; from oocysts sporulated at 144 hr, 4; from purified sporozoites, 5; from purified schizonts, 6; from purified merozoites, 7 and 8; from chicken cells separated on a 30%, and 50% percoll layer using uninfected chicken ceca, respectively, 9; only pcr buffer, 10; genomic dna from sporulated oocysts . ), although the expression between mrna and protein did not always correlate . E. tenella actin gene showed the same results with those of eimeripain (fig . 2b), except for e. tenella genomic dna . Expression of eimeripain genes during the developmental stages of e. tenella . Detection of eimeripain (499 bp) (a) and e. tenella actin (350 bp) (b) specific rna . L; dna marker, 1; rna from unsporulated oocysts, 2; from oocysts sporulated at 48 hr, 3; from oocysts sporulated at 144 hr, 4; from purified sporozoites, 5; from purified schizonts, 6; from purified merozoites, 7 and 8; from chicken cells separated on a 30%, and 50% percoll layer using uninfected chicken ceca, respectively, 9; only pcr buffer, 10; genomic dna from sporulated oocysts . Endogenous form of eimeripain: in order to identify the endogenous form of eimeripain, anti - eimeripain antibodies were prepared using recombinant eimeripain and a peptide corresponding to the mature domain of eimeripain . By western blotting analysis, the two polyclonal antibodies generated against eimeripain recognized the recombinant eimeripain fusion protein as a 60 kda band (fig . 3.western blot analysis using antisera with anti - eimeripain (a) and anti - peptide against eimeripain mature domain (b). L: molecular weight marker, 1; sporulated oocysts, 2, purified sporozoites, 3 and 4; purified schizonts and merozoites of the second generation, 5; purified recombinant eimeripain . Arrows, arrowhead and arrows with broken line show 33 kda mature eimeripain, 54 kda promature eimeripain and 60 kda recombinant eimeripain protein, respectively ., lane 5), and thus, these epitopes were confirmed to be present within the amino acid sequence of eimeripain . Consequently, a 54 kda band of promature eimeripain, previously reported, was detected in all stages of sporulated oocysts, sporozoites, merozoites and schizonts examined with the antiserum against recombinant eimeripain (fig . A 33 kda band, which has been reported as the active form of eimeripain, was recognized only in sporulated oocysts and schizonts (fig . Unexpectedly, the 54 kda band of promature eimeripain was also detected in schizonts (fig . These results show that antisera against recombinant eimeripain and eimeripain mature domain recognize promature and mature eimeripain, respectively, and that mature eimeripain is undetectable or does not exist in invasive zoites like sporozoites and merozoites, but only in sporulated oocysts and developmental schizonts . Western blot analysis using antisera with anti - eimeripain (a) and anti - peptide against eimeripain mature domain (b). L: molecular weight marker, 1; sporulated oocysts, 2, purified sporozoites, 3 and 4; purified schizonts and merozoites of the second generation, 5; purified recombinant eimeripain . Arrows, arrowhead and arrows with broken line show 33 kda mature eimeripain, 54 kda promature eimeripain and 60 kda recombinant eimeripain protein, respectively . Endogenous localization of eimeripain in invasive zoites: promature eimeripain was detected in the cytoplasm of sporozoites and merozoites by incubating with the antiserum against recombinant protein (fig . 4.immunofluorescent staining of sporozoites (a) and merozoites (b) with antibody against promature eimeripain (arrows). Left and right figures show fluorescent and bright field photos . They were observed as several dots, but not all over the entire region of sporozoite and merozoite bodies, although these reactivities were not strong . The antibody against the eimeripain mature domain did not show any reactivity with these zoites (data not shown). Immunofluorescent staining of sporozoites (a) and merozoites (b) with antibody against promature eimeripain (arrows). Left and right figures show fluorescent and bright field photos . Scale bar is 10 m . Endogenous localization during asexual stages: during the second generation, in sections at 72 hr, 96 hr and 120 hr post - infection, promature eimeripain was detected only in early schizonts at around 10 m in size, as constellations of dots in the cytoplasm (fig . 5afig . 5.immunofluorescent staining of asexual stages, the second generation schizonts (a) and the third generation schizonts (b) with antisera against promature (upper photos) and mature (bottom photos) eimeripain . Figures a and d are small immature schizonts of the second generation, b and e are large immature schizonts, and c and f are mature schizonts . Arrowheads and arrows show smaller immature schizonts and larger immature ones of the third generation in fig . H. scale bar is 10 m . ); it was not detected in schizonts over 15 m (fig . 5b and 5c). On the other hand, mature eimeripain was observed over the entire cytoplasm surrounding the nuclei in small immature schizonts and in developmental second - generation schizonts of middle size (around 20 m) with the strongest reactivity (fig . 5d and 5e). In mature schizonts, this reactivity became weak and seemed to be present only between merozoites in the mature schizonts (fig . Reactivity of promature eimeripain was similar to that of the second generation, and this enzyme was observed as dots in the cytoplasm . Mature eimeripain was present as dots in the cytoplasm of small immature third schizonts and observed over the entire cytoplasm of large immature ones (fig . 5 g and 5h), but disappeared in mature third schizonts (data not shown). Immunofluorescent staining of asexual stages, the second generation schizonts (a) and the third generation schizonts (b) with antisera against promature (upper photos) and mature (bottom photos) eimeripain . Figures a and d are small immature schizonts of the second generation, b and e are large immature schizonts, and c and f are mature schizonts . Arrowheads and arrows show smaller immature schizonts and larger immature ones of the third generation in fig . Endogenous localization during the sexual stage: at the sexual stage, reactivities were found to be similar between promature and mature eimeripain (fig . 6.immunofluorescent staining of sexual stage with antibodies against promature (a) and mature (b) eimeripain . Arrowheads, arrows and asterisks show macro - gametocytes with a prominent wall forming body, micro - gametocytes and zygotes, respectively . Scale bar is 10 m .) And different from those of the asexual stages described above . Both types of eimeripain were observed in the wall - forming bodies and cytoplasm of immature macro - gametocytes, only in the wall - forming bodies of mature macro - gametocytes, and across the cytoplasm of micro - gametocytes . In zygotes signals were present over the entire cytoplasm, except in the nuclei . At the oocyst formation stage, the final step of sexual development, which is seen as a distorted shape, eimeripain was weakly observed in the cytoplasm around the inner oocyst wall as spots (fig . Immunofluorescent staining of sexual stage with antibodies against promature (a) and mature (b) eimeripain . Arrowheads, arrows and asterisks show macro - gametocytes with a prominent wall forming body, micro - gametocytes and zygotes, respectively . Eimeripain has been proposed as a candidate drug target to be exploited against chicken coccidiosis in the future [8, 10]. In those studies, a panel of inhibitors was tested against the enzyme and three new inhibitors were identified . Moreover, it was confirmed that eimeripain was expressed in the extracellular stage during sporulation; however, no other life cycle stage was examined for its expression so far . In the present study, we generated two antisera against eimeripain; one recognized promature eimeripain, while the other recognized eimeripain by western blotting . As a result, the eimeripain was detected as protein of promature at examined in all examined stages of sporulated oocysts, and purified sporozoites, merozoites and schizonts . While, mature protein was identified as 33 kda molecule only at sporulated oocysts and schizonts, but not sporozoites and merozoites . Probably, the epitope recognized by anti - peptide sera against eimeripain mature domain might be masked by the modification of carbohydrate chain or protein folding in promature eimeripain before maturation . Interestingly, this polyclonal antibody also identified a 54 kda band in schizonts, but did not react with sporozoites or merozoites . There is a possibility that purified schizonts represent many developmental stages from immature to mature second - generation schizonts, and thus, mature eimeripain of 54 kda might be contained before complete maturation . These results suggest that mature eimeripain do not exist in invasive zoites, but only in sporulated oocysts and developmental schizonts . By indirect immunofluorescence assay, the reactivities of promature eimeripain in sporozoites and merozoites were similar to that seen in transfected sporozoites, as previously reported, but mature eimeripain was not . In intracellular developmental stages, the promature enzyme was detected in the cytoplasm of early schizonts of the second and third generations, but disappeared during their respective developments . Meanwhile, mature protein was present in the entire cytoplasm, but later was not detected . Thus, the promature protein is present as dots in the early asexual schizonts and probably sporozoites and merozoites as well, and active eimeripain might spread throughout the cytoplasm during development or differentiation of schizonts . These findings tempt speculation that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca in addition to the extracellular stage . Cathepsins, which are cysteine proteases related to papain - like enzymes (clan ca, family c1), are major virulence factors expressed by apicomplexan parasites [1, 3, 11]. For instance, in toxoplasma gondii, antisense or inhibitors of cathepsin b can block the invasion of tachyzoites into host cells and cause abnormal rhoptry morphology . In higher eukaryotic cells, however, the lysosomal system of e. tenella, like that of toxoplasma gondii, has not been characterized . In our study, eimeripain was expressed in all examined intracellular stages at both mrna and protein levels . Although more detailed functional characteristics remain to be analyzed, this enzyme might correlate with asexual and sexual development with a fundamental biological function . Therefore, the results reported here offer evidence that compounds of inhibitors could be effective candidates for novel drug discovery against chicken coccidiosis.
Previous studies on lung sound analysis (lsa) in patients with bronchial asthma have mostly analyzed abnormal sounds and audible rales and have reported on the mechanism of lung sound generation . Lsa studies on bronchial asthma have generally reported sound amplification in a high - frequency range> 400 hz and the elevation of the median frequency f50 and/or f75, but these reports are mostly based on the analysis of rales, including wheezing.13 when rales are heard in asthma patients, it is reasonable to speculate that airway inflammation and narrowing are present . However, given that asthma is a chronic inflammatory disease of the airway, inflammation exists in the airway even under stable conditions with no attacks . There have been reports that breath sound frequency components were correlated with the extent of airway obstruction as assessed by an airway hyperresponsiveness test, even in the absence of wheezing.4,5 other studies have reported that inspiration sound pressure at 700 hz increased with the severity of bronchial asthma in children without wheezing, demonstrating a significant correlation of the sound pressure at 700 hz with the forced expiratory volume in 1 second / forced vital capacity (fev1.0/fvc) ratio and the maximal expiratory flow rate at 50% of the vital capacity (fef50%predicted).6 however, little information is available on the correlation between airway inflammation and lung sounds in bronchial asthma patients . We have previously reported that in asthma patients who are in an attack - free period and exhibit normal sounds based on conventional stethoscope auscultation, airway inflammation can be detected by lsa at a single point in the posterior lower lung field.7,8 however, existing evidence indicates that airway inflammation and sites of narrowing are found as an uneven mixture of normal and abnormal tissues rather than uniform lesions throughout the lung fields.9,10 in this study, we performed lsa at 7 points in the trachea and in the anterior and posterior chest regions in asthmatic subjects who were in an attack - free, stable condition and had normal stethoscopic findings . The aim of this work was to determine whether lsa can be used for the local diagnosis of abnormal sites (with airway inflammation) vs normal sites (without airway inflammation). Lung sound data from the 20 healthy volunteers presented in this article were previously published.11 all asthmatic subjects who first visited our hospital from september 2011 to november 2014 included in this study fulfilled the global initiative for asthma criteria.10 all included asthmatic subjects had a history of asthmatic symptoms, including recurrent cough, wheezing, or dyspnea, and had positive airway hyperresponsiveness, ie, a provocative concentration of acetylcholine (ach) causing a 20% decrease in fev1.0 (pc20) of <8000 g / ml . Airway reversibility (ie, reversible with at least 12% and 200 ml improvements in fev1.0 after bronchodilator therapy) was confirmed in 16 of the asthmatic subjects but not in the remaining 6, who exhibited normal lung function during their first visit to our hospital; these asthmatic subjects were diagnosed with bronchial asthma based on their medical history and pc20-positive results . Cases complicated with chronic pulmonary obstructive disease (copd) and asthmatic subjects with cardiopulmonary disease affecting pulmonary function were excluded . All asthmatic subjects retained normal diffusion capacity, and no asthmatic subjects had previously used inhaled or oral corticosteroids . Antiasthma drugs, including bronchodilators, were discontinued for at least 24 hours prior to this examination . The ethics committee of fukuoka national hospital approved the study protocol (protocol number 23 - 14), and all participants received verbal and written study information before providing their informed consent . Breath sounds were recorded using a custom - made recording system for lung sounds in a quiet room in the hospital . The recording system comprised 7 air - coupled microphones (ecm - pc60; sony, tokyo, japan), amplifiers, a pneumotachograph (sp-310; fukuda sangyo, chiba, japan), an analog - to - digital converter, and a personal computer . Microphones were attached to 7 sites on the chest wall (2 cm above the top of the episternum, bilaterally at the anterior second intercostal space on the midclavicular line, bilaterally on the back between the fifth vertebra and scapulas, and 3 cm above the lung base at the midscapular line) with a rubber - made attachment and double - sided adhesive tape (gain: 40 db for lung sound channels, 16 db for tracheal sound channel) (figure 1a). The breath sounds acquired by the 7 microphones were amplified and subsequently digitized with a 16-bit resolution at a sampling frequency of 12.5 khz per channel . The recording system was calibrated using a reference sound pressure (94 db [0 db = 20 pa], 1 khz). Airflow in the mouth was measured using the pneumotachograph and was digitized simultaneously (figure 1b). The subjects were instructed to breathe deeply through the mouth piece of the pneumotachograph for 30 seconds . The breathing maneuver was not strictly defined; therefore, it varied according to the subject . The recorded sound and airflow signals were analyzed using a custom - made computer program . The program visualized the 7 lung sound channels as a spectrogram and airflow curve, simultaneously . The program scanned all the airflow data to determine the inspiratory and expiratory phases (figure 1b). Thereafter, the program scanned the lung sound data sequentially with a hanning window of 2048 points (0.164 ms) with 50% overlap and then calculated the power spectra using a fast fourier transform . The program yielded 6 octave band sound pressure levels (50100, 100200, 200400, 400800, 8001600, 16003200 hz) as representative variables to characterize the lung sound spectra for each segment . Thereafter, data from noise - contaminated breaths were excluded by visually inspecting the sound spectrograms . The octave band sound pressure levels were averaged for the inspiratory (ispl) and expiratory phases (espl). The ratio of espl to ispl was calculated and expressed as a linear unit (e / i). Measurements of the flow volume curves, the fractional exhaled nitric oxide concentration (feno), the bronchial hyperresponsiveness to ach, and the induced sputum were performed in accordance with previously described procedures.7 the wilcoxon / kruskal wallis test was performed to compare the 3 groups of e / i values among the asthmatic subjects (stratified by v50%pred of 80% vs> 80%) and healthy volunteers for each recording point and each frequency band . For all significant differences, next, spearman s rank test was performed on the data from asthmatic subjects for correlating e / i, ispl, and espl values with spirometry, logpc20, and feno data in each frequency band . A receiver operating characteristic (roc) analysis was performed between healthy and asthmatic subjects to establish the cutoff value at each recording point . Lung sound data from the 20 healthy volunteers presented in this article were previously published.11 all asthmatic subjects who first visited our hospital from september 2011 to november 2014 included in this study fulfilled the global initiative for asthma criteria.10 all included asthmatic subjects had a history of asthmatic symptoms, including recurrent cough, wheezing, or dyspnea, and had positive airway hyperresponsiveness, ie, a provocative concentration of acetylcholine (ach) causing a 20% decrease in fev1.0 (pc20) of <8000 g / ml . Airway reversibility (ie, reversible with at least 12% and 200 ml improvements in fev1.0 after bronchodilator therapy) was confirmed in 16 of the asthmatic subjects but not in the remaining 6, who exhibited normal lung function during their first visit to our hospital; these asthmatic subjects were diagnosed with bronchial asthma based on their medical history and pc20-positive results . Cases complicated with chronic pulmonary obstructive disease (copd) and asthmatic subjects with cardiopulmonary disease affecting pulmonary function were excluded . All asthmatic subjects retained normal diffusion capacity, and no asthmatic subjects had previously used inhaled or oral corticosteroids . Antiasthma drugs, including bronchodilators, were discontinued for at least 24 hours prior to this examination . The ethics committee of fukuoka national hospital approved the study protocol (protocol number 23 - 14), and all participants received verbal and written study information before providing their informed consent . Breath sounds were recorded using a custom - made recording system for lung sounds in a quiet room in the hospital . The recording system comprised 7 air - coupled microphones (ecm - pc60; sony, tokyo, japan), amplifiers, a pneumotachograph (sp-310; fukuda sangyo, chiba, japan), an analog - to - digital converter, and a personal computer . Microphones were attached to 7 sites on the chest wall (2 cm above the top of the episternum, bilaterally at the anterior second intercostal space on the midclavicular line, bilaterally on the back between the fifth vertebra and scapulas, and 3 cm above the lung base at the midscapular line) with a rubber - made attachment and double - sided adhesive tape (gain: 40 db for lung sound channels, 16 db for tracheal sound channel) (figure 1a). The breath sounds acquired by the 7 microphones were amplified and subsequently digitized with a 16-bit resolution at a sampling frequency of 12.5 khz per channel . The recording system was calibrated using a reference sound pressure (94 db [0 db = 20 pa], 1 khz). Airflow in the mouth was measured using the pneumotachograph and was digitized simultaneously (figure 1b). The subjects were instructed to breathe deeply through the mouth piece of the pneumotachograph for 30 seconds . The breathing maneuver was not strictly defined; therefore, it varied according to the subject . The recorded sound and airflow signals were analyzed using a custom - made computer program . The program visualized the 7 lung sound channels as a spectrogram and airflow curve, simultaneously . The program scanned all the airflow data to determine the inspiratory and expiratory phases (figure 1b). Thereafter, the program scanned the lung sound data sequentially with a hanning window of 2048 points (0.164 ms) with 50% overlap and then calculated the power spectra using a fast fourier transform . The program yielded 6 octave band sound pressure levels (50100, 100200, 200400, 400800, 8001600, 16003200 hz) as representative variables to characterize the lung sound spectra for each segment . Thereafter, data from noise - contaminated breaths were excluded by visually inspecting the sound spectrograms . The octave band sound pressure levels were averaged for the inspiratory (ispl) and expiratory phases (espl). The ratio of espl to ispl was calculated and expressed as a linear unit (e / i). The investigator who analyzed the data was blinded to the related information . Measurements of the flow volume curves, the fractional exhaled nitric oxide concentration (feno), the bronchial hyperresponsiveness to ach, and the induced sputum were performed in accordance with previously described procedures.7 the wilcoxon / kruskal wallis test was performed to compare the 3 groups of e / i values among the asthmatic subjects (stratified by v50%pred of 80% vs> 80%) and healthy volunteers for each recording point and each frequency band . For all significant differences, next, spearman s rank test was performed on the data from asthmatic subjects for correlating e / i, ispl, and espl values with spirometry, logpc20, and feno data in each frequency band . A receiver operating characteristic (roc) analysis was performed between healthy and asthmatic subjects to establish the cutoff value at each recording point . The patient population had a mean age of 39.5 years and consisted of 7 males and 15 females . The patient population was significantly different from the healthy population in terms of the age and sex ratio (table 1). Figure 2 depicts the median e / i in each frequency band from the trachea (channel [ch]7), anterior chest (ch2), posterior upper (ch4), and posterior lower (ch6) recordings, respectively, in asthmatic subjects with v50%pred <80%, those with v50%pred 80%, and healthy volunteers . The e / i plots based on the anterior chest, posterior upper and lower (ch1ch6) recordings exhibited a negative peak centered at 100400 hz, as well as significantly higher e / i levels in the lower frequency bands up to 400 hz, for asthmatic subjects with v50%pred <80% compared with the healthy volunteers (p<0.05). A significant difference in e / i was observed in the bronchial asthma patients with v50%pred <80% vs 80% in the data obtained from the left and right anterior chest points (ch1 and ch2) and the left posterior upper and lower points (ch4 and ch6) in the frequency bands of 100200 hz and 200400 hz; asthmatic subjects with v50%pred <80% had significantly higher e / i values than did those with v50%pred 80% (p<0.05) (table 2). In contrast, the e / i plots based on the tracheal (ch7) recordings showed a positive peak in the frequency band of 400800 hz (figure 2). The e / i values monitored by tracheal recordings in asthmatic subjects with v50%pred <80% were significantly higher than those in the healthy volunteers in the bands of 50100 hz and 100200 hz (p<0.05) but not in other frequency bands (table 2). The e / i values monitored at 5 recording positions, excluding the trachea (ch7) and right posterior upper (ch3) points, were more strongly correlated with the spirometry, logpc20, and feno data than were the espl or ispl values . The strongest correlations of e / i with spirometry, logpc20, and feno were observed in the frequency bands of 100200 hz (low frequency [lf]) and 200400 hz (mid frequency [mf]). The e / i from the tracheal recording did not show a significant correlation with spirometry, logpc20, or feno . In contrast, the e / i from the left anterior chest (ch2) and left posterior lower (ch6) recordings exhibited a significant negative correlation with fev1.0/fvc, v50%pred, v25%pred, and logpc20, as well as a significant positive correlation with feno . The e / i from the left posterior upper (ch4) recording demonstrated a significant negative correlation with fev1.0/fvc, v50%pred, and v25%pred and a significant positive correlation with feno . The e / i from the right anterior chest (ch1) and right posterior lower (ch5) recordings showed a significant negative correlation with fev1.0/fvc, v50%pred, and v25%pred (table 3). The roc analysis of the e / i mf from each recording point in the healthy and bronchial asthma patient populations confirmed good sensitivity and specificity, with a cutoff of 0.50.51 for the anterior chest (ch1 and ch2) and dorsal upper (ch3 and ch4) recordings . Good sensitivity and specificity were also confirmed, with a cutoff of 0.40 and 0.47 for the right posterior lower (ch5) and left posterior lower (ch6) recordings, respectively (table 4). Table 4 summarizes the e / i mf data according to recording point for individual asthmatic subjects; values equal to and greater than the cutoff, as established by roc, are shown in bold . Most asthmatic subjects with feno 70 ppb had higher e / i mf values at all recording points as well as v50%pred <80% . Asthmatic subjects with feno <70 ppb and v50%pred <asthmatic subjects with feno 3869 ppb or those with feno <38 ppb and v50%pred 80% were divided into groups with lower vs higher e / i mf values according to the recording position . The patient population had a mean age of 39.5 years and consisted of 7 males and 15 females . The patient population was significantly different from the healthy population in terms of the age and sex ratio (table 1). Figure 2 depicts the median e / i in each frequency band from the trachea (channel [ch]7), anterior chest (ch2), posterior upper (ch4), and posterior lower (ch6) recordings, respectively, in asthmatic subjects with v50%pred <80%, those with v50%pred 80%, and healthy volunteers . The e / i plots based on the anterior chest, posterior upper and lower (ch1ch6) recordings exhibited a negative peak centered at 100400 hz, as well as significantly higher e / i levels in the lower frequency bands up to 400 hz, for asthmatic subjects with v50%pred <80% compared with the healthy volunteers (p<0.05). A significant difference in e / i was observed in the bronchial asthma patients with v50%pred <80% vs 80% in the data obtained from the left and right anterior chest points (ch1 and ch2) and the left posterior upper and lower points (ch4 and ch6) in the frequency bands of 100200 hz and 200400 hz; asthmatic subjects with v50%pred <80% had significantly higher e / i values than did those with v50%pred 80% (p<0.05) (table 2). In contrast, the e / i plots based on the tracheal (ch7) recordings showed a positive peak in the frequency band of 400800 hz (figure 2). The e / i values monitored by tracheal recordings in asthmatic subjects with v50%pred <80% were significantly higher than those in the healthy volunteers in the bands of 50100 hz and 100200 hz (p<0.05) but not in other frequency bands (table 2). The e / i values monitored at 5 recording positions, excluding the trachea (ch7) and right posterior upper (ch3) points, were more strongly correlated with the spirometry, logpc20, and feno data than were the espl or ispl values . The strongest correlations of e / i with spirometry, logpc20, and feno were observed in the frequency bands of 100200 hz (low frequency [lf]) and 200400 hz (mid frequency [mf]). The e / i from the tracheal recording did not show a significant correlation with spirometry, logpc20, or feno . In contrast, the e / i from the left anterior chest (ch2) and left posterior lower (ch6) recordings exhibited a significant negative correlation with fev1.0/fvc, v50%pred, v25%pred, and logpc20, as well as a significant positive correlation with feno . The e / i from the left posterior upper (ch4) recording demonstrated a significant negative correlation with fev1.0/fvc, v50%pred, and v25%pred and a significant positive correlation with feno . The e / i from the right anterior chest (ch1) and right posterior lower (ch5) recordings showed a significant negative correlation with fev1.0/fvc, v50%pred, and v25%pred (table 3). The roc analysis of the e / i mf from each recording point in the healthy and bronchial asthma patient populations confirmed good sensitivity and specificity, with a cutoff of 0.50.51 for the anterior chest (ch1 and ch2) and dorsal upper (ch3 and ch4) recordings . Good sensitivity and specificity were also confirmed, with a cutoff of 0.40 and 0.47 for the right posterior lower (ch5) and left posterior lower (ch6) recordings, respectively (table 4). Table 4 summarizes the e / i mf data according to recording point for individual asthmatic subjects; values equal to and greater than the cutoff, as established by roc, are shown in bold . Most asthmatic subjects with feno 70 ppb had higher e / i mf values at all recording points as well as v50%pred <80% . Asthmatic subjects with feno <70 ppb and v50%pred <asthmatic subjects with feno 3869 ppb or those with feno <38 ppb and v50%pred 80% were divided into groups with lower vs higher e / i mf values according to the recording position . We examined asthmatic subjects using a device that can monitor lung sounds simultaneously at 7 points and analyzed the data for differences according to recording point . The results demonstrated that e / i lf and e / i mf data were better suited as indicators of airway inflammation in bronchial asthma, with the lower anterior chest (ch2) or left posterior lower (ch6) points being the favorable sites for recording breath sounds . The results also indicated that by recording lung sounds at 7 points, inflammation sites could be localized within the airway . Airway narrowing in asthma is thought to be caused by an increase in airway smooth muscle tone and, to some extent, by airway wall thickening . Spirometry and airway resistance measurements are influenced primarily by larger airways because the bulk of airway resistance is located there . Small airways contribute little to these measurements because of the low velocity of air passing through them . This suggests that lung sounds are not generated in small airways but mainly in larger airways, ie, those from the trachea up to the nineth division of bronchioles.12,13 the intensity of lung sound pressure during breathing is influenced by respiratory flow volume and pulmonary filtering.1418 to minimize interindividual differences in the respiratory flow volume, we adopted the expiration - to - inspiration sound pressure ratio (e / i) as a measure . In this study, we examined the espl and ispl values in individuals with and without bronchial asthma by partitioning the lung sound data into 6 frequency bands . This approach revealed the highest sound pressure levels in the frequency range from 50 to 100 hz and a decreasing trend in the sound pressure level with increasing frequency . This decreasing pattern varied with recording position; the anterior chest, posterior upper, and posterior lower recordings demonstrated a linear, decreasing trend with increasing frequency, while the tracheal recording yielded a steep decline (from 400 up to 1600 hz) followed by a plateau (data not shown). In contrast, when e / i was plotted on the vertical axis against frequency (in hertz) on the horizontal axis, the plots based on the anterior chest and posterior upper and lower recordings exhibited a negative peak centered at the 100200 hz and 200400 hz bands (figure 2). Asthmatic subjects with more airway narrowing showed significantly higher e / i values in the 100200 hz and 200400 hz bands than did those with less severe airflow limitations or healthy volunteers . In the frequency range <100 hz, asthmatic subjects with more airway narrowing had significantly higher e / i values than did healthy volunteers, but no correlation was noted with other parameters, such as respiratory function (data not shown). These results can likely be attributed to muscle- and heart - associated noises, which are more pronounced in the frequency range <100 hz.19 we have previously used an analysis of single - point recordings in the lung base to show that e / i mf is best suited for evaluating airway inflammation in bronchial asthma.7,8 in this analysis with multiple - point recordings, we demonstrate that the e / i values in the frequency bands of 100200 hz and 200400 hz (e / i lf and e / i mf, respectively) were strongly correlated with airway narrowing, hyperresponsiveness, and inflammation when the lung sounds were recorded at the anterior chest and posterior upper and lower points . We did not find a significant correlation of the e / i data obtained at the right posterior upper (ch3) point with airway narrowing, hyperresponsiveness, or inflammation, and it is unknown whether this is due to recording position or to incidental events . The heart is located on the left lung side, the left bronchial tubes are narrower than the right bronchial tubes, and the left lung sounds are said to be stronger than the right lung sounds.2022 our finding of better correlations in the left lung field than in the right lung field may be attributed to lower noise levels and stronger lung sounds . However, the tracheal (ch7) recordings resulted in e / i frequency plots that were different from those generated from the other recordings; the data from the trachea did not correlate with any asthma - indicating factor, such as respiratory function, pc20, or feno . The results of this study confirmed that analyzing e / i values calculated based on tracheal recordings could not adequately support the diagnosis of bronchial asthma . Therefore, recordings in lung fields other than the tracheal and right posterior regions may be recommended for diagnosing bronchial asthma using e / i data . Bronchial asthma is a chronic inflammatory disease that involves the airway and leads to increased airway hyperresponsiveness . Because the location of airway inflammation varies from patient to patient, wang and xiong23 reported the presence of left - to - right differences in the vibration energy caused by the respiration of asthmatic subjects . When we summarized the e / i mf data from asthmatic subjects by recording position along with feno and v50%pred values (table 4), in asthmatic subjects, severe airway inflammation with feno 70 ppb was associated with high e / i mf values at all recording points in the anterior chest, posterior upper, and posterior lower regions . In these asthmatic subjects, these observations suggest that more generalized airway inflammation is present and that peripheral airway narrowing exists even during attack - free periods . Asthmatic subjects with feno <38 ppb and v50%pred 80% were divided into 2 groups: one with lower e / i mf values at recording positions except the trachea, and the other with higher e / i mf values depending on the recording position . Asthmatic subjects with low e / i mf values are considered to be free of airway inflammation, and those with high e / i mf values are likely to have local eosinophilic or neutrophilic airway inflammation wherein a high e / i is detected . These asthmatic subjects are likely to have airway inflammation unevenly in the bronchial tubes, and the location of airway inflammation may be detected by e / i mf monitoring . Asthmatic subjects with feno 3870 ppb, along with v50%pred 80% and low e / i mf values at all recording points, may have an elevated feno level due to upper respiratory tract inflammation, such as rhinitis, while maintaining normal respiratory function under stable asthmatic conditions . Asthmatic subjects with low feno and v50%pred levels and high e / i mf values are suspected to have neutrophilic airway inflammation . Further study is required to examine differences by position in a larger sample size . In our analysis, there were significant differences in age and sex between the healthy and bronchial asthmatic populations . Lung sound data from the healthy volunteers presented in this article were previously published11 and did not include immunoglobulin e data . However, these factors may not have had an impact on these results, as previous data from our laboratory suggested that sex and age do not affect the e / i lf or e / i mf values7,8 and the primary outcomes of this study were evidently clarified by the asthmatic subject data . More useful lsa parameters for the evaluation of airway inflammation in bronchial asthma are e / i lf and e / i mf, and breath sounds should be recorded at the anterior chest or left posterior lower position . E / i lf and e / i mf monitoring with 7-point lung sound recordings may be used to detect not only inflammation of the entire airway but also sites of local airway inflammation or of airway narrowing caused by airway inflammation.
Venous malformation is defined as malformations comprised of slow - flowing, abnormal dilated veins, and venous network . Clinically venous malformations (vm) are present at birth and tend to grow steadily in proportion to the somatic growth of the child . Venous malformations are congenital lesions that affect boys and girls equally with a reported risk of developing other conditions within a specified period of time . The occurrence rate is 1 - 2 per 10,000 births and 0.1 - 1% of a population are found to have this condition . Histologic and histochemical studies show that abnormalities are formed by small and large dysplastic post - capillary, thin - walled vascular channels with patchy deficiency of mural smooth muscle . A 28-year - old male was referred to the oral and maxillofacial clinic for evaluation of multiple swellings on the left side of the face and inside his mouth . The patient first noticed the swelling over the temple when he was 11-years - old . As the boy grew, the swelling also proportionately increased . Multiple poorly defined swellings were noted on the left side of the head and neck region around the temple, cheek, and submandibular region [figure 1a and b]. Similar lesion was noted in the supraclavicular and scapular region [figure 2a and b]. The swelling was pulsatile and increased in size the when patient was standing (dependent position). Intraorally lobulated swelling was noted on the floor of the mouth in the left sublingual region . The lesion measured 3 4 cm, filling the entire floor of mouth, and purplish discoloration was also noted over the swelling [figure 3a and b]. (a) photograph (front view) shows swelling over the temple region and (b) photograph (side view) reveals well defined swelling over temple (large arrow) diffused swelling in the cheek, and submandibular region (small arrow). (a) photograph shows a well - defined swelling measuring 1 1 cm in the left supraclavicular region and (b) photograph of the scapular region shows a diffused swelling . (a) intraoral photograph shows swelling in the floor of mouth filling the entire floor of mouth (arrow) and a small swelling in the buccal mucosa that has a purplish hue (small arrow). (b) intraoral photograph of the labial sulcus reveals a purplish swelling (arrow). Ultrasonography of the submandibular region showed a well - defined hypoechoic lesion having anechoic areas with septations, showing flow inside the lesion . Ultrasonography of the submandibular region with 7 mhz small parts transducer shows heterogeneous hypoechoic lesion with slow flow and hyperechoic foci suggestive of calcifications (arrow). Magnetic resonance imaging (mri)-t2-weighted fat suppression, post - contrast axial images showed hyperintense lesions, which are well - defined with no flow voids . All the lesions in the submandibular, cheek, and temple region had similar findings [figure 5]. Direct puncture phlebogram of the left frontal scalp region revealed pooling of contrast into cavernous spaces draining into external jugular vein [figure 6]. (a) axial section at the submandibular region shows well - defined hyperintense lesion (arrow) in the floor of mouth . (b) axial section at the level of cheek shows well - defined hyperintense lesion in the cheek region (arrow). Phlebography of the lesion in the temple region shows filling of the entire lesion and draining to the regional area (arrow). Histopathological examination revealed several thin - walled venous channels lined by flattened endothelium, supported by a dense uninflamed fibrous connective tissue stroma . Hematoxylin and eosin stained specimen (40) shows irregular venous channels (large arrow) lined by thin endothelium . Venous malformation (vm) is the second most common vascular anomaly of the head and neck after hemangioma . Venous malformations can occur anywhere in the body but are most frequently seen in the head and neck (40%), extremities (40%), and trunk (20%). Vms are slow - flow vascular anomalies composed of ectatic venous channels that will continue to grow throughout the patient's lifetime . They grow slowly in size with age, but their growth may be exacerbated following trauma, sepsis, or hormonal changes and they do not regress spontaneously . Both men and women are equally affected . Vascular malformations are believed to be the result of a congenital error of vascular morphogenesis that occurs between the 4 and 10 weeks of intrauterine life . Vascular malformations have a quiescent endothelium and are considered to be localized defects of vascular morphogenesis, likely to be caused by dysfunction in pathways regulating embryogenesis and vasculogenesis . These lesions vary in color depending on depth of involvement and range from mild detectable color change to deep purple color . These lesions fill when the patient is standing and are compressible, which helps to distinguish them from lymphatic malformations on physical examination . Areas frequently involved in the head and neck region are masseter, temporalis, tongue musculature, as well as oral and airway mucosa . Soft tissue lesions are most frequently facial in location, with the buccal region being the most common site followed by the mandibular space, sublingual space, tongue, and orbit . Intraosseous calvarial involvement is most frequent in the frontoparietal region and the mandible is the most frequent location within the facial skeleton . There may, however, be no visible manifestations with deeper lesions . In our case, the first anatomopathologic classification of vascular lesions based on the microscopic appearance was developed by virchow and wegner . Mulliken and glowacki (1982) developed a biological classification of vascular anomalies that included physical findings, clinical behavior, and cellular kinetics and classified them as hemangiomas and vms . A more recently updated classification of vascular anomalies by international society for the study of vascular anomalies is now widely used [table 1]. Classification of vascular anomalies (issva 2007) plain radiographs have limited role in the investigations as they can identify only phleboliths . Ultrasound (us) is often the initial investigation to evaluate vascular malformations and it may characterize and define the extent of more superficial lesions . On gray - scale imaging, venous malformations can appear as hypoechoic or heterogenous lesions with anechoic structures visible in 50% of cases . In addition, the doppler flow is generally monophasic low velocity flow, and in some cases flow is only discernible with compression and release of the lesion . They typically appear as isointense or hypointense lesions on t1-weighted images, but could be hyperintense depending on the presence of intralesional fat . Lesions are typically lobulated, which gives them the characteristic bunch of grapes configuration . Septations and rounded signal voids corresponding to phleboliths are additional distinguishing features . In t2-weighted or inversion recovery sequences, vms demonstrate high - signal intensity.this imaging modality is used to determine the full extent of the lesion and its relationship to adjacent vital structures . Gradient echo sequences reveal areas of low signal corresponding to calcification or hemosiderin or thrombosis . T1-weighted post contrast imaging demonstrates homogenous or heterogeneous enhancement, and dynamic contrast - enhanced mri has increased the specificity of venous malformation diagnosis . Vms are best demonstrated by direct phlebography, which fills the sinusoidal spaces and any anomalous veins, allowing assessment of the size and extent of the lesion . In the present study, phleboliths were seen on plain radiography and us features were suggestive of vascular anomaly with slow flow rate . Whereas, the mri features were suggestive of venous malformation, which was confirmed by direct phlebography where the lesion was seen draining the regional vein . Venous malformations usually are associated with syndromes like proteus syndrome and blue rubber bleb nevus (bean) syndrome . Multiple treatment options exist for venous malformations, including conservative measures such as head of bed elevation and compression, laser therapy, sclerotherapy, and surgery . Conservative management of venous malformations is usually reserved for smaller isolated asymptomatic lesions and is also important in controlling the growth and symptoms . Elevation of the head of the bed is important as it decrease hydrostatic pressure in the malformation, which can lead to expansion and can also decrease symptoms of airway obstruction, swelling, and pain that are experienced . Sclerotherapy remains a good option for the treatment of venous malformations in the head and neck . Sclerotherapy involves percutaneous injection of a substance to induce inflammation and thrombosis of the lesion, which then will lead to more long - term fibrosis and hopefully decrease or eliminate the expansion of the lesion . Sotradecol foam or ethibloc (glue), or the sclerosant is mixed with fibrin glue or ethyl cellulose, bleomycin (pingyangmycin) and picibanil (ok-432) have recently been used as sclerosants in asia with promising results . Large cervicofacial venous malformations present a much greater challenge, and one must be prepared to use multimodal therapy to keep the lesion under control . These lesions generally cannot be cured as doing so would leave devastating functional and cosmetic results . Therefore, therapy is used to control growth, maintain cosmesis, and decrease symptoms . Venous malformations are either superficial or deep veins that are abnormally formed and dilated . A thorough examination and investigation of the condition is needed to establish the exact extension of the condition and plan proper treatment.
After the development of fiber - reinforced composite posts, adhesive resin cement systems are a good option for restoring endodontically treated teeth . However, the development of adhesive materials that form a continuous, strong and durable interface with tooth root is still a challenge . A complete filling of the root canal is expected to form a monoblock, a solid mass without gaps that seals the root perfectly and remains stable in the oral environment . According to this concept, the monoblock is strong enough to support mastication and dissipates resistance capacity throughout the remaining tooth structure . This is theoretical, however, and currently available adhesives do not provide a hermetic, leak proof seal . Conventional dual - cure resin cements are indicated for luting procedures because they have low solubility, high mechanical quality and adhesive properties . The characteristics of the dual - cure cements are independent and complementary to those of light - activated chemical cements, which makes them ideal for deep cavities such as the root canal . The use of dual - cure cements requires pretreatment of the tooth root with an adhesive system . Until a few years ago, most adhesive systems available were applied in a 3-step procedure, which were later combined into 2 steps and more recently, into a single self - etching application step . However, acid resinous monomers present in the surface layer of the 2-step and self - etching systems can weaken the adhesive binding to root dentin . The permeability of simplified adhesives therefore results in water movement, even in root - treated dentin . Recently developed self - adhesive resin cements do not require pretreatment of the dentin . Because these cements do not use an adhesive system, they drastically reduce the number of application steps, shortening clinical treatment time and decreasing technique sensitivity since it minimizes procedural errors throughout the treatment phases . Self - adhesive cements contain multifunctional phosphoric acid methacrylates that are reputed to react with the hydroxyapatite of hard tooth tissue . However, some studies suggest that self - adhesive cements have limited capacity to diffuse and decalcify the underlying dentin effectively . Some reasons for this limitation are: 1) high viscosity, which may rapidly increase after acid - base reactions, and 2) a neutralization effect that may occur during setting, resulting in the release of water and of alkaline filler that raises ph level and buffering components of the smear layer . These materials are relatively new and detailed information on their composition and adhesive properties is limited . This is commonly caused by the inefficiency of the adhesive system or of the self - adhesive resin cements and occurs at distinct levels of the root canal . The apical portion of the root is particularly vulnerable to debonding because in this area it is difficult to eliminate the smear layer or remaining sealer / gutta - percha, control moisture and apply the adhesive . In addition, dual - cure resin cements have limited ability to diffuse light across the entire length of resin cement, thereby compromising the degree of polymerization and conversion . Interfacial discontinuity is commonly attributed to resin shrinkage because the strength of polymerization contraction often exceeds dentin adhesiveness, forming interfacial gaps at sites with weakened bonds along the dentin surface . Root canal geometry is also unfavorable for resin bonding, as evidenced by the ratio of bonded to unbounded surface . With respect to clinical treatment with glass fiber posts, however, some authors assert that dentin bonding is more related to the dislocation resistance promoted by sliding friction and surface roughness than to shrinkage stress or canal geometry . Discontinuity in binding interface is a serious failure in endodontic procedures because pull off or debonding may shorten treatment duration and cause bacterial proliferation and infiltration . The interface formed by the main resin cements currently used we therefore evaluated the continuity of dentin - cement interface formed by dual - cure and self - adhesive cements . We hypothesized that, despite the complex application, dual - cure cements form a more continuous interface compared to their self - adhesive counterpart . Because geometry and environmental conditions are quite irregular along the root portions, we tested a second hypothesis that the interfacial continuity formed is different in the cervical, medium and apical portions of the root . For the present study we selected 40 single - rooted human teeth that had been extracted for orthodontic or periodontal reasons after approval by the research ethics committee (approval #753/2006). They were cleaned and stored in a 0.9% solution of thymol and their crowns were cut to a standard root length of 14 mm . The roots were instrumented up to a #45 k file (maillefer, dentsply maillefer ind . Petrpolis, rio de janeiro, brazil) at the working length, and they were irrigated with 2% chlorhexidine digluconate solution (clorhexidina, fgm - dental products ltd . Petrpolis, rio de janeiro, brazil) and sealer 26 root canal sealer (sealer 26, dentsply ind . Petrpolis, rio de janeiro, brazil) using the hybrid thermoplastic technique described by tagger (1984). The coronal access of the roots were sealed with temporary restorative cement, coltosol (coltosol, vigodent s / a ind . E com, rio de janeiro, brazil) and stored in a bottle with moistened foam in a microprocessor controlled incubator used for cell culture and bacteriology (model q326m2, callmex - products and services laboratory, florianpolis, sc, brazil) at 37c for 7 days . The samples were prepared at 10 mm, using a #3 largo drill (maillefer, dentsply maillefer ind . Petrpolis, rio de janeiro, brazil), compatible to the glass - fiber post reforpost #01 (20 mm length, 0.7 mm, 1.1 mm) (reforpost, angelus ind . The glass - fiber posts were tested into the root canal prepared and then cut in 14 mm length . The teeth and their posts were separated randomly into 4 groups of 10 samples according to the cement used . Londrina, pr, brazil) was applied on the post surface during 1 min . The hydrophobic adhesive adper scotchbond plus multi - purpose (adper scotchbond plus multi - purpose, 3 m espe, sumar, sp, brazil) was applied to the post, light - curing for 20 s, using radii curing light (radii led curing light, sdi, bayswater, victoria, australia) at an intensity of 1200 mw / cm . Chemical compositions of the resinous cements and adhesive systems gpdm - glycerol dimethacrylate dihydrogen phosphate tegdma - triethylenglycol dimethacrylate bisema - ethoxylated bisphenol a glycol dimethacrylate group ac: allcem conventional dual - cure resin cement (allcem, fgm - dental products ltd ., joinville, sc, brazil), and group arc: relyxtm arc conventional dual - cure resin cement (relyxtm arc, 3 m espe, sumar, sp, brazil). The root dentin was conditioned for 15 s with 37% phosphoric acid gel (cond ac 37, fgm - dental products ltd ., joinville, sc, brazil); it has been washed with distilled water and dried with an endodontic aspirator and absorbent paper points . The adper scotchbond plus multi - purpose adhesive system (adpertm scotchbondtm plus multi - purpose, 3 m espe, sumar, sp, brazil) was applied in the following sequence: first, the activator was applied with the microbrush, and after 30 s the excess was removed with absorbent paper; the same process was then repeated with the primer and catalyst . The cement was manipulated and inserted into the canal with centrix syringe and needle headed (centrix, dfl ind . E com sa, rio de janeiro, rj, brazil). The posts were then inserted, the excess cement removed and the remaining cement cured for 40 s on the occluded surface with a radii curing light (radii led curing light, sdi, bayswater, victoria, australia) at an intensity of 1200 mw / cm . Group u100: relyx u100 universal self - adhesive resin luting cement (relyx u100, 3 m espe, sumar, sp, brazil), and group mxc: maxcem elite dual self - adhesive resin luting cement (maxcem elite, kerr corporation, orange, ca, usa). On the cementing procedure, the treatment of the root canal with etching agent was omitted . Instead, the root canal was washed on distilled water; the cement was inserted with centrix syringe and needle headed (centrix, dfl ind . Com . The posts were inserted, the excess cement was removed and the remaining cement cured for 40 s on the occluded surface, with a radii curing light . The posts and canals were sealed with opallis composite resin (opallis, fgm, joinville, sc, brazil), and the samples were stored for 72 hours at 100% humidity and 37c in a microprocessor - controlled incubator used for cell culture and bacteriology (model q326m2, callmex - produtos e servios para laboratrio, florianpolis, sc, brazil). After storaging, the roots were sectioned with an isomet low speed saw (isomet, buehler ltd ., the samples were cleaned with 10% liquid phosphoric acid for 10 s, washed and submitted to ultrasound (ultra cleaner 1400a, unique, indaiatuba, so paulo, brazil) for 10 min . To obtain realistic results, the present study used positive epoxy resin replicas of the samples after root sectioning to preserve the interface integrity between the different resin cements and the canal wall dentin . They were then dried with an air jet, negative molds were produced using addition silicone (adsil, vigodent, rio de janeiro, rj, brazil), and positive replicas were made with epoxy resin (epxi, redelease, so paulo, sp, brazil). Two marks were made in the positive replicas in the region of the root dentin 3 and 6 mm from the cervical end with the aid of a scalpel for guidance when taking measurements . The replicas were cleaned in an ultrasonic bath for 10 min . And metalized, and electron micrographs of each third of the root were taken using a scanning electron microscope (sem), operating at 15 kv (jeol jsm-5410, jeol ltd, tokyo, japan) (35x and 200x magnification). The image analyst program was used to combine the electron micrographs making up each root to measure the length of the interfacial continuity (figure 2). The combined electron micrographs (35x magnification) were then analyzed with autocad 2002 software, and the following measurements were taken the length of each third of the root and the sum of the interfacial continuity . The integrity of the interface in each third was then expressed as the percentage of continuous (gap - free) interface . The percentage of the continuous interface along the entire cement / radicular dentin interface was also calculated (figure 3). Figure 4 shows what was considered interfacial continuity and figure 5 shows a misfit . Scanning electron micrographs of the cervical (c), medium (m) and apical (a) thirds combined using image analyst software . Original magnification, 40x measurements taken with the autocad 2002 software: total length (t) of the thirds and lengths of the attached sections (c, m, a). Original magnification, 40x representative electron micrograph of the third; interfacial adaptation (c = cement; d = dentin; p = post; a = interface continuity). Original magnification, 200x electron micrograph of a representative third; interfacial misfit (c = cement; d = dentin; p = post; g = gap). The samples were prepared at 10 mm, using a #3 largo drill (maillefer, dentsply maillefer ind . Petrpolis, rio de janeiro, brazil), compatible to the glass - fiber post reforpost #01 (20 mm length, 0.7 mm, 1.1 mm) (reforpost, angelus ind . The glass - fiber posts were tested into the root canal prepared and then cut in 14 mm length . The teeth and their posts were separated randomly into 4 groups of 10 samples according to the cement used . Afterwards, the silane coupling agent (silano angelus, angelus ind . E com . Londrina, pr, brazil) was applied on the post surface during 1 min . The hydrophobic adhesive adper scotchbond plus multi - purpose (adper scotchbond plus multi - purpose, 3 m espe, sumar, sp, brazil) was applied to the post, light - curing for 20 s, using radii curing light (radii led curing light, sdi, bayswater, victoria, australia) at an intensity of 1200 mw / cm . Chemical compositions of the resinous cements and adhesive systems gpdm - glycerol dimethacrylate dihydrogen phosphate tegdma - triethylenglycol dimethacrylate bisema - ethoxylated bisphenol a glycol dimethacrylate group ac: allcem conventional dual - cure resin cement (allcem, fgm - dental products ltd ., joinville, sc, brazil), and group arc: relyxtm arc conventional dual - cure resin cement (relyxtm arc, 3 m espe, sumar, sp, brazil). The root dentin was conditioned for 15 s with 37% phosphoric acid gel (cond ac 37, fgm - dental products ltd ., joinville, sc, brazil); it has been washed with distilled water and dried with an endodontic aspirator and absorbent paper points . The adper scotchbond plus multi - purpose adhesive system (adpertm scotchbondtm plus multi - purpose, 3 m espe, sumar, sp, brazil) was applied in the following sequence: first, the activator was applied with the microbrush, and after 30 s the excess was removed with absorbent paper; the same process was then repeated with the primer and catalyst . The cement was manipulated and inserted into the canal with centrix syringe and needle headed (centrix, dfl ind . E com sa, rio de janeiro, rj, brazil). The posts were then inserted, the excess cement removed and the remaining cement cured for 40 s on the occluded surface with a radii curing light (radii led curing light, sdi, bayswater, victoria, australia) at an intensity of 1200 mw / cm . Group u100: relyx u100 universal self - adhesive resin luting cement (relyx u100, 3 m espe, sumar, sp, brazil), and group mxc: maxcem elite dual self - adhesive resin luting cement (maxcem elite, kerr corporation, orange, ca, usa). On the cementing procedure,, the root canal was washed on distilled water; the cement was inserted with centrix syringe and needle headed (centrix, dfl ind . Com . The posts were inserted, the excess cement was removed and the remaining cement cured for 40 s on the occluded surface, with a radii curing light . The posts and canals were sealed with opallis composite resin (opallis, fgm, joinville, sc, brazil), and the samples were stored for 72 hours at 100% humidity and 37c in a microprocessor - controlled incubator used for cell culture and bacteriology (model q326m2, callmex - produtos e servios para laboratrio, florianpolis, sc, brazil). After storaging, the roots were sectioned with an isomet low speed saw (isomet, buehler ltd . The samples were cleaned with 10% liquid phosphoric acid for 10 s, washed and submitted to ultrasound (ultra cleaner 1400a, unique, indaiatuba, so paulo, brazil) for 10 min . To obtain realistic results, the present study used positive epoxy resin replicas of the samples after root sectioning to preserve the interface integrity between the different resin cements and the canal wall dentin . They were then dried with an air jet, negative molds were produced using addition silicone (adsil, vigodent, rio de janeiro, rj, brazil), and positive replicas were made with epoxy resin (epxi, redelease, so paulo, sp, brazil). Two marks were made in the positive replicas in the region of the root dentin 3 and 6 mm from the cervical end with the aid of a scalpel for guidance when taking measurements . The replicas were cleaned in an ultrasonic bath for 10 min . And metalized, and electron micrographs of each third of the root were taken using a scanning electron microscope (sem), operating at 15 kv (jeol jsm-5410, jeol ltd, tokyo, japan) (35x and 200x magnification). The image analyst program was used to combine the electron micrographs making up each root to measure the length of the interfacial continuity (figure 2). The combined electron micrographs (35x magnification) were then analyzed with autocad 2002 software, and the following measurements were taken the length of each third of the root and the sum of the interfacial continuity . The integrity of the interface in each third was then expressed as the percentage of continuous (gap - free) interface . The percentage of the continuous interface along the entire cement / radicular dentin interface was also calculated (figure 3). Figure 4 shows what was considered interfacial continuity and figure 5 shows a misfit . Scanning electron micrographs of the cervical (c), medium (m) and apical (a) thirds combined using image analyst software . Original magnification, 40x measurements taken with the autocad 2002 software: total length (t) of the thirds and lengths of the attached sections (c, m, a). Original magnification, 40x representative electron micrograph of the third; interfacial adaptation (c = cement; d = dentin; p = post; a = interface continuity). Original magnification, 200x electron micrograph of a representative third; interfacial misfit (c = cement; d = dentin; p = post; g = gap). Friedman and kruskal - wallis tests with an 5% were used to determine whether there were any statistically significant differences in the results for the resin cements tested . Friedman test revealed no statistically significant differences among the thirds of the roots for each group: group ac (p=0.1496), group arc (p=0.1496), group u100 (p=0.6703) and group mxc (p=0.7985). Mean percentage of the interfacial continuity resin cements (%) and standard deviation varying the cervical, medium and apical thirds comparison between columns: same capital letters indicate no statistically significant difference . The kruskal - wallis and dunn tests revealed a statistically significant difference between cements for each of the three thirds of the roots . Groups ac, arc and u100 showed better performance than group mxc for cervical thirds (p=0.0001), medium thirds (p=0.0001) and apical thirds (p=0.0013) (table 1). Taking into account the overall interfacial continuity of the cements, it was observed that u100>ac> arc> mxc (figure 6). Corroborating the first hypothesis of the present study, both the dual - cure resin cements tested in vitro produced a more continuous interface than the self - adhesive maxcem, which formed a discontinuous interface with many gaps . However, contradicting this same hypothesis, the interfacial continuity produced by u100 self - adhesive cement was as satisfactory as that formed by the dual - cure cements . In relation to root portion, we rejected the hypothesis concerning different cement adhesion in the cervical, medium and apical root regions . For the experimental assays, we used a reliable technique involving epoxy resin replicas, which avoided a possible influence of shrinkage under vacuum in scanning electron microscopy . A number of variables can compromise cement adhesion, such as root dentin morphology, humidity, adhesive system capabilities, compatibility of adhesive system and dual - cured luting cement, deep light cure capabilities, light transmissibility of fiber posts, polymerization shrinkage and relief of shrinkage stress . The use of an adequate resin cement system is particularly important for cement adhesion because it directly affects the quality of the tooth - luting interface . Dual self - adhesive resin cements generally have better adhesion than self - adhesive resin cements, suggesting that composite materials significantly influence the formation of the tooth - luting interface . Corroborating this hypothesis t, both dual self - adhesive resin systems tested here (allcem and relyx arc) exhibited good adaptation and low gap formation (ac=81.3% and arc=80.4% interfacial continuity; table 1). Conventional dual - cure resin cements can be used in adhesive systems that require pretreatment of the root with phosphoric acid as etching solution followed by the application of a 2- or 3-step adhesive system . We used a 3-step system, and the quality of this product undoubtedly contributed to the good results obtained for groups ac and arc . After the etching solution completely dissolves the smear layer and creates a partially demineralized zone of dentin, the 3-step adhesive system is applied using an activator followed by a primer and a catalyst (adper scotchbond multi - purpose plus). The 3-step etch & rinse adhesive system increases the interfacial adaptation of dual - cure luting cement because it increases adhesive penetration into dentinal tubules, thereby improving the pattern of dentin hybridization . The catalyst layer of the 3-step system (ph=5.7) is not composed of solvents that could allow a few droplets to transude the polymerized adhesive and compromise cement adhesion . In addition, the 3-step adhesive system lacks acid monomers, which ensures total compatibility with the dual - cure luting cement . Other adhesive systems, such as 2-step and one - step - self - etch, have uncured acidic resin monomers in the oxygen inhibition layer that are incompatible with the tertiary amines (initiator component) involved in the polymerization mechanism of dual - cure resin cements . Decreased ph is another disadvantage of adhesive systems containing acid monomers because it weakens bond strength . Conversely, the 3-step system provides effective binding between the adhesive agents and the dental structure . The catalyst used, scotchbond multi - purpose plus, contains a common initiator for self - curing resins, benzoylperoxide (bpo), which reacts with the tertiary amines of the self - cure and dual - cure resin cements and helps dual polymerization of the dual - luting cement along the root . Other advantages of the 3-step adhesive system is that no light curing is required before post cementation, overcoming an obstacle in curing deep regions . In addition to pretreatment, the composition of dual - cure resin cements unquestionably contributed to cementing quality . These resin cements contain cross - linking monomers such as bis - gma, which produce polymers of high mechanical quality because of their high molecular weight, low polymerization contraction and rapid hardening . Dual - cure resin cement also contains the monomer diluent triethylene glycol dimethacrilate (tegdma), which has high flexibility and low polymerization contraction . Tegdma also provides a high degree of conversion and has hydrophobic properties that prevent substantial water uptake after curing . Compared to bis - gma, tegdma allows better clinical handling owing to its low viscosity . A redox reaction of benzoyl peroxide of dual - cure resin cements with aromatic tertiary amines underlies the setting mechanism, a process that is compatible with the 3-step adhesive system, as mentioned before . Similarly to the conventional dual - cure cements tested here, the u100 self - adhesive cement exhibited good performance (table 1). Earlier studies reported satisfactory results using relyx unicem cement, which is chemically identical to the relyx u100 we tested, differing only in the application procedure . Relyx unicem produces good marginal continuity, but due to its high viscosity, does not form an obvious dentin hybrid layer and thus achieves limited infiltration . Therefore, both relyx u100 and relyx unicem are highly adapted to the substrate and can optimize physical interactions such as van der waals forces, hydrogen bridges and charge transfer, which enhance micromechanical retention and chemical bonding . The multifunctional, phosphoric acid modified methacrylate monomers of u100 (ph<2) demineralize root dentin as well as infiltrate the substrate and react with the hydroxyapatite of hard tissues . Thus, the micromechanical retention associated to the chemical adhesion to hydroxyapatite provides self - adhesiveness to the u100 cement (3 m espe technical information). This chemical interaction produces water, which accelerates neutralization of phosphoric - acid methacrylate, basic fillers and hydroxyapatite . The system likely gained water resistance, and although water and buffering of the smear layer may have reduced desmineralization capacity, the effectiveness of the u100 cement was not compromised, as reported in earlier study . Overall, we consider that relyx u100 cement is the best of the tested cements because, in addition to the good results it obtained, its application is significantly easier than that of conventional dual care cements . In contrast to u100, maxcem self - adhesive cement performed poorly, corroborating earlier studies . Maxcem contains several hydrophilic adhesive monomers of low molecular weight, such as the glycerol phosphate dimethacrylate (gpdm), which provide the necessary wettability for adhesion to dentin substrate . The cement is purportedly anhydrous prior to mixing, but the technical specifications of this product do not inform the way this monomer is initially hydrolyzed, the initial ph or the proprietary redox activator system . This may account for the discontinuous tooth - cement interface, with many premature failures, that we found in the mxc group . Maxcem has limited potential for removal or modification of the smear layer and low resin infiltration into the underline dentin . Therefore, it does not form a dentin hybrid layer that promotes the micromechanical interlocking with dentin collagen fibrils . The thick smear layer formed neutralizes the initial low ph of maxcem thereby impairing its performance . Therefore, despite the excellence of maxcem monomers as bonding agents of good compatibility with the humid dentin substrate, they do not provide an effective cementation in deep areas of high humidity . We however have not found cementation differences in the wettest and driest portions of the root, and the bad results of maxcem may derive from poor - quality of the polymer formed . For practical reasons we have not investigated chemical set cements . But some concerns should be made on these materials because they are widely used in endodontic treatments despite the application failures reported in other studies . These cement sets have a long setting time, with a prolonged gelation that possibly reliefs shrinkage stress via resin flow . This long setting time, however, allows moisture diffusion from the dentin through the hydrophilic primer, creating water blisters or droplets along the interface with the slow polymerizing resin cements . Moisture contamination reduces bond strength and facilitates leaching of water - soluble components from the resin . This process is as serious as the incompatibility of adhesive systems and may further contribute to break down the bond, as show in both in vitro and in vivo studies of endodontically treated teeth . Some studies on the adhesive resistance of cements show that binding is stronger in the cervical portion and weaker in the apical portion of the root . Some authors suggest that samples of low bond strength have a high number of gaps, but the relationship between bond strength and bond integrity has not yet been proved . In accordance with other authors, we in fact found that the experimental groups had a same pattern of continuity interface throughout the apical, medium and cervical portions of the root . We expected to find a higher continuity in the cervical portion of the root because the curing light absorbed along the canal decreases significantly light incidence in the medium and apical portions . This is minimized when light - transmitting posts are used because they improve polymerization in deep areas of canal roots . In our study, however, we used opaque posts and did not found that drops in light incidence have affected interfacial continuity . The cervical third of the root had higher cement thickness, but variations of this parameter did not affect interfacial continuity as well . The major implication of a high cement thickness is the shrinkage risk with consequent debonding and gap formation in the resin cement / dentin interface . In the present study, differences in cement thicknesses were rather related to the uneven root anatomy than to interface quality . Continuity of dentin / cement interface is necessary to promote sliding friction, which according to many authors is the main factor for adhesive resistance in the fiber post / resin cement system and for micromechanical interlocking . As already observed by practitioners in routine treatments, our results show that the cements currently used provide good cementation but do not form a complete bonding and a continuous interface along the length of the root channel . Complete fiber luting is important because it prevents post displacement by frictional retention and bacterial leakage, thereby enhancing the longevity of coronal restorations in endodontically treated teeth . According to schwartz (2006) and tay, loushine, lambrechts, et al . Therefore, more attention should be devoted to the recently developed low - shrinking resin composites that have been used only for tooth restorations . We therefore suggest the adaptation of these resins to endodontic treatments such as post luting . This is a promising resin to prevent gap formation in the dentin - adhesive interface . Based on the results of this in vitro study, we conclude that: 1 - the cements allcem, relyx arc and relyx u100 provide good cementation with 80% continuity . Maxcem elite does not produce a satisfactory cementation (only 47% interfacial continuity); 2 - cementation quality provided by the different cement types is similar among the cervical, medium and apical portions of the tooth roots; 3 - in practical terms, relyx u100 provides the best cementation because combines good cementation and easy application; 4- because the cements tested do not provide a complete interfacial continuity, we suggest the development of novel materials using low - shrinking resin composites that are already used for restoration purposes.
Ectopic thyroid tissue is a common abnormality and results from abnormal embryologic development and migration of the thyroid gland . Such tissue is usually found along the path of descent of the thyroid gland in the anterior midline of the neck . A lingual thyroid is the most common presentation of thyroid ectopy along with thyroglossal duct remnants . Ectopic thyroid tissue has been described in other parts of the head and neck such as the submandibular [3, 4] and parotid salivary glands . There are case descriptions of thyroid tissue identified in diverse locations such as the axilla, trachea [710], adrenal, small intestine, and porta hepatis . The most frequent noncervical location for ectopic thyroid tissue is the thoracic cavity [1418]., the authors describe a case of ectopic anterosuperior mediastinal thyroid mass excised through a cervical incision and the subsequent investigation to exclude malignancy . An 80-year - old nonsmoker female patient presented with an incidental finding of a mediastinal mass on mri scan for investigation of vertigo (figures 1 and 2). Thyroid stimulating hormone (tsh) and free thyroxine (ft4) were within the normal range, and routine preoperative blood tests were normal . An anteroposterior chest radiograph demonstrated deviation of the intrathoracic trachea around the mass (figure 3). A subsequent ultrasound scan demonstrated a superior mediastinal mass separate from the inferior pole of the right thyroid lobe measuring 4.8 cm in maximal diameter (figure 4). The first sample was inadequate (thy1), the second sample demonstrated only colloid and a few follicular cells . Since malignancy could not be definitively ruled out, the mass was excised through a cervical incision . Under general anesthesia, the patient was placed in the supine position with the neck extended, prepared, and draped . Endotracheal laryngeal nerve monitoring was used with nim contact emg endotracheal tube and nim - response 2.0 monitor (medtronic usa, inc.6743 southpoint drive north, jacksonville, florida, usa, 32216 - 0980). Through a standard 5 cm midline cervical incision, the right thyroid lobe was dissected, found to be grossly normal, and excised . The right recurrent laryngeal nerve was identified in the normal position, confirmed functioning with nerve stimulation at 2 ma, and preserved . Separate and inferior to the right thyroid lobe a discrete encapsulated mass was identified in the superior, mediastinum . This extended across the anterior surface of the trachea to the left side, adjacent to the thymus . The mass was separated from the thyroid gland by fat, in the same tissue plane but with no identifiable connection to the cervical region . The mass was excised through the cervical incision . During excision of the mass, a double vascular pedicle, arising from inferiorly in the mediastinum, histopathologic evaluation of the excised mediastinal specimen demonstrated a 5.3 cm by 2 cm thyroid tissue mass with large oedematous loose areas and foci of calcification and fibrosis (figure 5). The excised right thyroid lobe had a nodular architecture composed of follicles of varying sizes with focal lymphoid aggregates.thyroid function tests at 3 months were normal . Further investigation with whole body radioiodine i123 scintigraphy demonstrated expected residual uptake in the remaining left thyroid lobe and surgical bed but no uptake suggestive of other ectopic thyroid tissue or metastatic disease . Ultrasound surveillance of a 9 mm nodule in the remaining left lobe demonstrated no change in size over 6 months . In humans, the thyroid gland begins to develop at the 24th day of gestation . The thyroid is the first endocrine gland to develop and originates from between the first and second branchial arches . An invagination of endodermal epithelial cells begins at the midline of the developing pharyngeal floor forming a diverticulum . This site, known as the foramen caecum, lies between the tuberculum impar (median tongue bud) and the hypobranchial eminence (copula). The foramen caecum can be observed in adults as a small pit at the base of the tongue where the tongue is divided into an oral anterior two - thirds and pharyngeal posterior third by the sulcus terminalis . The initial path of descent of the bilobed thyroid diverticulum is anterior to the pharyngeal gut, the hyoid bone, and the laryngeal cartilages . At the same time embryological studies have shown that the strap muscles pull downward on the hyoid bone during development causing a forward tilt . This tilt pulls the tract posteriorly and causes it to be hooked up behind the hyoid bone . This explains the importance of excision of the median portion of the hyoid bone in the sistrunk procedure to excise a thyroglossal duct cyst . The gland reaches its final location anterior to the trachea by the 7th week gestation . By this stage, it has acquired a median isthmus and two lateral lobes . As it descends, it is joined by the ultimobranchial bodies which form the parafollicular c cells . Thyroid function begins at around the end of the 3rd month when the follicular cells commence production of colloid and follicles appear . During migration, from the 5th week gestation, the thyroid is still connected to the tongue by the thyroglossal duct . In normal development, this tubular structure subsequently obliterates entirely at approximately the 8 to 10th week . In some individuals, however, abnormalities in the embryologic development and migration of the thyroid gland can result in ectopic thyroid tissue . These remnants can appear at any point along the migratory path and are always located at or near the midline . The majority of remnants are found at, or just inferior to, the body of the hyoid bone as a thyroglossal cyst . Remnants can also be found in the tongue base as a lingual thyroid or close to the thyroid cartilage . The inferior end of the thyroglossal duct may fail to obliterate, and in at least half of individuals, a pyramidal lobe of the thyroid can be seen to persist . This pyramidal lobe itself may be attached to the hyoid bone or may be incorporated into a thyroglossal duct cyst . Ectopic thyroid tissue in other locations is rare and generally the subject of single case reports . The most important diagnosis to exclude is metastatic lymph node deposits of well - differentiated thyroid carcinoma . Indeed, it is generally accepted that early reports of lateral aberrant thyroid tissue may represent papillary thyroid carcinoma metastases . Another hypothesis is that a thyroid nodule may become detached from the gland . In this case, the vascular supply should come from branches of the superior or inferior thyroid artery . There are reports of thyroid deposits in the soft tissues of the neck representing surgical implantation of thyroid neoplasms . In one case, infiltrating thyroid tissue in muscle and fibrous tissue presented 3 years after major blunt trauma to the neck . The tissue resembled that in a disrupted thyroid nodule present in the gland itself and was regarded as traumatically implanted . There are, however, a number of ectopic thyroid masses reported which do not have features consistent with these theories and are considered truly developmental . Animal studies suggest that ectopic location of thyroid tissue may be related to vascular development . The normal arterial supply of the thyroid gland consists of paired superior and inferior thyroid arteries . The superior thyroid artery is generally considered to be present in 100% of cases, and its absence has only been reported once . An unusually large superior thyroid artery may replace the contralateral vessels or the inferior thyroid artery . The inferior thyroid artery when unusually small or absent may be replaced or supplemented by a thyroidea i m a artery . Published reports place the incidence of a thyroidea i m a artery between 2% and 12% . In the case the ectopic mass was separate from the thyroid gland but in the same anatomical plane . Careful histological examination of the mass and the excised ipsilateral thyroid lobe demonstrated no evidence of neoplasia, and exhaustive investigations showed no evidence of thyroid malignancy elsewhere . The authors therefore conclude that this represents developmentally ectopic sequestered thyroid tissue in the anterior mediastinum . Despite this, the authors suggest that abnormally situated thyroid tissue, other than that in the central neck along the path of embryological descent of the thyroid gland, should be excised for careful histological analysis in order to exclude metastatic disease.
Pregnant women and neonates are at greater risk for influenza - related complications than the general population [1, 2]. Most institutions and organizations recommend that all pregnant women receive the trivalent inactivated influenza virus vaccine [38]. Such endorsements rely on the immunogenic response of the mothers, the lack of teratogenicity, and the contrandication of immunization in children younger than six months [7, 911]. Despite the broad recommendation to vaccinate pregnant women against influenza, coverage is still limited . A survey held by the centers for disease control and prevention involving women who were pregnant from october 2011 to january 2012 showed that only half of the respondents had been vaccinated and fewer than 10% had received the vaccine before giving birth . Similar patterns were found in previous studies . Although there is clear evidence of the efficacy of the influenza vaccine for the general population, to our knowledge, a systematic approach with regard to the evidence of the therapeutic effects of influenza vaccination in pregnant women is lacking . Our objective is to review the effects of influenza vaccination in preventing influenza - related outcomes in pregnant women and their infants . We selected randomized controlled trials or cohort studies that assessed the effects of inactivated influenza vaccine in preventing influenza - related outcomes in pregnant women and their offspring compared with placebo, other vaccines, or no vaccines . We excluded studies that assessed monovalent vaccines, such as the h1n1 influenza vaccine, because they are used for specific epidemic situations . We searched for eligible studies in the following databases (from inception to september 2013): medline, embase, scopus, centre for reviews and dissemination (crd), cochrane central register of controlled trials (central), metaregister of current controlled trials (mcrt), latin american and caribbean center on health sciences information (lilacs), and scientific electronic library online (scielo). References to relevant publications in the field were also screened to identify potentially eligible studies . There were no restrictions on language, length of followup, publication date, or publication status . We used the following search terms to search medline (via pubmed) and adapted the strategy for the other databases: (influenza, human[mesh] or influenza[tiab] or human flu[tiab] or influenza[tiab] or influenzas[tiab] or grippe[tiab] or flu[tiab] or cold[tiab]) and (influenza vaccines[mesh] or influenza vaccines[tiab] or vaccine[tiab] or vaccine[tiab] or vaccines[tiab]) and (mothers[mesh] or mothers[tiab] or pregnant women[tiab] or pregnant[tiab]) and (infant[mesh] or infant[tiab] or infants[tiab] or infant, newborn[mesh] or newborns[tiab] or fetus[mesh] or fetus[tiab] or foetus[tiab] or fetal[tiab] or pregnancy). Two independent reviewers (eb and labl) selected the studies by assessing titles and abstracts and extracted the data . The extracted data consisted of the following: year, country, study design, gestational age, type of vaccine, posology, comparators, sample size, followup, and outcomes . When necessary, we contacted the corresponding authors for additional information . To assess the risk of bias of randomized controlled trials (rct), we used the cochrane collaboration tool, which includes judgments about the sequence generation, allocation sequence concealment, blinding, incomplete outcome data, selective outcome reporting, and other sources of bias . For observational studies, we evaluated the following: eligibility criteria, measurements of exposures and outcomes, control of confounding, and followup . We assessed the quality of evidence for each relevant outcome with the grading of recommendations assessment, development, and evaluation (grade) [17, 18]. For this evaluation, we separated the bodies of evidence into experimental and observational centered on rct and cohort studies, respectively . Following the grade approach, then, we assessed the evidence against five items that could decrease its quality: limitations (risk of bias), inconsistency, indirectness, imprecision, and publication bias . After assessing these items, the resulting quality of evidence could be rated as high, moderate, low, or very low . When distinct levels of quality were available for the same outcome, we considered the experimental design (rct) evidence in rating the quality . The final judgments regarding the risk of bias and evidence quality were achieved by consensus . The primary outcome was the incidence of influenza - like illness, which was defined as fever and either cough or sore throat . For infants' outcomes, we defined small for gestational age as a weight below the 10th percentile and prematurity as birth before a gestational age of 37 complete weeks . We extracted the estimates along with 95% confidence intervals (95% ci) according to the data available in the original studies (relative risk (rr), odds ratio (or), or hazard ratio (hr)). If reported, we only considered the adjusted estimates and did not perform further calculation . We attempted to perform meta - analyses using random effects models, if numerical data from studies allowed a summarization . Twenty - three records were selected for full - text assessment, and nine were included in the review . Were related to eight unique studies that enrolled 182,820 pregnant women and 182,246 neonates [3442]. We identified one rct conducted in bangladesh and seven cohort studies performed in the united states . The trivalent inactivated vaccine was the most common intervention and was assessed in seven studies [3541]. Newborns were not vaccinated . Only the rct had an active control group (pneumococcal vaccine). Nearly the entire sample of pregnant women came from three retrospective cohorts [35, 37, 41]. The length of followup of each outcome varied among studies and lasted up to 36 weeks . Some studies adjusted their results for confounding factors, such as the women's age, week of delivery, infant's gender, and gestational age . Observational studies that compared baseline characteristics of the groups showed that most variables did not differ between the groups . Some studies showed that vaccinated women had a worse profile than unvaccinated women, which were of higher risk for complications, were older, and had higher body mass index, higher parity, and more multiple gestation . We could not perform meta - analysis because the studies used different measures of association for the same outcome; the estimates are presented as available in the studies . Mothers who received the influenza vaccine had a lower incidence of influenza - like illness, as did their infants (high - quality evidence), but there was no difference found for influenza - like illness with fever higher than 38c (moderate - quality evidence). A lower incidence of influenza in infants, as confirmed by laboratory tests, was also observed (moderate - quality evidence). Very - low - quality evidence showed no difference between comparisons with regard to the incidence of upper respiratory infection, hospitalization, and medical visits for influenza - like illness in mothers and infants . Two studies found no difference in the incidence of hospitalization for influenza - like illness in infants [35, 37]; in one study, the reduction in the rate of hospital admission was significant . With regard to medical visits for influenza - like illness in infants, the observational studies showed no significant differences [35, 37, 40], and the rct showed a reduction in such rate (moderate - quality evidence). For the outcomes prematurity and small for gestational age, conflicting results were found across the studies . One single cohort indicated significant reduction in stillbirth and neonatal death among the influenza - vaccinated group (moderate - quality evidence). We did not assess the incidence of adverse reactions because the included studies did not systematically evaluate this outcome . In general, influenza vaccination had no association with local or minor systemic effects, fever, apgar score at one minute, hyperbilirubinemia, or major malformations . The influenza vaccine was found to reduce the risk of influenza - like illness in mothers and infants as well as the risk of laboratory - confirmed influenza in infants . Adverse reactions were not systematically assessed across the studies, but there was no evidence of increase in clinically relevant risk related to influenza vaccination during pregnancy . A big cohort study that focused on the safety of trivalent inactivated influenza vaccine, however, did not find any increased risk of adverse events and adverse obstetric events in the vaccinated mothers, when compared to unvaccinated pregnant women [43, 44]. Other factors can prevent influenza in infants such as the effect of breast - feeding and immunization of all the infant's close contacts, also known as cocooning . To avoid confounding and enable comparison, groups should be set by randomization . In the present review, however, only one rct was identified and included . Although most observational studies performed multivariate analyses, residual confounding may remain even after adjustment because this statistical procedure cannot control for all biological variabilities . Women receiving influenza vaccine would have more medical attention and be healthier than unvaccinated pregnant women; thus, the result found would be attributed to the health profile of vaccinated women rather than to the vaccine itself . However, some studies reported that vaccinated women were at high risk during gestation, and this difference was also statistically controlled [37, 42]. Studies consistently reported that the patients and the clinicians made the decision about taking influenza vaccine or not . Surveys about attitudes and beliefs of these actors regarding influenza vaccination in pregnancy show that the proportion of people who do not believe vaccine is safe is still high [48, 49]. Another limitation of our review is the absence of the systematic reporting of adverse reactions . Although the incidence of adverse reaction was not shown to be a concern in the included studies, this lack of evidence may inadvertently lead to the conclusion that this risk is minimal or nonexistent . Individuals with egg allergy, for example, require caution when receiving trivalent inactivated influenza vaccine . Previous narrative reviews concluded that influenza vaccination is safe, that there has been no evidence of teratogenicity, and that many countries recommend influenza vaccination among women with both healthy and high - risk pregnancies [9, 5156]. The role of education of patients and doctors in increasing adherence to maternal vaccination was also emphasized [5759]. One systematic review assessed the benefits and dangers of the influenza vaccine in special populations pregnant women included but limited the eligible studies to rcts only . Apprehensions about the use of thimerosal - containing influenza vaccines, based on theoretical risk of harm to the fetal brain, were widely spread in scientific and lay communities during the past years [55, 62]. Subsequent research proved that no causal relation existed between immunization with vaccine containing thimerosal preservative including exposure during pregnancy and neuropsychological outcomes [63, 64]. The most recent report of the global advisory committee on vaccine safety of the world health organization considered that available evidence strongly supports the safety of the use of thimerosal as a preservative for inactivated vaccines . It is expected that with the availability of higher - quality evidence, such concerns can be demystified . Attending to claims for more evidence [66, 67], several rcts assessing influenza vaccination in pregnancy are planned and some are ongoing [6877]. Such rcts focus on different populations, such as hiv - positive mothers, and factors that interfere with immunization coverage . We expect that, following the publication of these trials, the availability and quality of the evidence will radically improve . Additionally, the issue about the comparability between the vaccinated and unvaccinated groups will be more properly addressed . Maternal immunization for influenza significantly reduced the incidence of influenza - like illness in women and infants . For clinical practice, further studies should address this lack of evidence and enhance the overall quality of the outcomes.
High - throughput techniques have become common in the study of these alterations . However, the analysis of this type of data is challenging . One of the main difficulties is in sorting out the alterations that drive tumorigenesis from those that are only byproducts of the high number of divisions cancer cells undergo and have no effect on the cancerogenic phenotype . Moreover, the existence of different types of alteration makes the detection of causative ones even more difficult . Hence, it is clear the need for approaches to analyze and integrate cancer genomics data . Intogen integrates high - throughput data related to different types of alterations taking place in cancer such as copy number alterations, point mutations and transcriptomic changes from many independent studies to identify the genes and modules (e.g. Kegg pathways, go terms) more significantly altered in different tumor types (1). The data in intogen consists of publicly available cancer genomic studies collected from databases such as gene expression omnibus (geo) (3), arrayexpress (4), cosmic (5), progenetix (6), the sanger cancer genome project (http://www.sanger.ac.uk/genetics/cgp/) and the data portal of the cancer genome atlas (7). Each study contains results from high - throughput analyses of a number of human primary tumor samples compared to normal cells (normal cells of the same tissue in the case of expression) related to one or more types of cancer for a specific alteration . At the first step, all the samples in the study are annotated with appropriate terms from international classification of disease for oncology (icd - o) (8): a topography term indicating location in human body and, if available, a morphology term describing histological classification . Studies are also annotated with the platform(s) used for the experiment . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units, which correspond to a set of assays in one experiment annotated with the same topography and morphology . Analysis units are also created with assays in one experiment annotated with the same topography and any morphology (figure 1). Analysis units. Table 1 summarizes the number of studies, experiments and analysis units included in the current version of intogen (v03). Each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units in two ways, 1) according to the topography and the morphology, and 2) according to the topography (with the morphology annotated as any morphology). Table 1.summary of the data content in intogen (v03)alteration typemain data sourcesnumber of independent studiesnumber of experimentsnumber of analysis unitstranscriptomicgeo118122243arrayexpresstcgagenomic (copy number)progenetix188188343sanger cancer genome projecttcgatotal306310586 data annotation and classification . Each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units in two ways, 1) according to the topography and the morphology, and 2) according to the topography (with the morphology annotated as any morphology). Summary of the data content in intogen (v03) in intogen framework, the analysis is performed at different levels: on one side each experiment is analyzed independently (experiment level) and those experiments classified with the same topography and morphology terms are combined (combination level). Also on the other side, the analysis is performed at the level of genes (gene level) and at the level of modules (module level). A module is defined by a set of genes with some biological property in common, we currently analyze gene ontology (go) modules, kegg pathway modules, modules derived from genes sharing a transcription factor - binding side (tfbs) in their promoter and genes sharing microrna target motifs in their 3-utr [see ref . First, within each analysis unit, we identify genes altered in more samples than expected by chance using oncodrive [see ref . The results for the same gene are combined statistically across the analyzed experiments classified with the same topography and morphology terms using the weighted z - method (9). If the study contains enough samples (at least 20) for which morphology type information is known, then detection of significant alterations can be done at the level of topography and the level of morphology . The limit of 20 samples was setup to increase the reliability of results, as we consider that smaller number of replicates in a large - scale study can lead to anomalous conclusions (1). In this way, alterations specific to certain morphology type can be identified as well as those common to the topography of the cancer in general . After significantly altered genes are detected, enrichment analysis is done to find significantly altered modules (e.g. Biological processes or pathways) per experiment . In the same way as before, the results for the same module are combined across studies that analyze the same cancer type . Each analysis unit (set of assays from the same study using the same platform and annotated with the same icd - o terms) is analysed to detect the significantly altered genes . The experiment results with the same icd - o terms are combined both at the gene level and at the module level . For methods details each analysis unit (set of assays from the same study using the same platform and annotated with the same icd - o terms) is analysed to detect the significantly altered genes . The experiment results with the same icd - o terms are combined both at the gene level and at the module level . For methods details see (1). As can be expected, the interpretation of these highly inter - related results requires powerful visualization methods . The browser of intogen facilitates the exploration and intuitive visualization of results at different levels (available at: http://www.intogen.org), while the biomart portal (2) (available at: http://biomart.intogen.org) allows complex queries and facilitates the bulk download of the all analysis results . In intogen biomart portal, users can query for three types of data . For each type, there is a database; intogen experiments, intogen combinations and intogen oncomodules (table 2). Table 2.databases and data sets in the biomart of intogendatabasesdata setsdescriptionexperimentsgene genomic alterationsrecurrence and significance of genomic alteration (gain and loss) for each gene at the level of experimentsgene transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each gene at the level of experimentskegg genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each kegg pathway at the level of experimentskegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each kegg pathway at the level of experimentsgo genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each go term at the level of experimentsgo transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each go term at the level of experimentstfbs genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each tf at the level of experimentstfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each tf at the level of experimentsmirna genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each mirna at the level of experimentsmirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each mirna at the level of experimentscombinationsgene genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each gene at the level of combinations (tumor types and subtypes)gene transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each gene at the level of combinations (tumor types and subtypes)kegg genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each kegg pathway at the level of combinations (tumor types and subtypes)kegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each kegg pathway at the level of combinations (tumor types and subtypes)go genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each go term at the level of combinations (tumor types and subtypes)go transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each go term at the level of combinations (tumor types and subtypes)tfbs genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each tf at the level of combinations (tumor types and subtypes)tfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each tf at the level of combinations (tumor types and subtypes)mirna genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each mirna at the level of combinations (tumor types and subtypes)mirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each mirna at the level of combinations (tumor types and subtypes)oncomodulescombinationssets of genes significantly altered in each cancer type and subtypeexperimentssets of genes significantly altered in each experiment databases and data sets in the biomart of intogen in intogen experiments database, there are data sets for genomic and transcriptomic alterations . Users can query the results of the recurrence analysis for these alterations at the level of genes or modules, such as kegg pathways and go categories, for each experiment included in intogen . For both types of data sets, the results can be filtered in many different ways . Here are a few examples: genes annotated with a list of go ids, genes in a specific chromosomal band, a list of selected entrez / ensembl gene ids . The user can also filter by significance level and the results can be restricted to experiments done by specific authors, performed on a specific platform type, etc . The columns in the results can be determined by the selections done in attributes section . For experiment - level data, there are a number of statistics derived from the analysis that can be retrieved such as the number of samples in the experiment, the expected / observed number of alterations and p - values etc . Finally, a table that contains the selected attributes for the genes or modules is retrieved . In intogen combinations database, users can query the results for combinations, that is, the integration of results from experiments annotated with the same icd - o terms . This database includes data sets for genomic and transcriptomic alterations for genes and modules . The filters and attributes works in a similar way as in experiment database but without publication nor platform attributes and filters, and including the results attributes specific to the combination method . In intogen oncomodules database, there are two data sets, one for combinations and one for experiments . Each data set contains lists of genes that are significantly altered in a specific combination of icd - o terms or in a specific experiment . Again the user can filter the results in a variety of ways, with the significance level he / she likes, according to certain characteristics of genes, for a cancer type and, in the case of oncomodules at the level of experiments, for author or platform type . The data in intogen consists of publicly available cancer genomic studies collected from databases such as gene expression omnibus (geo) (3), arrayexpress (4), cosmic (5), progenetix (6), the sanger cancer genome project (http://www.sanger.ac.uk/genetics/cgp/) and the data portal of the cancer genome atlas (7). Each study contains results from high - throughput analyses of a number of human primary tumor samples compared to normal cells (normal cells of the same tissue in the case of expression) related to one or more types of cancer for a specific alteration . At the first step, all the samples in the study are annotated with appropriate terms from international classification of disease for oncology (icd - o) (8): a topography term indicating location in human body and, if available, a morphology term describing histological classification . Studies are also annotated with the platform(s) used for the experiment . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units, which correspond to a set of assays in one experiment annotated with the same topography and morphology . Analysis units are also created with assays in one experiment annotated with the same topography and any morphology (figure 1). Analysis units. Table 1 summarizes the number of studies, experiments and analysis units included in the current version of intogen (v03). Each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units in two ways, 1) according to the topography and the morphology, and 2) according to the topography (with the morphology annotated as table 1.summary of the data content in intogen (v03)alteration typemain data sourcesnumber of independent studiesnumber of experimentsnumber of analysis unitstranscriptomicgeo118122243arrayexpresstcgagenomic (copy number)progenetix188188343sanger cancer genome projecttcgatotal306310586 data annotation and classification . Each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . An experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . Analysis units in two ways, 1) according to the topography and the morphology, and 2) according to the topography (with the morphology annotated as in intogen framework, the analysis is performed at different levels: on one side each experiment is analyzed independently (experiment level) and those experiments classified with the same topography and morphology terms are combined (combination level). Also on the other side, the analysis is performed at the level of genes (gene level) and at the level of modules (module level). A module is defined by a set of genes with some biological property in common, we currently analyze gene ontology (go) modules, kegg pathway modules, modules derived from genes sharing a transcription factor - binding side (tfbs) in their promoter and genes sharing microrna target motifs in their 3-utr [see ref . First, within each analysis unit, we identify genes altered in more samples than expected by chance using oncodrive [see ref . The results for the same gene are combined statistically across the analyzed experiments classified with the same topography and morphology terms using the weighted z - method (9). If the study contains enough samples (at least 20) for which morphology type information is known, then detection of significant alterations can be done at the level of topography and the level of morphology . The limit of 20 samples was setup to increase the reliability of results, as we consider that smaller number of replicates in a large - scale study can lead to anomalous conclusions (1). In this way, alterations specific to certain morphology type can be identified as well as those common to the topography of the cancer in general . After significantly altered genes are detected, enrichment analysis is done to find significantly altered modules (e.g. Biological processes or pathways) per experiment . In the same way as before, the results for the same module are combined across studies that analyze the same cancer type . Each analysis unit (set of assays from the same study using the same platform and annotated with the same icd - o terms) is analysed to detect the significantly altered genes . The experiment results with the same icd - o terms are combined both at the gene level and at the module level . For methods details each analysis unit (set of assays from the same study using the same platform and annotated with the same icd - o terms) is analysed to detect the significantly altered genes . The experiment results with the same icd - o terms are combined both at the gene level and at the module level . For methods details as can be expected, the interpretation of these highly inter - related results requires powerful visualization methods . The browser of intogen facilitates the exploration and intuitive visualization of results at different levels (available at: http://www.intogen.org), while the biomart portal (2) (available at: http://biomart.intogen.org) allows complex queries and facilitates the bulk download of the all analysis results . In intogen biomart portal, users can query for three types of data . For each type, there is a database; intogen experiments, intogen combinations and intogen oncomodules (table 2). Table 2.databases and data sets in the biomart of intogendatabasesdata setsdescriptionexperimentsgene genomic alterationsrecurrence and significance of genomic alteration (gain and loss) for each gene at the level of experimentsgene transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each gene at the level of experimentskegg genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each kegg pathway at the level of experimentskegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each kegg pathway at the level of experimentsgo genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each go term at the level of experimentsgo transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each go term at the level of experimentstfbs genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each tf at the level of experimentstfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each tf at the level of experimentsmirna genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each mirna at the level of experimentsmirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each mirna at the level of experimentscombinationsgene genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each gene at the level of combinations (tumor types and subtypes)gene transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each gene at the level of combinations (tumor types and subtypes)kegg genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each kegg pathway at the level of combinations (tumor types and subtypes)kegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each kegg pathway at the level of combinations (tumor types and subtypes)go genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for each go term at the level of combinations (tumor types and subtypes)go transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for each go term at the level of combinations (tumor types and subtypes)tfbs genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each tf at the level of combinations (tumor types and subtypes)tfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each tf at the level of combinations (tumor types and subtypes)mirna genomic alterationsrecurrence and significance of genomic alterations (gain and loss) for putative targets of each mirna at the level of combinations (tumor types and subtypes)mirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations (upregulation and downregulation) for putative targets of each mirna at the level of combinations (tumor types and subtypes)oncomodulescombinationssets of genes significantly altered in each cancer type and subtypeexperimentssets of genes significantly altered in each experiment databases and data sets in the biomart of intogen in intogen experiments database, there are data sets for genomic and transcriptomic alterations . Users can query the results of the recurrence analysis for these alterations at the level of genes or modules, such as kegg pathways and go categories, for each experiment included in intogen . For both types of data sets here are a few examples: genes annotated with a list of go ids, genes in a specific chromosomal band, a list of selected entrez / ensembl gene ids . The user can also filter by significance level and the results can be restricted to experiments done by specific authors, performed on a specific platform type, etc . The columns in the results can be determined by the selections done in attributes section . For experiment - level data, there are a number of statistics derived from the analysis that can be retrieved such as the number of samples in the experiment, the expected / observed number of alterations and p - values etc . Finally, a table that contains the selected attributes for the genes or modules is retrieved . In intogen combinations database, users can query the results for combinations, that is, the integration of results from experiments annotated with the same icd - o terms . The filters and attributes works in a similar way as in experiment database but without publication nor platform attributes and filters, and including the results attributes specific to the combination method . In intogen oncomodules database, there are two data sets, one for combinations and one for experiments . Each data set contains lists of genes that are significantly altered in a specific combination of icd - o terms or in a specific experiment . Again the user can filter the results in a variety of ways, with the significance level he / she likes, according to certain characteristics of genes, for a cancer type and, in the case of oncomodules at the level of experiments, for author or platform type . Query #1 use a list of genes to check whether they have been significantly gained or lost in the topology breast . Databasedata setsfiltersattributesdatabase: intogen combinationsgene genomic alterationsgenes: i d list limit by a file with ids (ensembl, entrez etc. )genes> ensembl> gene ensembl idicd - o topography and morphology: breast; any morphologygenes> ensembl> gene symbolreferences> external references> entrez gene idresults> genomics> gain p - valueresults> genomics> loss p - value the result of high - throughput analysis is usually a list of genes such as genes significantly deregulated in an expression experiment . As resources are limited, for downstream analysis, this gene list must be prioritized . One way to do this is to check if the individual genes are altered in any way in the panel of cancer experiments in intogen . In query 1, the user can download the combined results for genomic alterations filtering them with their gene list by clicking on the i d list limit box and specifying the type of i d they use . For example, in order to filter using gene symbols such tp53 and rb1, in the filters sections, the user should check the i d list limit box and select the user can filter using a number of ids such as go i d, refseq, etc . In the figure 3 a screenshot of the web interface selecting the attributes for this query the user should click on the results button on the upper - left black bar, the count button gives the number of rows that match the query and the new button allows to start a new query . The user should click on the results button on the upper - left black bar, the count button gives the number of rows that match the query and the new button allows to start a new query . Databasedata setsfiltersattributesintogen oncomodulescombinations: oncomodulestype of alteration: gaingenes> ensembl> gene ensembl idicd - o topography and morphology: lung; any morphology databasedata setsfiltersattributesintogen combinationsgene transcriptomic alterationsgenes: i d list from the previous querygenes> ensembl> gene ensembl idicd - o topography and morphology: lung; any morphologygenes> ensembl> gene symbolresults> transcriptomic> upregulation p - valueresults> transcriptomic> downregulation p - value there are different types of alterations taking place in cancer . It is important to cross - check the relative contributions of different alteration types . With query 2, the user will get a list of ensembl genes gained in lung, nos; any morphology experiments . The user can also retrieve the identifier he / she chooses such as gene symbols, entrezgene i d, etc by changing the attributes for genes . With query 3, the user can use the list from the previous query, to filter the results for transcriptomic alterations . Query #4 compare the genomic alterations in brain cancer in general and two specific morphology types; ependymoma and astrocytoma . Databasedata setsfiltersattributesintogen combinationsgene genomic alterationsicd - o topography and morphology: brain; any morphologygenes> ensembl> gene ensembl idicd - o topography and morphology: brain; astrocytoma, nosgenes> ensembl> gene symbolicd - o topography and morphology: brain; epedymoma, nosresults> genomics> gain p - valueresults> genomics> loss p - value since the dissection of brain cancer into intrinsic subtypes has prognostic value, it has been the interest of experimental scientist to find gene list that can distinguish cancer subtypes . With query 4, the user can download the genomic alterations in brain cancer and those for specific mophologies, epedymoma and astrocytoma . In the filters section icd - o, multiple icd - o terms can be selected by clicking while keeping the control key down for windows machines and the command key down in mac machines . Query #5 compare the expression level of the genes annotated with go cell cycle term in different breast cancer experiments . Databasedata setsfiltersattributesintogen experimentsgene transcriptomic alterationsicd - o topography and morphology: breast; any morphologygenes> ensembl> gene ensembl idfilters: i d list limit by go i d: go:0007049genes> ensembl> gene symbolresults> transcriptomic> upregulation, p - valueresults> transcriptomic> downregulation, p - valueresults> transcriptomic> upregulation: total number of samples while enrichment with modules is informative, the user can also get the results for the genes in a module . By comparing the two results, it is possible to see which genes from the pathway are more likely to determine the activity of the pathway in different experiments of the same cancer type . With query 5, the user can filter the results from all breast studies for the genes with cell cycle annotation and compare the studies . To filter the results for gene with a specific go i d, in the filters section, activate i d list limit box and select go term id as the type of identifier . Databasedata setsfiltersattributesintogen experimentskegg pathway transcriptomic alterationsicd - o topography and morphology: prostate gland; any morphologykegg pathway idkegg nameresults> transcriptomic> upregulation p - valueresults> transcriptomic> downregulation p - value while cancers from different patients show extensive heterogeneity in terms of the specific genes altered, the set of biological processes / pathway affected by these alterations are similar . Enrichment analysis of sets of genes with a specific biological property is very useful to detect such patterns . With query 6, the user can retrieve the results for the pathways from kegg for different experiments that study breast cancer . Query #7a retrieve a table that lists the analysis units for transcriptomic alterations in intogen . Databasedata setsfiltersattributesintogen experimentskegg pathway transcriptomic alterationsnone selectedicd - o: topography and morphologyexperiment: publication authors, publication year, pubmed i d, publication title, experiment idplatform: platform title query #7b retrieve a table that lists the analysis units for genomics alterations in intogen . Databasedata setsfiltersattributesintogen experimentskegg pathway genomic alterationsnone selectedicd - o: topography and morphologyexperiment: publication authors, publication year, pubmed i d, publication title, experiment idplatform: platform title in order to retrieve the list of analysis units in intogen, the user has to perform two queries, one for transcriptomic alterations and the other for genomic alterations . It is important to select appropriate attributes to describe the analysis units, use no filters and retrieve the unique results only (click on unique results only). Intogen is a cancer analysis tool designed to facilitate the integration, analysis, exploration and interpretation of oncogenomic data . In addition to its browser, its biomart interface provides access to high - throughput data related to genomic and transcriptomic alterations taking place in different types of cancers . A unique feature of intogen is that it provides analysis at different levels of integration . The user can compare the results for individual experiments to those obtained by merging the experiments studying the same cancer type . A major feature of the biomart interface is that it facilitates bulk download of the data . We will continue adding new data from public databases as well as cancer projects such as tcga (5) and icgc (10). Another advantage of using intogen is that the data downloaded can directly be analyzed in gitools (11) (http://www.gitools.org), which is a stand - alone tool designed for the analysis and visualization of high - throughput data . For example, one can easily perform enrichment analyses on intogen data with modules or gene sets from various biomart portals to explore large - scale patterns in cancer genomics data (see http://help.gitools.org/xwiki/bin/view/tutorials/ for examples). With cheaper and faster sequencing technologies being available continuously, resources like intogen that allow the integration, visualization and interpretation of large amount of oncogenomics data will gain importance . We continuously work on improvements and updates on the system to be able to incorporate the data obtained using sequencing technologies . With more high - quality data and new analysis methods incorporated into intogen, spanish ministry of science and technology (saf2009 - 06954); agaur fellowship of the catalonian government (to g.g . ). Funding for open access charge: spanish ministry of science and technology (saf2009 - 06954).
Limited mouth opening is a common health problem that interferes with eating, speech and oral hygiene . The causes of limited mouth opening include trauma, infection, temporomandibular joint disorders (tmds), neurological disorders, rheumatoid arthritis, drugs, tumors, hyperplasia of the coronoid process, etc . This condition can interfere with various medical treatments that require access to the oral cavity . Masticatory muscle tendon - aponeurosis hyperplasia (mmtah), a rare cause of limited mouth opening, is a new disease entity which has been recently established [24]. At present, most clinicians are not yet aware of this disease, leading to the risk of misdiagnosis as other diseases, such as masseter muscle hypertrophy and tmds . In february 2013, a 39-year - old woman presented with limited mouth opening suspected by her dentist to be caused by tmd . She could not open her mouth widely since her elementary school days, and restricted mouth opening gradually developed . Oral examination revealed no pain on motion, tenderness, clicking or injury of the temporomandibular joint . The lower jaw moved smoothly, but unaided maximal mouth opening was only 27 mm (supplementary video 1). Thick aponeurosis of the anterior aspect of the masseter muscle was noted bilaterally (fig . 1). On maximal mouth opening, intraoral palpation along the anterior border of the masseter muscle confirmed a hard cord - like structure, consistent with the findings on mri . Thick masseter muscle aponeurosis and coronoidectomy, performed under general anesthesia, increased maximal mouth opening from 29 to 53 mm . Although the coronoid process did not interfere with the zygomatic bone on mouth opening, the coronoidectomy was needed to completely resect the temporal muscle tendon from the posterior of the coronoid process . Mmtah was designated as a new disease entity by the japanese society for oral and maxillofacial surgeons in 2005 . However, most clinicians throughout the world are probably unaware of the existence of mmtah, potentially leading to misdiagnoses such as tmd . For example, resection of a portion of the masseter muscle was previously reported not to improve limited mouth opening in a patient with bilateral masseter muscle hypertrophy . Although both mmtah and masseter muscle hypertrophy present with muscle hypertrophy, these two diseases differ substantially because masseter muscle hypertrophy is not accompanied by limited mouth opening . The aforementioned case is thus suspected to be mmtah . A diagnosis of typical mmtah requires bilateral thick aponeuroses on the anterior aspect of the masseter muscle as confirmed by intraoral palpation and mri, as well as slowly progressing limited mouth opening, which often develops in childhood . Mmtah is frequently accompanied by a square mandibular configuration with mandibular angle hypertrophy due to the hyperplastic aponeurosis and tendon . The limited mouth opening associated with mmtah is due to extension disturbance of the muscles, resulting from hyperplasia of tendons and aponeuroses, which appear normal histopathologically . We have performed the procedure described above followed by mouth - opening training in 34 patients since 2000 . The ratio of mmtah among about 1000 persons in japanese elementary and junior high schools was 1% (unpublished data). Anesthesiologists have also reported problems in laryngoscopy during the induction of anesthesia in patients undergoing surgery for mmtah . Recently, lehman et al . Reported that four female patients who had presented with limited mouth opening and lateral and protrusive movements within normal limits had been treated by bilateral coronoidectomy with the significant improvement of limited mouth opening . . When clinicians notice limited mouth opening on oral examination, they should be knowledgeable about diseases associated with limited mouth opening and a square mandibular configuration, such as mmtah.
Understanding the association between psychological affective disturbances and anthropometric parameters, including body mass index (bmi), is important, because of the potential preventive effects of weight reduction on different aspects of women's lifestyles such as psychopathological states . In this regard, the relationship between obesity and psychological disorders has been widely assessed, and recent evidence - based findings have consistently emphasized associations between eating behavior and both depression and anxiety . The relationship between weight and depression, suicidal ideation, or suicide attempts, has been investigated mainly in those classified as overweight or obese . Moreover, both ends of the weight spectrum have been shown to increase the risk of future depression outcomes and suicide . These researches may be more important in women due to physiological and endocrinological fluctuations in different periods of their life including; menstrual, pregnancy, and postmenopausal periods . In addition, the relationship between dietary restraint and bmi has been found in several longitudinal studies on adolescents, especially in women . In general, research has shown that women with higher socioeconomic status are more concerned about their weight and fitness indicating a probable association between restrained eating and anthropometric indices . Several studies have done to find an association of adult weight gain and obesity with subscales of eating behavior characteristics in women . Some of them found high levels of habitual disinhibiting were associated with substantial weight gain and obesity in older women european countries . But regarding to the difference in cultural and nutritional habits in different population and as to the best of our knowledge, no previous study have done among iranian woman to find relation of weight gain psychological factors among iranian employer women . The present study aimed to predict bmi based on psychological factors including; depression, anxiety, dietary restraint, and nutritional habits and behaviors . In this regard, we considered women with sedentary jobs, in order to control for the confounding effects of dynamic active conditions . In this context, a reduction of bmi may effectively prevent the progression of psychological defects and vice versa . In a cross - sectional study, 200 women aged more than 25 years, working on sedentary and less standing jobs in isfahan, iran, including banks, social security organizations, were entered in the study . Subjects with bmi higher than 30 kg / m were not included . To assess the severity of depression symptoms, the beck depression inventory (bdi - ii) was used . Each answer was scored on a scale of 03 with the cut off points of 013: minimal depression; 1419: mild depression; 2028: moderate depression; and 2963: severe depression . Higher total scores indicated more severe depressive symptoms . According to previous population - based studies on the bdi - ii - persian version, this tool has a high internal consistency (cronbach's = 0.87), and acceptable test - retest reliability (r = 0.74). Anxiety levels were assessed with the state - trait anxiety inventory (stai) developed by charles spielberger . This psychological inventory is based on a 4-point likert scale and consists of 40 questions on a self - report basis . The stai measures two types of anxiety including; state anxiety or anxiety about an event, and trait anxiety, or anxiety levels as a personal characteristic . The reliability and validity of the farsi adaptation of spielberger's state - trait inventory were shown indicated to be high (cronbach's coefficient of 0.93 and content validity of 83.0%). To assess nutritional habits, a self - administered questionnaire was designed which included biological, behavioral, and cultural factors, as well as some nutritional - related items such as; place of eating, eating time, speed of eating, and overeating . Score equal to 15 or higher indicated proper nutritional habits, while lower scores showed the existence of poor nutritional habits that were not conducive for maintaining proper body weight and fitness . To assess dietary restraint status, the ruderman questionnaire was used; it contained 10 items related to attitudes about; eating, frequency of dieting, and weight fluctuations . The scores of this tool varied from 0 (without response) to 4 or 5 (with the highest response). The reliability of this questionnaire in our study population was evaluated, and a cronbach's coefficient of 0.68 was achieved . After obtaining permission from the relevant governmental organizations, a mechanical column scales (seca weighing scale; 700, germany) measured their body weight with an accuracy of 100 g and their height to the nearest 0.5 cm . Bmi was calculated as weight in kilograms divided by the square of the height in meters (kg / m). The results were presented as mean standard deviation for the quantitative variables, and they were summarized by absolute frequencies and percentages for the categorical variables . The pearson's correlation coefficient test was applied to assess the correlation between the quantitative variables . Multivariable linear regression analysis was used for predicting bmi on different psychological components . For the statistical analysis, a statistical software spss (version 19.0) for windows (spss inc ., chicago, il, usa) was used . With respect to the baseline information; 36.5% of the individuals were aged 2535 years, 43.5% were aged 3645 years, 18.0% were aged 4655 years, and 2% were aged more than 55 years . With regard to their marital status, the overall bmi mean in the studied women was 25.07 4.54 kg / m (range: 1449 kg / m). In terms of their psychological states; the mean score of depression severity was 10.9 7.4 (range: 037), the average score of trait anxiety was 44.77 9.99 (range: 2272), and the average score of state anxiety was 42.35 8.97 (range: 1267). The mean score of 3.08 10.16 (range: 3121) was obtained for nutritional habits and a mean score of 11.02 4.49 (range: 023) for dietary restraint . A pearson's correlation coefficient [table 1] showed a reverse correlation of depression severity as well as anxiety levels with nutritional habit means, indicating that severe symptoms of depression and trait anxiety were both significantly correlated to poor nutritional habits (b = 0.18, b = 0.16, respectively). This analysis also found a reverse correlation between nutritional behavior and dietary restraint (b = 0.015). To assess the relationship between bmi and each of the psychological components using a multivariate regression model [table 2], only two components of nutritional habits (b = 0.19, p <0.001) and dietary restraint (b = 0.51, p <0.001), could effectively predict bmi in women; while depression and anxiety items had low predictive values for predicting bmi . In total, these four variables could predict 34% of the variance of the dependent indicator (bmi). Correlations between different psychological components and bmi multivariable linear regression analysis for predicting bmi on different psychological components the current study examined the value of a range of psychological components including; depression, anxiety, nutritional behavior, and dietary restraint, in order to predict bmi as a main indicator of obesity among a sample of iranian women with sedentary jobs . Our results demonstrated the hypothesis that there is an association between nutritional habits and dietary restraint with bmi in women aged higher than 25 years having sedentary jobs and working in some governmental organizations with a low workload . Several studies have been conducted on girls and younger women in developed countries, which have revealed an association between restrained eating and higher bmi, however, those sub - groups generally contained women with healthier diets containing lower intakes of energy, carbohydrates, and proteins; while in our study population, the main focus was directed at women with higher ages and considerably lower physical activity in their workplace, so in this sense, our population was different from other studied population . A previous systematic review has described employees with lower social positions as having higher stress levels and higher body weights, and these patterns were more apparent in women than in men . Several studies have reported a link between stress and bmi levels . However, in a meta - analysis of longitudinal studies on stress and adiposity, it was established that stress promotes weight gain . Work stress has also been associated with an increased bmi . In a follow - up study of a group of male and female employees, their findings showed increased alcohol consumption and decreased vegetable consumption in workers with low job control . On the other hand, in a group of low - income young mothers, longitudinal studies can provide insights into the direction and potential nature of associations among these variables . It is plausible that the obesity is a consequence of stress, for example reflecting the use of maladaptive coping strategies such as comfort eating or excessive sedentary behaviors . Previous studies have reported that chronic stress is associated with binge or comfort type eating, reduced physical activity levels, and increased sedentary behaviors . Preferences for more palatable, higher fat, energy dense foods have also been associated with stress . Thus, it can be concluded that for women with sedentary jobs, who hold certain psychological attitudes, including; inappropriate dietary restraint and poor dietary behavior, this combination can result in a high bmi, and as a consequence related complications such as cardiovascular disorders are significantly elevated . This is of particular importance when we know that the iranian women's diet is currently undergoing negative changes, and fitness levels are falling . Thus, the association between weight and bmi along with the previously mentioned psychological components will provide valuable information . On the other hand, improving dietary patterns and mental health in this sub - group of women, along with dietary regime modifications that result in improvements of bmi, are essential in order to prevent predictable health hazards . In this regard, some authors believed that lifestyle modification programs for prevention and treatment of adult - onset obesity currently focus on reducing situational and emotional overeating; and hence a stronger emphasis on strategies that target habitual overeating can be warranted . This information could be valuable for identifying women in these types of conditions who might be at risk for obesity or its exacerbation due to underlying abnormal psychological attitudes . Contrary to our expectations, the severity of anxiety and depression was not significantly correlated with bmi in this study . Previous longitudinal studies have reported that depression in women is correlated with the development and persistence of high bmi levels . A recent review of eleven studies has suggested that depressive symptoms in women were associated with an approximately two- to three - fold increased risk of subsequent weight increase, when assessed after a period of 115 years . However, the results in these studies mainly focused on young women and consistent with our findings, little or poor association was found in the rates of obesity and mood disorders between the ages of 35 and 55 years . It seems that bmi can be potentially and widely affected by women's hormonal fluctuations . Furthermore, other confounders such as lifestyle and socioeconomic factors including; education level and family income, may play a crucial role in the interaction between obesity and psychological factors . First, the study sample was composed of women employed in government organizations and completion of the questionnaire was done in their workplace . Due to the special conditions of the workplace including; time limits, job stress, responsibilities, and client referrals, it is possible that there was not enough attention paid to complete these questionnaires . In addition, the impact of nutritional habit, physical activity, and smoking was not considered in the study design . Our study demonstrates the high value of nutritional habits and dietary restraint in predicting bmi status in women with sedentary jobs in governmental organizations in iran . However, predicting bmi through the use of depression and anxiety status assessments was not demonstrated in our survey.
Since the first practical implementation of electrospray ionization (esi) with mass spectrometry reported by yamashita and fenn in 1984, a number of different atmospheric pressure ionization (api) techniques have been developed . Many api techniques require extensive sample preparation as well as costly consumables to obtain mass spectra . One of the most cost effective and widely available media for chemical separation and analysis is paper . It has been shown that an electro - osmotic flow similar to that of static electrospray can be utilized to interface a triangle of wetted paper to a mass spectrometer (ms); this form of analysis is called paper spray (ps) [49]. The use of a chromatographic medium such as paper allows for the analysis of complex mixtures since separations will occur as the analyte is being driven along the paper medium . Separating analytes on a paper medium provides the benefits of reduced probability of ion suppression and rapid analysis . The paper medium allows for analysis of complex samples such as biological tissue and dried blood spots which are not suitable for traditional infusion techniques due to their complex matrices [4, 7]. One limitation to ps, like many ms techniques that utilize incomplete chromatographic separations is the detector being utilized to perform the analysis . When dealing with complex samples or unknown samples, there is a need for high - quality data in order to determine what is being analyzed . Fourier transform ion cyclotron resonance (ft - icr) mass spectrometers are powerful tools for analysis of complex mixtures due to their unsurpassed capability to obtain broadband high - resolution mass spectra with high mass accuracy . Utilizing ps with a fourier transform ion cyclotron resonance (ft - icr) mass spectrometer as a detector allows for rapid analysis of both unknowns and/or complex mixtures . In this present work, we show the suitability of a 12 tesla ft - icr ms to obtain high - resolution broadband mass spectra utilizing a coffee filter paper spray medium . High - resolution mass spectra were first obtained for polypropylene glycol (ppg) and polyethylene glycol (peg) standards to test the source . Second, urine and calf lung surfactant extract (clse) were analyzed to test the ps source for analysis of complex mixtures not suitable for direct infusion . All solvents used were hplc grade obtained from sigma aldrich (st . Louis, mo, usa). Ppg was obtained in the form of a tuning standard from applied biosystems (bedford, ma, usa). Peg standard was made by dissolving 1 mg of peg 2000 obtained from sigma aldrich (st . Louis, mo, usa) in 1 ml of hplc grade water . Coffee filter, wegmans brand (rochester, ny, usa), was cut by hand into an isosceles triangle with dimensions of approximately 5 mm (base) by 10 mm (height). Samples for ppg and peg ps analysis were prepared by wetting the coffee filter triangle with 10 l of the standard solutions . The wet filter paper samples were allowed to dry under ambient conditions before ps analysis was performed . Mass spectra were then obtained by wetting the dried paper triangle with 10 l of 50: 50 methanol: water and using the method described in section 2.5 . (amherst, ny, usa). Prior to analysis, the sample was stored in chloroform in the refrigerator . Samples for clse analysis were produced by wetting a coffee paper triangle with 2 l of clse and allowing the sample to dry under ambient conditions before ps analysis was performed . Mass spectra were then obtained by wetting the paper with 10 l of 50: 50 methanol: water using the instrument method described in section 2.5 . Urine samples were collected from two healthy adult males into sterile polypropylene centrifuge tubes (vwr scientific) and immediately placed in the freezer . The samples were collected in accordance with the health sciences institutional review board at the university at buffalo (hsirb no . Prior to analysis, the samples were thawed to room temperature and 1 ml was pipetted into 2 ml sample vials (agilent, santa clara, ca, usa) for use . Ten l was injected directly onto a triangular coffee filter and allowed to dry under ambient conditions . Once dry, the filter paper was attached to the mass spectrometer via a copper clip and rewetted using 50: 50 methanol: water for ps analysis using the instrument method described in section 2.5 . High - resolution spectra were obtained on a 12 t bruker solarix ft - icr ms (bruker daltonics inc ., the positive potential was obtained by grounding the paper triangle using a copper clip and applying a negative voltage at the inlet to the mass spectrometer (figure 1 shows an image of the source). Mass spectra were obtained at various mass to charge ranges with a pre - ft - icr - trap hexapole accumulation time of 0.400 seconds and an ft - icr trap accumulation time of 0.025 seconds . All mass spectra were obtained in the positive mode at a spray voltage of 2 kv . Polymeric standards were utilized to determine the suitability of ps with high - resolution ft - icr ms . In ft - icr ms the resolution of the mass spectra is relative to m / z as well as the number of data points that can be obtained during the detection process . High - resolution spectra require a time domain signal stable for longer than several hundred milliseconds . Polyethylene glycol and polypropylene glycol produce a broad m / z range of ions ideal for testing the suitability of ps with high - resolution mass spectrometry . High - resolution mass spectra of peg and ppg are displayed in figures 2 and 3, respectively . Identifies of the oligomer ion detected and their associated errors is given in tables 1 and 2, respectively . Signal - to - noise obtained for peg and ppg after tuning spectra was in excess of 100/1 with absolute intensities that varied shot - to - shot by less than 20% . This allowed ample time for adjustment of the filter paper towards the inlet of the mass spectrometer so as to optimize instrument setup and analysis . Utilizing polymers of known m n values (provided by the manufacturers; polydispersity not given) such as peg 2000 and ppg 2000 provides a detection window several hundred m / z wide for broad mass accuracy calibrations for ft - icr ms . Calf lung surfactant extract (clse), which is commercially available in drug form as infasurf, is used to treat respiratory distress syndrome . Clse is obtained by doing a total lipid extraction on the lavage fluid from a slaughtered calf lung [11, 12]. Phosphatidylcholine (pc) makes 79% of the molar distribution of lipids and consists of a choline head group attached to the phospholipid with varying degrees of unsaturation in the fatty acid chains . Based on the optimized setup determined from previously analyzing the standards, clse was then analyzed . Ps mass spectra of clse are given in figures 4 and 5, and the identities with associated error provided in table 3 . Due to the complex nature of urine, many small molecules and metabolites were detected at varying intensities using ps with ft - icr . Ps mass spectra, showing numerous lipids and other metabolites obtained from the analyzed urine specimens of two healthy adult male subjects, are shown in figure 6; an inset of one of these ps mass spectra is shown in figure 7 while the other is shown in figure 8 . A few of the metabolites identified in the 27-year - old volunteer using the exact mass capability of ft - icr are provided in table 4 . Metabolites identified in the 44-year - old volunteer using the exact mass capability of ft - icr are given in table 5 . Of note, the 44-year - old volunteer, who was taking prescribed vitamin d supplements, showed indications of the vitamin d metabolite 25-hydroxyvitamin d2 - 25-glucuronide in the urine . Interestingly, both adult male subjects had indications of one or more glucuronides in urine . This may signal a particularly strong propensity for glucuronides to ionize via the ps ionization mechanism; this capability of ps should be further explored . We developed a ps source for bruker ft - icr instrumentation that can be utilized for a broad range of chemical species . The ps source was tested on a range of samples and determined the source was suitable for both polymer and biological samples . The ps source provided an api medium for the analysis of complex mixtures while mitigating the possibility of cross contamination due to the disposable nature of coffee filter paper.
Streptococcus agalactiae (s. agalactiae) is also known as group b streptococcus (gbs), and causes invasive disease in neonates and pregnant women . However, the incidence of invasive gbs disease among non - pregnant adults has recently increased, especially among elderly persons . Nevertheless, invasive gbs disease is rare among non - pregnant immunocompetent adults, and most patients have a chronic underlying disease, such as diabetes mellitus, neurological disease, renal failure, malignancy, or liver disease,,, . S. agalactiae is also a rare cause of infective endocarditis (ie), and most cases require early surgery because of serious complications, such as major emboli or heart failure with rapid heart valve destruction,,, . Identification of the causative pathogen is essential for the diagnosis and appropriate treatment of ie, although blood culture - negative endocarditis accounts for 2.531% of all cases of endocarditis . One of the most common causes of blood culture - negative endocarditis is the administration of antimicrobial agents before performing the blood cultures . In this report, we describe our experience with a case of rapid progressive culture - negative ie in an immunocompetent man, which was caused by s. agalactiae, based on the results from 16s ribosomal rna (16s rrna) gene sequencing using the resected valve . A 43-year - old previously healthy japanese man presented with a 3-week history of a high - grade fever (> 39 c), rigor, and lumbago . He was treated for a common cold using cefditoren pivoxil, although his family physician did not perform blood cultures . The patient was subsequently referred to our hospital because his symptoms did not resolve . On the first day, chest computed tomography revealed bilateral lower lobe consolidation with pleural effusion . Therefore, we performed two sets of blood cultures and started treatment using ceftriaxone (2 g once daily), based on a preliminary diagnosis of community - acquired pneumonia . One week after his first visit, he was admitted to our hospital after he developed dyspnea with or without effort (body temperature: 36.5 c, heart rate: 117 beats / min, blood pressure: 98/55 mmhg, respiratory rate: 20 breaths / min, and oxygen saturation: 95% in room air). Chest radiography revealed a butterfly shadow with cardiomegaly and bilateral pleural effusion, which was consistent with congestive heart failure . Transthoracic echocardiography revealed a 12 10-mm vegetation on the aortic valve and severe aortic regurgitation (fig . 1). A detailed medical interview revealed that he had undergone dental care without tooth extraction at 1 month before his presentation to our hospital . At the admission, we started treatment using ampicillin / sulbactam (12 g daily in four doses) and gentamicin (180 mg daily in three doses) instead of ceftriaxone for native valve endocarditis, and the patient immediately underwent emergency aortic valve replacement . The resected aortic valve was bicuspid (fusion of the right and left coronary cusps), and both leaflets of the bicuspid aortic valve (bav) were ruptured by the vegetation (fig . No organisms were detected from a total of four sets of blood cultures that were performed before the surgery . . Therefore, we performed 16s rrna gene sequencing using the resected valve, which revealed bacterial dna that was 99% identical to the sequence of s. agalactiae . Magnetic resonance imaging on day 8 also revealed spondylodiscitis in the l5 and s1 vertebrae and in the l5s1 disc space (fig . 3). Therefore, we diagnosed the patient as having ie that was caused by s. agalactiae and complicated by lumbar spondylodiscitis . On day 29, the antimicrobial treatment was changed to ampicillin monotherapy (12 g daily in six doses). On day 43 after the surgery, the intravenous ampicillin was changed to oral amoxicillin (1500 mg daily in three doses) to treat the spondylodiscitis . The patient was discharged from our hospital on day 51, and subsequently completed the 106-day regimen of antimicrobial treatment . First, s. agalactiae should be considered a potentially causative pathogen in cases of rapid progressive ie, even among non - pregnant immunocompetent adults . Although gbs is a rare cause of ie, gbs endocarditis is associated with a poor prognosis (a mortality rate of 3556% because of major emboli or heart failure), compared to ie that is caused by other streptococci (e.g., the viridans group); therefore, early surgery is recommended for gbs endocarditis,,, . In the present case, the man did not exhibit the typical risk factors for gbs disease, although we discovered that he had undiagnosed congenital bav . Similarly, most cases of bav are not detected until the onset of infection or calcification, despite bav being the most common congenital cardiac abnormality (found in 12% of the population). The second important clinical issue from the present case is that antimicrobial stewardship should be considered, as the administration of antimicrobial agents before performing blood cultures is a major cause of culture - negative ie . Given that blood culture - negative ie accounts for 2.531% of all ie cases, it remains challenging to diagnose and appropriately treat . Up to 61% of culture - negative ie resulted from administration antimicrobial agents before performing blood cultures . Thus, genetic testing (e.g., 16s rrna gene sequencing) can be useful for identifying the causative pathogen in patients with culture - negative ie . Furthermore, 16s rrna gene sequencing using the resected heart valve specimen is an important tool for complementing the diagnosis and therapeutic management of culture - negative ie, . One reported case of culture - negative ie was caused by s. agalactiae identified using 16s rrna gene sequencing, although the patient died from progressive heart failure despite receiving antimicrobial treatment without surgery . To the best of our knowledge, the present case is the first successfully treated case of ie that was caused by s. agalactiae identified using 16s rrna gene sequencing in a non - pregnant immunocompetent adult . Although gbs has been considered completely susceptible to penicillin, an increasing number of japanese isolates exhibit gbs with reduced penicillin susceptibility (prgbs). The minimum inhibitory concentration (mic) of penicillin g for prgbs is 0.251.0 g / ml, . However, the clinical significance of isolated prgbs remains unclear, and most isolates are from elderly individuals respiratory tissue specimens . In the present case, the patient was successfully treated using antimicrobial therapy (ampicillin / sulbactam and gentamicin) and surgery . Therefore, although we could not perform antimicrobial susceptibility testing, we selected ampicillin to replace the ampicillin / sulbactam as definitive therapy for the ie, after we had identified the s. agalactiae using 16s rrna gene sequencing . S. agalactiae can causes rapid progressive ie, even among non - pregnant immunocompetent adults . Furthermore, antimicrobial stewardship is important, as the administration of antimicrobial agents before performing blood cultures is a major cause of culture - negative ie . Therefore, although s. agalactiae is a rare cause of rapid progressive ie, it should be considered as a potentially causative pathogen, even among non - pregnant immunocompetent adults . The increasing incidence of prgbs with elevated -lactam mics may make it increasingly difficult to select an appropriate antimicrobial treatment for gbs endocarditis.
After their biosynthesis at the endoplasmic reticulum, secreted proteins are transported to the golgi complex, where they are post - translationally modified and sorted for secretion, plasma membrane delivery, or delivery to prelysosomes . The golgi contains multiple subcompartments, termed cis (early), medial (middle), and trans (late) cisternae; each of these subcompartments houses different sets of glycosyltransferases and other enzymes . Proteins enter the golgi at the cis compartment and exit at the trans compartment, but how they move from one cisterna to the next is still being determined . Two possible models are widely discussed [1 - 3]. According to the cisternal maturation (or progression) model, cargo remains in a given compartment and different enzymes arrive there to convert a cis cisterna into a medial one or a medial cisterna into a trans cisterna . Alternatively, cargo moves from one golgi compartment to the next, encountering different enzymes in each subsequent compartment until it reaches the trans cisterna, where it is then sorted into carriers bound for post - golgi destinations . This second class of model could use vesicles to transport cargo from one compartment to the next or compartment - connecting tubules through which cargo could pass . Conversion of one golgi compartment into another by high - resolution, live cell video microscopy . A limitation of those studies is that one of the compartment markers that was monitored is a peripheral membrane protein that is likely to reversibly bind to and release from the golgi surface . The situation may be more complex in mammalian cells, where golgi cisternae are stacked tightly together, unlike yeast; it is hard to imagine a single cisterna moving from one side of the well - stacked structure to the other . Nevertheless, large procollagen cargo traverses the golgi without ever leaving a cisterna, in support of a maturation model . To complicate matters, membrane tubules have been detected between golgi cisternae under conditions of active secretion; this scenario would permit cargo movement from one side of the stack to the next without maturation or vesicle transfer . Important new clues to how golgi compartments might mature come from a study of golgi - localized, ras - related, rab family gtpases in yeast . Rab gtpases are localized to different membrane compartments and catalyze the formation of functionally distinct, membrane microdomains that are important for transport vesicle formation, vesicle motility, and vesicle (or compartment) docking and fusion . Rab gtpases help early endosomes mature into later endosomes by a process called rab conversion . The early endosomal rab5 protein recruits a specific guanine nucleotide exchange factor (gef) that activates rab7 . Rab7 then recruits rab7-specific effectors to that compartment, thereby converting an early endosome into a late endosome . This type of rab cascade (figure 1b) was first described for a yeast golgi rab, ypt32p, recruiting the gef for the subsequent acting sec4p rab . (a) endoplasmic reticulum, golgi, and endosome membranes are capable of homotypic fusion and fission . (b) rab cascades occur when sequentially acting rabs recruit guanine nucleotide exchange factors (gefs) and gtpase - activating proteins (gaps) to membranes . Raba recruits a gef that will convert the subsequent acting rab to its active form . Rabb can then recruit a gap that will inactive the previous acting rab, thereby removing it from the newly formed, second compartment . Rivera - molina and novick have now used live cell video microscopy to detect rab conversion at the yeast golgi: they see compartments containing the early golgi rab, ypt1p, convert into a compartment containing the late golgi rab, ypt32p . (although the light microscopy method employed could not resolve structures smaller than about 200 nm, the images were nevertheless highly compelling .) The process involves the recruitment of ypt32p by the gtpase - activating protein (gap) that inactivates ypt1p: gyp1p . Upon inactivation, ypt1p becomes a substrate for removal from membranes by another protein, gdi (gdp - dissociation inhibitor). The removal of rabs from the membranes makes this work subject to one of the same limitations of the previous studies; nevertheless, these markers permitted the authors to detect an important molecular transformation . The data provide a direct molecular mechanism for compartment inter - conversion at the golgi, reminiscent of maturation in the endocytic pathway . Golgi compartments were seen to be dynamic, undergoing a certain amount of fission and fusion . In some cases (30%), a ypt32p compartment appeared to fuse to a ypt1p compartment to yield a mixed compartment or a mixed compartment appeared to undergo segregation and fission to yield separate ypt1p and ypt32p compartments . What this means is that, yes, yeast golgi compartments undergo apparent maturation by rab conversion, and at the same time, cargo may get the fast track from one compartment to the next by intermittent cisternal fusion and fission events (figure 2a, b). Importantly, although a compartment will seem to mature, it is actually forming from a stable predecessor . Early golgi has the capacity to fuse with other early golgi cisternae and so on . Fission is favored in the absence of microtubules or when golgin proteins are depleted from cells . Glycosyltransferases are localized to cis (red), medial (yellow), and trans (orange) golgi compartments but rarely have a perfectly sharp distribution . Thus, for example, cis - golgi homotypic fusion events can occur with another cis compartment (step 1) or perhaps a medial (yellow) compartment (or both). Similarly, a medial cisterna may be able to fuse with a trans cisterna (orange). In this manner, a large cargo may be able to encounter all golgi - processing enzymes without entering a transport vesicle, indicated by fusion steps (arrows) 1, 2, and then 3 . The ability of golgi cisternae to undergo fission and fusion has been known since the 1970s: simple, nocodazole - triggered depolymerization of microtubules causes the mammalian golgi to fragment into mini - stacks that disperse throughout the cytoplasm, and drug washout leads to rapid stack reassembly (figure 2a). This indicates that the golgi is capable of fission as soon as microtubules are lost and of fusion with itself as soon as microtubules repolymerize . Compartment collisions likely enhance fusion, and cytoskeletal motor proteins that decorate the golgi and connect it to both microtubules and actin cables are sure to contribute to both fusion and fission events, as is true in the endocytic pathway (figure 1a, c). Are intercisternal fusion / fission connections required for membrane traffic? Transport is only partially blocked in nocodazole - treated cells and this condition favors stack fission . But homotypic fission and fusion are likely much more prevalent than previously anticipated because cellular depletion of any one of many different golgi proteins (golgins) generates mini - stacks that are clustered near the microtubule - organizing center . Such transient fusion and fission could yield the tubules that have been detected in electron micrographs of mammalian cell golgi complexes . Fission and fusion would make it possible to accommodate extra - large cargoes, such as collagen, that are too big to fit into conventional transport vesicles . As is well established for the endocytic pathway, rab gtpases would define specific subdomains and retain specific golgi enzyme subsets there . Compartments would be defined by their distinct rab gtpases, and adjacent cisternae might fuse at some frequency that allows cargoes to encounter sequentially acting, processing enzymes . In a mixed compartment, rab gefs and gaps would segregate individual rabs into separate regions that would be segregated upon the simple action of a membrane - associated, cytoskeletal motor protein to drive fission . Order within the stack would be maintained by the relationship between specific rabs and their cognate activators (gefs) and inactivators (gaps), which are designed to permit rabs to function in a sequential cascade . Indeed, the proteins that stack the cisternae may use a rab cascade to achieve their position in the stack . At the trans golgi, rabs would also initiate the process by which specific cargoes are collected into distinct transport carriers and delivered to their final destinations . Validation and clarification of this model will require defining which rabs build which specific golgi enzyme microdomains and determining the specific molecular interactions that permit fission, fusion, and enzyme organization . Vesicles are likely to be involved in golgi transport: we know that cop - i - coated vesicles collect kdel receptors for delivery back to the endoplasmic reticulum; in this case, we can postulate that such vesicles bud from a rab gtpase - organized, functional membrane microdomain . The same proteins that drive vesicle targeting and fusion may also participate in cisternal docking and fusion to permit protein transport across this central cellular compartment . The mechanism by which depletion of any one of at least 10 different golgin proteins leads to mini - stack formation will likely tell us much about how proteins move through the mammalian golgi stack.
Major depressive disorder, especially in later life, has heterogeneous clinical characteristics and treatment responses . Classification of depression according to age of onset may be a useful approach to understanding its intricate and complex properties . Several studies of late - life depression have focused on age of onset between 40 and 60 years.1,2) however, although many studies have been conducted regarding differences between early - onset depression (eod) and late - onset depression (lod), only a few consistent differences between these disorders have been identified . Research has shown that eod is associated with more chronic features3) and a higher tendency toward suicidal behavior.4) in patients with eod, there are more commonly feeling of worthlessness, suicidal ideation and anxiety, whereas psychomotor retardation, lack of energy, and apathy tend to be more common in patients with lod.1,5) the person with lod have more problems in cognitive function, neurological abnormalities and increased medical comorbidity . Despite recent advances in psychopharmacologic treatments, 20% to 30% of patients with mood disorders experience inadequate responses to medication, often resulting in a trial of electroconvulsive therapy (ect).6) a large body of evidence suggests ect as an effective treatment especially for severe and treatment - refractory depression,7) and studies indicate that up to 90% of people with severe treatment - refractory depression improve dramatically with ect.8) with f - fluorodeoxy - d - glucose positron emission tomography - computed tomography (f - fdg pet / ct) we can obtain the status of brain metabolism of patients with neuropsychiatric disorders and the metabolic changes due to ect . Several studies have described the relationship between depressive disorder and brain metabolism . In this report, we described a case of 55-year - old female patient who suffered treatment refractory depression and cognitive deficit . The changes of brain metabolism before and after ect were presented by f - fdg pet / ct . A 55-year - old female patient who had suicidal attempt with organophosphate was treated in the department of nephrology for a while and transferred to the department of psychiatry . She had experienced no earlier episode of mood change, but two months prior to admission she started feeling depressed mood and feelings of guilt towards her family members . She had paranoid delusion that her husband had another partner and wanted to murder her for her insurance . The patient had feelings of hopelessness, helplessness, worthlessness, and poor self - esteem . A complete biochemical workup was negative, including cbc, thyroid hormones, hiv, and syphilis blood tests . She underwent full psychometric evaluation including wechsler test, cognition and memory function test, rey - kim memory test, minnesota multiphasic personality inventory - ii (mmpi - ii), sentence completion test, symptom check list-90-revised (scl-90-r), draw - a - person test, mini - mental state examination (mmse), clinical dementia rating (cdr), rorschach test, positive and negative syndrome scale (panss), and hamilton rating scale for depression (ham - d). Her ham - d score was 43, panss score was 113 and mmse was 20 . The differential diagnosis comprised initially psychotic mood disorder, neurodegenerative disease and iatrogenic delirium secondary to medication . The patient presented as medication - resistant during the course of duloxetine, mirtazapine, trazodone, aripiprazole, and quetiapine . Medications for 4 weeks showed no significant improvement of symptoms . To rule out organic brain damages, the patient underwent brain magnetic resonance imaging but there were no significant findings . For evaluating her cerebral function, f - fdg pet / ct scans were obtained with a biographmct 128 scanner (siemens healthcare, knoxville, tn, usa). She was intravenously injected with 185 mbq of f - fdg approximately 60 minutes before the imaging . The blood glucose level was <150.0 mg / dl before f - fdg injection . The patient was stable for 30 minutes prior to f - fdg injection and in the subsequent uptake phase . Each pet / ct scan after ct scanning, a pet scan was performed in the three - dimensional mode . A depressive episode was still suspected clinically and, because medication had little effect, it was progressively reduced . Modified bilateral ect was applied and the patient underwent 3 weeks of an index course ect (9 times). Thereafter, there was impressive improvement in the patient s global functioning with complete resolution of symptoms . Her ham - d score was 4, panss score was 34, and mmse was 29 . The final diagnosis was major depressive disorder of high severity with mood congruent psychotic features . After the course of ect, the patient was discharged on minimal dosage of mirtazapine, aripiprazole, and quetiapine . After discharge, she showed no signs of residual psychosis or mood disorder and greater emotional expressiveness and had no psychomotor retardation or cognitive deficit . Seven weeks after the last ect, the patient underwent f - fdg cerebral pet (fig . Acquired pre / post pet images were co - registered and the relative standard uptake value changes were estimated by image subtraction algorithm . The image was compared with the f - fdg cerebral pet that was obtained before ect procedure and showed recovery of cerebral metabolism without any evidence of a neurodegenerative process (fig . This case report presented the change of brain metabolism before and after ect procedure in a patient with treatment - refractory lod by pet / ct imaging . There was increased metabolic activity in cerebral cortex and basal ganglia during the course . In other studies, the patients with lod showed decreased cerebral glucose metabolism than the healthy group in multiple areas of brain, which might be the cause of cognitive impairment that commonly accompanies lod.9) the use of pet as a probe of the cerebral metabolic rate of glucose (cmr) in depressive patients appears especially promising, because depression is reportedly associated with changes in cmr in different cerebral regions and can be used to display the resting state metabolism of the brain.10) repeated pet provides an opportunity to explore changes in cmr associated with the use of ect in vivo . Thus, persistent changes in glucose metabolism patterns are expected to provide a reliable estimate of neuroanatomic metabolism.11) drevets et al.12) reported reduced cerebral blood flow (cbf) and metabolism in the anterior cingulate cortex (acc) ventral to the genu of the corpus callosum in patients with depression . And bell et al.13) reported decreased cbf in dorsomedial / dorsoanterolateral prefrontal cortex in patients with depression . Reduced glucose metabolism in bilateral anterior and posterior frontal areas represented the most consistent finding of previous studies, despite considerable methodological heterogeneities . The study with 6 depressive patients treated ect found that significant increases of cmr which were presented by pet in basal ganglia, brainstem and occipital lobe.14) some researchers found that significant decreased metabolism in left subgenual acc and hippocampal area in patients with depression; and ect led to increased metabolism in these areas.15) however, there are different results that the ect - induced seizures change from the site of initiation to other specific brain regions and decrease cbf in cingulate and left dorsolateral frontal cortex 30 minutes after induction of seizures.16) and nobler et al.17) have reported reduced regional cerebral metabolic rate for glucose in 10 patients with depression in post - ect state . Suwa et al.18) presented the synthetic view of these difference in metabolic change after ect . In those studies, they have shown that ect changed cbf and cerebral glucose metabolism which were decreased in depression patients . In our case, increases in glucose metabolism in the frontal, parietal, and occipital regions may have represented the antidepressant effect of ect . In this case, we described a patient who did not have any other history of mental disorder before and presented severe cognitive deficit and treatment - resistance . The patient presented a severe and diffuse cerebral glucose hypo - metabolism on f - fdg . Awata et al.19) have reported similar recovered regional cerebral blood flow patterns were shown at 2 weeks and 12 weeks after ect in depressive patients, because of that the post ect image was taken 7 weeks later after the last ect to minimize the effect of psychotropic agent and to evaluate the long - term treatment result . This case illustrates the potential of f - fdg brain pet to demonstrate the normalization of brain metabolism in major depressive episode in patients benefiting from ect . It also illustrates the efficacy of ect in the treatment of major depression and suggests that its clinical mechanism is related with changes in metabolic function . The multifactorial effect of numerous psychoactive drugs might have increased the severity of the deficits.20) we presented the cerebral metabolic changes in the patient with severe lod and cognitive disturbance . There were several studies reported cerebral metabolic changes during the ect in depressive patients, but the little of those researches concern with both lod and cognition . This case report is not only the evidence of classical view of ect in treatment resistant depression but also the treatment choice of lod with cognitive deficit . First, we described one female patient and were unable to compare with other patient(s) with the same conditions . Second, our patient was unipolar depression and it is not certain that the ect - induced brain metabolic change is replicable in bipolar depressions . Third, the patient had lod, therefore findings might differ in eod . Despite these limitations, fdg - pet represents a promising marker of neuronal cell functions and reflects an epiphenomenon of a complex and dynamic interaction of different neurobiochemical processes . Regardless of these methodological difficulties, research into this area highlights the potential of functional neuroimaging . Changes in metabolic patterns have been correlated with changes in behavioral variables reflecting the severity of the underlying disease; in addition, a link between changes in frontal activity and improvements in clinical symptoms was identified.17) further study should include more patients who have depressive episode, and focus on objectifying clinical improvement of depression after ect to allow a correlation with possible changes in cmr . Follow up pet scans after six or twelve months should be performed to assess possible long - term effects.21) clearly, a separation of patients with unipolar depression from patients with bipolar depression should be considered and larger samples of patients are needed to increase statistical power and account for heterogeneity in study cohorts.
While the recent strategies of lung - protective ventilation and proper fluid restriction therapy have reduced mortality in patients with acute lung injury (ali) and acute respiratory distress syndrome (ards), the mortality rate remains high . Although many pharmacological therapies have been attempted to treat ali / ards, no specific therapy has demonstrated a clear effect so far . Accordingly, mesenchymal stem cells (mscs) have been introduced as a possible therapy . Mscs are multipotent adult stem cells that have the ability to differentiate into many different cell lineages and the capacity for self - renewal (1). In early research on the use of mscs to treat ali / ards, the main mechanism of action of these cells seemed likely to involve the replacement of injured lung epithelium . However, subsequent studies have suggested that the most important therapeutic effect probably comes from the paracrine properties of mscs (2, 3). Thus far, the therapeutic potentials of mscs have primarily been supported by preclinical evidence and the need for clinical trials has been suggested (4). As there is a lack of clinical data, we here introduce a patient who was treated with mscs in the course of ards and subsequent pulmonary fibrosis . A 59-yr - old man with a history of pulmonary tuberculosis (tb) and no significant family history was diagnosed with idiopathic thrombocytopenia in june 2008 ., he developed a cough, sputum, and rhinorrhea and five days later displayed fever and dyspnea . His chest radiography and computed tomography (ct) scan showed multifocal patchy ground - glass opacities (ggos) in both lungs with underlying emphysema with large bullae in the left lower lobe and focal irregular nodular lesions in the right upper lobe that were presumed to be tb sequelae . After five days, he was transferred to our hospital, a tertiary referral center, and admitted to the medical intensive care unit . At admission, he had tachypnea with a fever of 39. he had progressive bilateral diffuse infiltrations on his chest radiography . His initial sao2 level was 75% and his pao2/fio2 (p / f) ratio was 166 mmhg . We started a course of methylprednisolone at a dose of 40 mg twice a day to treat a possible pneumocystis jiroveci pneumonia in conjunction with a continuing regimen of empirical antibiotics and an antiviral agent . However, no specific pathogen was identified in any specimen from this patient, including bronchoalveolar lavage, blood or sputum . Then, his chest radiography was stationary and his p / f ratio began to gradually improve . On hospital day (hd) 7, he underwent tracheostomy to enable early weaning from mv . However, he then developed hospital - acquired pneumonia and hydropneumothorax due to a ruptured bullae with bronchopulmonary fistula (bpf). He was conscious during this time but he could not communicate with the medical team or even his family members . On hd 87, he had a follow - up high - resolution ct, which showed aggravation of diffuse ggos and interlobular septal thickening in both lungs . This indicated the progression of pulmonary fibrosis as a post - ards sequelae . After discussing the possibility of administering mscs with his family, we commenced with a trial of this therapy . The umbilical cord blood (ucb)-derived mscs (ucb - mscs) were prepared as follows . Ucb - mscs were produced at the good manufacturing practice facility of medipost co., ltd . Quality control and quality assurance for the production of these cells were performed according to the standards of the korea food and drug administration . Flow cytometry analysis of expressed surface antigens showed that these cells were uniformly positive for cd29, cd44, cd73, cd105, and cd166 and negative for the hematopoietic lineage markers cd34, cd45, cd14, and hla - dr . The final ucb - mscs preparations used in the infusion were harvested from cell culture passage 6 and suspended at a final density of 7.510/1.5 ml in normal saline . On hd 114, the patient underwent the intratracheal administration of ucb - mscs at a target dose of 110/kg . Before the procedure, the ventilator was set to the pressure - controlled ventilation (pcv) mode of 26 cmh2o of inspiratory pressure (ip) and 0.45 of fio2 . There were no peri - procedural complications . The next morning (16 hr later), he was able to communicate effectively with our medical team, which had not been possible in the previous few months . After 24 hr, we changed the pcv to a pressure support mode with 18 cmh2o of support pressure with 0.4 of fio2 . After 48 hr, pcv was applied again with 22 cmh2o with 0.35 of fio2 . The dynamic compliance of his lung improved from a pre - procedure value of 22.7 to 26.5, 27.3, and 27.9 ml / cmh2o after 24, 48, and 72 hr, respectively . His p / f ratio subsequently increased from pre - procedural 191 to 328 mmhg on day 1 after the procedure and to 334 mmhg on day 3 (fig . 1). A follow - up chest radiography showed a slight decrease in bilateral lung infiltrates (fig . 2). On day 3, however, he suddenly experienced six generalized tonic - clonic seizures during the course of the day with a fever near to 39. he had no focal lateralizing signs . A brain ct was performed and showed no hemorrhage . We discussed the case with neurological specialists and determined that the current use of carbapenem was the most probable cause of his seizure . The weaning trial was restarted for our patient and the ip was reduced to 12 cmh2o with 0.3 of fio2 during the following week . However, he could not be weaned because his repeated infection by multidrug - resistant pathogens was not controlled . Eventually, he suffered septic shock due to empyema and died on hd 231 or day 118 after msc administration . Our current case involved a patient who developed ards due to pneumonia . He subsequently had post - ards pulmonary fibrosis . Weaning from mv was difficult as he did not improve for a long - period (four months). Although he failed to survive, his immediate improvement after cell infusion is worthy of mention . His mental status, his lung compliance, p / f ratio and his chest radiography all showed improvement over the course of at least three days . We speculate that these clinical, physiological and radiological improvements might be related to the paracrine properties of mscs, with immunomodulation, alveolar fluid clearance and regulation of lung protein permeability known as potential mechanisms (2). The main reasons for our failure to help this patient survive by mscs therapy are likely to be the following . First, his pulmonary fibrosis was so advanced that it could not respond to the stem cell therapy . Its effectiveness may depend mainly on paracrine activity rather than regeneration by stem cell engraftment . Thus, a long - term effect would not be expected due to his advanced stage of fibrosis . Second, his bpf - related repeat infections may have caused the lack of improvement in his lung compliance . Since the pathogens that were responsible for the infection were multidrug - resistant to current antibiotics, a proper antimicrobial treatment could not be performed in advance of the stem cell therapy . A third possible reason concerns the dose and the number of stem cells administered . We intratracheally infused 110/kg stem cells on one occasion but no study regarding the proper number of cells for intrapulmonary administration in humans has thus far been reported . Accordingly, our dose was based on that used intravenously in a trial in acute graft - versus - host - disease after allogeneic hematologic stem cell transplantation (5). We decided to infuse using a local route as we expected more intrapulmonary action and less systemic adverse effects than for an intravenous injection . However, our dose or the one - time - only administration may have been insufficient to produce a significant effect . This report has a number of notable limitations that hinder the interpretation of the definite effect of mscs in post - ards pulmonary fibrosis . First, we did not check the level of cytokines or other soluble factors that have been shown to be associated with the functions of mscs in preclinical studies . In addition, our interpretation in this case study relied on clinical parameters and radiological images only . Thus, it is difficult to precisely link the patient's short - term clinical improvement to the definite action of the mscs . Finally, even though the clinical studies of msc therapy for acute lung injury are currently underway, there is no definite evidence so far . Thus, our decision on the msc therapy may have some ethical problems although we had no other way as a salvage therapy for his fibrotic stage of ards . Our current findings suggest the possibility of using msc therapy in an ards patient and it is the first clinical case of ucb - mscs therapy ever reported . For a clear verification of msc therapy in ali,
Today, pharmaceutical business is among greatest businesses of the world . By spending a lot in order to produce new drug, in fact, while pharmaceutical industry is a sensitive one, it is an advantageous industry as well (1). Studies of drug status and recent anticipations indicate a progressive trend in world drug market, so that the rate of drug sale around world (world market) has reached 900 billion dollars in 2011 from 309 billion dollars in 1998 (around 2.9 times more) (2). It is expected that the drug world sale would go beyond 1 trillion (1043.4 billion) dollars in 2013, and reach 1.3 trillion dollars until 2020 (3). The drug world market during 1998 to 2008 had enjoyed an average growth of about 12%, while the rate is much lower in other industries . Naturally, the world market of pharmaceutical industry could be divided into four areas: united states, japan, europe, and the rest of the world (4). Iran s pharmaceutical industry share is around 2 billion of the 750 billion dollars, while turkey as one neighbor of iran has a share of 6 billion dollars (1). Drug expense share is averagely 30% of the whole health expenses among different world countries . Drug expenses of iran in 2011 have indicated an increase from 276 million to 829 million dollars for imported drugs . Generally, the ratio of rial sale of imported drugs to the drugs produced inside the country has changed from 0.28 to 0.71 in recent years (5). Since most world countries have joined world trade organization, iran as a developing country with no significant role and presence of non - oil economy in world economy should not be in isolation from world progress and evolutions (6). Therefore, iran s principal problem in this regard will be summarized in persistent and concentrated effort to make its membership possible with the least expense and the most profit . Hence precise recognition of effects and outcomes of membership in the organization will considerably help passing the membership path successfully (3). Therefore, studying effects of decreasing import tariff on major variants of drug field is a very important issue . Finally, we aimed to evaluate the impacts of trade openness on pharmaceutical industry using cge model . Model details associated with activities, production factor, and institutes are summarized in table 1 . Activities include agriculture, pharmaceutical industry, industry and mine sector, construction sector, and finally service sector which use two factors of labor and capital for their products . The model utilized include product associated trades, household and government consumption, saving, investment, and foreign business . It is assumed in this model that economic sectors use labor and capital as primary institutes for production . In order to realize the model, beside primary institutes, it is assumed that sectors use mediator institutes for production as well . For more convenience, it is assumed that in lower stage, additional value (composite primary factor) is obtained from composition of labor and capital with cobb - douglas type production function (equation 1) (7). Vaj = bj fdhjhjj as sectors index, h as index of primary production factors (labor and capital), vaj as additional value of jth sector, fdhj as demand for h th production factor by j sector, bj as efficiency parameter in production function, and bhj as share parameter in production function or production elasticity of jth sector to hth institutes ratio so that hj 1 and 1 hj are considered in this equation . Using leontief type production function in this stage, gross output is produced by combining additional value and mediator institutes (8). According to these two stages with regard to production, each sector maximizes its profit function . Yj = min (xijaxij, vajayj)in this equation, i as sectors index, yj as gross output of jth sector, axij as coefficient of minimum demand for mediator institutes of jth in order to produce a gross output unit of jth sector (technical coefficients of input - output), ayi as coefficient of minimum need for additional value in order to produce one unit of gross output, xij as ith sector production that is consumed as jth sector mediator are considered . According to these two stages xij = axij.yj j vaj = ayj.yj fdhj=hj.pnjwh.vaj h psj = ayj . Pnj+axij.pqi j pnj as additional value price of jth sector, wh as production factor wage, psj as supply price of jth sector, and pqi as goods price of jth sector are considered . It is assumed in this article that production factors (labor and capital) are in a state of equilibrium, and the supply of factors is constant (7). Hence change in tariffs does not change the whole demand of labor and capital, while just transmission of production factors from one sector to another takes place . In order to calculate private sector consumption (households), it is assumed that consumption basket is optimally selected by consumers (9). Their revenue is obtained from supply location of production factors (labor and capital) added by transmission payments of government to households and pure external moneys received . The favorability function is a cobb - douglas function which is maximized due to a budget equal to pure household revenue (household revenue minus direct tax amount and saving). Ci.pqi=ci (wh.fsh - taxdir - savhoh) in this equation, ci is considered as ith object household consumption, ci as share parameter in favorability function or each object share in household consumption basket so that 0 ci 1 and ci 1, fsh the amount of primary hth factor supply (exogenous variable) is dir revenue derived tax of the households . According to public sector consumption, it is assumed that government earns through implementation of sales tax, direct household income tax, and oil import and export tax . Taxind, j = txj.psj.yj taxdir = td.wh.fsh tariffj = tmj.pmj.mj gi.pqi=gi (taxdir+taxind, j+tariffj+eoil - savg in these equations, taxind, j is considered as indirect tax (sales tax), txj as sales tax rate, td as direct tax rate, tarifj as import tariff, tmj as import tariff rate, pmj as import domestic price, mj as import amount, gi as government expenditures in jth sector, savg as government saving, gi as share parameter of government expenditure in each sector, and eoil as oil export . Investment in every sector depends on total investment which is equal to total saving, and is obtained from private, public, and foreign savings together . Savhoh = shohwh.fsh savg = sg(taxind, i+tariffi+taxdir+eoil saving=(savh+savgov+exr.savf) saving = invest idi.pqi=i.invest in these equations, shoh is considered as mean tendency of private sector saving, sg as mean tendency of government saving, savf as foreign saving, saving as total saving, invest as total investment, idi as investment demand of ith sector, i as share parameter of ith sector investment, so that 0 i 1i and i 1 . In external world and foreign trade, it is assumed that the country is small i.e. The country has no effect on world prices and international market . Pei = pwei.exr pmi = pwmi.exr in these equations, pei is considered as domestic export price, pwei as world export price (exogenous variable), exr as foreign exchange rate . When the model is considered for an open economy, it is necessary to consider some precautions about alternating imported, exported and domestically supplied goods (12). In general equilibrium models, there is difference between imported and exported goods as well as goods produced for export and goods produced for domestic sale . It is assumed that imported and domestically supplied goods make composite good (argminton good). It is assumed that institutes are an incomplete alternate to domestic productions (13). The relation between imports and domestic production is indicated as a constant elasticity of substitution (ces) function (14). Qi=i(mi.mimi+di.dimi)1mi in this equation, qi is considered as composite good, di as domestically produced good, i as efficiency parameter in production function of composite good, mi as share parameters in argminton function, so that mi + di 0, mi as argminton function power or the parameter associated with substitution elasticity, so that mi=(i-1)i and i 1, ias argminton function elasticity is i =- d(mi - di)mi / di / d(pmipdi)pmi / pdi . Regarding the aim of problem maximization, demand functions for import and domestic production would be as equations number 20 and 21 . Mi=(imi.mi.pqi(1+tmi).pmi)11-mi.qi i di=(imi.di.pqipdi)11-emi.qi in this equation, pdi would be the price of domestically produced good . Equation number 22 indicates the relation between export and domestic production which is stated on a constant elasticity of transmission (cet) as well . Yi=(i(ei.eiei+di.diei)1ei i in this equation, ei is considered as the amount of export, qi as efficiency parameter of transmission function, ei and di as share parameters in transmission function, so that di + ei 0, ei, as transmission function power, or parameter associated with transmission elasticity, so that ei = (i + 1)/i, transmission elasticity as i = d(eidi)eidi / d(peipdi)peipdi regarding maximization problem, export supply and domestic good functions would be the equations number 23 and 24 respectively . Ei=(qiei.ei(txi+psi)pei)11-ei.yi di=(qiei.di(txi+psi)pdi)11-ei.yi associated prices are the modifying factors for supply and demand equality in each market in order to develop balance in four markets of labor, capital, composite good, and foreign exchange . Profit in labor market, composite good price in composite good market, and foreign exchange rate in foreign exchange market are mediating factors . Since there are uncountable solutions with similar relative prices, price normalizing equation is used in order to assure there is just one solution which is equilibrium solution . Price index is constant in this equation and other price changes are measured in proportion to this price which is shown in equation number 28(16). Pindex=wipqi in order to solve applied general equilibrium models, a complete set of statistics and data is needed . Since the aim of research is to study the effect of decrease in tariff on key variables of pharmaceutical products, the decrease has been studied gradually in two general scenario formats . Each scenario has been studied as 10, 30, 50, and 100% gradual decrease of custom tariff rate: 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products import on key variables of drug field10, 30, 50, and 100% decrease in tariff rate of other sectors (except pharmaceutical products) import on key variables of drug field 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products import on key variables of drug field 10, 30, 50, and 100% decrease in tariff rate of other sectors (except pharmaceutical products) import on key variables of drug field the results of scenarios simulation are summarized in table 2 . This table shows the impacts of tariff s cut for key variables during trade liberalization; decrease in import tariffs affects good import and services . Normally, increase in import of capital goods and mediator materials increases production, while good import and consumed services which decreases people demand for good and internal services decreases production . According to results of table 2, when tariff decreases in pharmaceutical products, each sector import will increase as a function of tariff rate in that sector according to equation number 16 . In fact, when tariff rate decreases, imported good price will decrease in the country which leads to increase in demand for export . Therefore, 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products in the first scenario will increase pharmaceutical product import by 0.2% on average, while decrease in tariff rate of agriculture sector (scenario 2) will decrease pharmaceutical product import by about 0.96% on average, and this variable will decrease by about 2.81% due to tariff rate decrease in industry and mine sector . Also, export variable has increased with decrease in tariff rate of pharmaceutical products in scenario 1 . This result is completely logical since according to its direct relation with supply (equation 22), increase in pharmaceutical products supply will undoubtedly increase drug export rate . Since most items of drug import are raw materials in iran, it is natural that increase in pharmaceutical raw materials would increase manufactured drugs, and become the main factor in drug export . In various studies performed in iran, trade liberalization has increased import variable of associated product while has decreased the product s import on the other side . It is worth noting that change percent and drug export growth is lower than drug import due to gradual decrease in tariff rate . Pharmaceutical product export indicates a 0.64% decrease due to decrease in tariff rate of agriculture sector, and 1.91% decrease due to decrease in custom tariff rate of industry and mine sector . According to results, the supply rate of pharmaceutical products has gradually increased by decrease in tariffs which is a logical result; since the rate of goods supplied in markets will increase by decrease in pharmaceutical product tariff and increase in drug import . Drug supply rate has decreased by 0.19% due to decrease in tariff rate of agriculture sector, and by 0.69% due to decrease in tariff rate of industry and mine sector . Since labor market has been considered in equilibrium state and labor supply in entire economy has been assumed to be constant in the current study, any change in tariffs would not change in total employment, while will change employment in the considered sectors . Therefore, if employment decreases in a sector, it means that labor has been transmitted to other sectors . Various studies indicate that one negative effect of globalization in developing countries is employment reduction in drug field; since due to competitiveness, these countries have to use new equipments and technology on one hand which reduces labor rate, and production of some pharmaceutical products will face problems due to lack of competitive advantage on the other hand which might lead to stopping drug production or unskillful labor unemployment . Results of the current study indicated that in the first scenario, gradual reduction of tariff rate will not make any change in labor employment in drug field due to model conditions; while in the second scenario, decrease in tariff rate of agriculture sector will reduce labor employment in drug field by 0.14% as compared to the first scenario . In the second scenario, decrease in tariff rate of industry and mine sector has led to a mean 0.35% decrease in labor employment of this field . However, according to capital factor, there is a mean 0.12% increase in capital factor employment in the first scenario, and about a mean 0.82% decrease due to decrease in tariff rate of agriculture sector, and about a mean 0.13% decrease in capital factor in drug field due to decrease in tariff rate of industry and mine sector . Investment in each sector directly depends on total investment and reversely depends on composite good price in each sector . In the first scenario, decrease in custom tariff of pharmaceutical products will increase the capital rate by a mean of about 0.1%, and in the second scenario, decrease in custom tariff rate of agriculture sector will increase capital rate by about 0.05%, and the same effect in industry and mine sector will lead to 0.13% increase . Therefore, trade liberalization will increase capital rate in all scenarios . The model results indicated that decrease in custom tariff rate in the first scenario shows a mean 0.13 increase which is a thoroughly logical result, since household drug consumption will increase due to decrease in tariffs of pharmaceutical products and import increase, while in the second scenario, drug consumption rate will decrease by a mean of 0.52% of household consumption of pharmaceutical products due to decrease in tariffs of agriculture sector, and household drug consumption will 1.57% decrease due to decrease in tariffs of industry and mine sector . Since the aimed function of this study model has been maximization in favorability function of society people, and this function indicates total society welfare, in the first scenario, total society welfare shows 0.63%, 0.64%, 0.96%, and 0.16% increase respectively with regard to the basic amount . However, according to effect of tariff decrease in agriculture sector, total society welfare has 0.83%, 0.60%, and 0.38% decreased respectively, and about 0.15% increased due to complete elimination of tariffs of agriculture sector . However, according to effect of tariff decrease in industry and mine sector, total welfare has 2.23%, 1.69%, and 1.15% decreased respectively, and 0.16% increased due to complete elimination (100%) of tariff in this sector . Therefore, total society welfare will increase with regard to basic year due to complete tariff elimination in other sectors (except pharmaceutical products), while society welfare will increase by an average of 0.09% due to gradual decrease in tariffs of pharmaceutical products . Sensitivity of results as compared to argminton elasticity on import variable of different economic sectors has been surveyed in this study . Argminton function in cge model indicates supply for composite good which is in fact a function with constant elasticity of transmission of demand for import and internal productions . Since this parameter evaluation has been selected exogenously from other studies, in order to model correctitude and define sensibility of results as compared with difference in this elasticity, it is assumed that the scenario of 50% decrease in tariff has taken place in all economic sectors, and argminton elasticity changes from 50 to 150% of primary elasticity is in 25% intervals . Results demonstrate that import amount has increased in elasticity s lower than basis, while it has decreased in elasticities higher than basis . Table 3 shows the elasticity of substitution, elasticity of transformation that is so important for sensitivity analysis . Values of behavioral elasticity s are usually estimated outside the model or taken from other studies . Considering the elasticity s drug in this research is critical and that is difference, we used it for sensitivity analysis . Although, cge models are standard in the world and shows usually a multi - sector model based on real world data (e.g. Social accounting matrix table or i - o table) (17) of one or several national economies to model the interactions of individual households and other agents on interdependent markets but cge model maybe give different results when we reduced tariff (14).in iran and brazil increased export and import aftershock of tariff cut but the quantity of increasing depends on structure of pharmaceutical sector . In iran, the most of drug s import are material raw that with reduction of tariff, volume of imports increased that this is natural . So, all of results in cge models depends on real data in the countries . Generally in iran, however, there are many studies related to different economic sectors some of which would be mentioned . According to precise searches of researchers up to mid 2012, four studies in the framework of general equilibrium in health field has been performed out of iran which has been in england (nottingham university) brazil, japan, and ghana . The study in england has been performed by dr martin rotten (2009) in form of phd thesis named economic effect of providing health care: evaluation of a cge model for england in 2005.in this thesis, nine economic sectors including agriculture, mine, pharmaceutical products, medical equipment, financial sector, defense sector, health sector, and other services has been considered, and production factors have been divided into two groups of work force (skilful and unskillful) and capital (10). Households are also divided into five groups including employed households who have children, unemployed households who have children, unemployed households with children, employed households with children, and finally those who are retired . The data of general equilibrium model is obtained from british sam, 2005, and the alternative and transmission elasticity is considered to be 2 . Four scenarios have been considered in the thesis including: a) effects of 10% increase in government expenditure on health care, b) 20% increase in prices for economic sectors, c) 10% increase in expert forces of medical fields from other countries, d) 10% increase in efficiency of production factors in health field . Results indicated about 8%, 1%, 7%, and eventually 5% improvement in health status by the first to fourth shock respectively . The scenarios have significantly affected on patient expectation list as well, so that there will be 15%, 3%10% and 15% change in patient expectation list respectively . The other study associated with trade liberalization has been performed by francisco (2003) on brazil labor using computable general equilibrium approach (18). 48 goods and two production factors, labor and capital have been used in this study . The first scenario is to impose custom tariff rate of 1990 for brazil economy structure in 1996 . Results of this study in different scenarios demonstrate that pharmaceutical products export would decrease 2.408% as well as 0.575 for drug pharmaceutical import in performing the model for the first scenario . The second shock will cause 15.7% increase in pharmaceutical export and 0.26% decrease in pharmaceutical import . Finally, pharmaceutical export will increase 9.66% and this would be 5.24% for pharmaceutical import in the third scenario . According to results of this study, the general society welfare increases by decrease in tariffs . The third study named tax and subsidy policies of medical services and pharmaceutical industry has been performed in the international university of japan by kato in 2003 using computable general equilibrium analysis . Decrease in import tax (custom tariff) of drugs will increase import and export and general society welfare . It is also worth to note that implementing the policy of decreasing custom tariff will cause import growth to be higher than export (19). The fourth study named government expenditure effect on health, economic growth in wealthy country using computable equilibrium model by ernest indicated that government expenditure has significant effect on health followed by economic growth . Results indicate that generally, tariff decrease in globalization process will increase imports of this sector in the present conditions of economic structure and pharmaceutical sector of the country . It is obvious that in each economic sector, those products which have production stability will have competitiveness and relative competitive advantage . However, since our pharmaceutical sector is strongly dependant on raw material import, competitiveness in this sector is reduced . Therefore, one significant way to increase competitiveness of pharmaceutical sector in the country is to decrease dependability on raw materials so that to decrease final price of such products by increasing efficiency and adopting suitable supports in the framework of one applied policy in pharmaceutical sector . Finally, the most important efforts that should be made by iran s pharmaceutical industry to increase exports include: promotion of drugs produced in accordance with gmp and dmf preparation guidelines and regulations on the pharmaceuticalobtain international confirmations and verifications such as fda, who and etc.develop skills and international expertise in the field of marketing for increasing of drug s exportestablish mechanisms and institutions needed for driving and reducing the risk of export services, such as transportation and insurance claims services exporters, especially in export markets in the region, central asia, africa and so on.strategic planning for export markets promotion of drugs produced in accordance with gmp and dmf preparation guidelines and regulations on the pharmaceutical obtain international confirmations and verifications such as fda, who and etc . Develop skills and international expertise in the field of marketing for increasing of drug s export establish mechanisms and institutions needed for driving and reducing the risk of export services, such as transportation and insurance claims services exporters, especially in export markets in the region, central asia, africa and so on . Strategic planning for export markets finally, pharmaceutical factories should compare their product with the global reference product, using qualitative studies before it s supplied . Also, iranian, pharmaceutical factories should attempt to offer various documents in the form of drug master file (dmf) international protocols to the ministry of health of target countries in order to provide proof of quality and register our pharmaceutical products . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
Based on the previous researches 70% of post - surgical patients are facing with moderate to severe pain . Post - operative nausea and vomiting (ponv) are also distressing with a prevalence of 20 - 30%, depending the type of surgery and patient - related factors . These problems are not only discomforting, but also may cause dehydration, electrolyte disturbances, delayed recovery, and delayed hospital discharge, if prolonged . Opioids and non - steroidal anti - inflammatory drugs remain the most common treatments for post - operative pain . However, many of these drugs are expensive, and all are associated with complications . Ondansetron is associated with headache, diarrhea, and transient increase in hepatic transaminase level . To avoid these side effects, there are certain points in the body, which are known as pressure points and are capable to transmit the energy . By pressing these points, muscle spasm goes away, blood circulation and the body's vital energy improve, and calm the nerves . Several studies have been conducted on the effects of acupressure on post - operative pain, nausea, and vomiting in variety of surgeries . (1991) in children undergoing strabismus correction, studied the effect of p6 acupressure on ponv . However, fan et al . Has reported that this method reduced the severity of ponv . Felhendler and lisander (1996) have also reported that acupressure was effective for pain relief after arthroscopy . Lanwei acupoint also known as le7 acupoint or the extra point33 is located below the right knee with a distance of 2 cun from the st . This point had been known as a useful acupoint for reducing pain of acute and chronic appendicitis . However, no study on the effect of le7 acupressure on post - appendectomy pain is available . We assumed that acupressure on this point may be effective in reliving post - appendectomy pain, nausea, and vomiting . Therefore, the present study was conducted with the aim of assessing the effect of acupressure on le7 acupoint on pain, nausea, and vomiting of patients after appendectomy which is the most common emergency abdominal surgery . If effective, this method may be suggested as a complementary treatment to reduce post - appendectomy pain, nausea, and vomiting . A single - blind randomized controlled trial was undertaken to evaluate the effects of le7 acupressure in relieving pain, nausea, and vomiting post appendectomy . The study was conducted on 70 post - appendectomy patients referred to a general surgical ward in a university hospital in the first half of 2012 . Sample size was calculated based on data obtained from a pilot study on 14 patients (7 in each group). After 1 h acupressure on le7 point and measuring the pain intensity, the difference between experimental and control groups was 0.44 (sd = 1.181). Based on the above parameters and selecting the significance level to be less than 0.05 and a power of 0.8, 34 subjects were estimated to be needed in each group . Then, we selected 35 patients in each group . Samples were selected consecutively among the patients admitted for appendectomy and patients who entered the study were randomly assigned into the groups [figure 1]. The overall design of the study (without specifying the allocated group) explained to the patients . The criteria for inclusion of the participants were: the desire to participate in the research, no injury or lesion in the region of the acupoint of le7, aged between 15 years and 70 years, being in the list of appendectomy surgery, and receiving general anesthesia . Those with the following conditions were not selected: a recent history of disorders associated with acute or chronic nausea and vomiting (such as gastrointestinal and ear disorders), experiencing nausea and vomiting during the last 24 h, the drug and alcohol addiction, a known neurological or psychiatric disease, and the previous use of acupressure or acupuncture techniques . The exclusion criteria were: occurring unforeseen complications during surgery and anesthesia, prolonged surgical time (more than 2 h), receiving drugs outside anesthesia protocol for the routine appendectomy patients, and fever above 38c . The first section was the demographic data form and had six questions about the patient's age, gender, height, weight, level of education, and the study group the patient was belonged to . It also had two questions about the length of surgical incision (in millimeters) and the duration of anesthesia (in minutes). Second section was a visual analog scale (vas) used for recording the severity of nausea . Descriptors were placed at each end of the line (0 = absence of nausea and vomiting and 100 = the worst condition). The observer asked the patient about the severity of nausea and marked an x on place that corresponded with severity of nausea . Nausea with a score of 70 was classified as severe, between 35 and 70 as moderate, and less than 35 as mild . If there was retch or vomiting more than 5 times each hour, it was considered as severe vomiting, between 3 times and 5 times as the average and less than 3 times as mild . A vas was also included in the third part for recording the severity of pain . Descriptors are placed at each end of the line (0 = no pain and 100 = the most unimaginable pain). Patients were asked to mark an x on place that corresponded with severity of pain . Acupressure was applied using a special band called acuband (psiband, american design, made in china). The technique was started after the full patient's consciousness (so that the patient is aware of the location, time, and person). All the patients were anesthetized using thiopental sodium (5 mg / kg), atracurium (0.6 mg / kg), and fentanyl (2 mcg / kg) and then the anesthesia continued using a gas mixture of o2 + n2o (each: 50%), morphine (0.1 mg / kg) and isofeluran (1 mac [minimum alveolar concentration]). All the patients were transferred to the surgical ward by a specially trained team to eliminate the effect of patient movement and handling of pain, nausea, and vomiting . Initially, the severity of pain, nausea, and vomiting were assessed and recorded . Then the acuband was fastened by the second researcher . In the experimental group the acuband was fastened on the right leg in such a way that the button located exactly on the le7 acupoint . Then, the pulses below the point were checked to ensure that the acuband would not interfere with the blood flow . The patient's foot was also examined for the possibility of impaired venous return . In order to keep the patients and the staff blind to the group the patient was belonged to, an acuband also used in the control group, but the band was fasten loosely and in a way that the button placed right in opposite side of the le7 point (as sham point). The occurrence and the severity of pain, nausea, and vomiting were assessed and recorded in the two groups hourly and up to 7 h. the acubands was preserved for 7 h. however, they were loosened for 10 min every 2 h and then tightened . For patients who suffered from severe nausea and vomiting during this period, anti - emetic medication (metoclopramid, 10 mg) was administered and the nausea was considered as severe one . An analgesic medication (pethidine, 1 mg / kg) also administered if the pain score was six or higher . This study was approved by the research council and the research ethics committee in our university of medical sciences . All the subjects had been informed about voluntary participation in the research, non - disclosure of personal information, and offered a written informed consent form to be signed before entering the study . All subjects have been assured that they will receive all the necessary care and pain medications if the intervention had no effect . The researchers were mindful of the need to pay close attention to ethical considerations and the welfare of the participants . The spss software (statistical package for the social sciences) version 11.5 used for data analysis . Independent sample t - test was used to compare the mean of pain and nausea severity of two groups . The mean of pain intensity before the intervention was compared to the average of pain intensity in the next 7 h. also the mean of the nausea intensity was compared with average of nausea intensity in the next 7 h. paired t - test was used to compare the mean of pain intensity of each group before and total mean of pain at post intervention hours . A p value of less than 0.05 was considered to be significant for all tests . A single - blind randomized controlled trial was undertaken to evaluate the effects of le7 acupressure in relieving pain, nausea, and vomiting post appendectomy . The study was conducted on 70 post - appendectomy patients referred to a general surgical ward in a university hospital in the first half of 2012 . Sample size was calculated based on data obtained from a pilot study on 14 patients (7 in each group). After 1 h acupressure on le7 point and measuring the pain intensity, the difference between experimental and control groups was 0.44 (sd = 1.181). Based on the above parameters and selecting the significance level to be less than 0.05 and a power of 0.8, 34 subjects were estimated to be needed in each group . Samples were selected consecutively among the patients admitted for appendectomy and patients who entered the study were randomly assigned into the groups [figure 1]. The overall design of the study (without specifying the allocated group) explained to the patients . The criteria for inclusion of the participants were: the desire to participate in the research, no injury or lesion in the region of the acupoint of le7, aged between 15 years and 70 years, being in the list of appendectomy surgery, and receiving general anesthesia . Those with the following conditions were not selected: a recent history of disorders associated with acute or chronic nausea and vomiting (such as gastrointestinal and ear disorders), experiencing nausea and vomiting during the last 24 h, the drug and alcohol addiction, a known neurological or psychiatric disease, and the previous use of acupressure or acupuncture techniques . The exclusion criteria were: occurring unforeseen complications during surgery and anesthesia, prolonged surgical time (more than 2 h), receiving drugs outside anesthesia protocol for the routine appendectomy patients, and fever above 38c . The first section was the demographic data form and had six questions about the patient's age, gender, height, weight, level of education, and the study group the patient was belonged to . It also had two questions about the length of surgical incision (in millimeters) and the duration of anesthesia (in minutes). Second section was a visual analog scale (vas) used for recording the severity of nausea . Descriptors were placed at each end of the line (0 = absence of nausea and vomiting and 100 = the worst condition). The observer asked the patient about the severity of nausea and marked an x on place that corresponded with severity of nausea . Nausea with a score of 70 was classified as severe, between 35 and 70 as moderate, and less than 35 as mild . If there was retch or vomiting more than 5 times each hour, it was considered as severe vomiting, between 3 times and 5 times as the average and less than 3 times as mild . A vas was also included in the third part for recording the severity of pain . Descriptors are placed at each end of the line (0 = no pain and 100 = the most unimaginable pain). Patients were asked to mark an x on place that corresponded with severity of pain . Acupressure was applied using a special band called acuband (psiband, american design, made in china). The technique was started after the full patient's consciousness (so that the patient is aware of the location, time, and person). All the patients were anesthetized using thiopental sodium (5 mg / kg), atracurium (0.6 mg / kg), and fentanyl (2 mcg / kg) and then the anesthesia continued using a gas mixture of o2 + n2o (each: 50%), morphine (0.1 mg / kg) and isofeluran (1 mac [minimum alveolar concentration]). All the patients were transferred to the surgical ward by a specially trained team to eliminate the effect of patient movement and handling of pain, nausea, and vomiting . Initially, the severity of pain, nausea, and vomiting were assessed and recorded . Then the acuband was fastened by the second researcher . In the experimental group the acuband was fastened on the right leg in such a way that the button located exactly on the le7 acupoint . Then, the pulses below the point were checked to ensure that the acuband would not interfere with the blood flow . The patient's foot was also examined for the possibility of impaired venous return . In order to keep the patients and the staff blind to the group the patient was belonged to, an acuband also used in the control group, but the band was fasten loosely and in a way that the button placed right in opposite side of the le7 point (as sham point). The occurrence and the severity of pain, nausea, and vomiting were assessed and recorded in the two groups hourly and up to 7 h. the acubands was preserved for 7 h. however, they were loosened for 10 min every 2 h and then tightened . For patients who suffered from severe nausea and vomiting during this period, anti - emetic medication (metoclopramid, 10 mg) was administered and the nausea was considered as severe one . An analgesic medication (pethidine, 1 mg / kg) also administered if the pain score was six or higher . This study was approved by the research council and the research ethics committee in our university of medical sciences . All the subjects had been informed about voluntary participation in the research, non - disclosure of personal information, and offered a written informed consent form to be signed before entering the study . All subjects have been assured that they will receive all the necessary care and pain medications if the intervention had no effect . The researchers were mindful of the need to pay close attention to ethical considerations and the welfare of the participants . The spss software (statistical package for the social sciences) version 11.5 used for data analysis . Independent sample t - test was used to compare the mean of pain and nausea severity of two groups . The mean of pain intensity before the intervention was compared to the average of pain intensity in the next 7 h. also the mean of the nausea intensity was compared with average of nausea intensity in the next 7 h. paired t - test was used to compare the mean of pain intensity of each group before and total mean of pain at post intervention hours . A p value of less than 0.05 was considered to be significant for all tests . This study was undertaken on 70 patients in the age range 15 - 70 years . No significant difference was observed between the two groups in terms of age, body mass index, duration of anesthesia and the incision length [table 1]. Demographic data of study groups the mean of pain intensity in the le7 group was less than the control group after the intervention (p = 0.02) [table 2]. A significant deference was also observed between the mean pain severity of the le7 group before the intervention and the total mean of pain severity in the seven post - operative hours (p = 0.018). However, such difference was not significant in the control group (p = 0.32). Figure 2 also shows that the mean of pain intensity was lower in le7 group till the 6 h after the surgery . Mean (sd), estimated difference (95% confidence interval) and p values for pain intensity in the two groups before and after the intervention the mean and standard deviation of the pain severity in the seven post - operative hours in total, 35 patients (18 in the le7 group and 17 in the control group) experienced post - operative nausea . Also, no significant difference was observed between the means severity of post - operative nausea in the two groups [table 3 and figure 3]. Mean (sd), estimated difference (95% confidence interval) and p values for severity of nausea in the two groups before and after the intervention the mean and standard deviation of the nausea severity in the seven post - operative hours also 31 patients (16 in the le7 group and 15 in the control group) experienced post - operative vomiting . From the total patients who experienced vomiting, four ones in the le7 group and 7 patients in the control group had mild vomiting while the severity of vomiting was moderate in the other ones . In the present study the intensity of post - operative pain was lower in le7 group than the control groups . Studies have been conducted on the effects of acupressure on pain have reported different results . However, another study reported that the method was not successful on pain reduction after abdominal surgeries . It is noteworthy that in previous studies on the effect of acupressure on pain, researchers applied the method on points that were fully known to be effective in traditional chinese medicine . For example, lee stimulated the sp6 point that painless labor is one of its applications in acupuncture . In the present study we examined the effect of pressure on le7 acupoint that had been reported to be effective in reducing pain of acute and chronic appendicitis but, no study was available to assess its acupressure on post appendectomy patients . On the other hand, the effects observed in this study, may be due to the connections between the skin's dermatomes . In the present study, the incidence and the severity of post - operative nausea did not significantly differ in the two groups . Several studies have been conducted to evaluate the effects of acupressure on ponv (although not in post - appendectomy patients). Two studies have reported that this technique was not effective for nausea and vomiting after cholecystectomy and strabismus correction . However, some of the studies showed that this method has significantly reduced the severity of nausea and vomiting after adeno - tonsillectomy and laparoscopic surgeries . While no study on the effects of acupressure on le7 point is available, the studies that used p6 or other acupoints for ponv have not been reached to a definitive conclusion . The present study showed that acupressure on le7 acupoint was effective on post - appendectomy pain while its effect on ponv did not differ with the control group . It seems that nurses can be trained to use acupressure on le7 acupoint as a remedy for post - appendectomy pain . However, further investigations are recommended to confirm the results of the present study . In the present study, acupressure was started when the patients recovered from anesthesia and findings showed a considerable reduction in the severity of post - operative pain . However we suggest further studies with starting acupressure at the onset of pain or nausea and vomiting . Second, we assessed the patients pain, nausea and vomiting only for 7 h. third, our study was single blinded . Therefore, it is recommended to repeat a larger, double - blind study with longer period of assessment of pain and ponv . Also amount of medication received in each group did not check in the present study . It is suggested that the amount of medication to be controlled in the future studies.
Garcin s syndrome was first defined in 1926 by raymond garcin, who defined the syndrome paralytique unilateral global des nerfs craniens (1). Garcin s syndrome is characterized by (1): (i)the presence of unilateral palsies of the cranial nerves,(ii)no sensory or motor long tract disturbance,(iii)no intracranial hypertension and(iv)an osteoclastic lesion in the skull base the presence of unilateral palsies of the cranial nerves, no sensory or motor long tract disturbance, no intracranial hypertension and an osteoclastic lesion in the skull base patients reported to have garcin s syndrome do not always have unilateral palsies of all cranial nerves; thus, this syndrome was redefined as the presence of at least seven ipsilateral cranial nerve palsies . He noticed a mass in his left buccal sulcus, and both his general medical practitioner and general dental practitioner prescribed a total of three courses antibiotics . As there was no resolution he was referred to his local oral and maxillofacial unit for further investigation . By this time he was also complaining of left sided deafness and diplopia . On extra - oral examination he had an obvious soft tissue swelling emanating from his parotid, and extending buccally towards the mental foramen . An orthopantomogram showed a widening of the inferior dental nerve canal and signs of bony resorption . Left acoustic (viii) and facial (vii) cranial nerve palsies occurred initially, followed by trigeminal (v), abducens (vi) glossopharyngeal (xi), vagal (x), accessory (xi), and hypoglossal (xii) left sided cranial nerve palsies . Seven of the 12 cranial nerves were involved in this patient . Nevertheless, his muscle tone, power, sensation, and coordination were normal in all four extremities . Gadolinium - enhanced mri of the brain showed an extra - axial enhancing lesion, which enveloped the hemi - mandible at the level of the left inferior alveolar nerve . The lesion was 3.8 cm at its maximal thickness, and eroded the cortex and the mandible . This revealed a left sided parotid mass extending posteriorly to the mandibular angle, anteriorly beyond the mental foramen and postero - superiorly to the skull base . An incisional biopsy was taken which was histologically reported as non - hodgkin s lymphoma . Following discussion at the head and neck mdt, the patient was referred to the lymphoma team . A rare case of garcin s syndrome presented with progressive multiple unilateral cranial nerve palsies due to non - hodgkin s lymphoma . To our knowledge, only one case of garcin s syndrome due to non - hodgkin s lymphoma has been previously reported . (3) it is more frequently caused by malignant disease; however it can be caused by benign disease . Garcin s syndrome is reported widely as a result of metastatic spread from a primary tumour . There are only three cases of garcin s syndrome being caused by a primary lesion within the head and neck reported within the last forty years . These were caused by a chemodectoma (4) and a meningioma (5). Benign causes of garcin s syndrome include pachymeningitis secondary to otitis media (6) and a large internal carotid artery aneurysm (7).our case is interesting as it was caused both by a primary lesion, and it is the only non - hodgkin s lymphoma case to have been found pre - mortem and as a primary lesion intra orally . On admission to our hospital, the patient had palsies of seven left cranial nerves, which satisfied the clinical picture of garcin s syndrome . The initial diagnosis in this case was bell s palsy which is frequently encountered in clinical practice . Multiple cranial nerve palsies are rare; however the clinician must be aware of such cases to make sure early accurate diagnosis is achieved . In conclusion, multiple cranial palsies are rare and often present initially as a single cranial nerve palsy . In our case of garcin s syndrome the patient presented with a facial nerve palsy and an accurate diagnosis of non - hodgkins lymphoma at this stage may have prevented the development of this syndrome . The diagnosis can only be a made with a high clinical suspicion and appropriate diagnostic imaging.
Allergic rhinitis (ar) is a recurrent or chronic allergen specific, ige - mediated hypersensitivity disorder affecting the nasal lining and characterized by nasal congestion, rhinorrhea, sneezing, nasal itchiness, and/or postnasal drip . Limited studies on ar exist and epidemiological studies based on the allergic rhinitis and its impact on asthma (aria) criteria are lacking . According to aria guidelines, allergic rhinitis is defined if two or more symptoms of rhinorrhea, nasal itching, nasal blockage, or sneezing are present in a patient for at least one hour per day for 4 days or more a week and also for 4 or more weeks a year . Based on duration, symptoms are intermittent (<4 days / week or <4 weeks / year) or persistent (> 4 days / week or> 4 weeks / year). Severity grading is either mild or moderate - severe based on the absence or presence of sleep disturbance and impairment in daily activities, school, and work, respectively . Skin prick test (spt) is a standardized test widely used in the diagnosis of suspected cases of ige - mediated allergy . Generally accepted indications for spt include allergic rhinitis, asthma, atopic dermatitis, suspected food allergies, latex allergy, and conditions in which specific ige is suggested to play a role in the pathogenesis . It provides information about the presence of specific ige to protein and peptide antigens (allergens). Identification of common aeroallergens in an area is necessary, in order to educate the patient on what allergens to avoid and also help find the best formulation of allergen immunotherapy for effective ar treatment . To date, there has been no information regarding the common allergen from the federal capital territory (fct) of nigeria, abuja . The aim of this study is to identify the clinical profile of ar patients according to aria guidelines and investigate the common allergens in abuja, nigeria . This cross - sectional study was conducted for a period of 18 months (march 2014 to september 2015). Sample population was based on the ar patients referred to the allergy clinic, affiliated to medicaid radiodiagnostic center, wuse 2, abuja . Patients were consecutively recruited (convenient sampling method) and fall within the age range of 5 to 65 years . They were patients with a positive history of nasal inflammation (at least 2 or more of the following symptoms: rhinorrhea, sneezing, nasal blockage, nasal itchiness, and postnasal drip) for at least one - year duration . Patients' symptoms were categorized as sneezers - runners and blockers based on the predominant complaint . Patients whose chief complaints include sneezing, rhinorrhea, and itchy eyes and nose were classified as sneezers - runners, while those with nasal blockage, postnasal drip, and difficulty with breathing were classified as blockers . Spt was performed on patients who have stopped taking antihistamines at least 5 days before the test, while patients with severe dermatographism were excluded from the study . A total of 22 allergens were used in this study; these allergens make up the subtropical prick test batch of alk - abello, denmark . These include tree pollen (oak, pecan, black willow, pine, cypress, red cedar, and box elder), weed pollen (pigweed, ragweed, and plantain), grass pollen (bermuda, bahia, johnson grass, and grass mix (meadow, orchard, timothy, june, rye, and redtop)), house dust mites (dermatophagoides pteronyssinus and dermatophagoides farinae mix), molds (alternaria tenuis, c. cladosporioides, penicillium mixed, and aspergillus mixed), animal dander (cat hair and dog epithelium), and cockroach extracts . Of the pollen allergen used in this study, about 80% of the plants are present in abuja environment . Spt was performed according to international guidelines as a one - time test done on two forearms with lancets and allergens (alk - abello skin prick test kit, berge alle, 2970 hrsholm ., histamine hydrochloride (1%) and normal saline (0.9%) were used as positive and negative controls, respectively . The patients' data was classified according to the aria guidelines and spt results were analysed in allergen - clusters of tree pollen, weed pollen, grass pollen, house dust mites (hdm), molds, animal dander, and cockroach extracts . Spss 16 software (chicago illinois) was used in the analysis and p value of less than 0.05 was considered significant . A total of 96 new patients with suspected allergic rhinitis presented at the allergic clinic within the study period . Only 74 patients from these had a positive spt result and were enrolled into the study . Twenty - one respondents (28.4%) were categorized as children (5 to 17 years of age) with a male: female ratio of 1.3: 1 . The majority of the study population resided in abuja municipal area council (amac) territory which is basically the city center . The prevalence of asthma, urticaria, and conjunctivitis as comorbidity was lower when compared with comorbidity such as hypertension . Positive family history of atopy was seen in 56.8% of patients and 20.2% of the subjects had animal contact within their environment . According to predominant symptoms, the proportion of sneezers - runners was higher than blockers sneezers - runners tend to have persistent ar, while blockers symptoms were more intermittent (p = 0.007). Based on aria guidelines, most patients (67.2%) had moderate - severe ar (intermittent and persistent) and this was significantly related to animal exposure (p = 0.035) and not to age, gender, or family history of atopy . There was a significant association between ar severity and the predominant complaints (table 2) by the patients (p = 0.005). Moderate - severe persistent ar was more common among sneezers, while moderate - severe intermittent ar was common with the blockers . There was no significant association between ar severity and the presence of asthma (p = 0.26) or family history of atopy (p = 0.19). House dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). Weed pollen allergen yielded the least (7.4%), while animal dander and fungi allergen both came to 13.1% each (figure 1). Also, house dust mites was significantly related to a positive family history of atopy (p = 0.035). There was no significant difference between the positive skin tests and gender as well as history of asthma . Furthermore, no relationship was observed between the allergens tested and duration of ar (intermittent and persistent), as shown in table 3 . Most of the patients with a positive spt were in the persistent ar category (66.2%). The number of allergens which produced a positive skin response from each patient was closely distributed . The highest was reaction to 3 or more allergens (35.1%) followed by reaction to 2 allergens (33.8%) and then to one allergen (31.1%), as shown in table 4 . Statistical analysis did not reveal a relationship between ar severity and skin prick test reactivity . The prevalence of ar is increasing worldwide, yet it remains underdiagnosed and undertreated especially in developing countries . A self - reported survey of ar among adult nigerians observed a prevalence of 29.6% and a mean age of 29.3 years which was close to the mean age observed in this study . The number of patients in this study with concomitant asthma is lower than other nigerian studies [7, 8] and this could be due to small sample size or poor awareness of asthma symptoms by the respondents . . Observed a low level of awareness of asthma by patients in nigeria, such that most people with asthma symptoms do not present to the physician but prefer unorthodox means of medical care . About a third of the children (33%) in this study reported persistence or recurrence of rhinitis symptoms after adenoidectomy . The slight female predominance observed in our adult ar patients is in consonance with the findings in malaysia and india [3, 5]. We also corroborated the study that showed a higher male predominance of ar among children . We recorded a higher proportion of sneezers - runners to blockers similar to the findings by lee et al . And shah and pawankar but different from study by deb et al . Sneezers having persistent ar and more blockers having intermittent ar (p = 0.007). We could not establish any relationship between these predominant symptoms and the aeroallergens used in this study . Blockers were more sensitized to hdm, house dust, and fungi, while this study observed that a majority of ar patients were categorized as moderate - severe persistent ar, according to aria classification, while the least were mild intermittent ar . This has been observed by most studies [3, 5, 12] with warm climate like nigeria . A constantly high environmental temperature and humidity could lead to a persistently high concentration of indoor and outdoor allergens all year round . There could also be a selection bias, since patients would more likely present for treatment when their condition is severe and persistent . No relationship was established between the type of ar and the allergen to which the patients were sensitized . This was similarly observed in a national, cross - sectional study of ar patients in mexico . The most common aeroallergen was house dust mites, followed by tree pollen as was seen in other spt studies in nigeria [8, 1416]. However, there are no ar studies in relation to tree pollen sensitivity to compare with . This study highlights the importance of pollen allergens among ar patients living in nigeria, a tropical country with high humidity . This supports an earlier observation of increased tree pollen sensitization in tropical environment and emphasizes the need for increased research in this aspect . Further studies are needed to record the season of pollination of the different pollens found in abuja and to correlate these findings with the timing of symptoms in sensitized patients . Thus a patient with positive sensitization to house dust mites could also be sensitized against pollen, insect, or fungi allergens . This supports the argument that time of exposure (seasonal or perennial) does not properly define ar patients and also creates difficulty with regard to immune therapy via hyposensitization . In addition, there is need for the use of spt that incorporates wide variety of allergens within a specific environment in order to avoid skipping some of the sensitive allergens attributed to each individual ., most ar patients presenting for treatment in abuja, nigeria, have moderate - severe persistent ar and show similar spt sensitization pattern with other countries having similar climatic conditions . Sensitization patterns are not related to aria classification or any predominant ar symptoms but rather may rely on the environmental condition of study area and genetic makeup of the study population.
Most cases were reported in patients with chronic liver disease and cancer, and cases of s. algae infection in end - stage renal disease (esrd) patients are few . The problems related to clinical practice are that little is known about the etiology of s. algae infection in esrd patients and that this bacterium is often confused with shewanella putrefaciens, although these two species may have different pathogenicities in humans . We herein report a case of s. algae bacteremia in a patient with esrd and review previous case reports on shewanella infection in esrd patients . A 71-year - old man was admitted to our hospital in mid - july because of a 5-day history of nausea and abdominal pain . He had been diagnosed with chronic kidney disease (ckd) due to chronic glomerulonephritis and had had an arteriovenous fistula in his left arm in preparation for renal replacement therapy since two month prior to the onset . He had been given an erythropoiesis - stimulating agent (esa; darbepoetin alpha 180 g / month) for renal anemia; he was not on any iron supplements . The laboratory data 1 month before the onset of symptoms showed hemoglobin of 6.9 g / dl, fe of 141 g / dl, transferrin saturation (tsat) of 76.6%, and ferritin of 704 ng / ml, findings that were indicative of dysregulated iron metabolism . He had eaten sliced raw fish or sashimi of mackerel and squid three days before the manifestations of nausea and abdominal pain, for which he visited the outpatient unit, where he was diagnosed with infectious enterocolitis and was given oral fosfomycin . However, his symptoms worsened, and he was admitted to our hospital five days after the onset of symptoms . A physical examination showed that his blood pressure was 160/70 mmhg, heart rate 83 beats / min, and body temperature 39.5. he had abdominal tenderness without rebound tenderness . Laboratory tests on admission showed a white blood cell count of 3,700 /l with 83% segmented neutrophils, hemoglobin of 7.6 g / dl, mean corpuscular volume (mcv) of 93.4 fl, mean corpuscular hemoglobin (mch) of 30.2 pg, mean corpuscular hemoglobin concentration (mchc) of 32.5 g / dl, and platelet count of 156,000 /l . Serum chemistries showed serum sodium 134 meq / l, potassium 5.0 meq / l, chloride 91 meq / l, calcium 5.0 mg / dl, phosphorus 11.7 mg / dl, magnesium 1.6 mg / dl, uric acid 11.8 mg / dl, blood urea nitrogen 166.9 mg / dl, creatinine 23.0 mg / dl, total protein 6.8 g / dl, albumin 2.8 g / dl, aspartate aminotransferase 27 u / l, alanine transaminase 19 u / l, lactate dehydrogenase 882 u / l, alkaline phosphatase 210 u / l, -glutamyl transpeptidase 19 u / l, creatine kinase 1,809 u / l, and c - reactive protein 11.7 mg / dl . Abdominal computed tomography (ct) revealed intestinal swelling and fluid collection, compatible with infectious enterocolitis (figure). A, b: abdominal computed tomography on admission revealed intestinal swelling and fluid collection, compatible with infectious enterocolitis . Two sets of blood cultures were collected before cefmetazole (1 g / day) was started . On the second hospital day, he went into cardiac arrest; cardiopulmonary resuscitation resulted in the return of spontaneous circulation . Antimicrobial therapy was changed to meropenem (2 g / day). On the third hospital day, gram - negative bacteria appeared in both sets of the blood culture obtained on admission and were identified as s. putrefaciens 4 days later . Ceftazidime (2 g / day) was given based on the susceptibility test (table 1). Mic: minimum inhibitory concentration his condition improved; therefore, continuous hemodiafiltration was switched to hemodialysis 3 times per week . On the 13th hospital day, the antimicrobial agent was changed to levofloxacin and continued for 1 week . He was discharged on the 26th hospital day without any complications and continued maintenance hemodialysis . The bacterium recovered from the blood culture was further analyzed because the patient's condition had deteriorated . A 16s rrna gene sequence analysis revealed that the bacterium was s. algae s. algae is a rare causative pathogen of bacteremia, skin and soft tissue infection, or hepatobiliary infection . We reported a case of s. algae bacteremia in an esrd patient and demonstrated the probable link between raw seafood consumption and shewanella infection . This patient had iron overload that was related to a dysregulated iron metabolism caused by esrd . Raw seafood consumption by an esrd patient may be a risk factor for shewanella infection . Shewanella species are gram - negative bacteria that are mainly isolated from the marine environment . This species was once named pseudomonas putrefaciens; it was originally classified in the family vibrioneceae until the 1990s, when it was reclassified as genus shewanella (1). Most case reports of shewanella infection have described an association with exposure to seawater; such contact can cause ear infection, skin or soft tissue infection, and bacteremia (1 - 3). Two species, s. putrefaciens and s. algae, have been reported to be pathogenic in humans (1). It is often difficult to distinguish between these two species, because automated identification systems only include the database of s. putrefaciens, but not s. algae (2). Therefore, some authors have suggested that the previously reported s. putrefaciens infections may have actually been caused by s. algae, which is thought to be more virulent (3). The accurate distinction between these two species requires a 16s rrna gene sequence analysis or phenotypic tests; s. algae can only grow at 42 and in 6% nacl (1). In the present case, an automated identification system (microscan walkaway 96 plus, siemens healthcare diagnostics, tokyo, japan) initially determined the bacterium to be s. putrefaciens; however, a 16s rrna gene sequence analysis confirmed the bacterium to be s. algae . Shewanella infection can be fulminant and fatal and is often misdiagnosed as vibrio vulnificus infection, because of the similarities in their clinical manifestations (2,4). Patients with underlying diseases, such as hepatobiliary disease or malignancy, were reported to be predisposed to shewanella infection, as well as to v. vulnificus infection (3); the mortality rate of shewanella bacteremia was increased in these patients (5). Shewanella species belong to the microflora of the marine environment, and exposure to seawater was reported to be one of the risk factors for shewanella infection, especially in temperature regions (3). Therefore, more attention should be paid to the pathogenicity of this bacterium, especially in patients with underlying diseases who are exposed to these environments . There have been seven previously published case reports on shewanella infection in esrd patients (4,6 - 11) (table 2). Two cases were reported to be caused by s. algae and four cases by s. putrefaciens; however, only two cases were further analyzed by a biochemical procedure . The most common route of infection was skin ulcer (3 cases), followed by catheter - related infection (2 cases); no case was related to oral intake of seafood . In the present case, the onset of symptoms was related to seafood consumption, and abdominal ct scan showed signs of infectious enterocolitis . Therefore, we concluded that the shewanella infection in this patient was probably caused by dietary intake of raw fish or sashimi . This was the first case of s. algae infection that might have been transmitted orally in an esrd patient . Esrd: end - stage renal disease, flux: flucloxacillin, gm: gentamicin, pipc: piperacillin, cpfx: ciprofloxacin, cez: cefazolin, lvfx: levofloxacin, mino: minocycline, cfpm: cefepime, caz: ceftazidime, mepm: meropenem, vcm: vancomycin, doxy: doxycycline, drpm: doripenem, amk: amikacin although the reason why the esrd patient was predisposed to shewanella infection is still unclear, dysregulated iron metabolism may be a risk factor for this patient . Iron is not freely available, since it binds to transferrin in the blood (12). Pseudomonas and vibrio species have the ability to produce siderophore, which plays an important role in supplying iron to bacteria (12,13). Siderophore has a high affinity to iron and is able to displace iron from transferrin . S. algae was reported to produce siderophore and was capable of absorbing iron into its body (14). Patients with hepatobiliary diseases who are susceptible to s. algae often had the complication of iron overload . The association between shewanella infection and hepatobiliary diseases may be due to iron overload (5). In the present case, the patient did not have hepatic dysfunction but did have esrd with a dysregulated iron metabolism; his anemia was esa - resistant, and there seemed to be excessive iron levels, although he was not taking any iron supplement . It was suspected that uremia of the patient contributed to the esa - resistant anemia, because the patient was just starting renal replacement therapy, and the serum creatinine level on admission was extremely high . Recent studies have demonstrated that hepcidin, which reduces iron release from reticuloendothelial and hepatocyte stores, plays an important role in the disordered iron metabolism of uremia including esa resistance (15,16). The serum hepcidin level is related to the residual renal function and elevates in ckd and in esrd patients (15,17). Serum hepcidin can be removed by renal replacement therapy (18), and insufficient hemodialysis is known to cause esa - resistant anemia (19). Therefore, we speculated that the esrd, especially the accompanying iron overload, might have been a risk factor for shewanella infection in this patient . To our knowledge, no previous report has described the status of iron metabolism in the esrd patients with shewanella infection . The limitation of this report was that the data for serum ferritin and tsat on admission were not available . However, we measured both of these markers at one month before the onset, when the patient was in a stable condition without acute inflammation or infection . Iron status should be evaluated based on ferritin and tsat in ckd and esrd patients (19,20). The ferritin level and tsat of the patient in the present study were compatible with iron overload (21,22). In addition, we were unable to recover s. algae from a stool culture on the eighth hospital day, when the patient had already received courses of an antimicrobial agent to which the bacterium was susceptible . We could not collect a stool sample at an earlier time, such as at admission, because of the patient's critically ill condition . In conclusion, we reported a case of s. algae bacteremia that was probably caused by oral intake of raw fish in an esrd patient . In addition, a dysregulated iron metabolism in esrd may be a risk factor for shewanella infection.
Strains in this study were jm837 (leu132 h-), jm1262 (pil1-mcherry::natr h-), jm1293 (fhn1::kanmx6 ura4-d18 leu132 ade6-m210 h) and jm1461 (sle1::kanmx6 ura4-d18 leu132 ade6-m210 h). Expression from plasmid pjm512 (prep41-pil1-gfp) was induced by growth in the absence of thiamine for 36 hours at 32c . To observe endogenous pil1-mcherry rods, strain jm1262 was grown on ye4s plates at 32c for 3 d. cells were scraped from the plate and immediately imaged . Electron microscopy: for thin sectioning, cells carrying plasmid pjm512 (prep41-pil1-gfp) or control plasmid pjm211 (prep41-gfp) were grown in the absence of thiamine for 36 hours at 32c, and then prepared and imaged as previously described . For immunolabeling of pil1 bundles, fixations and digestion were the same except for the following changes: initial fixation of 3% pfa/1% gta was used instead of 3% gta/1% pfa . Samples were incubated in 0.05 m glycine in nacac buffer for 30 min to remove excess aldehydes . Additionally, 1% p - phenylenediamine (ppd) was included in each etoh solution . Four additional 85% etoh/1% ppd rinses over 1 hour were performed, and then pellets were resuspended in a 2:1 mixture of 85% etoh/1% ppd: lr white medium resin . This suspension was placed on a rotator for 2 hours at room temperature and then resuspended in 1:1 85% etoh/1% ppd: lr white medium resin, rotated for 1 hour, and then kept at 4c overnight . The following day, samples were resuspended in 1:2 85% etoh/ 1% ppd: lr white medium resin rotated for 1 hour at room temperature and then resuspended in 100% lr white medium resin . Solution was changed until clear, and then 3 more changes over 6 hours rotating at room temperature were performed before placing at 4c overnight . Samples were warmed to room temperature and 4 changes of 100% lr white medium resin were made with 1 hour each change . Samples were transferred to gelatin capsules and left for 4 h at room temperature, then polymerized at 50c for 24 h. thin sections (7080 nm) were placed on g300 nickel - coated grids (electron microscopy sciences). Grids with sections were washed in pbs for 5 min, put in block solution of pbs + 5% bsa (sigma - aldrich) for 15 min, incubated for 1 hour in 1/25th dilution of rabbit anti - gfp, washed 6 times for 2 min, incubated for 30 min in a 1/25th dilution of goat anti rabbit 6 nm gold - conjugated secondary antibody (25104; electron microscopy sciences), then washed 6 times for 2 min with 2 additional washes in pbs and 2 final washes in distilled water . Washes and antibody dilutions were in pbs + 0.1% bsa (sigma - aldrich) at rt . Grids were stored at room temperature and stained with 2% aqueous uranyl acetate for 4 min and reynold's lead citrate for 10 sec . Light microscopy: fluorescence microscopy was performed as previously described using a deltavision imaging system (applied precision). For filipin staining, cells expression from plasmid pjm512 was induced by growth in the absence of thiamine for 20 hours at 32c; filipin (sigma f9765) was added at a final concentration of 5 g / ml to live cells . This work was funded by national institutes of health grant gm099774 (to j.b.m .) And t32-gm008704 (to r.k . ).
Educational evaluation (ee) is a formal process performed to evaluate the quality of effectiveness and/or value of a program, process, goal or curriculum.12 it deals with data collection and assessment of the progress of academic programs.34 by considering some principles related to educational measurements and data collection, ee may result in a better understanding of such programs.57 during the past thirty years, theorists have presented numerous methods of evaluation . Worthen and sanders2 mentioned that more than 50 different evaluation approaches has been developed in recent decades . Among these, methods based on internal criteria are known as the ones that can interpret the scientific, educational, and therapeutic authenticity of different educational groups.48 this is greatly welcomed by the academic community and is widely spread to all universities in the world . That is because this method provided a scientific, appropriate, precise, timely, and valid basis regarding the interpretation of decision making system quality and programming for its promotion and development.3 such a method was successfully carried out in four medical education groups at supervisory and expansion of medical education council secretariat of ministry of health, treatment and medical education of iran in 1995.8 ee has its most effect, value, and results when it can provide needed information to individuals which are directly related, as well as those who may be benefited from its results.346 educating dental professionals consists of theoretical and practical (clinical, paraclinical, and laboratory) courses, differing in duration, and educational curriculum among different countries . It might vary from 4 years (e.g., in india, turkey, and russia) to 6 years or more (e.g., in iran consisting of 2 years of only basic medical sciences and 4 + years of dentistry courses). Due to numerous practical educational units in dentistry education and with regard to expensive but very critical protocols for infection control, a great deal of resources is consumed in governmental universities of iran over training every general practitioner with a degree of doctorate of dental surgery . On the other hand, the quality of dental services plays an important role in public health . Considering these issues, dentistry education needs to be cost - effective in terms of optimizing its quality . To date, the pace of advancements in dentistry necessitates a continuous revision of educational programs by officials to achieve new expectations of educational system and determine or update the policies.9 proper evaluation and research in education are accounted as scientific instruments for moving along with these developments in order to achieve improvements in education, health, and treatment qualities.911 such advancements may depend on education of faculty members and panels and their cooperation, elimi - nation of shortcomings, and approximation of components and educational instruments to standard indices.9 aim and mission of this educational groups are training and tutoring students of undergraduate and residency courses in order to gain complete ability for diagnose and treatment of patients needing this kind of treatments, so that residents can provide treatment and disease prevention services with a desirable quality after learning these courses.10 this study was conducted in year 2010 to assess the efficacy of educational programs provided at dental school of tehran university of medical sciences to estimate and address the limitations and strengths . Apart from educational groups dedicated to deliver basic medical sciences to dental students, dentistry school of tehran university of medical sciences includes twelve only - dentistry educational groups (table 1). The objective of this descriptive cross - sectional study was to assess the quality of education provided at this school, compared with the standards . The following aims would be fulfilled: 1) determining the educational and research needs of group directors; 2) determining those of other faculty members; 3) defining such needs of under- and post - graduate students; 4) surveying attitudes of graduated doctors; 5) surveying regarding research and educational facilities as well as residency resources; 6) determining educational procedures; 7) surveying patients regarding their satisfaction from received dental cares; 8) distinguishing human resources; and 9) determining poor fields, needing to be pushed . Dentistry school of tehran university of medical sciences educational groups in 2010 through this descriptive cross - sectional study, the efficacy of provided education by mentioned 12 departments were assessed in 13 fields . Research society, educational groups including group director and sub - societies which include faculty teachers, students, alumni, human, and support recourses were completely involved in this survey . These included aims and missions of groups, management and organization, scientific board, students, human resources and support, educational, research, health and treatment spaces, educational, diagnostic, research, and laboratory instruments, educational, research, health, and treatment programs, process of teaching and learning, evaluation and assessment, and satisfaction of alumni and patients . Each field was evaluated through the following steps: 1) establishment of standards; 2) data collection; 3) determining the importance of components; and 4) analyzing the collected data . Inspection, interview, and checklists were to evaluate educational, research, health, and treatment spaces of dentistry sections and educational, research, laboratory and diagnostic instruments . First questionnaire was used to gather the feedback of panel director and faculty scientific members to establish the coefficient of desired factors . Another questionnaire was utilized to evaluate 11 fields by multiple - choice questions based on a 5-point likert scale . Measurement instruments, evaluated factors and data collection sources we tried to establish study variables in line with objectives and research questions in providing data collection instruments . For this reason, before preparation of data collecting instrument, a table was created that precisely identified each research question's variables and based on that the instruments were generated . Afterward, in order to increase the validity, questionnaires were reviewed by experts and the straight and vague questions were addressed.12 considering the reliability of data collecting instrument, after preparation of questionnaires according to arranged subjects, confusions about some questions were identified and removed, taking the use of a pilot study in a 15-individual group and interviews with academic board members in educational groups . Eventually, the final data collecting instrument was designed . In order to determine the coefficient of each of the 13 criteria, feedback forms were utilized . Besides, through interviews and delphi technique the academic board members opinions were recorded and the importance of each criterion was determined . Results indicated that all academic board members have given equal coefficient values to all questions . After determination of evaluated factors and sources of gathering related data, criteria for each factor were designated (e.g., an evaluated factor was management and organization of group, and a criterion related to this factor was group director). A marker was created for each of these criteria . In order to perform this, a) specificities of desirable condition were described; b) a marker of desirable condition was set, c) for assessment of goal achievement, the criteria condition was compared to the desirable condition marker . In order to analyze likert multiple - choice questions, scores 1 to 5 were respectively assigned to highest and lowest scores . Utility rate was determined by the percentage of the related index . To facilitate the assessment of components and evaluated factors, the desirability level of each factor was classified based on the score percentage: desirable, more than 75%; relatively desirable, 50 - 75%; and not desirable, less than 50% (table 3). Average educational evaluation results of educational groups in dentistry school of tehran university of medical sciences in 2010 descriptive statistics were calculated using spss10 . Ee results were analyzed based on swots (strengths, weaknesses, opportunity, and threats) method in evaluated educational groups . Educational evaluation committee accepted fluency and clarity of the missions and aims of educational groups in undergraduate and graduate courses . Revision of aims was suggested in three areas of knowledge, attitude, and practice . The quality of departments educational systems were relatively desirable (55.98) in all 13 fields . For management fields, average results were relatively desirable (52.9%) based on specificities of desirable condition . In this field, 72% of group members believed that group managers have acceptable scientific and educational background, 55% were aware of group manager selection criteria and 90% of academic field members were satisfied with the organization and management abilities of the group manager . In evaluation on academic board, mean age of educational group members was 43.9 3.2 years old and mean teaching background was 14.4 3 .9 years . Most of academic board members were men and all of them were assistant professors and officially occupied . 80% of academic board members were involved in research projects; more than 80% were mentoring specialty course thesis; and 50% were satisfied with workspace condition . An important specificity of this group was that they were involved in programming of theoretical and applied education of students in undergraduate and residency courses . Mean ee result in academic board field was relatively desirable (56.92%). In the field of learners, all residents were asked about their association, correlation with academic board, study duration, and educational, research activities, and student projects . Desirability in the field of human resources and support in sections educational, treatment, research, audiovisual, library, diagnostic laboratory, radiology, and facilities was 54.58%, considered relatively desirable . And in both fields of educational, research, health, and treatment spaces and educational, research, diagnostic, and laboratory facilities ee results were relatively desirable (50.36% and 51.55% respectively). In the field of educational, research, teaching process and learning courses, nearly all academic board members (91%) 82% of group members believed that aims and mission of educational courses in groups were of their interests . Most of the group members (91%) believed that cultural revolution committee headings were applied in educational programming and 82% of them were involved in both basic and clinical teachings . These fields had the average of 58.09% and 60.16% respectively and considered relatively desirable . In the field of graduates, mean age of individuals which have entered the university in year 1997 and graduated in year 2003 was 26.3 and 80% were men . Considering the service condition, most graduates (66.7%) were spending their duty project . Regarding their satisfaction, this field was in a relatively desirable (58.72%) condition . In the field of patients, more than 63% of patients of general (undergraduate) ward and 73% of patient which referred to residency wards stated that their reason for selecting this university was that they trusted in the precise and effective treatment delivered by the students at this university . After comparison of results to the desirability criteria regarding the trust of service takers to precise treatment and a good referring size of the ward, this fields condition was considered relatively desirable (61.32%). The evaluated educational groups have given suggestions about quality improvement in education, research, health, and treatment in four levels (group, faculty, university, and ministry) considering their strength and weaknesses.10 the mean of educational evaluation in departments of educational school of dentistry tehran university of medical sciences, 2010 . Ee can study and assess the educational programs utilizing standards, predetermined aims, or educational quality236710 after such assessments, it is possible to address these shortages in educational system and arrange an efficient educational system.11314 ee of educational programs is an important and basic task of medical universities.1 rate of occupational capabilities and medical alumni performances in order to offer educational and research programs, health and treatment services with the aim of supplying and improving society members health is related to the rate of educational programs realization.1 if educational programs are not well designed and performed, it can impose irreparable damage and harmful social, economical, and cultural effects on individuals, society, alumni, and also faculty and finally university's credit.1 considering the importance of education and its major role in improving nearly all aspects of societies, educational programs should be carefully probed to elucidate shortcomings and advantages in order to improve programs as well, teaching methods such as workshops, seminars, presentations, interactive teaching methods which may involve the students and thus increase their learned topics and introducing outlines of the lessons in the first place.16 however, unfortunately most teachers are not trained regarding teaching techniques and are not completely ready to undertake educational responsibilities.1718 an effective way of improving this shortcoming is conducting courses in teaching techniques and skills.19 evaluating the draw backs, preferences, and priorities of teachers teaching skills, as well as other educational shortcomings may enable the program directors of such courses to canalize the materials to which is more needed by teachers and educational groups.20 universities may determine their position in national and international levels to further improve their programs.2122 for example, india, as a country with the highest number of medical universities and thus the most number of medical faculty members, has developed such programs called national teachers training centers (nttc). These programs train teachers to gain skills, and to find certain teachers as group directors and leader, in long - term . Such programs are globally called faculty development.23 the overall ee findings in educational groups were relatively desirable (mean score = 55.98%). Based on the findings, activities of group managers, educational management, and academic board members in these groups were performed in order to improve the procedure of assessment . Results of other national studies shows that school of medicine,13 school of nursing and midwifery,24 and school of rehabilitation25 had average 75.3%, 80.4% and 77.8%, respectively and the quality of education, research, and treatment were desirable . Was started with implementation of a pilot ee study in six educational groups in 1996.26 results showed that ee in iranian culture would lead to improvement.26 30 national medical educational groups in medical universities of the country implemented the ee project.27 farzianpour and bazargan in 1999 revealed that ee is the best measurement index for evaluation of university hospitals.27 in 2004, fifteen basic sciences and clinical educational groups of tehran university of medical sciences have reported their ee results desirable.28 saberian et al29 from school of midwifery and nursing of semnan described results of ee in surgery ward desirable in international congress of educational evaluation in 2004 which was held in edinburgh . Olyaei et al14 from rehabilitation school noted that the results of their ee was 76.2% and was desirable . Farzianpour et al28 from tehran university of medical sciences, and harden et al30 from dundee university of england had positive attitudes towards ee and improvement of education and research in clinical fields . Researches in world's educational system report that ee is an effective way to find out the strengths and weaknesses of an educational system.142734 universities might follow standards of education evaluation so that the result could be better comparable . However, diverse techniques have been described for educational evaluation.35 these included expertise - oriented, management - oriented, and objectives - oriented.35 kirkpatrick36 has introduced a 4-step assessment . The application of theoretical knowledge of learners in their practice would be assessed . Finally the impact of program on the institution and community would be evaluated . Until conducting a unified method, international comparisons would be difficult to perform . Quality of patient care and students learning were the best fields (respectively 61% and 60%). Educational aims and objectives, and research and educational spaces had the poorest results (respectively 49% and 50%).
There has been growing interest during the last decade in the development of fluorescent molecular sensors for cations and anions in solution [1 - 8]. Especially, fabricating fluorescent materials for the detection of zinc cation has drawn much more attention [9 - 13], as zinc not only plays important roles in human bodies, but also closely relates to severe pathological diseases such as alzheimer s and parkinson s diseases . So far, much study has been done for the detection and real - time localization of zn(ii). Yasuhiro shiraishi s group has synthesized a quinoline polyamine conjugate as a fluorescent chemosensor for quantitative detection of zn(ii) in water . Maarten merkx et al . Used chelating fluorescent protein chimeras for ratiometric detection of zn(ii) in living cells and the detection range was from 10 nm to 1 mm . Jinshi ma and coworkers have synthesized several bis(pyrrol-2-yl - methyleneamine) ligands as fluorescent sensor for zn(ii), and their results revealed that the ligands exhibit excellent fluorescent properties . However, much of the work was just based on organic molecules as fluorescent chemosensors . For a few practical applications the attachment of the fluorescent units to a solid support has advantages like the possibility of recovering the materials for their repetitive use . For this point, scientists chose silica nanoparticles, nanosized boehmite particles and silicon nanowires to support fluoresence ligands as fluorescence sensors . And these materials exhibit excellent selectivity and sensitivity to sense metal ions . In this study, we chose multi - walled carbon nanotubes (mwnts) as fluorescent support . Since its discovery, the fluorophore in this study is glycine - n-8-quinolylamide (gnq) molecule, in light of the fact that the 8-aminoquinoline derivatives could effectively coordinate with specific metal ions . The quinoline group has been covalently grafted to the surface of the mwnts that can behave as recognition center for metal ions depending on its actual protonation state . We find that this new material (mwnts - gnq) has high selectivity and sensitivity to detect zn(ii), and the sensitivity is down to 0.2 m ., which is about the same as the silica nanoparticles - supported fluorescence sensors . For other sensing materials, the fluorescence enhancement selectivity is not only for zn(ii) but also for cd(ii), which may reduce selectivity when they are used just for zn(ii) detection . On the other hand, the fluorescence enhancement of mwnts - gnq is only for zn(ii). As the use of organic inorganic hybrid materials for bio - application has become a hot subject in the research field currently [28 - 31], mwnts - gnq may be used to build nanosensor devices to sense directly in intracellular environment, because carbon nanotubes (cnts) can penetrate into cells and almost have no toxicity to organism . The multi - walled carbon nanotubes (mwnts, diameters: 2040 nm, purity: 9598%) prepared by the catalytic decomposition of ch4 were provided by shengzhen nanotech port ltd . Glycine - n-8-quinolylamide (gnq) was synthesized according to the known method . In a typical experiment, 300 mg pristine - mwnts were added to a 180 ml 3:1 mixture of concentrated h2so4and hno3 . The mixture was treated in an ultrasonic bath (40 khz) for 20 min and stirred at 60 c for 4 h under reflux . Then, the mixture was vacuum - filtered through a 0.22 m millipore polytetrafluoroethylene membrane and washed with distilled water until ph of the filtrate was 7 . The filter cake was dried under vacuum at 40 c for 24 h to obtain mwnts - cooh . The acid - treated mwnts - cooh of 200 mg was reacted with 20 ml socl2for 24 h under reflux, and then the residual socl2was removed by the reduced pressure distillation, the solid was washed with anhydrous thf several times until the brown - colored supernatant became to colorless . The remaining solid acyl chloride - functionalized mwnts (mwnts - cocl) was dried under vacuum at 20 c for 24 h. functionalization of mwnts mwnts - cocl of 100 mg was dispersed in 10 ml anhydrous chloroform and the mixture was sonicated for 20 min to create a homogeneous suspension . The mixture was added with 50 mg gnq under a nitrogen atmosphere, and then immersed in an oil bath at 70 c accompained by mechanical stirring for 24 h. the resulting reaction medium was vacuum - filtered through a 0.22 m polycarbonate membrane three times to yield mwnts - gnq . Fourier transform infrared (ftir) spectrometer (bruker ifs66/s), dupont-1090 thermal gravimetric analysis (tga) instrument and gmbh varioel elementar analysensyteme were used to characterize the materials . Perkin elmer ls 55 spectrofluorimeter was used to obtain the fluorescence spectra of the fluorescence material . The multi - walled carbon nanotubes (mwnts, diameters: 2040 nm, purity: 9598%) prepared by the catalytic decomposition of ch4 were provided by shengzhen nanotech port ltd . In a typical experiment, 300 mg pristine - mwnts were added to a 180 ml 3:1 mixture of concentrated h2so4and hno3 . The mixture was treated in an ultrasonic bath (40 khz) for 20 min and stirred at 60 c for 4 h under reflux . Then, the mixture was vacuum - filtered through a 0.22 m millipore polytetrafluoroethylene membrane and washed with distilled water until ph of the filtrate was 7 . The filter cake was dried under vacuum at 40 c for 24 h to obtain mwnts - cooh . The acid - treated mwnts - cooh of 200 mg was reacted with 20 ml socl2for 24 h under reflux, and then the residual socl2was removed by the reduced pressure distillation, the solid was washed with anhydrous thf several times until the brown - colored supernatant became to colorless . The remaining solid acyl chloride - functionalized mwnts (mwnts - cocl) was dried under vacuum at 20 c for 24 h. functionalization of mwnts mwnts - cocl of 100 mg was dispersed in 10 ml anhydrous chloroform and the mixture was sonicated for 20 min to create a homogeneous suspension . The mixture was added with 50 mg gnq under a nitrogen atmosphere, and then immersed in an oil bath at 70 c accompained by mechanical stirring for 24 h. the resulting reaction medium was vacuum - filtered through a 0.22 m polycarbonate membrane three times to yield mwnts - gnq . Fourier transform infrared (ftir) spectrometer (bruker ifs66/s), dupont-1090 thermal gravimetric analysis (tga) instrument and gmbh varioel elementar analysensyteme were used to characterize the materials . Perkin elmer ls 55 spectrofluorimeter was used to obtain the fluorescence spectra of the fluorescence material . Figure 1shows the infrared spectra of a gnq and b mwnts - gnq . In the ftir spectrum of gnq,, the characteristic peaks of amino groups in the spectrum of mwnts - gnq disappeared, demonstrating that the amino groups on gnq have reacted with acyl chloride groups on the surface of mwnts . A new peak that appeared around 1686 cmis attributed to the amide carbonyl (c = o) stretch . Another new peak at 1629.87 cmis attributed to secondary amide band which accompanies the absorption at 1686 cm . Another peak at 1082.47 cmattributed to (c n) has also been found, and it has obviously been enhanced . Ftir spectra ofagnq andbmwnts - gnq the tga curves of mwnts - cooh and mwnts - gnq were recorded on a dupont-1090 thermogravimeter in ar atmosphere at the heating rate 10 c / min from 20 c to 500 c (fig . 2a, there is a continuous weight lose of the mwnts - cooh, and the amount of the weight loss is about 10% typical for acid - functionalized mwnts . 2b, the major weight loss happened in the temperature range from 200 c to 450 c due to the degradation of the gnq grafted to the mwnts . The content of the gnq grafted to the mwnts is about 12 wt%, which is similar to the calculation result of the elemental microanalysis (table 1). Tga ofamwnts - cooh andbmwnts - gnq elemental microanalysis for mwnts - gnq the fluorescence spectra of gnq and mwnts - gnq are shown in fig . Fluorescence spectrum of gnq (1 10 m) and mwnts - gnq (1 10 m). Ex = 324 nm gnq is expected to be a stronger coordinating agent because it is a tridentate ligand . When gnq is selectively coordinated with metal ions, the fluorescence from gnq this phenomenon can be utilized to construct a material for the detection of metal ions based on mwnts (scheme 2). Accordingly, titration of various metal ions in the presence of mwnts - gnq in methanol solution was performed, and the results are summarized in fig ., it is observed that the intensity of fluorescence from mwnts - gnq containing zn(ii) is much higher than that of other metal ions . This is very nice because under many conditions (e.g., physiological conditions) various metal ions may exist at certain concentrations compared to zn(ii). Fluorescent chemosensor (mwnts - gnq) for detection of zn(ii) relative fluorescence intensity of mwnts - gnq (1 10 m) in the presence of variouse metal ions alone (1 10 m). Methanol solution . Ex = 324 nm it was reported that most of the previous zn(ii) sensors do not exhibit good selectivity to these metal cations (for instance, the selectivity in many cases is close to 1:1 for zn(ii):cd(ii)). This may bring trouble to certain applications where co(ii), ni(ii), cu(ii), or cd(ii) may interfere (e.g., in environmental science). Herein, mwnts - gnq shows 2.8-fold fluorescence enhancement for zn(ii) versus just minimal fluorescence enhancement for cd(ii) and ni(ii). From these results, it is evident that mwnts - gnq have a high selectivity to zn(ii). As for selectivity of mwnts - gnq to metal ions, when zn(ii) forms a complex with mwnts - gnq with a suitable radius and an electronic structure 3d4 s, the electron - transfer process of mwnts - gnq is forbidden, and an extended electron conjugation system is formed synchronously . This conjugation system is involved in an internal charge transfer process from the ligand donor to the zn(ii) acceptor, and simultaneously inhibits the excited - state proton transfer and photo - induced electron transfer that strongly suppress the fluorescence of mwnts - gnq . Thus zn(ii) considerably enhances the fluorescence of mwnts - gnq . To further characterize the performance of the sensing material for zn(ii), a series of comparison experiments were carried out with mwnts - gnq . Because zn(ii) always coexists with cu(ii) or cd(ii), titration addition of 1 10 m cu(ii) and cd(ii) to mwnts - gnq solution containing zn(ii) when cu(ii) was added to the solution, it led to about 97.5% quenching of the total fluorescence intensity, probably because cu(ii) formed some complex with gnq group, resulting in quenching of fluorescence as reported . But cd(ii) almost had no influence on the fluorescence intensity of the mwnts - gnq solution containing zn(ii), because the electronic structure of cd(ii) is fairly similar to that of zn(ii). To resolve the problem of fluorescence quenching by cu(ii), a masking agent of na2s2o3 was chosen . As is well known that s2o3 can form a very stable complex with cu(ii), the coordination number is three, and na(i) almost has no influence to fluorescence intensity of mwnts - gnq . The results are shown in fig . 5, from which we can see that the masking agent almost does not affect the experimental results except in the case when the system contains zn(ii) and cu(ii). When three stoichiometry s2o3 were added to the zn(ii) and cu(ii) solution, the fluorescence intensity was greatly enhanced . Although, compared to the solution only containing zn(ii), the intensity was a little weaker, na2s2o3 is still a good masking agent to mask cu(ii). We thus conclude that the presence of cd(ii) does not affect the sensitivity of mwnts - gnq for zn(ii) detection, and na2s2o3 could be used as a masking agent when cu(ii) coexists in the system . Relative fluorescence intensity of mwnts - gnq (1 10 m) or mwnts - gnq (1 10 m) containing s2o3(3 10 m) in the presence of zn(ii) (1 10 m) and interfering ions with cu(ii) (1 10 m) or cd(ii) (1 10 m), respectively . Ex = 324 nm the sensitivity of fluorescence enhancing from mwnts - gnq by zn(ii) was further investigated, and the results are shown in fig . 6b), which suggested the 1:1 stoichiometry of the ligand to the zinc ions . Because zn(ii) desires a square planar geometry when coordinated, while the three nitrogen on mwnts - gnq can only provide a tridentate ligand, the fourth coordination can come from the solvent methanol oxygen . It can be seen from fig . 6b that when the concentration of zn(ii) is lower than 0.2 m, the relative fluorescence intensity is about 1 . But when the concentration of zn(ii) is higher than 0.2 m, the relative fluorescence intensity is also increased . It can be expressed by the following formula:(1) wherein, ais the value of the fluorescence intensity, cis the concentration of zn(ii), and the range ofcis from 0.2 m to 10 m in this study . So, it is evident that the detection limit for zn(ii) is established at 0.2 m under the experimental conditions in our study . Afluoresence spectra of mwnts - gnq (1 10 m) with zn(ii),brelative fluoresence intensity of mwnts - gnq at different concentration of zn(ii). We have prepared a new organic inorganic hybrid sensing material based on cnts as support and glycine - n-8-quinolylamide as fluorescent center . The results of the fluorescence characterization show that the composite has a highly selective and sensitive (0.2 m) detection for zn(ii), and reveal that ratiometric zn(ii) sensing is possible with fluorophore chemically modified carbon nanotubes . This novel fluorescent material may be used as a fluorescent device in intracellular environment for the detection of zn(ii).
Epigenetics are widely implicated in tumor initiation and progression by different modifications of dna and histones . There is a growing body of evidence demonstrating the importance of histone modification in silencing tumor suppressor genes . Among the various histone modifiers, histone acetyltransferases (hats) and histone deacetylases (hdacs) are two reversible enzymes regulating histone acetylation status, attaching or removing acetyl groups from the lysine residues in histone tails . Hats prevent the positive lysine residues from interacting more closely with the dna phosphate backbone, resulting in an open chromatin state, whereas the hdacs remove acetyl groups, resulting in a closed configuration, which blocks the access of the transcription machinery to dna, suppressing gene expression . To date, 18 mammalian hdac isoforms have been identified in humans; 11 of them (class i, ii, iv) are zn - dependent hdac enzymes (classical hdacs). Class i hdac, comprising hdac 1, 2, 3, and 8, are homologous to yeast rpd3 (reduced potassium dependency-3) protein; class ii members include hdac 4, 5, 6, 7, 9, and 10 and are structurally related to yeast hda 1 (histone deacetylases 1). Class iii hdacs, called sirtuins (sirt17), require nad for their activity . Hdac1 mainly localizes in the nucleus due to lack of the nucleus export signal . The knockout of hdac1 in mice caused proliferation defects and development retardation because of an arrest of cell growth that is associated with upregulation of the p21 and p27 (cyclin - dependent kinase inhibitors). A recent study showed that hdac1 largely accumulated in mature areas of differentiated tumor on the mice with teratoma, indicating that hdac1 is a possible biomarker of benign teratoma . Hdac3 is one of the most frequently upregulated genes in human cancers and is involved in each of the three major targets of cancer therapy: cell cycle control, differentiation, and apoptosis . There are many direct links between hdac3 and various tumor types . In colon cancer cells hct116 and caco-2, silencing of hdac3 expression resulted in growth inhibition, a decrease in cell survival, and increased apoptosis through stimulating p21 promoter activity and expression . In hela cells, the majority of cellular hdac3 is found to associate with smrt and n - cor complexes, and knockdown of hdac3 resulted in hyperacetylation of histone h3 and apoptosis . In metastatic breast cancer cell mda - mb-231, hdac3 efficiently inhibited creb3-enhanced nf-b activation . Moreover, high hdac3 expression is also associated with gastric cancer, glioma, renal cancer, liver cancer, and chronic lymphocytic leukemia . On the basis of the above evidence, developing hdacis more specifically against hdac1/3 may prove to be a worthwhile goal . In the past 10 years, over 490 clinical trials of more than 20 hdacis candidates have been initiated, culminating in the approval of two antitumor drugs vorinostat (saha) and romidepsin (fk228). Recently, development of class or isoform selective hdacis has drawn increased attention . Although selective hdacis were hypothesized to have fewer side effects than other pan - hdacis, their therapeutic advantages have yet to be confirmed clinically . Hdacis are classified into different classes depending on their chemical structures, namely, hydroxamates, benzamides, aliphatic acids, cyclic tetra peptides, electrophillic ketones, and miscellaneous groups, among which hydroxamates are considered as the most common hdacis . Although some class i selective, hdac6 (class iib) selective, and class iia selective hydroxamates inhibitors have been reported (figure 1), hydroxamates are generally thought to have limitation in selectivity against desired hdac isoforms due to their very strong chelating ability with zinc ion . To the best of our knowledge, most of the subclass i selective inhibitors in research are benzamides hdacis, such as ms-275 (30), mgcd0103 (31), ci994 (32), and so on (figure 1). Additionally, recent study demonstrated aryl substituents in the o - phenylenediamine of benzamide hdacis led to dramatic improvements in potency and selectivity for hdac1 and 2 versus hdac3 (figure 1). Recently, we have embarked on development of n - hydroxycinnamamide - based hdacis; in our previous study, compound (s, e)-tert - butyl (1-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)-2-methoxyphenoxy)-3-(1h - indol-3-yl)propan-2-yl)carbamate (14a) exhibited modest hdac inhibitory . Here in this article, we report the design, synthesis, and biological evaluation of a new series of selective hydroxymates hdacis describing their structure activity relationship (sar) studies, isotype - selectivity, and anticancer activities . Several compounds show an excellent effect in proliferation inhibition against u937, hel, kg1, hl60, k562, pc-3, a549, mda - mb-231, hct116, and mcf-7 cell lines . In vivo antitumor assay in u937 xenograft model showed compound 11r is a potent, orally active hdaci . We designed and synthesized a library of cinnamamide derivatives based on the lead compound 41 (14a) (figure 2). The library was designed to fully explore the influence of variation in the methoxy group of ferulic acid and the cap group . Synthesis of 3 was started with l - try by methyl ester protection and boc - protection . Reduction of 3 by lialh4 led to 4, which was further subjected to mitsunobu reaction with 6 and gave 7 . Intermediate 8, the n - deprotected product of 7, reacted with different carboxylic acids or boc - protected amino acids by tbtu - mediated amide formation to afford 9b9z . Compounds 9b9z were converted to hydroxamic acid compounds 11a11z by nh2ok in dry methanol . Reagents and conditions: (a) ch3cocl, ch3oh, 95%; (b) (boc)2o, et3n, ch2cl2, 90%; (c) lialh4, anhydrous thf, 86%; (d) pph3, dead, anhydrous thf, 55%; (e) acoet / hcl, 80%; (f) carboxylic acids or boc - protected amino acids, tbtu, et3n, anhydrous ch2cl2 (6065%); (g) nh2oh, koh, anhydrous ch3oh, (3040%). Compound 4 was treated with ch3i and phase transfer catalyst tbabr in h2o / thf containing koh to get the methyl protected 12 (scheme 2). Subsequent reaction of 12 with 6 through mitsunobu reaction in the presence of pph3 and dead afforded compound 13 . Finally, the hydroxamic acid group was achieved in usual conditions to get compound 14 . Reagents and conditions: (a) ch3i, koh, tbabr, h2o, thf, 50%; (b) pph3, dead, anhydrous thf, 55%; (c) nh2ok, anhydrous ch3oh, 30% . Scheme 3 shows the preparation of secondary amine 21a21c . Intermediate 8 was mixed with benzaldehyde, 4-chlorobenzaldehyde, or 4-fluorobenzaldehyde in anhydrous ch3oh, respectively, then nabh4 was added to give 18a18c . Reagents and conditions: (a) benzaldehyde (4-chlorobenzaldehyde or 4-fluorobenzal dehyde), et3n, nabh4, anhydrous ch3oh, 85%; (b) (boc)2o, et3n, ch3oh, 65%; (c) nh2ok, anhydrous ch3oh, 3040%; (d) acoet / hcl, 70% . Unlike scheme 1, scheme 4 used 3-hydroxycinnamic acid instead of 4-hydroxycinnamic acid to give another new series of compounds 27a27c . The reagents and conditions of scheme 4 are the same as those in scheme 1 . Reagents and conditions: (a) ch3cocl, ch3oh, 95%; (b) pph3, dead, anhydrous thf, 55%; (c) acoet / hcl, 80%; (d) carboxylic acids, tbtu, et3n, anhydrous ch2cl2 (6065%); (e) nh2oh, koh, anhydrous ch3oh, (3040%). In our previous study we found the lead compound 41 displayed modest inhibition toward hdacs . At the beginning of our present research, we made an attempt to find a new lead compound by modifying the r1 and r2 groups of 41 (table 1). We changed the r2 group of 41 by replacing the methoxy group with h to get compound 10a (scheme 1) and changed the r1 group of 10a with a methyl group to get compound 14 (scheme 2). We used hela cell extract as the enzyme source to efficiently screen our target compounds . The result in table 1 showed that compound 10a exhibited slightly increased hdac inhibitory activity compared with 41 and 14 . This indicated that the methoxy group in r2 and methyl group in r1 were not necessary, so we selected 10a as the lead compound for further optimization and modification . Assays were performed in replicate (n 2); data are shown as mean sd . In the following work, we designed an analogue to probe the effect of the n - substituent by replacing the boc group of 10a with other functional groups (scheme 1). First, we synthesized compounds 11a11o with different substituents including boc - protected amino acids, carboxylic acids, sulfo acids, and several anti - inflammatory acids (table 2). It was obvious that the compounds with the structure of acid amide substituents were more potent than those with sulfamide substituents (table 2). For example, the ic50 value of 11 g in hela extract was 1703.4 nm, much lower than compound 11h (> 5000 nm). The same result was also shown in compounds 11e and 11f . Additionally, compound 11i was more potent than 11j, which indicated that a short side alkyl chain might advance hdac inhibitory activity . Among compounds 11a11o, the most potent compounds, 11e and 11k, with ic50 values of 15.4 and 20.8 nm, respectively, were far more potent than saha (120.8 nm). The common element they all share is a simple benzamide as their n - substituent, so further modification focused on compound 11e . Assays were performed in replicate (n 2); data are shown as mean sd . Next, we synthesized 11p and 11q, which had different alkyl chain lengths relative to 11e . Activity data showed 11e with the shortest side chain had the best activity, which also agreed with the conclusion shown in 11i and 11j, that a short side alkyl chain may promote their activity . Then we synthesized compounds 11r11y (scheme 1) to probe the effect of substituent in benzene ring of benzamide (table 3). Surprisingly, the para - substituted compounds 11r, 11w, 11x, and 11y showed a marked increase in the hdac inhibition potency compared with 11e, the ic50 values of 11r, 11w, 11x, and 11y were 5.6, 6.7, 20.4, and 4.8 nm, respectively, however, the ortho - substituted, metha - substituted, and disubstituted compounds 11s, 11 t, 11u, and 11v exhibited inferior activity relative to 11e . The result showed para - substituents on a benzene ring have an obvious promoting effect on hdac inhibition . Assays were performed in replicate (n 2); data are shown as mean sd . Scheme 4 was designed to evaluate the activity of 3-hydroxycinnamide - based series . We modified some of the most potent compounds, 11e, 11r, and 11w, to achieve compounds 27a27c . The hdac inhibition result in table 4 showed compounds 27a27c exhibited poor activity compared with 11e, 11r, and 11w, which revealed that 4-hydroxycinnamide - based series were superior to the 3-hydroxycinnamide - based series . Then 21a21c were designed to improve water solubility and chemical stability by replacing the amide of 11e, 11r, and 11w with a secondary amine . Compounds 21a21c showed mild inhibition, and the activity was 15 times lower than 11e, 11r, and 11w . The possible explanation for why compounds 11r, 21c, and 27c with similar structure exhibited quite different activity was described in the molecule docking section . Assays were performed in replicate (n 2); values are shown as mean sd . In order to explore the hdac isoform selectivity profile, we chose the most potent compounds 11e, 11p, 11r, 11w, and 11y described in the above discussion, as well as secondary amine analogue 21c and 3-hydroxycinnamide - based analogue 27c to conduct enzyme inhibitory assays against hdac1, hdac2, hdac3, and hdac6 . Results presented in table 5 reveal that compounds 11e, 11r, 11w, and 11y exhibited dual selectivity against hdac1/3, among which compound 11r exhibited the most obvious dual selectivity, particularly against hdac3 . Ic50 value of 11r against hdac3 was 3.9 nm, which was 80-fold and 130-fold lower than that of hdac6 and hdac2, respectively . The positive control, saha, had almost no selectivity toward these hdac isoforms . To ascertain the selectivities of our compounds across the broader family of hdac isoforms, we next profiled representative 11r against hdac8 (class i), hdac4 (class iia), and hdac11 (class iv). Compound 11r also displayed low micromolar activity against hdac8, hdac4, and hdac11 compared to the low nanomolar activity against hdac1 and hdac3 (table 6). Assays were performed in replicate (n 2); the sd values are <20% of the mean . Assays were performed in replicate (n 2); the sd values are <20% of the mean . Compounds 11e, 11p, 11r, 11w, and 11y with the most potent hdacs inhibitory activity were selected to test their antiproliferative activity against 10 types of solid or hematological tumor cell lines, which were most frequently used in evaluating hdacis (table 7). Data showed cell lines hl60, mcf-7, and a549 had lower sensitivity with our tested compounds than the other cell lines . All the tested compounds exhibited much more superior antiproliferative potency than saha against u937, k562, hel, kg1, mda - mb-231, pc-3, and hct116 cells . Compounds 11e, 11r, 11w, and 11y, which displayed the highest hdac enzyme inhibitory potency, also exhibited the best antiproliferative activity . Among these analogues, compound 11r exhibited the highest potency, and human leukemic monocyte lymphoma u937 seemed to be the most sensitive cell line to our hdacis . Furthermore, cytotoxicity of 11r against human umbilical vein endothelial cells (huvec) was tested in our lab, and the ic50 value was 61.48 6.17 m, which revealed our compounds selectivity over nontransformed cells when compared with tumor cells . Assays were performed in replicate (n 2); the sd values are <20% of the mean . Although the hdac inhibitory activity of our hdacis was confirmed in cell - free assays, the results were not as clear in cell based assays . In order to investigate the hdac inhibitory activity of our hdacis in cells further, we chose compounds that were used in evaluating hdac isoform - selectivity to conduct western blot assay in u937 cell . Intracellular levels of acetylated histoneh3, acetylated histoneh4 (known substrates for hdac1, 2, and 3), and acetylated -tubulin (a known substrate for hdac6) were compared with saha and lbh589 (one of the most potent pan - hdacis) as reference compounds (figure 3). Results show compounds 11e, 11p, 11r, 11w, and 11y can markedly increase the level of acetylated histoneh3 and acetylated histoneh4, which were much superior to saha, while comparable to lbh589 . However, the levels of acetylated -tubulin of these compounds, especially 11r, were much lower than lbh589 . These data demonstrated that some of our compounds showed lower potency against hdac6 than class i hdacs, which is in agreement with the isoform selectivity data observed in cell - free assays . Western blot analysis of acetylated tubulin, acetylated histone h3, acetylated histone h4, and pro - caspase3 in u937 cell lines after 24 h treatment with compounds at 1 m using saha (sa) and lbh589 (lbh) as positive control . As our compounds displayed hdac1/3 dual selectivity and some studies reported that knockdown of hdac1 and hdac3 could increase the percent of apoptotic cells in carcinoma cells, we also evaluated the level of procaspase3 (inactive form of apoptotic effector caspase3) in u937 cells (figure 4). Cleavage of pro - caspases3 results in the production of an active effector, caspase3, which can cleave essential structural proteins such as cytokeratins, nuclear lamins, and also an inhibitor of caspase - activated dnase (icad), which liberates the dnase (cad) to digest chromosomal dna and cause cell death . We can see from the result that our compounds 11e, 11k, 11r, 11w, and 11y dramatically reduced the level of procaspase3, which was most likely due to the fact that procaspase3 was cleaved to the active form (caspase3), which could promote apoptosis of u937 cells . At the same time, we performed flow cytometry analysis to confirm the effect of these compounds in inducing apoptosis . In this assay, we chose compounds 11r and 11w as representative compounds and saha as the positive control . Results demonstrated that 11r, 11w, and saha can significantly induce apoptosis in a time - dependent and dose - dependent manner . At the same concentration (1 m), 11r and 11w induced 61.76% and 64.25% cell apoptosis, respectively, which were much higher than that of saha (26.83%). Detailed activity evaluation and apoptosis mechanism studies are currently underway in our laboratory . Induction of apoptosis at 12 and 24 h by compounds 11r, 11w, and saha in different concentrations in u937 cells . Having demonstrated that 11r had ideal in vitro activity, we next set out to assess its tumor growth inhibitory effects in subcutaneous cell lines and primary tumor derived xenograft models . Before we conducted the in vivo study, we developed hplc methods to briefly study the stability of 11r in artificial gastric juice, artificial intestinal juice, rat liver homogenate, and human plasma . Compound 11r was incubated under each condition at 37 c for 24 h, extracted, and analyzed by hplc . We observed that 11r was stable in artificial gastric juice, artificial intestinal juice, rat liver homogenate, and human plasma (figure 5). Stability of compound 11r in artificial gastric juice, artificial intestinal juice, rat liver homogenate, and human plasma . Points were achieved after 0, 1, 2, 4, 6, 8, and 24 h, respectively, and values are shown as mean sd . Because of the great stability of 11r, it was feasible to evaluate its in vivo activity orally . U937 cells (5 10) were subcutaneously implanted in the right flanks of female nude mice (balb / c - nu). When tumor size reached about 100 mm, mice were randomized to five per group and were treated with compound 11r or saha (100 mg / kg / day) by oral gavage for 16 days . Tumor growth inhibition (tgi) and relative increment ratio (t / c) were calculated at the end of treatment to reveal the antitumor effects in tumor weight and tumor volume, respectively . Compound 11r demonstrated potent in vivo oral antitumor activity with higher tgi value (55.1%) and lower t / c value (37%) than saha (tgi = 32.1%; t / c = 47%). In the mice group treated by 11r, no significant body weight loss and no evident toxic signs in liver and spleen were detected (figures 6 and 7). Antitumor activity comparison of 11r and saha against u937 human tumor xenografts implanted in mice, expressed as mean tumor volume . Figure 8a revealed the docked conformation of compounds 11r, 21c, and 27c . The docked conformations of 11r and 21c were very similar in the linker and zbg, while they were quite different in the aromatic ring and indole ring of cap group . Moreover, conformation of 27c was different from 11r and 21c, leading to diverse binding modes shown in figure 8c e . Figure 8c, d showed 11r and 21c had similar docking mode and hydrogen bonds; the greater potency of 11r might be amenable to the stiffening of the scaffold introduced by the amide bond, which forces the lipophilic p - chlorophenyl moiety to yield closer van der waals interactions with the protein rather than projecting itself toward solvent as in 21c . Furthermore, in cap group, hydrogen bonds formed between 21c and residues asp92 and asp93 had a longer bond length (2.19 and 1.97) than 11r (1.95 and 1.59), which also might be factors that made 11r more effective against hdac3 than 21c . Compared with 11r (figure 8c), compound 27c (figure 8e) could form two diverse hydrogen bonds with amide n h of phe200 and imidazole n h of his172 through carbonyl oxygen atom and etheric oxygen atom, respectively, but at the same time, 27c lost two hydrogen bonds with asp92 in the cap group and tyr298 around zinc ion . In addition, the carbonyl oxygen of 27c had the same distance with 11r to zinc ion (2.1), but the hydroxyl oxygen of 27c had a longer distance to the zinc ion (4.4) than 11r (1.9), which indicated that 27c could only form a weak monodentate interaction with zinc, while 11r could form a strong bidentate interaction with zinc . This might be the reason why 27c was much less potent than 11r (figure 8b). (a) docked conformational alignment modeling of compounds 11r, 21c, and 27c . For clarity, hdac3 (derived by modification of pdb code 4a69 using tripos sybyl 8.0) used for docking is not shown . (b) proposed docked mode of 27c and 11r for binding zinc . (c e) proposed binding preferences comparison of compounds 11r (c), 21c (d), and 27c (e) at hdac3 . The zinc ion is shown as a red sphere, and the dashed lines stand for the hydrogen bonds (atom types: h, white; n, blue; o, red). For the first time, n - hydroxycinnamamide - based derivatives have been shown to be potent dual hdac1/3 selective hdacis . The representative compound 11r showed low nanomolar ic50 values against hdac1 (11.8 nm) and hdac3 (3.9 nm), with micromolar or submicromolar ic50 values against hdac2, hdac8, hdac4, hdac6, and hdac11 . In addition, a few of the examples of the new series in antiproliferative study exhibited high potency against several solid or hematological cells, even though antiproliferative activity of the compounds seemed inferior to their hdac inhibition activity . For example, antiproliferative activity of 11r was about 10 times higher than saha, while its hdac inhibition activity was about 100 times higher than saha . We supposed 11r had poor transcellular permeability; this speculation was confirmed by parallel artificial membrane permeation assay (pampa) conducted on 11r and saha (data not shown). Compound 11e, 11r, 11w, and 11y increased histone h3 and h4 acetylation in the same level of lbh589 (one of the most potent hydroximates hdacis in clinical) in u937 cell line . At the same time, some of the compounds decreased the level of pro - caspase3, which was consistent with the result of flow cytometry analysis in inducing apoptosis . Compound 11r was evaluated for its in vivo antitumor study in a u937 xenograft mice model and showed higher efficacy compared to the approved drug saha . More detailed studies of the antitumor profiles of these promising compounds are underway in our lab . Using 11r as lead, continued studies are underway to search for more promising hdacis with improved transcellular permeability and isoform selectivity . H nmr spectra were obtained on a bruker drx spectrometer at 300 mhz with tms as an internal standard, in parts per million, and j in hertz . High - resolution mass spectrometry was performed by shandong analysis and test center in jinan, china . All reactions were monitored by tlc using 0.25 mm silica gel plates (60gf-254). Flash chromatography was accomplished using the automated combiflash rf system from teledyne isco and was done using silica gel of 200300 mesh . All tested compounds are> 95% pure by hplc analysis, performed on an agilent 1100 hplc instrument using an ods hypersil column (5 m, 4.6 mm 250 mm) according to one of the following methods . Method a: compounds 10a, 11a11x, and 27a27c were eluted with 35% acetonitrile/65% water (containing 0.4% formic acid)65% acetonitrile/35% water (containing 0.4% formic acid) over 20 min, with detection at 290 nm and a flow rate of 1 ml / min . M potassium dihydrogen phosphate / phosphoric acid (ph = 3.0) over 20 min, with detection at 290 nm and a flow rate of 1 ml / min . The temperature of the column was 25 c, and quantity of injection was 20 m . (s)-methyl 2-amino-3-(1h - indol-3-yl)propanoate hydrochloride (2), (s)-methyl 2-((tert - butoxycarbonyl)amino)-3-(1h - indol-3-yl)propanoate(3), and (s)-tert - butyl(1-hydroxy-3-(1h - indol-3-yl)propan-2-yl)carbamate (4) were synthesized as described previously . P - coumaric acid (16.4 g, 100mmol) was dissolved in 200 ml of meoh, then acetyl chloride (24 g, 300mmol) was added dropwise at 0 c; the mixed solution was refluxed at 75 c for 5 h. the solvent was evaporated under vacuum; the product was dissolved with etoac, washed by saturated nahco3 solution (2 100 ml), 1 m aqueous citric acid (2 100 ml), and brine (2 100 ml), dried over mgso4 overnight, and evaporated under vacuum to obtained product compound 6, a white solid (15.6 g, 87.5%). H nmr (300 mhz, dmso - d6) 3.69 (s, 3h), 6.43 (d, j = 16.2 hz, 1h), 6.81 (d, j = 8.7 hz, 2h), 7.547.59 (m, 3h), 10.01 (s, 1h). H nmr (300 mhz, dmso - d6) 3.72 (s, 1h), 6.54 (d, j = 15.9 hz, 1h), 6.836.86 (m, 1h), 7.047.05 (m, 1h), 7.15 (d, j = 7.5 hz, 1h), 7.207.25 (m, 1h), 7.60 (d, j = 15.9 hz, 1h), 7.62 (s, 1h). Esi - ms m / z: 179.2 [m + h]. Compound 4 (2.9 g, 10mmol) was taken up in thf (50 ml), then (ch3ch2ch2ch2)4n(br) (tbabr, 0.5 g) was added followed by ch3i (3.5 g, 25mmol). The reaction was stirred overnight, thf was evaporated under vacuum, and the residue was extracted with etoac (3 50 ml). The organic layer was washed with saturated nahco3 solution (2 100 ml), 1 m aqueous citric acid (2 100 ml), and brine (2 100 ml) and dried over mgso4 overnight . H nmr (300 mhz, dmso - d6) 1.35 (s, 9h), 2.602.73 (m, 1h), 2.842.91 (m, 1h), 3.223.40 (m, 2h), 3.583.67 (m, 1h), 3.72 (s, 3h), 4.64 (t, j = 5.4 hz, 1h), 6.53 (d, j = 8.1 hz, 1h), 7.697.07 (m, 2h), 7.15 (t, j = 7.8 hz, 1h), 7.37 (d, j = 8.1 hz, 1h), 7.60 (d, j = 7.8 hz, 1h). Compound 4 (5.8 g, 20mmol), 6 (4.2 g, 24mmol), and pph3 (7.9 g, 30mmol) were dissolved in anhydrous thf, and then dead (5.2 g, 30mmol) was added dropwise at 0 c; the mixture was stirred for 4 h, and then thf was evaporated . The crude material was purified via flash chromatography to afford the compound 7, a white solid (5.4 g, 60%). H nmr (300 mhz, dmso - d6) 1.36 (s, 9h), 2.853.00 (m, 2h), 3.70 (s, 3h), 3.954.07 (m, 3h), 6.51 (d, j = 15.9 hz, 1h), 6.936.99 (m, 4h), 7.08 (t, j = 7.4 hz, 1h), 7.13 (d, j = 2.1 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.56 (d, j = 7.8 hz, 1h), 7.63 (d, j = 15.9 hz, 1h), 7.66 (d, j = 8.4 hz, 2h), 10.82 (d, j = 1.5 hz, 1h). Compound 7 was dissolved in a solution of etoac (8 ml) saturated by dry hcl gas . The filtered precipitate was washed by diethyl ether to give the compound 8, a white solid powder (3.8 g, 83%). H nmr (300 mhz, dmso - d6) 3.103.24 (m, 2h), 3.71 (s, 3h), 3.713.75 (m, 1h), 4.07 (dd, j = 5.7 hz, j = 10.5 hz, 1h), 4.21 (dd, j = 5.7 hz, j = 10.5 hz, 1h), 6.53 (d, j = 15.9 hz, 1h), 6.977.01 (m, 3h), 7.11 (t, j = 6.9 hz, 1h), 7.27 (d, j = 2.4 hz, 1h), 7.38 (d, j = 8.1 hz, 1h), 7.597.64 (m, 2h), 7.70 (d, j = 8.7 hz, 2h), 8.40 (s, 2h), 11.05 (s, 1h). Esi - ms m / z: 351.5 [m + h]. Pale yellow oil . Esi - ms m / z: 351.5 [m + h]. To a solution of p - toluene sulfonic acid (0.34 g, 2mmol) in anhydrous ch2cl2 was added 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (tbtu, 0.71 g, 2.2mmol), followed by et3n (0.3 g, 3mmol). After 30 min, compound 8 (0.85 g, 2.2mmol) was added followed by tea (0.3 g, 3mmol). Four hours later, the solution of ch2cl2 was washed with 1 n hcl (2 30 ml), saturated na2co3 (2 30 ml), and brine (2 30 ml), dried over mgso4 overnight, and the solvent was evaporated under vacuum . The crude product was purified by recrystallization with etoac and petroleum ether to achieve a white pure solid (0.51 g, 50% yield). H nmr (300 mhz, dmso - d6) 2.30 (s, 3h), 2.85 (dd, j = 6.0 hz, j = 14.4 hz, 1h), 3.01 (dd, j = 8.1 hz, j = 14.4 hz, 1h), 3.603.65 (m, 1h), 3.70 (s, 3h), 3.86 (d, j = 4.8 hz, 2h), 6.50 (d, j = 16.2 hz, 1h), 6.76 (d, j = 8.7 hz, 2h), 6.91 (t, j = 7.4 hz, 1h), 7.007.06 (m, 2h), 7.20 (d, j = 8.1 hz, 2h), 7.247.30 (m, 2h), 7.567.62 (m, 5h), 7.99 (d, j = 7.2 hz,1h), 10.77 (d, j = 1.5 hz, 1h). H nmr (300 mhz, dmso - d6) 0.730.80 (m, 6h), 0.961.00 (m, 2h), 1.041.11 (m, 4h), 1.301.42 (m, 2h), 2.102.17 (m, 1h), 2.863.03 (m, 2h), 3.70 (s, 3h), 3.984.07 (m, 2h), 4.344.42 (m, 1h), 6.51 (d, j = 15.9 hz, 1h), 6.926.95 (m, 3h), 7.07 (t, j = 7.5 hz, 1h), 7.14 (d, j = 2.4 hz, 1h), 7.33 (d, j = 7.8 hz, 1h), 7.58 (d, j = 9.0 hz, 1h), 7.64 (d, j = 17.0 hz, 1h), 7.67 (d, j = 9.0 hz, 2h), 7.94 (d, j = 7.8 hz, 1h), 10.82 (d, j = 1.5 hz, 1h). H nmr (300 mhz, dmso - d6) 2.08 (s, 3h), 3.27 (s, 3h), 3.083.16 (m, 2h), 3.70 (s, 3h), 4.114.22 (m, 2h), 4.554.64 (m, 1h), 6.49 (d, j = 15.9 hz, 1h), 6.74 (t, j = 7.8 hz, 1h), 6.83 (d, j = 7.8 hz, 1h), 6.916.99 (m, 4h), 7.037.09 (m, 3h), 7.207.26 (m, 2h), 7.34 (d, j = 8.1 hz, 1h), 7.577.64 (m, 5h), 8.57 (d, j = 7.8 hz, 1h), 9.39 (s, 1h), 10.84 (d, j = 1.5 hz, 1h). H nmr (300 mhz, dmso - d6) 2.903.06 (m, 2h), 3.43 (s, 2h), 3.71 (s, 3h), 4.01 (d, j = 5.4 hz, 2h), 4.274.35 (m, 1h), 6.52 (d, j = 15.9 hz, 1h), 6.916.96 (m, 3h), 7.08 (t, j = 7.5 hz, 1h), 7.10 (d, j = 2.1 hz, 1h), 7.177.27 (m, 5h), 7.34 (d, j = 8.1 hz, 1h), 7.57 (d, j = 7.8 hz, 1h), 7.64 (d, j = 16.2 hz, 1h), 7.67 (d, j = 8.7 hz, 2h), 8.30 (d, j = 7.8 hz, 1h), 10.83 (d, j = 1.5 hz, 1h). The solution of compound 8 (0.76 g, 2mmol) in ch3oh was added et3n (0.22 g, 2.2mmol) for neutralization, followed by the addition of methanol (0.21 g, 2mmol); the resulting solution was stirred for 2 h and the solvent evaporated . The residue was dissolved in anhydrous ch3oh, nabh4 (0.15 g, 4mmol) was added slowly at 0 c, the mixture was stirred at room temperature overnight, and after the reaction finished, the reaction was quenched by adding ice water slowly . Ch3oh was evaporated, and the residue was extracted by etoac (3 30 ml). The organic layer was washed with brine (2 30 ml) and saturated na2co3 (2 30 ml) and dried over mgso4 overnight . The solvent was evaporated under vacuum to get crude product 11a, a colorless oil (0.79 g, 90%). H nmr (300 mhz, dmso - d6) 2.95 (d, j = 6.3 hz, 2h), 3.143.18 (m, 1h), 3.70 (s, 3h), 3.87 (s, 2h), 3.94 (d, j = 5.1 hz, 2h), 6.50 (d, j = 15.9 hz, 1h), 6.92 (t, j = 7.5 hz, 1h), 6.95 (d, j = 8.7 hz, 2h), 7.06 (t, j = 7.4 hz, 1h), 7.14 (d, j = 2.4 hz, 1h), 7.167.22 (m, 1h), 7.267.33 (m, 5h), 7.43 (d, j = 7.8 hz, 1h), 7.63 (d, j = 15.9 hz, 1h), 7.65 (d, j = 8.7 hz, 2h), 8.99 (s, 1h), 10.83 (s, 1h). Et3n (0.61 g, 6mmol) followed by (boc)2o (0.78 g, 3.6mmol) were added to the solution of compound 11a (0.79 g, 1.8mmol) in ch2cl2 . The resulting solution was washed by saturated nahco3 solution (2 30 ml), 1 m aqueous citric acid (2 30 ml), and brine (2 30 ml) and dried over mgso4 overnight . The solvent was evaporated under vacuum to get crude product 12a, a colorless oil (0.92 g, 95%). H nmr (300 mhz, dmso - d6) 1.431.47 (m, 9h), 2.903.16 (m, 2h), 3.70 (s, 3h), 4.004.07 (m, 2h), 4.314.37 (m, 2h), 4.56 (m, 1h), 6.49 (d, j = 16.2 hz, 1h), 6.776.80 (m, 2h), 6.98 (t, j = 7.8 hz, 1h), 7.09 (m, 2h), 7.127.17 (m, 1h), 7.207.24 (m, 4h), 7.35 (d, j = 7.8 hz, 1h), 7.417.47 (m, 1h), 7.567.64 (m, 3h), 10.84 (s, 1h). Koh (28 g, 509mmol) and nh2ohhcl (23.35 g, 343mmol) were dissolved, respectively, in 70 and 120 ml of meoh to get solution a and solution b. next, solution a was added dropwise to solution b. after filtering the precipitate (kcl), a nh2ok solution was obtained . Compound 7 (0.45 g, 1 mmol) was dissolved in the nh2ok solution and stirred overnight . The residue was acidified with 1 n hcl to a ph 34 and then extracted with etoac (3 20 ml). The organic layer was washed with brine (2 30 ml) and dried over na2so4 overnight . The crude material was purified via flash chromatography to afford the compound 10a (0.15 g, 30% yield). H nmr (300 mhz, dmso - d6) 1.37 (s, 9h), 2.793.00 (m, 2h), 3.904.04 (m, 3h), 6.33 (d, j = 15.6 hz, 1h), 6.927.00 (m, 4h), 7.08 (td, j = 0.9 hz, j = 7.6 hz, 1h), 7.13 (d, j = 1.8 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.42 (d, j = 15.6 hz, 1h), 7.48 (d, j = 8.7 hz, 2h), 7.56 (d, j = 7.8 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c25h29n3o5 [m + h] 452.2180, found 452.2182 . Retention time: 18.9 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.39 (s, 9h), 2.853.10 (m, 2h), 3.483.57 (m, 2h), 3.96 (m, 2h), 4.314.35 (m, 1h), 6.33 (d, j = 15.9, 1h), 6.996.90 (m, 4h), 7.08 (t, j = 6.9 hz, 1h), 7.14 (d, j = 2.1 hz, 1h), 7.33 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.59 (d, j = 7.8 hz, 1h), 7.98 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.85 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h32n4o6 [m + h] 509.2395, found 509.2392 . Retention time: 7.4 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.37 (s, 9h), 1.681.82 (m, 2h), 2.42 (t, j = 7.8 hz, 2h), 2.973.04 (m, 2h), 3.36 (s, 3h), 3.904.05 (m, 3h), 4.254.42 (m, 1h), 6.32 (d, j = 15.6 hz, 1h), 6.906.97 (m, 4h), 7.08 (t, j = 6.9 hz, 1h), 7.20 (d, j = 2.1 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.41 (d, 15.9 hz, 1h), 7.48 (d, 8.4 hz, 2h), 7.60 (d, j = 7.8 hz, 1h), 7.99 (d, j = 7.5 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h), 10.85 (s, 1h). Hrms (ap - esi) m / z calcd for c30h38n4o6s [m + h] 583.2585, found 583.2486 . Retention time: 10.55 min, eluted with 47% acetonitrile/53% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.36 (s, 9h), 2.913.05 (m, 2h), 3.513.54 (m, 2h), 3.934.02 (m, 3h), 4.314.38 (m, 1h), 4.84 (t, j = 6.3 hz, 1h), 6.33 (d, j = 15.6 hz, 1h), 6.65 (d, 8.1 hz, 1h), 6.926.97 (m, 3h), 7.08 (t, j = 6.9 hz, 1h), 7.16 (d, j = 2.1 hz, 1h), 7.34 (d, j = 8.4 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.59 (d, j = 7.8 hz, 1h), 7.96 (d, j = 8.1 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.85 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c28h34n4o7 [m + h] 539.2500, found 539.2496 . Retention time: 5.3 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.01 (d, j = 5.7 hz, 3h), 1.37 (s, 9h), 2.923.05 (m, 2h), 3.863.94 (m, 4h), 4.344.36 (m, 1h), 4.78 (d, j = 5.4 hz, 1h), 6.276.35 (m, 2h), 6.916.97 (m, 3h), 7.08 (t, j = 7.2 hz, 1h), 7.16 (d, j = 1.8 hz, 1h), 7.317.48 (m, 4h), 7.60 (d, j = 7.8 hz, 1h), 7.96 (d, j = 7.8 hz, 1h), 8.96 (s, 1h), 10.65 (s, 1h), 10.83 (s, 1h). Hrms (ap - esi) m / z calcd for c29h36n4o7 [m + h] 553.2657, found 553.2658 . Retention time: 6.5 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.12 (d, j = 6.6 hz, 2h), 4.044.21 (m, 2h), 4.544.65 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.936.99 (m, 3h), 7.08 (td, j = 0.9 hz, j = 7.4 hz, 1h), 7.19 (d, j = 2.1 hz, 1h), 7.34 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.437.55 (m, 5h), 7.64 (d, j = 7.8 hz, 1h), 7.85 (d, j = k7.5 hz, 2h), 8.53 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h25n3o4 [m + h] 456.1918, found 456.1916 . Retention time: 12.6 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 2.31 (s, 3h), 2.85 (dd, j = 6.0 hz, j = 14.4 hz, 1h), 3.01 (dd, j = 8.1 hz, j = 14.4 hz, 1h), 3.603.62 (m, 1h), 3.84 (d, j = 4.8 hz, 2h), 6.32 (d, j = 15.9 hz, 1h), 6.76 (d, j = 8.7 hz, 2h), 6.92 (td, j = 0.9 hz, j = 7.4 hz, 1h), 7.007.06 (m, 2h), 7.21 (d, j = 8.1 hz, 2h), 7.27 (d, j = 10.2 hz, 1h), 7.30 (d, j = 8.1 hz, 1h), 7.41 (d, j = 15.9 hz, 1h), 7.44 (d, j = 8.7 hz, 2h), 7.59 (d, j = 8.4 hz, 2h), 8.00 (d, j = 6.9 hz, 1h), 8.99 (s, 1h), 10.66 (s, 1h), 10.78 (d, j = 1,8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h27n3o5s [m + h] 506.1715, found 506.1744 . Retention time: 22.9 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.14 (d, j = 6.9 hz, 2h), 3.994.12 (m, 1h), 4.184.28 (m, 1h), 4.564.67 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.947.00 (m, 3h), 7.09 (t, j = 7.2 hz, 1h), 7.20 (d, j = 2.1 hz, 1h), 7.35 (d, j = 8.1 hz, 1h), 7.377.43 (m, 2h), 7.477.52 (m, 4h), 7.66 (d, j = 7.8 hz, 1h), 7.727.78 (m, 4h), 7.96 (d, j = 8.4 hz, 2h), 8.60 (d, j = 6.9 hz, 1h), 10.67 (s, 1h), 10.84 (s, 1h). Hrms (ap - esi) m / z calcd for c33h29n3o4 [m + h] 532.2192, found 532.2231 . Retention time: 5.0 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 2.852.91 (m, 1h), 2.983.05 (m, 1h), 3.643.75 (m, 1h), 3.823.91 (m, 1h), 6.29 (d, j = 15.9 hz, 1h), 6.75 (d, j = 8.7 hz, 2h), 6.91 (t, j = 7.8 hz, 1h), 7.01 (t, j = 7.8 hz, 1h), 7.10 (d, j = 2.1 hz, 1h), 7.24 (d, j = 8.1 hz, 1h), 7.307.45 (m, 5h), 7.487.53 (m, 2h), 7.647.68 (m, 4h), 7.77 (d, j = 8.4 hz, 2h), 8.17 (d, j = 7.2 hz, 1h), 8.99 (s, 1h), 10.65 (s, 1h), 10.80 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c32h29n3o5s [m + h] 568.1861, found 568.1901 . Retention time: 5.1 min, eluted with 65% acetonitrile/35% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 0.69 (t, j = 7.2 hz, 3h), 0.79 (t, j = 7.5 hz, 3h), 1.231.47 (m, 4h), 1.912.02 (m, 1h), 2.903.04 (m, 2h), 3.99 (d, j = 5.4 hz, 2h), 4.364.44 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.95 (d, 8.4 hz, 2h), 6.98 (td, j = 0.9 hz, j = 7.1 hz, 1h), 7.08 (td, j = 0.9 hz, j = 7.6 hz, 1h), 7.14 (d, j = 2.1 hz, 1h), 7.33 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.6 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.59 (d, j = 7.8 hz, 1h), 7.97 (d, j = 8.1 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h), 10.82 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c26h31n3o4 [m + h] 450.2387, found 450.2389 . Retention time: 10.9 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 0.730.80 (m, 6h), 1.971.20 (m, 6h), 1.301.46 (m, 2h), 2.122.18 (m, 1h), 2.863.03 (m, 2h), 3.98 (d, j = 5.1 hz, 2h), 4.344.42 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.95 (d, j = 8.7 hz, 2h), 6.98 (td, j = 0.9 hz, j = 6.8 hz, 1h), 7.08 (td, j = 0.9 hz, j = 7.2 hz, 1h), 7.14 (d, j = 2.1 hz, 1h), 7.33 (d, j = 7.8 hz, 1h), 7.42 (d, j = 15.6 hz, 1h), 7.49 (d, j = 8.7 hz, 2h), 7.58 (d, j = 7.8 hz, 1h), 7.63 (d, j = 9.0 hz, 1h), 7.94 (d, j = 8.1 hz, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c28h35n3o4 [m + h] 478.2699, found 478.2699 . Retention time: 24.5 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.14 (d, j = 6.9, 2h), 4.084.22 (m, 2h), 4.594.70 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.866.91 (m, 2h), 6.967.00 (m, 3h), 7.09 (t, j = 7.4 hz, 1h), 7.18 (d, j = 2.1 hz, 1h), 7.35 (d, j = 8.1 hz, 1h), 7.377.42 (m, 2h), 7.49 (d, j = 8.4 hz, 2h), 7.64 (d, j = 7.8 hz, 1h), 7.92 (dd, j = 1.5 hz, j = 8.1 hz, 1h), 8.87 (d, j = 8.1 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.85 (d, j = 1.8 hz, 1h), 12.37 (s, 1h). Hrms (ap - esi) m / z calcd for c27h25n3o5 [m + h] 472.1867, found 472.1868 . Retention time: 17.2 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 2.08 (s, 3h), 2.27 (s, 3h), 3.053.12 (m, 2h), 4.084.20 (m, 2h), 4.554.67 (m, 1h), 6.32 (d, j = 15.9 hz, 1h), 6.74 (t, j = 7.5h, 1h), 6.83 (d, j = 8.4 hz, 1h), 6.916.98 (m, 4h), 7.037.09 (m, 3h), 7.237.26 (m, 2h), 7.34 (d, j = 8.1 hz, 1h), 7.41 (d, j = 15.9 hz, 1h), 7.47 (d, j = 8.1 hz, 2h), 7.64 (t, j = 7.2 hz, 2h), 8.58 (d, j = 8.1 hz, 1h), 8.97 (s, 1h), 9.40 (s, 1h), 10.65 (s, 1h), 10.84 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c35h34n4o4 [m + h] 534.1023, found 575.2653 . Retention time: 10.2 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 0.810.86 (m, 6h), 1.241.31 (m, 3h), 1.711.85 (m, 1h), 2.352.41 (m, 2h), 2.843.05 (m, 2h), 3.593.62 (m, 1h), 3.92 (m, 1h), 3.963.98 (m, 1h), 4.264.36 (m, 1h), 6.33 (dd, j = 4.5 hz, j = 15.9 hz, 1h), 6.847.08 (m, 6h), 7.137.19 (m, 3h), 7.427.61 (m, 5h), 8.14 (t, j = 6.4 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h), 10.83 (s, 1h). Hrms (ap - esi) m / z calcd for c33h37n3o4 [m + h] 540.2857, found 540.2856 . Retention time: 7.7 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.351.42 (m, 3h), 2.893.08 (m, 2h), 3.743.80 (m, 1h), 3.853.86 (m, 3h), 3.924.00 (m, 2h), 4.294.39 (m, 1h), 6.31 (d, j = 15.9 hz, 1h), 6.84 (d, j = 8.7 hz, 2h), 6.947.09 (m, 3h), 7.117.51 (m, 8h), 7.62 (d, j = 7.8 hz, 1h), 7.677.75 (m, 3h), 8.23 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.85 (s, 1h). Hrms (ap - esi) m / z calcd for c36h33n3o5 [m + h] 564.2493, found 564.2495 . H nmr (300 mhz, dmso - d6) 2.903.06 (m, 2h), 3.43 (s, 2h), 3.99 (d, j = 5.1 hz, 2h), 4.274.38 (m, 1h), 6.34 (d, j = 15.9 hz, 1h), 6.916.97 (m, 3h), 7.08 (td, j = 0.9, j = 7.4 hz, 1h), 7.11 (d, j = 2.1 hz, 1h), 7.177.28 (m, 5h), 7.35 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.50 (d, j = 8.7 hz, 2h), 7.58 (d, j = 7.8 hz, 1h), 8.31 (d, j = 7.8 hz, 1h), 8.99 (s, 1h), 10.66 (s, 1h), 10.84 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c28h27n3o4 [m + h] 470.2074, found 470.2074 . Retention time: 12.8 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 2.41 (t, j = 8.1 hz, 2h), 2.81 (t, j = 7.8 hz, 2h), 2.853.02 (m, 2h), 3.94 (d, j = 5.1 hz, 2h), 4.284.38 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.96 (d, j = 8.7 hz, 2h), 6.07 (t, j = 6.9 hz, 1h), 7.037.09 (m, 2h), 7.137.35 (m, 5h), 7.34 (d, j = 7.8 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.50 (d, j = 8.7 hz, 2h), 7.58 (d, j = 7.8 hz, 1h), 8.06 (d, j = 8.1 hz, 1h), 8.99 (s, 1h), 10.67 (s, 1h), 10.83 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c29h29n3o4 [m + h] 484.2236, found 484.2231 . Retention time: 17.4 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.11 (d, j = 6.6 hz, 2h), 4.064.11 (m, 1h), 4.144.20 (m, 1h), 4.544.61 (m, 1h), 6.32 (d, j = 15.6 hz, 1h), 6.936.98 (m, 3h), 7.08 (t, j = 6.9 hz, 1h), 7.18 (d, j = 2.1 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.55 (d, j = 8.7 hz, 2h), 7.63 (d, j = 7.8 hz, 1h), 7.88 (d, j = 8.7 hz, 2h), 8.63 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c27h24cln3o4 [m + h] 490.1526, found 490.1528 . Retention time: 6.0 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.003.13 (m, 2h), 4.10 (d, j = 5.4 hz, 2h), 4.494.57 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.957.00 (m, 3h), 7.10 (t, j = 7.4 hz, 1h), 7.22 (d, j = 2.1 hz, 1h), 7.29 (dd, j = 1.8 hz, j = 7.5 hz, 1h), 7.327.40 (m, 3h), 7.427.50 (m, 4h), 7.63 (d, j = 7.8 hz, 1h), 8.66 (d, j = 8.1 hz, 1h), 8.98 (s, 1h), 10.67 (s, 1h), 10.87 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h24cln3o4 [m + h] 490.1528, found 490.1528 . Retention time: 10.8 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.13 (d, j = 6.9 hz, 2h), 4.064.11 (m, 1h), 4.144.21 (m, 1h), 4.544.65 (m, 1h), 6.48 (d, j = 15.9 hz, 1h), 6.946.99 (m, 2h), 7.037.08 (m, 1h), 7.127.19 (m, 2h), 7.277.32 (m, 2h), 7.347.55 (m, 4h), 7.65 (d, j = 7.8 hz, 1h), 7.837.85 (m, 2h), 8.53 (d, j = 8.1 hz, 1h), 9.06 (s, 1h), 10.72 (s, 1h), 10.83 (s, 1h). Hrms (ap - esi) m / z calcd for c27h24cln3o4 [m + h] 490.1528, found 490.1528 . Retention time: 18.8 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.033.16 (m, 2h), 4.104.19 (m, 2h), 4.514.60 (m, 1h), 6.33 (d, j = 15.6 hz, 1h), 6.936.99 (m, 3h), 7.08 (t, j = 6.9 hz, 1h), 7.18 (d, j = 2.1 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.7 hz, 2h), 7.63 (d, j = 7.8 hz, 1h), 7.81 (d, j = 1.8 hz, 2h), 7.83 (d, j = 1.8 hz, 1h), 8.77 (d, j = 7.8 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h), 10.85 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c27h23cl2n3o4 [m + h] 534.1023, found 524.1138 . Retention time: 10.1 min, eluted with 51% acetonitrile/49% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.003.13 (m, 2h), 4.084.10 (m, 2h), 4.474.58 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.956.99 (m, 3h), 7.10 (t, j = 7.2 hz, 1h), 7.21 (d, j = 2.1 hz, 1h), 7.31 (d, j = 8.1 hz, 1h), 7.36 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.48 (dd, j = 2.1 hz, j = 8.1 hz, 1h), 7.50 (d, j = 8.2 hz, 2h), 7.63 (d, j = 7.8 hz, 1h), 7.67 (d, j = 2.1 hz, 1h), 8.72 (d, j = 8.1 hz, 1h), 8.99 (s, 1h), 10.66 (s, 1h), 10.86 (s, 1h). Hrms (ap - esi) m / z calcd for c27h23cl2n3o4 [m + h] 524.1138, found 524.1138 . Retention time: 14.2 min, eluted with 43% acetonitrile/57% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.11 (d, j = 6.9 hz, 2h), 4.054.20 (m, 2h), 4.524.60 (m, 1h), 6.32 (d, j = 15.9 hz, 1h), 6.936.99 (m, 3h), 7.08 (t, j = 6.9 hz, 1h), 7.18 (d, j = 2.1 hz, 1h), 7.267.33 (m, 3h), 7.41 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.7 hz, 2h), 7.63 (d, j = 7.8 hz, 1h), 7.897.94 (m, 2h), 8.56 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c27h24fn3o4 [m + h] 474.1824, found 474.1824 . Retention time: 12.0 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 3.11 (d, j = 6.9 hz, 2h), 4.064.20 (m, 2h), 4.514.61 (m, 1h), 6.32 (d, j = 15.6 hz, 1h), 6.936.98 (m, 3h), 7.08 (td, j = 0.9, j = 7.8 hz, 1h), 7.17 (d, j = 2.1 hz, 1h), 7.36 (d, j = 8.1 hz, 1h), 7.41 (d, j = 15.6 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.66 (d, j = 9.3 hz, 1h), 7.69 (d, j = 1.8 hz, 2h), 7.81 (d, j = 1.8 hz, 2h), 8.63 (d, j = 7.8 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.83 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h24brn3o4 [m + h] 534.1023, found 534.1021 . H nmr (300 mhz, dmso - d6) 3.10 (d, j = 6.9 hz, 2h), 3.80 (s, 3h), 4.024.20 (m, 2h), 4.524.63 (m, 1h), 6.33 (d, j = 15.9 hz, 1h), 6.937.00 (m, 5h), 7.08 (td, j = 0.9 hz, j = 7.4 hz, 1h), 7.17 (d, j = 2.1 hz, 1h), 7.34 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.9 hz, 1h), 7.49 (d, j = 8.4 hz, 2h), 7.64 (d, j = 7.8 hz, 1h), 7.85 (d, j = 8.7 hz, 2h), 8.38 (d, j = 8.1 hz, 1h), 8.98 (s, 1h), 10.66 (s, 1h), 10.82 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c28h27n3o5 [m + h] 486.2023, found 486.2023 . Retention time: 10.1 min, eluted with 47% acetonitrile/53% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.36 (s, 9h), 2.832.99 (m, 2h), 3.72 (s, 3h), 3.934.04 (m, 3h), 6.33 (dd, j = 4.5 hz, j = 15.9 hz, 1h), 6.95 (d, j = 8.4 hz, 2h), 6.977.02 (m, 2h), 7.107.15 (m, 2h), 7.367.42 (m, 2h), 7.49 (d, j = 8.4 hz, 2h), 7.58 (d, j = 7.8 hz, 1h), 8.97 (s, 1h), 10.66 (s, 1h). Hrms (ap - esi) m / z calcd for c26h31n3o5 [m + h] 466.2336, found 466.2332 . Retention time: 13.8 min, eluted with 47% acetonitrile/53% water (containing 0.4% formic acid). H nmr (300 mhz, dmso - d6) 1.151.25 (m, 9h), 2.903.06 (m, 2h), 3.974.06 (m, 2h), 4.314.40 (m, 2h), 4.56 (s, 1h), 6.31 (d, j = 15.6 hz, 1h), 6.766.79 (m, 2h), 6.98 (t, j = 7.2 hz, 1h), 7.017.07 (m, 2h), 7.097.25 (m, 5h), 7.327.43 (m, 5h), 8.96 (s, 1h), 10.64 (s, 1h), 10.84 (s, 1h). Esi - ms m / z: 541.4 [m + h]. A pale yellow oil, 80% yield . A pale yellow oil, 85% yield . A pale yellow powder, 30% yield . H nmr (300 mhz, dmso - d6) 3.15 (d, j = 8.4 hz, 2h), 4.064.11 (m, 1h), 4.164.22 (m, 1h), 4.544.64 (m, 1h), 6.48 (d, j = 15.6 hz, 1h), 6.946.99 (m, 2h), 7.08 (t, j = 7.6 hz, 1h), 7.127.14 (m, 2h), 7.19 (d, j = 2.4 hz, 1h), 7.30 (d, j = 8.1 hz, 1h), 7.34 (d, j = 7.8 hz, 1h), 7.38 (d, j = 15.9 hz, 1h), 7.427.54 (m, 3h), 7.65 (d, j = 7.8 hz, 1h), 7.827.85 (m, 2h), 8.52 (d, j = 8.1 hz, 1h), 9.05 (s, 1h), 10.71 (s, 1h), 10.82 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c27h25n3o4 [m + h] 456.1918, found 456.1919 . Retention time: 11.5 min, eluted with 46% acetonitrile/54% water (containing 0.4% formic acid). A pale yellow powder, 28% yield . H nmr (300 mhz, dmso - d6) 3.053.19 (m, 2h), 4.054.11 (m, 1h), 4.134.20 (m, 1h), 4.524.61 (m, 1h), 6.48 (d, j = 15.6 hz, 1h), 6.946.98 (m, 2h), 7.08 (t, j = 7.2 hz, 1h), 7.117.18 (m, 3h), 7.267.34 (m, 4h), 7.42 (d, j = 15.9 hz, 1h), 7.64 (d, j = 7.5 hz, 1h), 7.897.94 (m, 2h), 8.54 (d, j = 7.8 hz, 1h), 9.04 (s, 1h), 10.70 (s, 1h), 10.80 (s, 1h). Hrms (ap - esi) m / z calcd for c27h24fn3o4 [m + h] 474.1824, found 474.1825 . A pale yellow powder, 33% yield . H nmr (300 mhz, dmso - d6) 3.093.12 (m, 2h), 4.064.20 (m, 2h), 4.544.61 (m, 1h), 6.48 (d, j = 15.9 hz, 1h), 6.946.99 (m, 2h), 7.08 (t, j = 7.4 hz, 1h), 7.127.14 (m, 2h), 7.18 (d, j = 2.1 hz, 1h), 7.277.34 (m, 2h), 7.42 (d, j = 15.6 hz, 1h), 7.55 (d, j = 8.7 hz, 2h), 7.64 (d, j = 7.5 hz, 1h), 7.88 (d, j = 8.7 hz, 2h), 8.62 (d, j = 7.8 hz, 1h), 9.05 (s, 1h), 10.71 (s, 1h), 10.82 (d, j = 1.5 hz, 1h). Hrms (ap - esi) m / z calcd for c27h24cln3o4 [m + h] 456.1918, found 456.1919 . Retention time: 17.2 min, eluted with 40% acetonitrile/60% water (containing 0.4% formic acid). This method was the same as the general procedure for the preparation of 8 and 25 . A pale yellow oil, 65% yield . H nmr (300 mhz, dmso - d6) 3.173.27 (m, 1h), 3.413.47 (m, 1h), 3.67 (s, 1h), 4.124.17 (m, 1h), 4.254.27 (m, 1h), 4.36 (s, 2h), 6.38 (d, j = 15.6 hz, 1h), 6.916.99 (m, 3h), 7.10 (t, j = 7.2 hz, 1h), 7.22 (d, j = 2.4 hz, 1h), 7.37 (d, j = 7.8 hz, 2h), 7.427.50 (m, 6h), 7.647.68 (m, 2h), 9.72 (s, 1h), 9.92 (s, 1h), 10.31 (s, 1h), 11.05 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h27n3o3 [m + h] 442.2125, found 442.2125 . H nmr (300 mhz, dmso - d6) 3.173.26 (m, 1h), 3.413.48 (m, 1h), 3.70 (s, 1h), 4.124.17 (m, 1h), 4.254.28 (m, 1h), 4.36 (s, 2h), 6.39 (d, j = 15.9 hz, 1h), 6.926.99 (m, 3h), 7.10 (t, j = 6.9 hz, 1h), 7.23 (d, j = 2.1 hz, 1h), 7.29 (d, j = 8.7 hz, 2h), 7.357.42 (m, 2h), 7.487.53 (m, 3h), 7.697.74 (m, 2h), 9.74 (s, 1h), 9.94 (s, 1h), 11.05 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h26fn3o3 [m + h] 460.2031, found 460.2033 . H nmr (300 mhz, dmso - d6) 3.173.26 (m, 1h), 3.413.47 (m, 2h), 4.114.16 (m, 1h), 4.244.27 (m, 1h), 4.37 (s, 2h), 6.38 (d, j = 15.9 hz, 1h), 6.926.99 (m, 3h), 7.11 (t, j = 7.2 hz, 1h), 7.23 (d, j = 2.1 hz, 1h), 7.37 (d, j = 8.1 hz, 1h), 7.42 (d, j = 15.6 hz, 1h), 7.487.53 (m, 5h), 7.70 (d, j = 8.4 hz, 2h), 9.75 (s, 1h), 9.69 (s, 1h), 10.30 (s, 1h), 11.05 (d, j = 1.8 hz, 1h). Hrms (ap - esi) m / z calcd for c27h26cln3o3 [m + h] 476.1735, found 476.1735 . Were conducted as previously described . In brief, 10 l of enzyme solution (hela nuclear extract, hdac1, hdac2, hdac3 . Or hdac6) was mixed with various concentrations of tested compound (50 l). The mixture was incubated at 37 c for 5 min, then luorogenic substrate boc - lys(acetyl)-amc (40 l) was added . After incubation at 37 c for 30 min, the mixture was stopped by the addition of 100 l of developer containing trypsin and tsa . Twenty minutes later, fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 390 and 460 nm, respectively . The inhibition ratios were calculated from the fluorescence intensity readings of tested wells relative to those of control wells, and the ic50 values were calculated using a regression analysis of the concentration / inhibition data . All cell lines were maintained in rpmi1640 medium containing 10% fbs at 37 c in a 5% co2 humidified incubator . Briefly, cells were passaged the day before dosing into a 96-well cell plate, allowed to grow for 12 h and then treated with different concentrations of compound sample for 48 h. a 0.5% mtt solution was added to each well . After incubation for another 4 h, formazan formed from mtt was extracted by adding 150 l of dmso rocking for 15 min . Briefly, u937 cells with different treatment were collected and lysed with lysis buffer (20 mm tris - hcl [ph 7.5], 1% np-40, 1 mm sodium vanadate, 1 mm edta, 1 mm egta, 50 mm naf, and 1 mm phenylmethyl sulfonylfluoride [pmsf]) for 30 min, then centrifuged for 15 min at 14 000 rpm at 4 c, and the supernatant was the whole - cell extracts . Total protein extracts (30 g per lane) were separated by 12% sds polyacrylamide gel electrophoresis and transferred onto pvdf membranes (cat . Membrane was blocked with 5% s36 milk in tbs - t (10 mm tris [ph 7.4], 150 mm nacl, and 0.1% tween 20) for 1 h at room temperature, then incubated with a 1:1000 or 1:2000 dilution of primary antibody overnight at 4 c . Then the membrane was washed three times and incubated at 1:2000 dilution of antimouse or antirabbit goat - hrp - conjugated secondary antibodies for 2 h at room temperature . Finally the membrane was washed another three times and developed by enhanced chemiluminescence (ecl, cat . U937 cells (1.5 10) were treated with different concentrations of 11r, 11w, or saha for 12 or 24 h. cells were harvested following incubation, washed twice in cold pbs, centrifuged, and resuspended in 1 annexin - binding buffer . Cells were diluted in 1 annexin - binding buffer to 1 10 cells / ml, preparing a sufficient volume to have 100 l per assay . Five microliters of alexa fluor 488 annexin v and 1 l of 100 g / ml pi were added to each 100 l of cell suspension ., 150 l of 1 annexin - binding buffer was added, mixed gently, and kept on ice . The stained cells were analyzed by flow cytometry, measuring the fluorescence emission at 530 and 575 nm (or equivalent) using 488 nm excitation . Alexa fluor 488 annexin v / dead cell apoptosis kit with alexa fluor 488 annexin v and pi for flow cytometry was used for this assay (invitrogen). Preparation of artificial gastric juice: 160 ml of water and 2 g of pepsin were added to 3.28 ml of diluted hydrochloric acid . Preparation of artificial intestinal juice: 1.36 g of sodium dihydrogen phosphate was dissolved in 100 ml of water, and the ph was adjusted to 6.8 using 0.4% naoh . Then 2 g of pancreatin was added, and the mixture was diluted to 200 ml with water . Preparation of rat liver homogenate: wistar rats (experimental animal center, shandong university, female, 200 20 g) were sacrificed after fasting 12 h. liver was homogenized in quadruple phosphate butter (ph 7.4) and centrifuged (4000 rpm), 10 min . Artificial gastric juice, artificial intestinal juice, rat liver homogenate, and human plasma were preincubated at 37 c prior to the addition of 50 l of stock solution of mutual prodrugs (1 mg / ml in ch3oh). Sample aliquots were taken for processing and analysis after 24 h incubation at 37 c . All of the samples underwent extraction using 600 l acetonitrile and were filtered (0.22 m) after shocking 30 s and centrifugation at 12 000 rpm, 10 min . Chromatographic analysis was achieved by hplc method on an agilent 1100 hplc instrument using a ods hypersil column (5 m, 4.6 mm 100 mm) according the following method: compound was eluted with 42% acetonitrile/58% water (containing 0.4% formic acid) over 20 min, with detection at 290 nm and a flow rate of 1 ml / min . The temperature of the column was 25 c, and the quantity of injection was 20 m . For in vivo antitumor efficacy research, 1 10 human histiocytic lymphoma cells (u937) were inoculated subcutaneously in the right flank of male athymic nude mice (56 weeks old, slac laboratory animal, shanghai). Ten days after injection, tumors were palpable, and mice were randomized into treatment and control groups (5 mice per group). The treatment groups received compound 11r (100 mg / kg / d) or saha (100 mg / kg / d) by oral administration, and the blank control group received equal volume of pbs solution containing 40% dmso . During treatment, subcutaneous tumors were measured with a vernier caliper every three days, and body weight was monitored regularly . After treatment, mice were sacrificed and dissected to weigh the tumor tissues and to examine the internal organ injury . Tumor growth inhibition (tgi) and relative increment ratio (t / c) were used as the evaluation indicators to reveal the antitumor effects in tumor weight and tumor volume, respectively . Tumor volumes (v) were estimated using the equation (v = ab/2, where a and b stand for the longest and shortest diameter, respectively). T / c was calculated according to the following formula: where rtv is the relative tumor volume = vt / v0 (vt, the tumor volume measured at the end of treatment; v0, the tumor volume measured at the beginning of treatment). Compounds were docked into the active site of hdac3 (pdb code 4a69) using tripos sybyl 8.0 . Before the docking process, the protein structure was treated by deleting water molecules, adding hydrogen atoms, fixing atom types, and assigning amber7 ff99 charges . The molecular structures were generated with the sybyl / sketch module and optimized using powell s method with the tripos force field with convergence criterion set at 0.05 kcal/(mol) and assigned charges with the gasteiger
It is seen with the use of lasers that do not make use of optical fibers for laser beam delivery, such as carbon - dioxide (co2) lasers which use articulated arm technology . They have optical fibers for delivery of the laser at the site of surgery, facilitating more precision and control . A 7-year - old boy was admitted for excision of a cystic swelling at the posterior aspect of the base of the tongue a few centimeters anterior to the valleculae . The smallest laser endotracheal tube (ett) available was of 7-mm outer diameter (od) and an internal diameter of 5.5 mm . After induction of anesthesia with propofol, followed by administration of suxamethonium intravenously (iv), nasotracheal intubation was done with this tube . Anesthesia was maintained oxygen in air, atracurium, intermittent boluses of propofol, and fentanyl iv . The saline - soaked throat pack was obstructing the field of vision of the surgeon and also restricting the outward pull of the tongue as cyst was large (3 3 cm) and placed posteriorly . The surgeon assured that adequate precautions would be taken to avoid bringing the diode laser delivery handle tip in contact with the ett . Toward the end of the surgical laser excision, the tip of the handle accidently slipped and touched the ett . We heard a very short pop like noise which lasted for about 12 s. the exhaled gas from the expiratory valve also smelt of smoke for about a minute or so . There was however no change in hemodynamics, drop in spo2 or reduction in air entry / chest expansion . As the surgery had been completed and we were able to ventilate the lungs, we reversed the neuromuscular blockade . After tracheal extubation, a flexible laryngopharyngoscope was passed to visualize the trachea up to the carina and fortunately everything appeared to be normal . The removed ett [figure 1] had a very small circular hole and the inner aspect of the tube was black distal to the hole [figure 2]. The cuff was intact but the tubing from the pilot balloon to the cuff was damaged, which is typically seen in patient recovered well and was shifted to postoperative, ward, monitored and given humidified oxygen and discharged next day . Diode lasers are used with low power setting and do not usually cause perforation or damage to polyvinyl chloride (pvc) ett coming in direct contact . Though studies have shown silicon and red rubber tubes to be more resistant to ignition compared to pvc, some studies have reported that pvc is less flammable than silicon or red rubber, having a flammability index of 0.26 vs. 0.19 for silicon and red rubber, respectively . Use of laser resistant material wrapped around pvc ett or double - cuffed silicon - coated ett is however more safe . Laser - resistant ett are bulkier and more rigid than conventional ett and thus need careful insertion to prevent mucosal abrasions . In the event of a blow - torch phenomenon, pvc tubes burn more vigorously and hydrochloric acid may be produced, which is a pulmonary toxin . In case of such an airway fire, oxygen should be temporary discontinued, damaged ett removed, trachea reintubated with a fresh ett, and the pharynx flushed with cold saline . A rigid or fiberoptic bronchoscopy examination should be done to look for damage and pieces of burned ett . Humidified oxygen, steroids, and antibiotics must be administered and at times controlled ventilation and tracheostomy may become necessary . Postoperatively, patient should be intensively monitored and a chest x - ray should be taken . Surgeons must ensure that laser energy is not reflected from smooth metal surfaces or directed at sensitive structures . Applying protective metal foil wraps to ett, such as aluminum or copper has been advocated. [35] the ett cuff can be filled with colored saline and placed far distally in trachea and the visible cuff covered with moistened cotton pledgets, which should be remoistened as needed . Laser resistant tubes are available for use during laser surgery . To reduce the obstruction in the surgical field, by airway instruments,
Hospitalization for acute exacerbation of chronic obstructive pulmonary disease (aecopd) is recognized as a major event in the natural history of chronic obstructive pulmonary disease (copd) due to its negative effect on lung function, survival, risk of readmission, and quality of life.16 the prolonged length of stay (los) may also indicate more fragile patients who need more attention from health personel.7 although only 10%15% of all patients with copd will experience severe exacerbations that require hospital admission, expenditures associated with hospitalization represent more than 70% of all copd - related medical care costs.8 until now, no established predictors of prolonged los are available, and there is no international accepted norm of how long a hospitalization due to aecopd should be . This is illustrated by the wide range of reported los (316 days).9,10 in the absence of a published definition for prolonged los, we defined a los longer than the 75th percentile as prolonged los in this article, which is in line with other studies.1113 a limited number of studies have published a heterogeneous set of variables that are associated with long hospital stay for aecopd . For example, social and demographic data,14,15 clinical variables,10,1618 comorbidities,11,1922 number and type of drugs used on admission,11 and day of the week of the admission16 are among the risk factors for long los . There is a lack of consistency in identified predictors of long los, which makes it difficult to determine which prognostic factors are most important . To the best of our knowledge, there are no established statistical models for predicting los based on variables collected at the time of hospital admission for aecopd . Thus, increased knowledge about predictors of prolonged los and earlier prediction of los might contribute to better patient treatment, optimal discharge planning, and shortening of the los, which will ultimately lead to cost savings for hospitals . The primary objective of this retrospective study was to identify predictors of prolonged los in patients hospitalized for aecopd . A secondary objective was to develop a predictive model for los based on available variables obtained at the time of admission for aecopd . The study included all patients with a discharge diagnosis of copd from march 2006 until december 2008, after introduction of electronic medical records at oslo university hospital, aker . Based on the international statistical classification of disease and related health problems, 10th revision (icd-10)23, we included both patients with copd (j43 or j44) as the main diagnosis, and patients with respiratory failure (j96) or pneumonia (j12j18) as the main diagnosis and copd (j43 or j44) as a secondary diagnosis . Although patients with different main diagnoses might have different los, we decided not to split the sample to keep it as big as possible . Another reason was that the diagnoses were set at the time of discharge and were therefore not available at the time of admission and could not be used as a predictor in this study . We chose to include the first hospitalization of all consecutive patients hospitalized for aecopd in this period, regardless of number of previous admissions . In total we extracted the following data from the computerized medical record at the time of hospital admission: demographic data, comorbidities, use of long - term oxygen therapy, and clinical data obtained in the emergency department (table 1). We registered a history of any of the following conditions as comorbidities: ischemic heart disease, cardiac arrhythmias, chronic heart failure, pulmonary heart disease or pulmonary hypertension, psychiatric disorder, general atherosclerosis, stroke, diabetes mellitus, other neurologic disorders than cerebral insult, any malignancy ever, osteoporosis, renal failure, or abuse of drugs or alcohol . We created a summary variable using the total number of these comorbidities, which again was categorized as 0, or 1 comorbidity . We analyzed the impact of both individual comorbidities and the dichotomized comorbidity groups, eg, with or without registered comorbidities, on los . We also extracted the value of the forced expiratory volume in 1 second (fev1) determined at the time closest to that of the admission, los, number of copd - related admissions during the previous 12 months before the current admission, and the day of the week of admission . The reason for dichotomizing weekday was because we considered that the natural time of discharge for those admitted between thursday and saturday would be before the following weekend, and thus that they might have a higher risk of a prolonged stay . In the final multivariate analysis, we used only dichotomized weekdays in order to reduce the number of variables in the prediction model . A copd - related admission was defined by discharge diagnoses from previous admissions using the same criteria as for the current admission . This study was considered as a quality assessment study by the regional committee for medical and health research ethic, south east norway (s-09079d, 2009/123) and approved by the local privacy ombudsman for clinical research, oslo university hospital (2011 - 12102). The later also approved that written consent from each patient was not necessary . Based on a literature review, we analyzed how 14 variables from our data set were related to los (table 1). Descriptive statistics are expressed as mean standard deviation (sd) or median (interquartile range [iqr]) values for continuous data, and number (%) for categorical data . Missing values for fev1, serum albumin level, and arterial carbon dioxide tension (paco2) were imputed from the other known variables using linear regression analysis; we did not impute missing values for the other variables . We used univariate and multivariate logistic regression analyses to assess predictors of a los> 11 days (corresponding to> 75th percentile). We then manually removed variables one by one while watching changes in the coefficients in order to arrive at a final parsimonious model . Stata software (version 10.1) was used for statistical analysis (stata corp, college station, tx, usa). In an attempt to establish a statistical model to predict whether or not a patient would be hospitalized for longer than the 75th percentile, receiver operating characteristic (roc) analysis was performed for independent predictors of a long los as determined by multivariate logistic regression analysis . The study included all patients with a discharge diagnosis of copd from march 2006 until december 2008, after introduction of electronic medical records at oslo university hospital, aker . Based on the international statistical classification of disease and related health problems, 10th revision (icd-10)23, we included both patients with copd (j43 or j44) as the main diagnosis, and patients with respiratory failure (j96) or pneumonia (j12j18) as the main diagnosis and copd (j43 or j44) as a secondary diagnosis . Although patients with different main diagnoses might have different los, we decided not to split the sample to keep it as big as possible . Another reason was that the diagnoses were set at the time of discharge and were therefore not available at the time of admission and could not be used as a predictor in this study . We chose to include the first hospitalization of all consecutive patients hospitalized for aecopd in this period, regardless of number of previous admissions . In total we extracted the following data from the computerized medical record at the time of hospital admission: demographic data, comorbidities, use of long - term oxygen therapy, and clinical data obtained in the emergency department (table 1). We registered a history of any of the following conditions as comorbidities: ischemic heart disease, cardiac arrhythmias, chronic heart failure, pulmonary heart disease or pulmonary hypertension, psychiatric disorder, general atherosclerosis, stroke, diabetes mellitus, other neurologic disorders than cerebral insult, any malignancy ever, osteoporosis, renal failure, or abuse of drugs or alcohol . We created a summary variable using the total number of these comorbidities, which again was categorized as 0, or 1 comorbidity . We analyzed the impact of both individual comorbidities and the dichotomized comorbidity groups, eg, with or without registered comorbidities, on los . We also extracted the value of the forced expiratory volume in 1 second (fev1) determined at the time closest to that of the admission, los, number of copd - related admissions during the previous 12 months before the current admission, and the day of the week of admission . The reason for dichotomizing weekday was because we considered that the natural time of discharge for those admitted between thursday and saturday would be before the following weekend, and thus that they might have a higher risk of a prolonged stay . In the final multivariate analysis, we used only dichotomized weekdays in order to reduce the number of variables in the prediction model . A copd - related admission was defined by discharge diagnoses from previous admissions using the same criteria as for the current admission . This study was considered as a quality assessment study by the regional committee for medical and health research ethic, south east norway (s-09079d, 2009/123) and approved by the local privacy ombudsman for clinical research, oslo university hospital (2011 - 12102). Based on a literature review, we analyzed how 14 variables from our data set were related to los (table 1). Descriptive statistics are expressed as mean standard deviation (sd) or median (interquartile range [iqr]) values for continuous data, and number (%) for categorical data . Missing values for fev1, serum albumin level, and arterial carbon dioxide tension (paco2) were imputed from the other known variables using linear regression analysis; we did not impute missing values for the other variables . We used univariate and multivariate logistic regression analyses to assess predictors of a los> 11 days (corresponding to> 75th percentile). We then manually removed variables one by one while watching changes in the coefficients in order to arrive at a final parsimonious model . Stata software (version 10.1) was used for statistical analysis (stata corp, college station, tx, usa). In an attempt to establish a statistical model to predict whether or not a patient would be hospitalized for longer than the 75th percentile, receiver operating characteristic (roc) analysis was performed for independent predictors of a long los as determined by multivariate logistic regression analysis . Of 599 patients discharged after aecopd, nine were excluded from further analysis due to missing data after our imputation of fev1, serum albumin level, and paco2 values . The final sample of 590 patients had a mean age of 73.210.8 years and 54% female . The mean los was 8.99.7 days, with a median of 6 days (iqr 3.511.0 days). Analysis, age, fev1, copd - related admission during the previous 12 months before the current admission, admission between thursday and saturday (here under, thursday and saturday), high paco2, high pulse rate, low serum albumin level, and having one of a selection of comorbidities (ischemic heart disease, heart failure, cardiac arrhythmia, diabetes, and stroke) were significantly associated with a los longer than 11 days (p<0.05, table 2). In multivariate analysis, only admission between thursday and saturday, high paco2, low serum albumin level, and having heart failure, diabetes, or stroke were independently associated with a long los (table 3). Admission between thursday and saturday increased the odds ratio (or) of a los longer than 11 days to 2.50 (95% confidence interval [ci] 1.663.77). The or for the presence of heart failure, diabetes, and stroke was 2.26 (95% ci 1.343.80), 1.90 (95% ci 1.073.37), and 1.83 (95% ci 1.043.21), respectively . An increase of 1 kpa in paco2 had an or of 1.26 (95% ci 1.121.41), and an increasing serum albumin level (or 0.92 [95% ci 0.880.97]) were associated with a shorter los . Of 599 patients discharged after aecopd, nine were excluded from further analysis due to missing data after our imputation of fev1, serum albumin level, and paco2 values . The final sample of 590 patients had a mean age of 73.210.8 years and 54% female . The mean los was 8.99.7 days, with a median of 6 days (iqr 3.511.0 days). Analysis, age, fev1, copd - related admission during the previous 12 months before the current admission, admission between thursday and saturday (here under, thursday and saturday), high paco2, high pulse rate, low serum albumin level, and having one of a selection of comorbidities (ischemic heart disease, heart failure, cardiac arrhythmia, diabetes, and stroke) were significantly associated with a los longer than 11 days (p<0.05, table 2). In multivariate analysis, only admission between thursday and saturday, high paco2, low serum albumin level, and having heart failure, diabetes, or stroke were independently associated with a long los (table 3). Admission between thursday and saturday increased the odds ratio (or) of a los longer than 11 days to 2.50 (95% confidence interval [ci] 1.663.77). The or for the presence of heart failure, diabetes, and stroke was 2.26 (95% ci 1.343.80), 1.90 (95% ci 1.073.37), and 1.83 (95% ci 1.043.21), respectively . An increase of 1 kpa in paco2 had an or of 1.26 (95% ci 1.121.41), and an increasing serum albumin level (or 0.92 [95% ci 0.880.97]) were associated with a shorter los . This study found that admission between thursday and saturday, high paco2, low serum albumin level, and having heart failure, diabetes, or stroke were independently associated with a long los . The statistical model had an area under the roc curve of 0.73, which we considered to be too low to allow the development of a meaningful predictive model . The impact of weekday at the time of admission on length of hospital stay has hardly been studied in copd given its impact on mortality; a large spanish study showed that patients admitted on the weekend for aecopd are more likely to die in the hospital compared to those admitted on weekdays.24 our finding that admission between thursday and saturday was associated with a los greater than the 75th percentile (ie, 11 days) pointed out another possible negative consequence of being admitted around the weekend for aecopd . Our finding is consistent with a spanish study demonstrating an association between weekend admission (friday sunday) and prolonged los.20 however, a long los was defined as 3 days in the spanish study, which is shorter than the median in the present study (6 days), and shorter than the commonly reported mean or median los of 611 days.7 furthermore, over 90% of the patient population in the spanish study was male, which stands in contrast to our patient population of more than 50% women . Our finding, together with the finding from the spanish study, indicates that prolonged los in connection to weekend admissions may be a more common phenomenon than we are aware of . This is supported by studies of other diseases that have also found that admission day of the week affects los.25,26 there are several possible explanations for this phenomenon, and they probably vary between specific health care systems . In our case natural discharge day of these patients was immediately before the next weekend, as indicated by the median los (6 days), and hence the discharge could be delayed by the discontinuity and reduction of the medical staff during weekends, as suggested by previous studies.16,22 this phenomenon might also be due to the reduced primary health care or support available from relatives following hospital discharge . Alternatively, patients admitted between thursday and saturday may have more severe aecopd than patients admitted on other weekdays, as has been suggested by some authors.27 association between comorbidity which is common in copd and los are less studied compared with their association with mortality under / after aecopd . The published results had either been concentrated on single diagnoses21,22 or number of comorbidities.11,19,20 in the present paper, most of the recognized comorbidities of copd have been assessed; we found heart failure, diabetes, and stroke to be independently related with prolonged los . Both heart failure and diabetes are common in patients with copd.28,29 since the exacerbation of copd and its treatment with beta - agonist and systemic steroids may worsen heart failure30 and diabetes,22 respectively, it is understandable that a prolonged stay might be needed to get these comorbid conditions under control . That a high paco2 measured in the emergency department was associated with a prolonged los is reasonable because paco2 and other blood - gas variables reflect the severity of respiratory failure . This finding is consistent with two previous studies16,31 but inconsistent with two others.25,26 the discrepant results of these studies may be due to differences between the patient populations, since patients needing intensive care and with infiltration on chest radiographs indicating pneumonia were excluded from the latter two studies . Our finding of an association of low serum albumin level with a long los is consistent with a british study demonstrating an association between a low serum albumin level and a los> 7 days.31 serum albumin level is a marker of nutritional status,32 with a low level being associated with higher long - term mortality in copd patients.2,33 albumin also forms a part of the acute - phase protein response, and hence low serum albumin level may reflect the deterioration of clinical status or increased persistent inflammation during aecopd.33 in the present study, we wanted to identify patients with prolonged los primarily because such patients may have a worse health status and hence an increased need for post - discharge support.34 in the absence of a standardized definition of prolonged los, we decided to use a los cutoff at the 75th percentile rather than at the median1113 because the potential benefit from improved discharge planning would be greater with increasing los . In univariate analysis, increasing age was significantly associated with a los longer than 11 days . This can, for example, be attributed to older patients being more fragile, having lower serum albumin values, having more comorbidities, or more severe copd exacerbations and higher paco2 . Therefore, age by itself was no longer significantly related to prolonged los, and the age effect was probably captured by the other variables . Some evidence supports that social circumstances affect los.15 however, in this data set, we had no feasible variable for social factors like income, class, or (earlier) profession . The area under the roc curve was 0.73, suggesting a poor to fair performance . Although the usefulness of a prediction model does not follow directly from how big the area under the roc curve, a level of 73% will in most cases be too low for the purpose of resource planning in a hospital, while a level close to 80% is usually considered sufficient for practical use.35 the present roc curve illustrates how one must trade sensitivity for specificity if we need to correctly identify 75% of the patients with a los> 11 days, we must accept that 50% of the patients with a los 11 are incorrectly classified . Alternatively, if we require that 75% of the patients with a los 11 be correctly classified, only approximately 50% of the patients with a los> 11 will be identified . In our opinion, these uncertainties are too large for the predictions to be useful in a clinical setting . There may be several reasons for the suboptimal performance of this model, with one possibility being the presence of a large random component that is independent of patient - related variables . Previous studies found that only 8%12% of los variation could be explained by clinical variables.18,36 a us study showed that hospital stays for patients hospitalized for aecopd were considerably extended beyond what was considered strictly necessary because physicians observed patients an extra day to be certain that patients could cope at home after discharge.16 such individual assessment is one of several random elements that are difficult to incorporate in a statistical prediction model . Additionally, local practice guidelines, hospital resources, and the organization of care may influence los.24,37 some limitations of this retrospective study should be discussed . The number of variables in the analyses was limited; hence, potentially important predictors might have been missed . We reduced the possibility of false - positive findings by limiting the number of variables before the analysis . Some medical records at the time of the admission were incomplete, for example, lacked information on comorbidities or results of tests actually being done on admission . This was partially compensated by imputing values for missing values for the variables that we thought would be most important this imputation contributed to reducing the uncertainty associated with missing data and avoided the exclusion of patients, which would have reduced the effect of the results of the study . In our case, we had no indication that the missing values for serum albumin, paco2 and fev1 were systematically missing . Those patients with incomplete data would be excluded, which could also introduce bias.38 finally, the study population was recruited from a single university hospital in oslo, which served as a primary hospital for a geographically defined area of the city . However, the included population was relatively large and unselected in that all patients with a discharge diagnosis of copd were included . Furthermore, both age and los in this population were comparable to those in previous larger studies from other countries, which support the external validity of our sample.24,31 in conclusion, we found that admission between thursday and saturday, paco2, serum albumin level, and comorbidities such as heart failure, diabetes, and stroke were associated with los . The reported findings may help physicians to identify patients at risk of a long los in the early stages of an aecopd admission, and thus introduce the possibility of offering better follow - up . However, we were unable to create a robust scoring model for risk stratification that would have been useful for discharge planning . The reasons for prolonged los are probably multidimensional, and future studies should focus on both patient- and non - patient - related factors.
Posterior circulation stroke (pcs) is less common than stroke involving the anterior circulation stroke (acs). Pcs, which can be infratentorial (ipcs) or supratentorial (spcs), account for 1015% of all types of strokes . The area includes brainstem, cerebellum, occipital lobes, and thalamus and is supplied by 2 vertebral arteries, 1 basilar artery, and 2 posterior cerebral arteries . Ipcs differs from acs in the area of etiology, clinical features as well as associated morbidity and mortality . Its manifestations may include brief or minor brainstem symptoms and are more difficult to diagnose than acs . Common infratentorial posterior circulation symptoms include visual disturbance, vertigo, and ataxia . Delayed or incorrect diagnosis of this stroke subset may have devastating consequences such as potentially preventable death or severe disability . Therefore, healthcare professionals in the emergency settings and outpatient clinics need to be acquainted with the features of the acute neurological deficits associated with ipcs . However, there is a scarcity of hospital statistics available for ipcs in nigeria . Against this background, we evaluated the clinical characteristics, risk factors, outcome, and predictors of outcome of pcs in northwestern nigeria . Out of all stroke patients seen from june 2007 to june 2013, persons with ipcs presenting in the emergency unit and a neurology clinic of a tertiary hospital in kano were recruited for the study . All the patients enrolled were assessed by clinicians experienced in the subtlety of stroke diagnosis, typing, and management . Classification of stroke into hemorrhagic and infarctive subtypes was based on computed tomography brain and/or brain magnetic resonance imaging (mri). The diagnosis of posterior circulation was made when the patient had a clinical stroke syndrome compatible with the involvement of posterior circulation territory and neuroimaging evidence . Patients with neuroimaging that showed recent infarction or hemorrhage in the acs, spcs, and other nonvascular lesions were excluded from the study . Patients with ambiguous clinical and/or radiological diagnosis of stroke were also excluded from the study . A detailed history was obtained from the patient or a close relative regarding the onset of stroke, demographic data, risk factors and family history, risk factors, clinical characteristics, stroke subtypes, and mortality . A detailed physical examination was carried out in accordance to the proforma, including vital signs and detailed examination of other systems . Each diagnosed patient was followed up for 30 days until either death at the hospital or discharge . Analysis of data was carried out using the statistical package for social sciences (spss) program for windows version 16.0 (spss inc ., chicago, il, usa). Mean and standard deviation were used to describe quantitative variables . Bivariate analysis was carried out using pearson chi - square or fisher exact test for categorical variables . Student's t - test was used to compare the means of continuous variables and scores on glasgow coma scale (gcs) were compared using a mann whitney u - test . Multiple logistic regression model was used and the covariates were adjusted for each independent (regression) variable to find independent predictors of in - hospital mortality . Ipcs accounted for 57 (9.6%) of 595 of all strokes seen in the study period . They comprised 44 males (mean age 47.8 17.7) and 13 females (mean age 46.3 13.7). Overall, their age ranged between 24 and 90 (mean age 47.4 16.7). The most common anatomical site affected was the cerebellum, which was seen in 33 (57.9%) patients [table 1]. Hypertension was the most common risk factor (86%) but the majority (54.4%) of the patients had more than one risk factor [table 2]. Headache and vertigo were the most common features accounting for 83.6% and 86.3%, respectively; however, none of the features in the study had significant association with the type of stroke as the p value was> 0.05 across all the clinical features [table 3]. Distribution of anatomical sites involved with infratentorial pcs distribution of the traditional risk factors across gender distribution of the clinical manifestations and their comparison across stroke types factors associated with death in the patients were hyperglycemia, high gcs, hemorrhagic stroke, and aspiration pneumonitis [table 4]. However, independent predictors of death in the study were hyperglycemia on admission and hemorrhagic stroke [table 4]. Relationship between common variables and mortality (unadjusted) and independent predictors of death (adjusted), ipcs accounted for about 10% of the total stroke patients seen during the study period . The variations may be largely ascribed to differences in methodological approaches and to the fact that the focus of the current study was an infratentorial stroke . The majority (67%) of the patients this finding, which was a reflection of the pattern of the type of stroke in the population studied, is compatible with reports from elsewhere . A study showed that out of the different anatomical sites involved in pcs, infratentorial ischemic stroke was the most common (63%). However, a study in which only brain mri was used for identification reported a relatively higher figure (70%). In the current study, this figure, though similar to what is obtainable in other developing countries, is lower than that reported in studies from the western countries . This disparity could be attributed to a higher incidence of stroke in the young adult in northwestern nigeria . In general, the risk factors for posterior circulation infarction do not differ from those for acs . The present study showed that hypertension, diabetes mellitus, smoking, and hypercholesterolemia were the most common risk factors ., caplan et al ., and sundar and mehetre the rarity of alcohol consumption, as a risk factor for ipcs, that was strikingly low when compared to the studies elsewhere probably reflects oddity of alcohol consumption in the predominantly muslim community where the study was conducted . Headache and vertigo were the clinical features mostly encountered in the patients . The relatively low frequency of visual impairment observed in the current study was because of the fact that the current study focused on infratentorial stroke thereby excluding occipital and thalamic strokes . The organization of the brainstem relative to its vascular supply tends to lead to a number of well - recognized syndromes when occluded . Consequently, infarctive lesions in the territory supplied by posterior inferior cerebellar artery territory may result in the lateral medullary syndrome (wallenberg's syndrome) that as exemplified by two of the patients in the current study, is characterized by ipsilateral facial sensory disturbances, nystagmus, dysphagia, dysarthria, contralateral dissociated hemi - sensory disturbances, and horner's syndrome . It should be noted, however, that none of the clinical features observed in the patients in the current study could distinguish between ischemic or hemorrhagic stroke . This figure was comparable to that (35%) reported by jones et al ., but it is higher than that reported by sundar and mehetre (17%), patrick et al . The care of patients with stroke within the limited health budgets of most african countries offers a major and increasing challenge, therefore, the higher figure recorded in current study can be partly ascribed to delayed presentation, inadequate health infrastructure, nonexistent of stroke unit, and inadequacy of intensive unit . Nonetheless, like in many hospital - based studies, the patients recruited during the study period may not be entirely representative of all stroke cases occurring in the community as those with severe stroke, such as primary brainstem and cerebellar hemorrhage would cause early death and hence, were less likely to have reached the hospital . As reported in studies elsewhere, hyperglycemia, low gcs score, hemorrhagic stroke, and aspiration pneumonitis were found to be associated with 1-month mortality in our study, nevertheless, hemorrhagic stroke type and hyperglycemia emerged as independent predictors of death . This observation may be attributed to the fact that intraparenchymatous bleeding in the posterior fossa is usually a medical emergency, as pontine hemorrhages are lethal in 6090% of cases, depending on the extent of hemorrhage and location . Moreover, this finding, which was consistent with the reports from studies conducted on stroke in general, has significant implication for treatment in resource - poor setting . Identification of predictors of death is of paramount importance for clinicians, so that specific therapies and management strategies can be applied to patients at high - risk of dying . It is noteworthy that none of the patients seen in the course of the study had thrombolytic therapy . Like other centers in nigeria, thrombolytic therapy for infarctive stroke one of the most important barriers to thrombolysis therapy in the developing world is a prehospital delay . The other factors militating against the use of thrombolytic therapy in developing countries includes financial constraints and lack of the necessary infrastructures . Therefore, until a cheaper thrombolytic agent and the proper infrastructure for utilization of thrombolytic therapy is available, in developing countries where such is not available, focusing on primary and secondary stroke prevention strategies will be potentially rewarding . In order to reduce the incidence, mortality, and morbidity associated with pcs furthermore, health education with an emphasis on control of other predisposing factors such as diabetes mellitus, avoidance of smoking and fatty foods, as well as regular exercise, should be embarked upon . Apart from the earlier mentioned limitation encountered in the course of this study, the absence of magnetic resonance angiography for further localization of the vascular territory involved in the ipcs is also worthy of mention . Nonetheless, the data generated in this study have implications for clinicians and could serve as the basis for a large hospital- and community - based study in nigeria . Independent predictors of death in the study were hyperglycemia at presentation and hemorrhagic stroke subtype.
The incidence of cerebrospinal fluid (csf)-related complications (i.e., csf leak, pseudomeningocele, and meningitis) after intradural spinal tumor surgery (ist) remains high following resection of intradural extramedullary spinal tumors (iest) and intradural intramedullary spinal tumors (iist) (1 - 17). Treating these complications is a vexing problem and often requiring prolonged postoperative bed rest, operative re - exploration, the placement of an external lumbar drain, and a prolonged use of antibiotics (18 - 30). Furthermore, these complications may lead to additional complications, and even the death of the patient . Despite mayfield s (18) and black s (5) recommendations many years ago to use a fat graft to prevent potentially life - threatening csf complications in patients undergoing posterior fossa and spinal surgery, the practice did not gain routine use for those undergoing surgery for ists . We were not able to find any reports of the use of intraoperative fat grafting in the ist literature or reports of any other efficient way that ensures preventing this vexing postoperative complication problem while csf - related complications of surgical ist series have been regularly reported in up to 18% of patients (1, 2, 4, 6, 7, 9, 11 - 17, 19 - 21, 25, 28 - 30). In our earlier practice, csf - related complications were similar to those reported by other authors (1 - 3, 6, 7, 9, 11 - 14, 17 - 19, 22, 23, 25, 26). As a consequence, this study was approved by the appropriate institutional review board and was a retrospective analysis of all patients treated operatively by the senior author for ists over 13-year period (september 2003 through september 2016). Medical records were reviewed for pre and postoperative exams, pre and postoperative magnetic resonance imaging (mri), and follow - up visits . In june 2005 was noted because this was when a patient with a sacral dumbbell schwannoma developed a large pseudomeningocele postoperatively, prompted us for the prospective application of autograft fat during dural closure for all other patients . The patients demographics, the tumors histology, the degree of tumor resection, and neurological outcomes were evaluated . All patients underwent postoperative mri scans with and without contrast in the hospital 2 months after surgery, yearly for 2 years, and then every 5 years during follow - up . Fat harvest and application technique after endotracheal intubation and line placement in the supine patient, the left paraumbilical area is prepared and draped (figure 1). The harvested fat is placed in antibiotic saline in a sterile cup until needed for closure . Once the tumor was resected, the dura was closed with 5 - 0 prolen stitches in a running fashion . If the incision was in the midline, the valsalva maneuver was done at 30 cm h20 for 510 seconds to ensure that it was watertight . If any area in the suture line leaked csf, an additional suture was placed there and a piece of fat tissue was cut and positioned inside the stitch, which was then tightened . If the dural incision was t - shaped (i.e., for dumbbell, intra / extradural, foraminal tumors), or y - shaped (i.e., for sacral canal / foraminal tumors), the dural incision in the midline and its t or y extension over the nerve root were closed in running fashion with 5 - 0 prolen . Multiple pieces of fat tissue were then incorporated into single additional dural sutures to achieve watertight closure and reinforce the dural suture . When suturing was complete, a layer of fat tissue 68 mm thick was placed over the entire exposed dura to obliterate the dead space that remained after a laminectomy or facetectomy . The fat graft also created small pressure onto the dural suture line, lessening the chance of csf seepage and pseudomenigocele . In other words, the graft prevented a potential low - pressure space into which csf may migrate and form a pseudomeningocele, later producing a csf leak . The muscle, fascia, subcutaneous tissue, and skin layers are then closed in the usual fashion (figures 24). Note the fat graft overlying the dura dorsally and enforcing the patch graft / dural suture line . The patient regained his full strength postoperatively and his balance problems resolved . A woman in her early 30s with an iist (hemangioblastoma) at c1 . B) postoperative post - contrast t1-weighted mri showing the tumor resection and collapse of the cyst . Note the fat graft overlying the dura dorsally . The patient s balance and swallowing problems resolved postoperatively . B) axial t2-weighted mri showing the left - sided tumor, which involved the c1c2 intervertebral foramen and compressed the spinal cord . Over the course of 13 years (september 2003september 2016), 40 patients with an ist were operated on by the senior author . Of these, 34 were iest, comprising 13 meningiomas, 14 schwannomas, 6 myxopapillary ependymomas, and 1 breast cancer metastasis . Six patients had iists: 3 astrocytomas (one high grade), 2 ependymoma, and 1 hemangioblastoma . There were 11 men (28%) and 29 women (72%) with ages ranging from 2489 years (mean 56 years). Thirteen patients (33%), all with iest, were older than 65 years . The length of hospital stay ranged from 2 to 8 days (mean, 4 days), and follow - up ranged from 1 to 36 months (mean, 45 months). Abbreviations: iest = intradural extramedullary spinal tumors; iist = intradural intramedullary spinal tumors; ist = intradural spinal tumors except for 5 patients with schwannomas who presented with pain and numbness in the involved nerve areas, all other patients presented with motor, sensory, sphincter, and neurological deficits in various combinations . Six patients (15%) had dumbbell tumors that extended into the corresponding intervertebral neural foramen; 5 of these had schwannomas and 1 had a meningioma . One patient, a woman in her mid 40s with a grade iii conus / cauda astrocytoma, died 3.5 years later as a result of disease progression and csf seeding throughout her neuraxis despite postoperative irradiation and chemotherapy . The third patient in the series, a man in his late 40s with perineal numbness and tingling, impotence, and severe low back pain harbored a dumbbell schwannoma at s1s2 and underwent resection . Ten days after surgery, he presented with a large subcutaneous swelling in the sacral area, consistent with a pseudomeningocele, which was verified with computed tomography . He underwent a second surgery, during which the dural closure was revised with a fat autograft along with external lumbar drainage for 5 days bed rest . All patients after this patient received a fat autograft at the time of dural closure, and no evidence of a csf leak or a pseudomeningocele was noted on physical exam or neuroradiologic follow - up in these other patients . No evidence of complications related to the fat harvest site was noted . On the second postoperative mri, 2 months after surgery, the fat tissue routinely showed signs of significant resorption, and the 1-year follow - up mri scans in all patients showed that the fat was totally reabsorbed . Efficacy of the technique in their classic work, yasargil and colleagues (34) reported the successful radical removal of ists using microsurgical techniques with favorable neurological outcomes . Since then, numerous other reports of series of ists with good results followed (1 - 9, 11 - 31, 35). Nonetheless, the incidence of csf - related complications (i.e., csf leak, pseudomeningocele, and meningitis) after ist tumor surgery remains high and in reports from major high volume centers ranges between 5% up to 18% (1 - 4, 6, 7, 9, 11, 13 - 17, 19 - 22, 25, 28 - 30). Treating these complications may often require prolonged postoperative bed rest, external lumbar drainage, operative re - exploration, and the prolonged use of antibiotics . These complications may frequently lead to additional complications, such as deep vein thrombosis, pulmonary embolism, pneumonia, urinary tract infection, skin breakdown, subdural or cerebellar hematomas, and even death . They also significantly increase medical expenses and the length of the hospital stay, which in turn may worsen the outcome scores of both the hospital and individual surgeons, as well as patient satisfaction surveys . Weber et al . (32) reported a 50% increase in mean hospital cost per case with csf - related postoperative complications after elective spinal surgery . The effect of csf - related complications on outcomes is particularly true as patients undergoing this surgery may be older (> 65 years) and have medical co - morbidities, as we found in one - third of the patients in our series . A higher number of elderly patients with ists who require surgical treatment has also been reported by sacko and colleagues (25). While such patients can have a successful surgery with radical tumor resection and frequently no neurological consequences, they may not tolerate the prolonged bed rest required to repair the csf - related postoperative complication . The additional complications mentioned earlier can also jeopardize an otherwise successful surgery and rehabilitation process . Two recent studies (10, 33) evaluated the efficacy of polyethylene glycol sealants in an attempt to decrease the rate of csf - related complications after intradural spinal surgery . Goodwin and colleagues (10) reported that csf leaks occurred in 5% of patients and meningitis occurred in 1% . Similarly, wright and associates (33) compared the use of polyethylene glycol sealant to the standard of care dural closure . Csf - related complications that required re - operation in the sealant treated group was 7% compared with 13% in the standard dural closure cohort . In addition, each cohort had additional 4% rate of pseudomeningocele formation that did not require re - operation . Thirty - five and 15 years ago, respectively, both mayfield (18) and black (5) described and recommended the use of a fat graft to prevent potentially life - threatening csf complications after posterior fossa and spinal surgery . However, we were not able to find any reports of the use of intraoperative fat grafting in the literature or reports of any other efficient way that ensures preventing this vexing postoperative complication problem . Our own experience in the surgical series of ists treated at our institution mirrored the experience of others . As a consequence, we prospectively adopted the intraoperative use of fat grafting after the third patient in our series developed a large pseudomeningocele, was readmitted to the hospital, and underwent a second surgery with revision of the dural closure and placement of an autologous fat graft and external lumbar drainage for 5 days . Since then, we have operated on an additional 37 patients with ists prospectively using this graft at dural closure and have not seen any csf - related complications in these patients . Technical considerations the spinal dura appears to be thickest dorsally in the midline and thins laterally, particularly along the dural sleeves of the spinal nerves . We use the running dural midline suture to achieve watertight closure after the tumor is removed and then use the valsalva maneuver to ensure watertight closure . Still, there is no guarantee that the suture line wo nt weaken or that a leak wo nt occur after the patient is mobilized postoperatively, when csf fully replenishes and patient engages in full, physiologic csf pressure challenges of the dural suture line . Csf may still seep into the low pressure dead space created after a laminectomy, leading to a pseudomeningocele or csf leak . We found that reinforcing the dural suture line with autologous fat is useful particularly in cases when the valsalva maneuver revealed csf seepage . In addition, the fat graft obliterates the dead space created by a laminectomy and muscle dissection, and creates gentle pressure to the dural suture line that may prevent the formation of a pseudomeningocele and a csf leak . In some cases, it may be impossible to achieve watertight dural closure, and these cases are particularly at risk for csf leak and therefore clearly benefit from this concept and technique: 1) an ist in a sacral location (one of our cases); 2) a craniospinal ist requiring a y - shaped dural incision and patch grafting (figures 2 and 3, 27% of cases in our series); 3) when the tumor invades the dura (e.g., meningioma) and necessitates dural excision to achieve radical resection and subsequent dural patching; and 4) if the tumor is a dumbbell iest extending into the intervertebral foramen and beyond (figures 4 and 5). Eighteen percent of patients in our series had dumbbell tumors (5 schwannomas and 1 meningioma) and after radical tumor resection, it was not possible to achieve a watertight dural closure without incorporating a fat graft into the sutures . Fat tissue was harvested at the beginning of surgery via a horizontal incision of approximately 2 cm while the patient was supine . It was routinely done on the left side to prevent confusion in the case of a future appendectomy or other abdominal surgery on the right . Fat deposits are abundant in the abdominal area even in patients with a low bmi . In our experience, the tissue harvest did not add any significant time or expense to surgery, nor did it lead to infection, hematoma, or cosmetic problems for our patients . In addition, fat autograft carries neither the risk of hypersensitivity reaction nor risk of infectious disease transmission . Finally, we favored a separate incision for the tissue removal, rather than harvesting fat from the subcutaneous tissue in the area of the primary incision . This approach prevents the development of additional tissue pouches in the primary surgical area that may favor a pseudomeningocele or hematoma formation and jeopardize wound healing . The reported incidence of csf related complications after surgery for ist remains high in published series . The prospective use of autologous fat grafting ensures watertight dural closure and obliterates the dead space created during surgical exposure, muscle dissection, and bone removal . This technique appears to significantly reduce, if not completely eliminate, postoperative csf - related complications in patients with ists, without adding any significant operative time, expense or complications.
The prevalence of obesity has risen dramatically in the united states . The rates for adults have more than doubled during the past 3 decades . Increases in obesity prevalence have been marked across all gender, race, and socioeconomic groups . Because of a relatively high prevalence, a rapidly increasing trend, and large social - group disparities, adult obesity is recognized as a major public health problem in the us [1, 2]. While obesity data for us adults are routinely available by age, gender, and race / ethnicity [1, 3], prevalence estimates for various immigrant and socioeconomic groups are less well known, particularly temporal obesity patterns among them [47]. The immigrant population in the us has increased four - fold in the last four decades [810]. In 2008, there were 38 million immigrants, an increase of 28.4 million since 1970 [810] given such a rapid population increase, analysis of obesity patterns among immigrants of various ethnicities assumes a special importance [4, 11]. In addition to ethnic and immigrant disparities, monitoring socioeconomic inequalities in health has long represented an important research and policy focus [1, 2]. Socioeconomic inequalities as well as immigrant differentials in health, life expectancy, and mortality from major causes of death have not only remained substantial in the us but have also increased over time [4, 1215]. Inequalities in chronic disease risk factors such as obesity, smoking, physical inactivity, and poor diet have contributed greatly to the persistence and/or widening of the health gradients [1, 12, 13, 16]. The purpose of this study was to describe national trends in immigrant and social class inequalities in the prevalence of obesity and overweight and to identify immigrant and social class groups who are at high risk of obesity and who have experienced substantial increases in their obesity rates . Specifically, we (1) estimate over time changes in obesity and overweight prevalence among 30 major immigrant groups stratified by race / ethnicity and length of immigration and among detailed education, occupation, and income groups, using large, nationally representative samples of us adults and (2) compare the magnitude of ethnic - immigrant and socioeconomic disparities in obesity and overweight prevalence among adults aged 18 over time . Temporal individual - level data on obesity, overweight, and selected socioeconomic, demographic, and behavioral characteristics were derived from the 1976 and 19912008 national health interview surveys (nhis) [17, 18]. The nhis, which is conducted by the national center for health statistics, uses a complex, multistage probability design and is representative of the civilian non - institutionalized population of the us [17, 18]. All data are based on self - reports, including height and weight information, and obtained via in - home person interviews [1, 17, 18]. Substantive and methodological details of the nhis are described elsewhere [1, 17, 18]. Annual trends in obesity and overweight prevalence and bmi were estimated for the overall immigrant and us - born groups and for five educational groups from 1991 to 2008 . To analyze trends over time by detailed ethnic - immigrant and socioeconomic characteristics, we pooled 4 years of the nhis data from 1992 to 1995 and 6 years of data from 2003 to 2008 . Aggregating data for several years in this fashion ensured sufficient sample sizes for analyzing patterns for groups stratified by ethnicity, immigrant status, and length of immigration . We could not use the 1991 nhis file in the pooled analyses because it lacked detailed ethnic and income groupings . The 1976 nhis, the earliest survey to collect height and weight data, was used to provide baseline estimates for various socioeconomic groups . Obesity and overweight differentials were analyzed for 323,627 adults in 19921995 and 154,649 adults aged 18 in 20032008 for whom information on bmi was available . Adult overweight was defined as a bmi 25 kg / m and obesity as a bmi 30 kg / m [1, 4, 19]. Note that the overweight category includes obese individuals . Immigrant status was defined on the basis of adults place of birth [4, 9, 11]. Us - born were those born in one of the 50 us states or washington, dc . Immigrants or foreign - born refer to those born outside these territories [4, 9, 11]. Race / ethnicity was classified into 11 major categories: non - hispanic whites, non - hispanic blacks, american indians / alaska natives, chinese, asian indians, filipinos, other asian / pacific islanders, mexicans, puerto ricans, cubans, and central and south americans, including other hispanics . The joint variable of ethnic - immigrant status included 30 categories, with each racial / ethnic group (except for american indians / alaska natives who are, by definition, a native group) divided into the us - born group, the recent immigrant group, and the long - term immigrant group . Although all puerto ricans are us citizens, those born in puerto rico and abroad were classified as immigrants for convenience . Following a previous study and given the health and socio - behavioral profiles by duration of residence, recent immigrants were defined as those who immigrated to the us in the previous 15 years, whereas long - term immigrants were those who immigrated to the us more than previous 15 years . In addition to ethnic - immigrant status, we considered the following socioeconomic and demographic factors that are known to influence obesity: age, gender, marital status, region of residence, educational attainment, family income / poverty status, occupation, and physical activity (pa) [4, 5, 7, 11, 19]. These covariates were measured as shown in tables 1 and 2.table 1observed (weighted) prevalence and adjusted odds of obesity (bmi 30) among 30 ethnic - immigrant groups aged 18 + years and by selected socioeconomic and demographic characteristics: the national health interview survey, 19922008covariates19921995 (n = 323,627)20032008 (n = 154,649)19922008prevalenceadjusted odds ratioprevalenceadjusted odds ratio% increase in prevalence%seor95% ci%seor95% ciduration of residence in the us (years) <15.70.80.410.300.558.11.50.270.180.4144.1 157.90.50.530.460.6110.80.70.350.290.4036.0 * 597.60.30.430.390.4814.60.60.420.370.4791.2 * 10149.70.40.570.520.6316.40.80.470.420.5368.4 * 15 + 13.10.30.720.680.7622.00.40.660.620.7067.9 * us - born15.60.11.00reference26.50.21.00reference70.6relative index of disparity36.122.2438.152.235.6gamma ()0.180.0080.210.00816.7non - hispanic white recent immigrants9.10.60.660.570.7710.50.90.410.340.5014.8 long - term immigrants11.30.40.740.680.8019.20.90.750.670.8369.6 * us - born14.20.11.00reference24.70.21.00reference73.3non - hispanic black recent immigrants11.91.00.820.680.9917.31.40.640.530.7946.1 long - term immigrants13.81.10.880.721.0825.71.80.950.791.1486.9 * us - born23.90.31.711.651.7736.10.41.601.531.6751.2american indian / alaska native22.41.41.581.321.8839.22.11.811.512.1775.2chinese recent immigrants1.10.30.070.040.132.30.80.080.040.17108.1 long - term immigrants2.20.50.140.090.223.20.90.110.060.1945.5 us - born5.91.60.540.320.918.42.10.400.230.6743.4filipino recent immigrants2.10.50.150.090.247.61.90.290.180.48256.5 * long - term immigrants4.80.90.320.220.4712.61.60.450.340.59164.2 * us - born11.32.71.030.631.6819.72.50.850.621.1574.7asian indian recent immigrants3.30.70.230.150.355.71.00.230.160.3375.7 long - term immigrant / us - born5.31.10.380.250.598.81.40.330.230.4765.6other asian and pacific islanders recent immigrants2.80.50.170.120.254.00.80.130.090.2040.4 long - term immigrants4.50.80.280.190.417.10.90.220.170.2960.2 * us - born7.91.00.630.500.8118.12.30.820.621.09129.9mexican recent immigrants12.80.70.750.670.8418.90.70.660.590.7447.9 long - term immigrants21.30.81.151.051.2630.80.81.030.941.1344.8 * us - born21.20.51.591.501.6934.60.81.641.511.7762.9puerto rican recent immigrants14.91.10.910.761.0827.63.01.180.891.5784.7 long - term immigrants22.41.21.221.071.4032.71.71.231.061.4446.2 * us - born14.20.81.100.961.2730.61.51.321.151.51116.2cuban recent immigrants12.50.90.670.570.7922.03.20.720.491.0475.4 long - term immigrants16.02.30.980.711.3724.21.80.840.691.0150.7 * us - born14.10.11.160.881.5324.12.91.130.821.5571.3central and south americans and other hispanics recent immigrants9.10.60.550.480.6314.60.90.480.420.5660.4 long - term immigrants13.30.70.760.680.8525.41.30.840.720.9791.6 * us - born14.80.71.151.021.2928.01.41.291.121.4989.6relative index of disparity39.512.1138.441.972.7gender male14.90.11.00reference25.40.21.00reference70.4 female15.10.10.960.940.9925.20.20.960.930.9966.9education (years of school completed) 0820.90.31.901.802.0128.00.61.631.511.7634.3 91119.00.31.711.631.7929.10.41.641.551.7452.9 * 1216.00.11.461.401.5128.20.31.551.471.6276.5 * 131513.60.21.341.291.3926.70.31.541.471.6195.7 * 16 + 10.30.11.00reference18.40.31.00reference78.8relative index of disparity54.950.2241.620.0124.3gamma ()0.190.0040.140.00426.3family income ($) <10,00020.00.51.621.511.7428.40.71.441.321.5842.3 10,00019,99918.70.31.561.481.6427.20.51.361.271.4545.3 * 20,00024,99917.40.31.451.381.5326.10.61.261.161.3649.7 * 25,00034,99916.40.31.391.311.4627.10.51.301.221.3965.1 * 35,00044,99915.50.21.301.251.3627.80.51.321.241.4179.6 * 45,00064,99914.80.21.231.181.2826.20.51.191.121.2677.7 * 65,000 + 11.70.21.00reference22.10.31.00reference88.5relative index of disparity39.340.3919.400.5250.7gamma ()0.140.0040.080.00542.9poverty status (ratio of family income to poverty threshold) <100%28.30.51.331.251.42 100199%28.30.41.291.221.36 200299%27.70.41.241.181.31 300399%27.40.41.221.161.29 400499%24.80.51.101.031.16 500%22.20.31.00referencerelative index of disparity19.160.22gamma ()0.090.004occupation professional / managerial12.50.21.00reference22.00.31.00reference75.9 sales / clerical / technical support13.70.20.970.931.0125.50.31.020.981.0786.2 * service16.50.31.051.001.1027.50.41.071.011.1366.8 * craft and repair15.40.30.940.890.9927.60.40.980.931.0478.8 * laborers17.70.21.061.011.1131.50.51.211.141.2978.4 * unemployed / not in labor force16.10.21.061.031.1022.70.61.000.921.0840.7relative index of disparity21.140.2121.850.093.4gamma ()0.110.0040.090.00418.2all chi - square tests for independence between each covariate (except gender) and obesity prevalence were statistically significant at p <.05 increases in obesity prevalence or changes in gamma or disparity indices during 19922008 were statistically significantly different from 0 (p <.05)adjusted by logistic regression for survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty statusrecent immigrants were those who immigrated to the us <15 years previous . Long - term immigrants were those who had been in the us 15 yearstable 2observed (weighted) prevalence and adjusted odds of overweight(bmi 25) among 30 ethnic - immigrant groups aged 18 + years and by selected socioeconomic and demographic characteristics: the national health interview survey, 19922008covariates19921995 (n = 323,627)20032008 (n = 154,649)19922008prevalenceadjusted odds ratioprevalenceadjusted odds ratio% increase in prevalence%seor95% ci%seor95% ciduration of residence in the us (years) <125.91.70.510.430.6037.7 3.30.430.320.5845.5 * 1532.80.90.660.600.7145.41.30.580.520.6538.3 * 5938.70.60.710.670.7649.91.00.580.530.6429.0 * 101441.20.90.790.740.8655.21.00.710.640.7833.9 * 15 + 48.10.50.890.850.9260.90.50.820.780.8726.7 * us - born48.30.21.00reference61.30.21.00reference26.9relative index of disparity18.891.3715.601.7317.4gamma ()0.090.0050.090.0060.0non - hispanic white recent immigrants39.01.30.810.730.9146.31.70.700.610.8118.5 long - term immigrants46.20.70.920.860.9758.01.10.910.830.9925.5 * us - born46.70.21.00reference59.60.21.00reference27.8non - hispanic black recent immigrants49.41.71.321.171.4951.41.90.830.710.974.1 long - term immigrants55.81.41.401.231.5967.71.91.351.121.6221.4 us - born58.40.31.791.741.8469.70.41.721.651.7919.3american indian / alaska native56.31.41.591.401.8069.62.01.581.311.9223.5chinese recent immigrants15.01.00.220.190.2619.82.30.190.140.2632.2 long - term immigrants21.32.20.280.220.3723.22.20.190.150.249.1 us - born24.62.30.500.410.6040.13.30.640.480.8563.3filipino recent immigrants23.81.60.420.350.5139.63.40.510.390.6766.8 long - term immigrants32.62.40.560.450.7147.72.40.590.480.7346.3 * us - born35.75.40.910.61.3854.53.51.030.771.3652.6asian indian recent immigrants26.41.90.460.380.5737.72.20.510.420.6242.5 long - term immigrant / us - born36.62.50.660.540.8146.02.90.590.470.7425.5other asian and pacific islanders recent immigrants18.91.10.290.250.3423.01.80.230.180.2921.8 long - term immigrants23.51.40.360.310.4234.41.60.330.290.3846.2 us - born35.91.20.790.700.8849.23.00.810.651.0337.1mexican recent immigrants49.00.91.211.121.3160.70.91.191.081.3023.8 long - term immigrants62.20.81.631.521.7473.90.81.601.451.7618.7 * us - born56.90.71.791.691.9069.30.71.811.681.9521.9puerto rican recent immigrants50.62.11.361.131.6359.33.41.190.901.5617.2 long - term immigrants61.41.21.511.361.6871.21.91.511.261.8216.0 * us - born46.91.41.301.161.4765.91.71.501.291.7540.7cuban recent immigrants55.32.11.281.091.5164.73.71.120.791.6017.1 long - term immigrants55.32.41.251.031.5164.82.41.040.861.2717.1 * us - born46.02.21.231.021.4856.53.41.120.841.4823.0central and south americans and other hispanics recent immigrants40.71.30.860.770.9554.41.40.860.760.9733.7 long - term immigrants49.41.11.020.921.1269.11.21.321.171.4939.9 * us - born47.81.11.261.161.3764.41.41.501.321.6934.8relative index of disparity23.920.7820.561.2614.0gender male57.00.21.00reference68.10.21.00reference19.6 * female39.10.20.450.440.4653.10.30.500.480.5135.8education (years of school completed) 0856.70.31.501.441.5666.00.61.391.291.4916.5 91152.10.31.441.381.4962.20.51.381.301.4519.5 * 1249.10.21.311.281.3563.50.31.401.351.4629.5 * 131544.80.21.231.201.2761.10.31.421.361.4736.4 * 16 + 42.30.31.00reference54.50.31.00reference28.9relative index of disparity15.860.0812.840.0719.0gamma ()0.130.0030.120.0047.7family income ($) <10,00048.40.81.241.161.3357.70.81.101.021.1919.2 * 10,00019,99949.70.41.231.181.2859.80.51.121.051.1920.4 * 20,00024,99950.40.41.221.181.2761.20.71.141.051.2221.6 * 25,00034,99949.30.41.181.141.2261.40.61.111.051.1824.5 * 35,00044,99949.50.31.191.161.2362.70.61.141.071.2126.7 * 45,00064,99948.90.31.151.121.1862.40.51.111.051.1727.6 * 65,000 + 45.00.21.00reference59.20.41.00reference31.6relative index of disparity8.390.273.160.1162.3gamma ()0.060.0030.010.00583.3poverty status (ratio of family income to poverty threshold) <100%58.60.61.121.061.18 100199%62.00.41.151.091.2 200299%62.30.41.121.071.17 300399%62.40.51.121.071.18 400499%62.20.51.121.061.19 500%59.40.41.00referencerelative index of disparity3.390.02 gamma ()0.010.004occupation professional / managerial45.40.21.00reference58.10.31.00reference28.2 sales / clerical / technical support43.10.20.980.951.0058.30.31.041.001.0835.4 * service48.60.41.071.031.1061.10.41.071.021.1225.7 * craft and repair58.10.41.051.021.0968.50.40.990.941.0417.8 * laborers55.80.31.091.051.1369.50.51.131.071.2024.6 * unemployed / not in labor force47.10.20.990.961.0150.40.70.910.850.977.1relative index of disparity12.870.108.430.0634.5gamma ()0.130.0030.130.0040.00all chi - square tests for independence between each covariate and overweight prevalence were statistically significant at p <.05 * increases in overweight prevalence or changes in gamma / disparity indices during 19922008 were statistically significantly different from 0 (p <.05)adjusted by logistic regression for survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty statusrecent immigrants were those who immigrated to the us <15 years previous . Long - term immigrants were those who had been in the us 15 years observed (weighted) prevalence and adjusted odds of obesity (bmi 30) among 30 ethnic - immigrant groups aged 18 + years and by selected socioeconomic and demographic characteristics: the national health interview survey, 19922008 all chi - square tests for independence between each covariate (except gender) and obesity prevalence were statistically significant at p <.05 * increases in obesity prevalence or changes in gamma or disparity indices during 19922008 were statistically significantly different from 0 (p <.05) adjusted by logistic regression for survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty status recent immigrants were those who immigrated to the us <15 years previous . Long - term immigrants were those who had been in the us 15 years observed (weighted) prevalence and adjusted odds of overweight(bmi 25) among 30 ethnic - immigrant groups aged 18 + years and by selected socioeconomic and demographic characteristics: the national health interview survey, 19922008 all chi - square tests for independence between each covariate and overweight prevalence were statistically significant at p <.05 * increases in overweight prevalence or changes in gamma / disparity indices during 19922008 were statistically significantly different from 0 (p <.05) adjusted by logistic regression for survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty status recent immigrants were those who immigrated to the us <15 years previous . Long - term immigrants were those who had been in the us 15 years educational attainment was measured both as a categorical variable (08, 911, 12, 1315, 16 years) and a continuous variable in terms of years of school completed . Annual family income was also measured both as a categorical variable and a continuous variable . The seven income strata for 20032006 were: <10,000, 10,00019,999, 20,00024,999, 25,00034,999, 35,00044,999, 45,00064,999, and 65,000 . The corresponding income strata for 19921995 were: <7,000, 7,00014,999, 15,00019,999, 20,00024,999, 25,00034,999, 35,00049,999, and 50,000 . The income categories were roughly comparable for the 19921995 and 20032006 periods, given an increase by a factor of about 1.3 in the consumer price index between 1995 and 2005 . Occupational class was defined in terms of 5 broad categories: professional and managerial occupations, sales / clerical and technical support occupations, service, craft and repair, and laborers . These occupational groups, derived from the major occupational groups defined by the census bureau, are consistent with previously defined social class positions of upper white collar, lower white collar, upper blue collar, and lower blue collar jobs [20, 21]. Professional and managerial occupations included executives, managers, administrators, engineers, architects, mathematical and computer scientists, teachers, writers, artists, and other professional specialty occupations . Sales / clerical and technical support occupations included technicians, health technologists, sales workers, computer equipment operators, secretaries, typists, and financial records processing, mail, message distributing, and other administrative support occupations . Service occupations included private household, protective service, food service, health service, cleaning and building service occupations, farm and other agricultural workers . Craft and repair occupations included mechanics, repairers, and those in construction, extractive trades, and precision production jobs . Laborers included machine operators, fabricators, assemblers, motor vehicle and material moving equipment operators, construction laborers, handlers, equipment cleaners, and helpers . In addition, a residual category for the unemployed and those outside the labor force was used . Pa level was measured by the number of times / week of vigorous activities of at least 10 min that caused heavy sweating or large increases in breathing or heart rate . The variable was coded as <1, 12, 34, 5 times / week of activity . Multivariate logistic regression was used to examine the association between the binary outcomes of obesity and overweight and selected socioeconomic and demographic factors . Least squares regression was used to model mean bmi . To account for the complex sample design of the nhis the two - sample t test was used to test the difference in prevalence between any two groups at one point in time or to test for change in prevalence between two time points for a specific group . The gamma () statistic, varying between 1 and 1, was used to measure the magnitude of the association between an ordinal covariate and obesity . An index of disparity (i d), which approximated in relative terms the average deviation of the rates from the rate for the best - off ethnic - immigrant or socioeconomic group, was used to summarize disparities across all social groups [16, 23]. The relative mean deviation index of disparity was calculated as:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{id}} = \, \left\ {{\left ({\sum\limits_{i} \|{o_{\text{ri}}} - o_{\text{rl}} \|/i} \right)/o_{\text{rl}}} \right\} \times 100;\quad o_{\text{rl}}> 0 $$\end{document}where ori is the obesity / overweight prevalence for the ith group, orl is the rate for the standard group or group with the lowest obesity / overweight prevalence, and i is the number of groups . A simulation method was used to estimate the standard error for i d . The obesity prevalence for the total us adult population aged 18 tripled from 8.7% in 1976 to 27.4% in 2008 . The overweight prevalence for all us adults increased from 36.9% in 1976 to 62.0% in 2008 . In 2008, 59 million us adults were obese and 134 million overweight . This represented an absolute increase of 47 million obese and 80 million overweight adults since 1976 . During 19912008, the obesity prevalence for us - born adults increased from 13.9 to 28.7%, whereas the prevalence for immigrants increased from 9.5 to 20.7% (fig . 1). The average annual rates of increase for the two groups were 4.5% and 4.6%, respectively . During 19912008, increases in overweight prevalence were equally marked among both us - born and immigrant adults, with the prevalence for the us - born rising from 45.7 to 62.7%, while that for immigrants rising from 39.6 to 58.4% . The mean bmi for the us - born increased from 25.24 in 1991 to 27.75 in 2008, while for immigrants it increased from 24.55 to 26.56 (fig . 1trends in obesity and overweight prevalence (%) among us adults by immigrant status and education, 19762008 trends in obesity and overweight prevalence (%) among us adults by immigrant status and education, 19762008 annual trends by educational attainment show persistent disparities in obesity and overweight prevalence and bmi (fig . 1). Educational gradients were more consistent and pronounced in 1976 and the 1990s than during the first decade of the 2000s . The rate of increase in obesity and overweight was greater for those with 12, 1315 and 16 years of education than for those with 08 and 911 years of education . During 19912008, the average annual rates of increase in obesity for the 5 (low to high) educational groups were 2.58, 3.63, 4.75, 5.54, and 5.05%, respectively . The corresponding rates of increase in overweight were 1.36, 1.90, 2.32, 2.90, and 2.25% . The immigrant groups varied substantially in their socioeconomic characteristics (fig . 2). They were also twice as likely to be without a high school diploma as the us - born . Socioeconomic achievement levels increased with increasing duration of residence in the us . During 20032008, less than 6% of mexican immigrants were college graduates, compared with 67% of recent asian indian immigrants . Less than 9% of mexican immigrants were employed in professional and managerial occupations, as compared with 52% of recent asian indian immigrants and 53% of us - born chinese . Poverty rates varied from a low of 5% for long - term filipino immigrants to a high of 33% for recent mexican immigrants.fig . 2selected socioeconomic characteristics (%) of 30 ethnic - immigrant groups, us adults aged 18 years and older, 20032008 selected socioeconomic characteristics (%) of 30 ethnic - immigrant groups, us adults aged 18 years and older, 20032008 table 1 shows increases in obesity prevalence between 19921995 and 20032008 for detailed ethnic - immigrant and socioeconomic groups . Regardless of ethnicity, all immigrant groups experienced a substantial increase in prevalence, with the increase being greater among the us - born population and longer - term immigrants . Tables 1 and 2 show considerable disparities in obesity and overweight prevalence by immigrant status and selected socioeconomic factors . The observed obesity prevalence in 20032008 ranged from 2.3% for recent chinese immigrants to 30.6% or higher for american indians, us - born blacks, mexicans, and puerto ricans, and long - term mexican and puerto rican immigrants (table 1). The overweight prevalence in 20032008 ranged from 19.8% for recent chinese immigrants to 70% or higher for american indians, us - born blacks and mexicans, and long - term mexican and puerto rican immigrants (table 2). Mean bmi in 20032008 varied from a low of 22.6 for recent chinese immigrants to a high for 28.9 for us - born blacks and american indians, and 28.6 for us - born mexicans (table 3). The summary index of disparity showed similar ethnic - immigrant disparities in obesity and overweight prevalence in 19921995 and 20032008.table 3observed and adjusted mean body mass index (bmi) among 30 ethnic - immigrant groups aged 18 + years and by selected sociodemographic characteristics: the national health interview survey, 19922008covariates19921995 (n = 323,627)20032008 (n = 154,649)19922008observedadjustedobservedadjustedincrease in bmibmisebmisebmisebmise%absoluteduration of residence in the us (years) <123.30.1724.10.1424.30.2924.80.284.31.0 1523.90.1024.50.0924.90.1125.30.114.11.0 5924.40.0524.50.0625.60.0925.50.095.11.3 101424.70.0924.80.0726.10.1025.80.115.91.5 15 + 25.40.0525.10.0426.80.0526.40.065.81.5 us - born25.60.0225.60.0227.40.0327.40.037.11.8non - hispanic white recent immigrants24.50.1124.80.1025.00.1525.50.152.00.5 long - term immigrants25.20.0725.00.0726.40.1126.50.115.11.3 us - born25.40.0225.30.0227.10.0327.10.036.91.8non - hispanic black recent immigrants25.40.1425.60.1325.90.2226.10.222.30.6 long - term immigrants26.00.1325.70.1327.70.2427.30.236.51.7 us - born27.00.0427.00.0428.90.0628.80.067.11.9american indian / an26.60.1826.60.1928.90.3228.70.318.62.3chinese recent immigrants22.00.1122.30.1022.60.2023.10.202.70.6 long - term immigrants22.80.1622.70.1623.10.1623.20.161.50.4 us - born23.20.2924.10.2224.30.2925.50.304.71.1filipino recent immigrants23.10.1323.60.1324.30.2524.80.225.31.2 long - term immigrants23.80.1623.90.1625.50.2025.50.206.81.6 us - born24.60.4625.50.3826.40.4026.90.377.41.8asian indian recent immigrants23.20.1623.60.1624.00.1624.80.163.60.8 long - term imm / us - born24.00.1624.10.1524.80.2125.10.203.30.8other asian / pacific islander recent immigrants22.30.1122.50.1122.90.1923.30.202.90.6 long - term immigrants23.00.1923.20.2024.20.2124.10.194.91.1 us - born24.20.1724.70.1525.90.3526.60.327.21.7mexican recent immigrants25.60.0925.60.0926.60.0926.70.104.11.1 long - term immigrants26.90.1126.20.0928.30.0927.50.105.11.4 us - born26.60.0826.80.0828.60.1128.80.117.42.0puerto rican recent immigrants25.50.1925.50.1827.30.3727.50.336.91.8 long - term immigrants26.90.1226.10.1228.10.2127.60.204.71.3 us - born25.30.1325.80.1328.30.2028.40.1912.03.0cuban recent immigrants25.60.1625.20.1627.20.3826.70.396.21.6 long - term immigrants26.00.3425.70.3127.50.3127.10.285.81.5 us - born25.30.2825.80.2727.20.4327.70.427.21.8central and south americans and other hispanics recent immigrants24.70.1124.80.0926.00.1226.00.135.11.3 long - term immigrants25.50.1025.10.1027.60.1327.00.148.22.1 us - born25.50.1025.90.0927.60.1927.90.188.12.1gender male26.10.0226.20.0227.60.0327.60.035.71.5 female24.90.0224.90.0226.90.0326.80.037.81.9education (years of school completed) 0826.50.0426.10.0427.80.0827.50.084.61.2 91126.10.0425.90.0427.60.0627.50.066.01.6 1225.70.0225.60.0227.70.0427.50.047.82.0 131525.20.0325.50.0227.40.0427.50.048.72.2 16 + 24.80.0224.90.0326.30.0326.40.045.81.5family income ($) <10,00026.00.0926.10.0827.60.1227.90.126.21.6 10,00019,99926.00.0426.00.0427.40.0727.60.075.71.5 20,00024,99925.80.0425.80.0427.40.0927.50.096.21.6 25,00034,99925.70.0425.70.0427.40.0727.40.076.81.7 35,00044,99925.60.0325.60.0327.60.0727.40.077.61.9 45,00064,99925.50.0325.50.0327.40.0627.30.067.51.9 65,000 + 25.10.0225.10.0326.80.0526.70.056.91.7poverty status (ratio of family income to poverty threshold) <100%27.60.0827.80.06 100199%27.70.0527.70.05 200299%27.50.0527.40.05 300399%27.50.0627.40.06 400499%27.20.0627.10.06 500%26.80.0426.80.04occupation professional / managerial25.20.0225.50.0226.80.0427.20.046.31.6 sales / clerical / tech support25.10.0225.40.0227.10.0427.30.048.32.1 service25.70.0425.60.0327.50.0627.40.067.11.8 craft and repair26.20.0325.40.0427.80.0527.10.056.11.6 laborers26.20.0325.60.0328.30.0727.70.078.12.1 unemployed / not in lf25.50.0225.60.0326.40.1027.10.103.60.9adjusted bmi was derived from fitted least square regression models that included survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty statusall percentage and absolute increases in bmi during 19922008 were statistically significantly different from 0 (p <.01) observed and adjusted mean body mass index (bmi) among 30 ethnic - immigrant groups aged 18 + years and by selected sociodemographic characteristics: the national health interview survey, 19922008 adjusted bmi was derived from fitted least square regression models that included survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty status all percentage and absolute increases in bmi during 19922008 were statistically significantly different from 0 (p <.01) the odds and prevalence of obesity and overweight, even after adjusting for sociodemographic factors, increased with increasing duration in the us (tables 1, 2). The obesity gradients by length of immigration were steeper in 20032008 than in 19921995 . Compared with the us - born, immigrants who had lived in the us for <1 year or 15 years had 73 or 34% lower odds of obesity in 20032008 and 59 or 28% lower odds of obesity in 19921995, respectively (table 1). Immigrants who had lived in the us for <1 year or 15 years had 57 or 18% lower odds of overweight than the us - born in 20032008 (table 2). During 19922008, the disparity indices suggest a slight increase in obesity differentials between us - born and immigrants of various durations . Compared with us - born whites, the odds of obesity were 59 and 25% lower for recent and long - term white immigrants, 36% lower for recent black immigrants, 5592% lower for us- and foreign - born chinese, asian indians, and filipino immigrants, 3452% lower for recent mexican and central / south american immigrants, respectively . However, us - born blacks, american indians, mexicans, and puerto ricans, and long - term puerto rican immigrants had 60, 81, 64, 32, and 23% higher odds of obesity, respectively than us - born whites (table 1). Compared with chinese immigrants, us - born blacks, mexicans, puerto ricans, and central / south americans, and american indians had 1419 times higher odds of obesity, whereas white, black, mexican, cuban, and central / south american immigrants had 413 times higher odds of obesity . Compared with chinese immigrants, all other ethnic - immigrant groups had 310 times higher odds of overweight (table 4).table 4age - sex - year and covariate - adjusted odds of obesity (bmi 30) and overweight (bmi 25) by ethnic - immigrant status (chinese immigrants used as reference group), us adults aged 18 + years: the national health interview survey, 20032008ethnic - immigrant groupobesityobesityoverweightoverweightage sex year adjusted modelcovariate adjusted modelage sex year adjusted modelcovariate adjusted modelor95% cior95% cior95% cior95% cinon - hispanic white recent immigrants4.502.837.184.332.716.913.782.994.793.702.924.69 long - term immigrants8.305.3412.907.815.0212.165.044.066.264.783.835.96 us - born11.917.7518.3110.466.7716.145.874.827.145.274.316.44non - hispanic black recent immigrants8.395.1813.576.734.1310.985.013.876.474.373.375.67 long - term immigrants11.777.4418.629.926.2515.767.856.0510.197.105.459.25 us - born21.3013.8832.6816.7410.8625.8010.308.4212.609.057.3611.12american indian / alaska native24.1115.1138.4818.9311.8230.319.747.4012.838.336.3111.00chinese immigrants (all)1.00reference1.00reference1.00reference1.00reference us - born3.591.767.344.162.028.593.002.064.373.372.304.93filipino recent immigrants3.101.596.023.031.575.862.752.013.742.691.973.66 long - term immigrants4.792.917.914.682.837.733.222.414.293.122.324.20 us - born9.545.6816.008.875.2814.885.554.007.695.413.867.58asian indian recent immigrants2.341.354.062.431.404.222.612.013.402.692.073.50 long - term immigrants / us - born3.341.935.773.431.985.943.052.244.153.112.284.24other asian and pacific islanders recent immigrants1.570.852.911.410.762.601.270.931.741.220.891.68 long - term immigrants2.621.564.422.311.373.901.871.482.371.741.372.21 us - born8.815.1914.938.625.1214.514.333.136.014.293.085.97mexican recent immigrants9.816.3415.176.894.4410.717.706.269.486.265.077.73 long - term immigrants15.159.8423.3110.827.0116.7110.328.3212.808.426.7710.48 us - born21.4313.7833.3217.1010.9926.6311.099.0213.639.547.7311.76puerto rican recent immigrants15.859.4526.5912.317.2820.807.285.2110.176.254.468.77 long - term immigrants16.7610.6526.3812.888.1420.369.267.1611.977.986.1210.39 us - born17.3011.0427.0813.828.7921.729.187.2111.687.916.1910.10cuban recent immigrants10.015.6217.817.484.2113.307.044.6810.605.913.918.94 long - term immigrants10.976.8317.608.765.4414.126.354.778.465.504.147.32 us - born13.287.8722.3911.786.9619.946.404.549.035.884.178.30central and south americans and other hispanics recent immigrants6.604.1710.465.053.178.035.354.266.724.533.595.72 long - term immigrants11.437.2817.958.755.5513.798.156.5010.226.955.528.75 us - born15.6410.2123.9413.528.7920.798.656.9110.837.886.279.91adjusted for survey year, age, gender, marital status, family size, region of residence, education, occupation, and poverty status age - sex - year and covariate - adjusted odds of obesity (bmi 30) and overweight (bmi 25) by ethnic - immigrant status (chinese immigrants used as reference group), us adults aged 18 + years: the national health interview survey, 20032008 adjusted for survey year, age, gender, marital status, family size, region of residence, education, occupation, and poverty status socioeconomic gradients in obesity, although substantial in each period, were less pronounced in 20032008 than in 19921995 and 1976 . The summary indices also indicate decreasing educational disparities over time . Between 1976 and 2008, obesity prevalence doubled for those with <9 years of education, while it increased 45 fold for those with a college education . Those with <9 years of education had 152% higher adjusted odds of obesity in 1976, 90% higher odds in 19921995, and 63% higher odds in 20032008 than those with a college degree . In terms of continuous education, each additional year of education was associated with 11% lower odds of obesity (or = 0.89; 95% ci = 0.890.90) and 7% lower odds of overweight (or = 0.93; 95% ci = 0.930.94) in 1976, 8% lower odds of obesity (or = 0.92; 95% ci = 0.920.92) and 6% lower odds of overweight (or = 0.94; 95% ci = 0.940.94) in 19921995, and 6% lower odds of obesity and overweight (or = 0.94; 95% ci = 0.940.95) in 20032008 . Each additional year of education was associated with a 0.17 point decrease in bmi in 20032008, the effect being significantly lower than the 0.19 point decrease in bmi in 19921995 (p <income gradients were steeper in 19921995 than in 20032006, with income disparities in prevalence, as measured by the summary indices, diminishing over time . During 20032006, those with family income <$10,000 had 44% higher odds of obesity than those with income $65,000; the roughly comparable odds in 19921995 were 62% higher for those with family income <$7,000 than for those with income $50,000 . A $5,000 increase in family income was associated with 7% (or = 0.93; 95% ci = 0.930.93) and 4% (or = 0.96; 95% ci = 0.960.96) lower odds of obesity and overweight, respectively in 19921995; in 20032006, the corresponding odds ratios were smaller: 0.97 (95% ci = 0.970.98) for obesity and 0.99 (95% ci = 0.980.99) for overweight . A $5,000 increase in family income was associated with a 0.08 point decrease in bmi in 20032006, the effect being significantly lower than the 0.14 point decrease in bmi in 19921995 (p the obesity prevalence for individuals in sales occupations quadrupled and for those in other occupations tripled between 1976 and 20032008 . In 20032008, after adjusting for education, income, and other demographic factors, service workers and laborers had 7 and 21% higher odds of obesity than those employed in professional/ managerial occupations . Although observed occupational inequalities in obesity were substantial, most of the occupational effects were accounted for by education and income differences . After adjusting for ethnicity and socioeconomic factors, physical inactivity was associated with 54% higher odds of obesity (or = 1.54; 95% ci = 1.461.62) and 36% higher odds of overweight (or = 1.36; 95% ci = 1.301.42) in 20032008 (data from the full model not shown). The obesity prevalence for the total us adult population aged 18 tripled from 8.7% in 1976 to 27.4% in 2008 . The overweight prevalence for all us adults increased from 36.9% in 1976 to 62.0% in 2008 . In 2008, 59 million us adults were obese and 134 million overweight . This represented an absolute increase of 47 million obese and 80 million overweight adults since 1976 . During 19912008, the obesity prevalence for us - born adults increased from 13.9 to 28.7%, whereas the prevalence for immigrants increased from 9.5 to 20.7% (fig . 1). The average annual rates of increase for the two groups were 4.5% and 4.6%, respectively . During 19912008, increases in overweight prevalence were equally marked among both us - born and immigrant adults, with the prevalence for the us - born rising from 45.7 to 62.7%, while that for immigrants rising from 39.6 to 58.4% . The mean bmi for the us - born increased from 25.24 in 1991 to 27.75 in 2008, while for immigrants it increased from 24.55 to 26.56 (fig . 1trends in obesity and overweight prevalence (%) among us adults by immigrant status and education, 19762008 trends in obesity and overweight prevalence (%) among us adults by immigrant status and education, 19762008 annual trends by educational attainment show persistent disparities in obesity and overweight prevalence and bmi (fig . 1). Educational gradients were more consistent and pronounced in 1976 and the 1990s than during the first decade of the 2000s . The rate of increase in obesity and overweight was greater for those with 12, 1315 and 16 years of education than for those with 08 and 911 years of education . During 19912008, the average annual rates of increase in obesity for the 5 (low to high) educational groups were 2.58, 3.63, 4.75, 5.54, and 5.05%, respectively . The corresponding rates of increase in overweight were 1.36, 1.90, 2.32, 2.90, and 2.25% . The immigrant groups varied substantially in their socioeconomic characteristics (fig . 2). They were also twice as likely to be without a high school diploma as the us - born . Socioeconomic achievement levels increased with increasing duration of residence in the us . During 20032008, less than 6% of mexican immigrants were college graduates, compared with 67% of recent asian indian immigrants . Less than 9% of mexican immigrants were employed in professional and managerial occupations, as compared with 52% of recent asian indian immigrants and 53% of us - born chinese . Poverty rates varied from a low of 5% for long - term filipino immigrants to a high of 33% for recent mexican immigrants.fig . 2selected socioeconomic characteristics (%) of 30 ethnic - immigrant groups, us adults aged 18 years and older, 20032008 selected socioeconomic characteristics (%) of 30 ethnic - immigrant groups, us adults aged 18 years and older, 20032008 table 1 shows increases in obesity prevalence between 19921995 and 20032008 for detailed ethnic - immigrant and socioeconomic groups . Regardless of ethnicity, all immigrant groups experienced a substantial increase in prevalence, with the increase being greater among the us - born population and longer - term immigrants . Tables 1 and 2 show considerable disparities in obesity and overweight prevalence by immigrant status and selected socioeconomic factors . The observed obesity prevalence in 20032008 ranged from 2.3% for recent chinese immigrants to 30.6% or higher for american indians, us - born blacks, mexicans, and puerto ricans, and long - term mexican and puerto rican immigrants (table 1). The overweight prevalence in 20032008 ranged from 19.8% for recent chinese immigrants to 70% or higher for american indians, us - born blacks and mexicans, and long - term mexican and puerto rican immigrants (table 2). Mean bmi in 20032008 varied from a low of 22.6 for recent chinese immigrants to a high for 28.9 for us - born blacks and american indians, and 28.6 for us - born mexicans (table 3). The summary index of disparity showed similar ethnic - immigrant disparities in obesity and overweight prevalence in 19921995 and 20032008.table 3observed and adjusted mean body mass index (bmi) among 30 ethnic - immigrant groups aged 18 + years and by selected sociodemographic characteristics: the national health interview survey, 19922008covariates19921995 (n = 323,627)20032008 (n = 154,649)19922008observedadjustedobservedadjustedincrease in bmibmisebmisebmisebmise%absoluteduration of residence in the us (years) <123.30.1724.10.1424.30.2924.80.284.31.0 1523.90.1024.50.0924.90.1125.30.114.11.0 5924.40.0524.50.0625.60.0925.50.095.11.3 101424.70.0924.80.0726.10.1025.80.115.91.5 15 + 25.40.0525.10.0426.80.0526.40.065.81.5 us - born25.60.0225.60.0227.40.0327.40.037.11.8non - hispanic white recent immigrants24.50.1124.80.1025.00.1525.50.152.00.5 long - term immigrants25.20.0725.00.0726.40.1126.50.115.11.3 us - born25.40.0225.30.0227.10.0327.10.036.91.8non - hispanic black recent immigrants25.40.1425.60.1325.90.2226.10.222.30.6 long - term immigrants26.00.1325.70.1327.70.2427.30.236.51.7 us - born27.00.0427.00.0428.90.0628.80.067.11.9american indian / an26.60.1826.60.1928.90.3228.70.318.62.3chinese recent immigrants22.00.1122.30.1022.60.2023.10.202.70.6 long - term immigrants22.80.1622.70.1623.10.1623.20.161.50.4 us - born23.20.2924.10.2224.30.2925.50.304.71.1filipino recent immigrants23.10.1323.60.1324.30.2524.80.225.31.2 long - term immigrants23.80.1623.90.1625.50.2025.50.206.81.6 us - born24.60.4625.50.3826.40.4026.90.377.41.8asian indian recent immigrants23.20.1623.60.1624.00.1624.80.163.60.8 long - term imm / us - born24.00.1624.10.1524.80.2125.10.203.30.8other asian / pacific islander recent immigrants22.30.1122.50.1122.90.1923.30.202.90.6 long - term immigrants23.00.1923.20.2024.20.2124.10.194.91.1 us - born24.20.1724.70.1525.90.3526.60.327.21.7mexican recent immigrants25.60.0925.60.0926.60.0926.70.104.11.1 long - term immigrants26.90.1126.20.0928.30.0927.50.105.11.4 us - born26.60.0826.80.0828.60.1128.80.117.42.0puerto rican recent immigrants25.50.1925.50.1827.30.3727.50.336.91.8 long - term immigrants26.90.1226.10.1228.10.2127.60.204.71.3 us - born25.30.1325.80.1328.30.2028.40.1912.03.0cuban recent immigrants25.60.1625.20.1627.20.3826.70.396.21.6 long - term immigrants26.00.3425.70.3127.50.3127.10.285.81.5 us - born25.30.2825.80.2727.20.4327.70.427.21.8central and south americans and other hispanics recent immigrants24.70.1124.80.0926.00.1226.00.135.11.3 long - term immigrants25.50.1025.10.1027.60.1327.00.148.22.1 us - born25.50.1025.90.0927.60.1927.90.188.12.1gender male26.10.0226.20.0227.60.0327.60.035.71.5 female24.90.0224.90.0226.90.0326.80.037.81.9education (years of school completed) 0826.50.0426.10.0427.80.0827.50.084.61.2 91126.10.0425.90.0427.60.0627.50.066.01.6 1225.70.0225.60.0227.70.0427.50.047.82.0 131525.20.0325.50.0227.40.0427.50.048.72.2 16 + 24.80.0224.90.0326.30.0326.40.045.81.5family income ($) <10,00026.00.0926.10.0827.60.1227.90.126.21.6 10,00019,99926.00.0426.00.0427.40.0727.60.075.71.5 20,00024,99925.80.0425.80.0427.40.0927.50.096.21.6 25,00034,99925.70.0425.70.0427.40.0727.40.076.81.7 35,00044,99925.60.0325.60.0327.60.0727.40.077.61.9 45,00064,99925.50.0325.50.0327.40.0627.30.067.51.9 65,000 + 25.10.0225.10.0326.80.0526.70.056.91.7poverty status (ratio of family income to poverty threshold) <100%27.60.0827.80.06 100199%27.70.0527.70.05 200299%27.50.0527.40.05 300399%27.50.0627.40.06 400499%27.20.0627.10.06 500%26.80.0426.80.04occupation professional / managerial25.20.0225.50.0226.80.0427.20.046.31.6 sales / clerical / tech support25.10.0225.40.0227.10.0427.30.048.32.1 service25.70.0425.60.0327.50.0627.40.067.11.8 craft and repair26.20.0325.40.0427.80.0527.10.056.11.6 laborers26.20.0325.60.0328.30.0727.70.078.12.1 unemployed / not in lf25.50.0225.60.0326.40.1027.10.103.60.9adjusted bmi was derived from fitted least square regression models that included survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty statusall percentage and absolute increases in bmi during 19922008 were statistically significantly different from 0 (p <.01) observed and adjusted mean body mass index (bmi) among 30 ethnic - immigrant groups aged 18 + years and by selected sociodemographic characteristics: the national health interview survey, 19922008 adjusted bmi was derived from fitted least square regression models that included survey year, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, family size, region of residence, education, occupation, and family income or poverty status all percentage and absolute increases in bmi during 19922008 were statistically significantly different from 0 (p <.01) the odds and prevalence of obesity and overweight, even after adjusting for sociodemographic factors, increased with increasing duration in the us (tables 1, 2). The obesity gradients by length of immigration were steeper in 20032008 than in 19921995 . Compared with the us - born, immigrants who had lived in the us for <1 year or 15 years had 73 or 34% lower odds of obesity in 20032008 and 59 or 28% lower odds of obesity in 19921995, respectively (table 1). Immigrants who had lived in the us for <1 year or 15 years had 57 or 18% lower odds of overweight than the us - born in 20032008 (table 2). During 19922008, the disparity indices suggest a slight increase in obesity differentials between us - born and immigrants of various durations . Compared with us - born whites, the odds of obesity were 59 and 25% lower for recent and long - term white immigrants, 36% lower for recent black immigrants, 5592% lower for us- and foreign - born chinese, asian indians, and filipino immigrants, 3452% lower for recent however, us - born blacks, american indians, mexicans, and puerto ricans, and long - term puerto rican immigrants had 60, 81, 64, 32, and 23% higher odds of obesity, respectively than us - born whites (table 1). Compared with chinese immigrants, us - born blacks, mexicans, puerto ricans, and central / south americans, and american indians had 1419 times higher odds of obesity, whereas white, black, mexican, cuban, and central / south american immigrants had 413 times higher odds of obesity . Compared with chinese immigrants, all other ethnic - immigrant groups had 310 times higher odds of overweight (table 4).table 4age - sex - year and covariate - adjusted odds of obesity (bmi 30) and overweight (bmi 25) by ethnic - immigrant status (chinese immigrants used as reference group), us adults aged 18 + years: the national health interview survey, 20032008ethnic - immigrant groupobesityobesityoverweightoverweightage sex year adjusted modelcovariate adjusted modelage sex cior95% cior95% cinon - hispanic white recent immigrants4.502.837.184.332.716.913.782.994.793.702.924.69 long - term immigrants8.305.3412.907.815.0212.165.044.066.264.783.835.96 us - born11.917.7518.3110.466.7716.145.874.827.145.274.316.44non - hispanic black recent immigrants8.395.1813.576.734.1310.985.013.876.474.373.375.67 long - term immigrants11.777.4418.629.926.2515.767.856.0510.197.105.459.25 us - born21.3013.8832.6816.7410.8625.8010.308.4212.609.057.3611.12american indian / alaska native24.1115.1138.4818.9311.8230.319.747.4012.838.336.3111.00chinese immigrants (all)1.00reference1.00reference1.00reference1.00reference us - born3.591.767.344.162.028.593.002.064.373.372.304.93filipino recent immigrants3.101.596.023.031.575.862.752.013.742.691.973.66 long - term immigrants4.792.917.914.682.837.733.222.414.293.122.324.20 us - born9.545.6816.008.875.2814.885.554.007.695.413.867.58asian indian recent immigrants2.341.354.062.431.404.222.612.013.402.692.073.50 long - term immigrants / us - born3.341.935.773.431.985.943.052.244.153.112.284.24other asian and pacific islanders recent immigrants1.570.852.911.410.762.601.270.931.741.220.891.68 long - term immigrants2.621.564.422.311.373.901.871.482.371.741.372.21 us - born8.815.1914.938.625.1214.514.333.136.014.293.085.97mexican recent immigrants9.816.3415.176.894.4410.717.706.269.486.265.077.73 long - term immigrants15.159.8423.3110.827.0116.7110.328.3212.808.426.7710.48 us - born21.4313.7833.3217.1010.9926.6311.099.0213.639.547.7311.76puerto rican recent immigrants15.859.4526.5912.317.2820.807.285.2110.176.254.468.77 long - term immigrants16.7610.6526.3812.888.1420.369.267.1611.977.986.1210.39 us - born17.3011.0427.0813.828.7921.729.187.2111.687.916.1910.10cuban recent immigrants10.015.6217.817.484.2113.307.044.6810.605.913.918.94 long - term immigrants10.976.8317.608.765.4414.126.354.778.465.504.147.32 us - born13.287.8722.3911.786.9619.946.404.549.035.884.178.30central and south americans and other hispanics recent immigrants6.604.1710.465.053.178.035.354.266.724.533.595.72 long - term immigrants11.437.2817.958.755.5513.798.156.5010.226.955.528.75 us - born15.6410.2123.9413.528.7920.798.656.9110.837.886.279.91adjusted for survey year, age, gender, marital status, family size, region of residence, education, occupation, and poverty status age - sex - year and covariate - adjusted odds of obesity (bmi 30) and overweight (bmi 25) by ethnic - immigrant status (chinese immigrants used as reference group), us adults aged 18 + years: the national health interview survey, 20032008 adjusted for survey year, age, gender, marital status, family size, region of residence, education, occupation, and poverty status socioeconomic gradients in obesity, although substantial in each period, were less pronounced in 20032008 than in 19921995 and 1976 . The summary indices also indicate decreasing educational disparities over time . Between 1976 and 2008, obesity prevalence doubled for those with <9 years of education, while it increased 45 fold for those with a college education . Those with <9 years of education had 152% higher adjusted odds of obesity in 1976, 90% higher odds in 19921995, and 63% higher odds in 20032008 than those with a college degree . In terms of continuous education, each additional year of education was associated with 11% lower odds of obesity (or = 0.89; 95% ci = 0.890.90) and 7% lower odds of overweight (or = 0.93; 95% ci = 0.930.94) in 1976, 8% lower odds of obesity (or = 0.92; 95% ci = 0.920.92) and 6% lower odds of overweight (or = 0.94; 95% ci = 0.940.94) in 19921995, and 6% lower odds of obesity and overweight (or = 0.94; 95% ci = 0.940.95) in 20032008 . Each additional year of education was associated with a 0.17 point decrease in bmi in 20032008, the effect being significantly lower than the 0.19 point decrease in bmi in 19921995 (p income gradients were steeper in 19921995 than in 20032006, with income disparities in prevalence, as measured by the summary indices, diminishing over time . <$10,000 had 44% higher odds of obesity than those with income $65,000; the roughly comparable odds in 19921995 were 62% higher for those with family income <$7,000 than for those with income $50,000 . A $5,000 increase in family income was associated with 7% (or = 0.93; 95% ci = 0.930.93) and 4% (or = 0.96; 95% ci = 0.960.96) lower odds of obesity and overweight, respectively in 19921995; in 20032006, the corresponding odds ratios were smaller: 0.97 (95% ci = 0.970.98) for obesity and 0.99 (95% ci = 0.980.99) for overweight . A $5,000 increase in family income was associated with a 0.08 point decrease in bmi in 20032006, the effect being significantly lower than the 0.14 point decrease in bmi in 19921995 (p <the obesity prevalence for individuals in sales occupations quadrupled and for those in other occupations tripled between 1976 and 20032008 . In 20032008, after adjusting for education, income, and other demographic factors, service workers and laborers had 7 and 21% higher odds of obesity than those employed in professional/ managerial occupations . Although observed occupational inequalities in obesity were substantial, most of the occupational effects were accounted for by education and income differences . After adjusting for ethnicity and socioeconomic factors, physical inactivity was associated with 54% higher odds of obesity (or = 1.54; 95% ci = 1.461.62) and 36% higher odds of overweight (or = 1.36; 95% ci = 1.301.42) in 20032008 (data from the full model not shown). Race / ethnicity, immigrant status, and social class have long been considered three of the most important axes of health and social stratification [1, 2, 21]. Health and social inequalities by these factors remain quite marked in the contemporary us [1, 4, 12, 20, 21]. Our study examined long - term trends and inequalities in obesity and overweight prevalence among adults from a wide range of social class and immigrants groups, using nationally representative annual cross - sectional samples of the us population . Although immigrant differentials in adult obesity have been examined previously in the us [4, 11, 25], immigrant disparities in obesity across all of the major racial / ethnic groups had not been explored . Furthermore, previous national studies had not examined a wide range of socioeconomic differentials in adult obesity using different socioeconomic measures, such as education, occupation, income, and poverty status [1, 57]. Decreasing social class gradients in obesity shown here are consistent with those reported for adult obesity trends in canada and england, where men and women in higher income or social class groups, despite having lower prevalence than their counterparts from lower income groups, have experienced faster increases in their obesity rates [2628]. Diminishing socioeconomic differentials in us adult obesity prevalence, along with steeper increases in obesity among higher socioeconomic groups over time, were also found using measured height and weight data from the nhanes [1, 5, 6]. Socioeconomic trends in us adult obesity rates, however, differ from us childhood obesity trends which indicate rising social inequalities in prevalence during this decade . Declining physical activity levels and increases in total energy intake may have contributed to rising trends in adult obesity [1, 29]. In analyses not shown here, we found that although increased pa was associated with reduced obesity risks in both immigrants and natives, adjusting for pa levels had little impact on the magnitude of ethnic - immigrant differentials in adult obesity . Indeed, because immigrants had higher inactivity levels, adjusting for pa only widened immigrant differentials in obesity . Although pa did partly account for socioeconomic differences in obesity risks during 20032008, the extent to which social class trends in physical inactivity and sedentary activities account for obesity trends is not known due to lack of temporal data . Ethnic - immigrant and social class differences in dietary factors in appendix table 5 might provide some insights into understanding the obesity differentials shown here . Immigrants in each racial / ethnic group have significantly lower total calorie and fat intake than the us - born . Moreover, immigrants likelihood of excess calorie and fat intake increases with increasing length of residence in the us . Contrary to expectation, the nhanes data show lower total calorie and fat intake among adults in lower ses groups . However, studies have found higher consumption of lower - quality diets and energy - dense foods and lower intakes of fruits and vegetables among lower ses groups [30, 31]. Income or education differences in energy density, fruit and vegetable intake, and other dietary outcomes have persisted or narrowed over time, with individuals in higher socioeconomic groups losing their relative advantage in diet quality in more recent times . These dietary trends appear to coincide with the social class trends in obesity reported here . Future studies need to directly assess the significance of dietary influences in explaining temporal changes in obesity risks and differentials between immigrant and social class groups . Positive immigrant selectivity in health, education, skills, and ambition has been suggested as a possible explanation for lower obesity risks among immigrants [4, 9]. Those migrating to the us in recent decades have come predominantly from latin america and asia, who tend to be healthier than those who remain in their countries of origin . Given the us immigration laws of the past four decades, most immigrants are chosen rather than randomly self - selected based primarily on their skill criteria . Asian immigrants, in particular, are a highly selective group with relatively high socioeconomic attainment levels as noted in fig . 2 . Immigrant patterns in obesity shown here are consistent with those observed for other health indicators, including smoking, breastfeeding, infant mortality, low birthweight, morbidity, mortality, and life expectancy [4, 9, 11, 32]. Immigrants have a significant advantage over the us - born in most health and behavioral outcomes, which tends to decrease with increasing acculturation levels or length of residence in the us [4, 9, 11, 32]. Consistent with previous research on acculturation and obesity risks among us and canadian immigrants[11, 25, 33, 34], our study showed increasing obesity rates with increasing duration of us residence in both 19921995 and 20032008 . Obesity and overweight prevalence estimates from nhis are derived from self - reported height and weight data, which may underestimate the actual prevalence among ethnic - immigrant and social class groups [1, 26]. In 20072008, for example, 26.8% of us adults aged 18 were classified as obese based on the nhis data, whereas the nhanes prevalence was 33.0% . However, the nhanes with its much smaller sample size does not permit detailed examinations of ethnic, immigrant, and socioeconomic disparities in obesity such as those shown here . Second, because of the cross - sectional nature of the nhis, the obesity impacts of socioeconomic variables and pa may have been misestimated . Third, dietary information in the nhis is lacking, and data on immigration and acculturation are limited . The survey does not collect information on legal status of immigrants as well as more direct measures of acculturation such as ethnic - cultural identity, social networks, and dietary preference [4, 9, 11, 33]. Finally, we did not examine if ethnic - immigrant and social class trends in obesity differed by gender; this should be examined in future studies . In conclusion, continued immigrant and socioeconomic disparities in prevalence will likely have substantial impacts on future obesity trends in the us . Immigrants in each racial / ethnic group generally had lower obesity risks than their us - born counterparts, with immigrants obesity risks increasing with increasing length of stay in the us . Considerable heterogeneity in risk was observed, with us - born blacks, mexicans, blacks, puerto ricans, and american indians having 1014 times higher obesity prevalence than chinese immigrants . Barring us - born and foreign - born chinese and asian indians, an average american was likely to be overweight . The overall obesity and overweight prevalence for us adults and an overweight prevalence of 70% or higher for some groups such as us - born blacks, american indians, mexican immigrants, and puerto rican immigrants rank among the highest in the world [19, 36, 37]. Clearly, the presence of such large ethnic - immigrant and social class disparities is a major reason for america s unfavorable international standing in obesity . Continued monitoring of disparities in obesity prevalence among immigrant and social class groups is, therefore, essential in tracking progress towards achieving the national goal of eliminating health inequalities [1, 2]. See table 5.table 5nutritional characteristics by ethnic - immigrant status and socioeconomic factors for us adults aged 20 years and older: the 20012006 national health and nutrition examination survey (nhanes)characteristicenergy intake in kcalscalorie intake 3500fat intake in gfat intake 120 gobservedadjustedprevalenceadjustedobservedadjustedprevalenceadjustedmeansemeanse%seor95% cimeansemeanse%seor95% ciduration of residence in the us (years) <52,002671,895587.01.60.490.260.936436337.62.60.290.140.57 592,1941102,0921009.52.30.690.381.2778577512.93.20.550.301.00 10192,1611022,0521019.42.30.720.361.4278575513.82.80.550.311.00 20 + 2,098672,154789.72.01.090.592.0277479412.11.70.610.380.99 us - born2,235302,2423010.40.61.00reference86186118.21.21.00referencenon - hispanic white us - born2,248152,2651511.20.41.00reference86186118.50.61.00reference immigrant2,177622,1805510.11.90.820.531.2981381314.42.00.700.500.98non - hispanic black us - born2,181242,1862111.30.80.940.791.1183184117.30.80.930.831.06 immigrant1,994621,921674.11.60.260.110.636736536.31.50.240.130.42mexican american us - born2,303412,2504113.71.41.030.771.3888287219.21.60.970.761.25 immigrant2,253282,1033210.71.00.510.380.6977173213.01.10.490.370.63other hispanic us - born2,100742,106554.71.50.340.180.6578479314.72.90.810.501.32 immigrant2,076652,017646.41.80.400.200.7873372310.92.20.450.280.71all other ethnic groups us - born2,285652,2266210.01.90.720.481.0787385217.22.30.810.581.13 immigrant1,939431,945454.71.10.370.220.636526525.81.30.250.150.42education (years of school completed) <122,106252,1862610.00.71.00reference77181113.40.71.00reference 122,260212,2601712.10.70.970.771.2286186118.90.91.211.011.44 13 + 2,245142,2181510.40.40.830.661.0485184117.70.61.090.931.28poverty status (ratio of family income to poverty threshold) <100%2,188242,2052510.80.81.00reference80183116.30.91.00reference 100199%2,149252,2112310.30.71.060.821.3680184114.70.80.920.741.13 200299%2,186272,1852311.30.91.090.831.4483183117.81.41.000.801.27 300399%2,263352,223339.80.90.820.621.0885283216.71.20.820.661.04 400499%2,260262,2232810.20.80.880.681.1387185118.51.20.940.761.16 500%2,295222,2552111.70.91.050.801.3788185119.80.80.980.791.20adjusted by weighted least squares or logistic regression for time period, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, education, and poverty statusduration of residence in the us was available only for the 20052006 nhanes nutritional characteristics by ethnic - immigrant status and socioeconomic factors for us adults aged 20 years and older: the 20012006 national health and nutrition examination survey (nhanes) adjusted by weighted least squares or logistic regression for time period, age, gender, ethnic - immigrant status (or race / ethnicity and length of immigration), marital status, education, and poverty status duration of residence in the us was available only for the 20052006 nhanes
Ventriculoperitoneal shunt (vps) surgery is the commonly used technique for the management of hydrocephalus in pediatric cases . A high range of complications has been reported following this procedure, which are troublesome either to the surgeon or to the patient . Extrusion of the peritoneal end of the shunt through intact abdominal wall is extremely rare and few cases have been reported in the literature . We are reporting two such rare cases of spontaneous extrusion of the peritoneal catheter through the intact abdominal wall . A 7-month - old female baby was admitted to neurosurgical department of s.c.b . Medical college and hospital with the vps catheter's distal end protruding through the intact abdominal wall [figure 1]. Three months earlier, she had undergone an installation of a medium - pressure vps for posttraumatic hydrocephalus . One week prior to her current admission, her mother noticed a painless, small blister, and erythema on the upper abdominal wall, which eroded and gave way to protrude a part of the peritoneal catheter . Spontaneous extrusion of the distal peritoneal catheter through the intact abdominal wall a 14-year - old boy presented with extrusion of peritoneal end of the vps catheter through the intact abdominal wall [figure 2]. Eight years back, he had undergone medium - pressure vps surgery for hydrocephalus caused by posterior fossa pilocytic astrocytoma . Five days prior to his current admission, he noticed erosion of the upper abdominal wall, through which a part of the peritoneal catheter extruded out . Extruded peritoneal end of the ventriculoperitoneal shunt catheter through intact abdominal wall on admission, both of them were conscious and had no neurological deficits . In both cases, the peritoneal catheter protruded from the upper abdominal wall near the epigastrium away from the previous surgical incision . After abdominal ultrasonography (usg) did not show any signs of intra - abdominal pathology, the shunts were removed immediately . The peritoneal catheter was disconnected from the valve chamber by a small skin incision placed behind the ear under local anesthesia and was pulled out from the abdominal wall . Computed tomography (ct) of the head revealed dilated ventricles on the postoperative day 3 . The patients were discharged after 8 days with the advice for periodical checkups . At 6-months follow - up a 7-month - old female baby was admitted to neurosurgical department of s.c.b . Medical college and hospital with the vps catheter's distal end protruding through the intact abdominal wall [figure 1]. Three months earlier, she had undergone an installation of a medium - pressure vps for posttraumatic hydrocephalus . One week prior to her current admission, her mother noticed a painless, small blister, and erythema on the upper abdominal wall, which eroded and gave way to protrude a part of the peritoneal catheter . A 14-year - old boy presented with extrusion of peritoneal end of the vps catheter through the intact abdominal wall [figure 2]. Eight years back, he had undergone medium - pressure vps surgery for hydrocephalus caused by posterior fossa pilocytic astrocytoma . Five days prior to his current admission, he noticed erosion of the upper abdominal wall, through which a part of the peritoneal catheter extruded out . Extruded peritoneal end of the ventriculoperitoneal shunt catheter through intact abdominal wall on admission, both of them were conscious and had no neurological deficits . In both cases, the peritoneal catheter protruded from the upper abdominal wall near the epigastrium away from the previous surgical incision . After abdominal ultrasonography (usg) did not show any signs of intra - abdominal pathology, the shunts were removed immediately . The peritoneal catheter was disconnected from the valve chamber by a small skin incision placed behind the ear under local anesthesia and was pulled out from the abdominal wall . Computed tomography (ct) of the head revealed dilated ventricles on the postoperative day 3 . The patients were discharged after 8 days with the advice for periodical checkups . At 6-months follow - up there have been a number of reports of complications relating to the peritoneal section of vps . Among them, perforation by the peritoneal catheter was reported to have occurred in the vagina, intestine, umbilicus, and the surgical scar of the abdominal wall . Others are transoral or transanal protrusion, extrusion through the scrotum, and formation of abdominal pseudocyst, etc ., spontaneous extrusion of the distal peritoneal catheter through the intact abdominal wall is very rare . The sharp tip at the distal end of the catheter is blamed for higher complication rates . Various hypotheses have been put forward regarding causes of spontaneous extrusion of the peritoneal catheter through the intact abdominal wall or chest wall . Etiology of early extrusion of the shunt may include focal wound dehiscence and infection, while delayed presentation may be attributed to ischemic necrosis of dermis overlying shunt components . Other factors that may contribute to shunt extrusion include poor host immunity, factors related to surgical technique, and bioreactivity of shunt components . It is therefore advised that the trocar should not be used, and the distal shunt should be placed under direct vision . The shunt can be removed directly without laparotomy only if one is sure that there is no evidence of either abdominal abscess formation or peritonitis . . Meningitis or ventriculitis secondary to retrograde migration of bacteria should be treated according to the culture - antibiogram results . If spontaneous extrusion of the distal peritoneal catheter through the intact abdominal wall is encountered, prophylactic antibiotics should be started without delay and the shunt should be removed completely . We suggest that the firm tip of the catheter coupled with its movements with respiration produced a hammer effect and eroded the abdominal wall caused local inflammation, and then extruded through the skin . Disconnection of the peritoneal catheter from the chamber and pulling out the extruded catheter through the abdominal wall is suggested as a simple and effective method of removal of the shunt.
Pyomyositis affects the massive skeletal muscles and is most frequently caused by staphylococcus bacteria (1). Necrotizing fasciitis is a localized spread of infection that follows events such as trauma or surgery (2), and which is commonly affected by group a streptococcus . Rhabdomyolysis comprehensively affects the skeletal muscle and, like pyomyositis and necrotizing faciitis, shows serum creatinine kinase elevation; however, rhabdomyolysis occurs as a systemic disease and is not associated with the focal manifestation of abscesses (3). The pathogenesis of infectious rhabdomyolysis is thought to be associated with the systemic or local metabolic changes related to a systemic or local infection (4); however, the mechanisms underlying its pathogenesis have not been established . In addition, gram - negative bacillus rhabdomyolysis is rare, and its microbiological pathology remains to be elucidated . We diagnosed the patient, a 64-year - old japanese man with multiple myeloma, with bacteremia caused by morganella morganii . He was admitted to our institute due to septic shock at the sudden onset of a febrile episode . His vital signs at admission were as follows: blood pressure, 82/60 mmhg; heart rate, 78/min; body temperature, 39.1; respiration rate, 20/min; and spo2, 95% (room air). The laboratory findings at the time of his diagnosis showed massive myolysis and a significantly elevated level of creatinine kinase (ck; 3,582 u / l); thereafter, an isotype analysis of the patient's ck confirmed that the elevated ck was derived from the patient's skeletal muscle (98.8%). The status of the patient's multiple myeloma, which had been diagnosed 5 years earlier, was stage iiia, igg type . He had undergone treatment with a variety of chemotherapies: melphalan (mp; 12 mg / day for 4 days + prednisolone 60 mg / day for 6 days, repeated triweekly), vad (vincristine 0.4 mg / m for 4 days as a continuous infusion + doxorubicin 10 mg / m for 4 days as a continuous infusion + dexamethasone 40 mg / day, days 1 - 4, 9 - 12, and 17 - 20, repeated monthly), bd (bortezomib 1.3 mg / m on days 1, 4, 8, and 11 + dexamethasone 40 mg / day for 4 days, repeated monthly), and thalidomide (100 mg / day). His last treatments were lenalidomide 20 mg / day, and igg gradually increased 6 months before the current infectious episode . After the patient's admission for sepsis caused by m. morganii, rehydration was immediately performed via central venous catheterization and a catecholamine infusion (dopamine 3 mg / kg / h) was administered; at the same time, antimicrobial treatment with meropenem (0.5 g three times a day) was initiated . Cervical to pelvis computed tomography (ct) was performed on the day of admission, but there were no clear findings explaining the patient's massive myolysis . On day 2 of the patient's hospitalization, the patient's vital sign were as follows: blood pressure, 61/45 mmhg; heart rate, 87/min; and body temperature, 36.8. we started hemodialysis combined with endotoxin absorption the same day . However, respiratory failure occurred, necessitating mechanical ventilation, on the night of day 2 . The patient died of multi - organ failure two days after undergoing intubation . Before his death, rhabdomyolysis developed day - by - day and was reflected in the remarkable elevation of the patient's ck level . The final ck value was 19,790 u / l (normal range: 40 - 200 u / l) (figure). We found that the m. morganii was sensitive to all of the broad spectrum beta - lactams, including carbapenems and cephalosporins, but only resistant to cefotiam, minomycin, and ciprofloxacin . His muscle damage manifested as with elevated levels of ck, aspartate aminotransferase (ast), alanine aminotransferase (alt), and lactate dehydrogenase (ldh) after the onset of the disease (day 1), with increasing leukocytes and c - reactive protein (crp). The days in the x - axis indicate the days after the patient s admission . The patient visited our outpatient clinic for a routine follow - up examination 6 days before his admission . Infectious rhabdomyolysis is caused by various pathogens including viruses, bacteria and fungi (3). The clinical entity of infectious rhabdomyolysis was established in 1982 (5), and a review (3) identified the predisposing risk factors for rhabdomyolysis: alcohol ingestion, compression injury, and generalized seizures . Regardless of the pathogen that causes infectious rhabdomyolysis, the underlying mechanism of the disease is metabolic . The most frequent differential diagnosis is necrotizing fasciitis, which occurs when the infection spreads into the deep fascial layers (2). Necrotizing fasciitis is generally caused by infection, and the initial entry site is sometimes trivial . In contrast, rhabdomyolysis is caused by the following two critical conditions: (1) physical distress (for example, distress due to or involving excessive exercise, seizure, muscle compression, hyperthermia, or hypothermia); and (2) systemic metabolic disorders (for example disorders due to hypoxia, acidemia, drug abuse, infection, or inflammation). The localization of a soft - tissue infection can be distinguished by systemic imaging, especially by sensitive mri (6). In the reported cases of rhabdomyolysis, the local radiological findings are faint . In the present case, our evaluation of the present case raised an important question: what is the microbiological mechanism underlying the development of rhabdomyolysis accompanied by systemic infection (3,7)? Thus, rhabdomyolysis is relatively common among patients with miscellaneous bacterial infections including, but not limited to, legionella (4), pneumococcal (8), and salmonella (7). We underscore that patients these types of infectious rhabdomyolysis share the following common findings: 1) the patients were immunocompromised due to malignancy or a severe wound; 2) precedent or concomitant acute renal failure were observed in the patient's clinical course; and 3) metabolic abnormalities such as dehydration or acidemia due to single / multi - organ failure existed as the underlying pathogenesis . Thus, ischemia or hypoxia caused by systemic infection are considered to be involved in the mechanism underlying the development of rhabdomyolysis . M. morganii is rarely pathogenic in humans, but it sometimes develops into an opportunistic infection in a compromised host . Although there are no reported cases of infectious rhabdomyolysis caused by m. morganii, arranz - caso et al . The authors noted that gram - negative bacterial pyomyositis can occur in an immunocompromised case followed by enteric organism translocation . Gram - negative bacterial infection does not favor pyomyositis, which is usually raised by gram - positive bacteria . Our case suggests that infectious rhabdomyolysis may be underdiagnosed as a comorbidity of gram - negative bacterial infections, even if the causative organism is an opportunistic pathogen such as m. morganii . Regarding myolysis / rhabdomyolysis complicated with a systemic bacterial infection and the accumulation of muscle damage the non - bacteremic mechanism includes alterations of systemic metabolism (i.e., hypoxia, acidosis) and an intracellular metabolism in situ (e.g., disturbances of glycolysis and oxidation in muscle cells) (7). The non - bacteremic mechanism of rhabdomyolysis accompanied by systemic infection is similar, regardless of the pathogen that is involved (3,7). The common mechanisms underlying muscle damage arise from a combination of hypoxia, dehydration, and acidemia in the tissue (4). In general, rhabdomyolysis develops during the course of systemic infection in up to 5% of bacterial and viral cases (7). Rhabdomyolysis is a specific term describing the excess leakage of enzymes derived from muscular tissue, which can result in multi - organ failure . In our patient, the apparent infection focus was not identified by repeated systemic ct . This negative finding caused a delay in our differentiation of the cause of the patient's rhabdomyolysis . We treated the patient with meropenem, to which the causative pathogen, m. morganii, is sensitive; however, an adequate antimicrobial therapy cannot always rescue a patient from advanced multiple - organ deterioration . Rhabdomyolysis should thus be recognized as a more aggressive clinical situation than local myolysis, and one that requires intensive care . Furthermore, non - specific myolysis / rhabdomyolysis can occur due to any pathogen (7); thus, it may be underdiagnosed during the infection . Myalgia and scant laboratory signs of myolysis / rhabdomyolysis (usually appearing as liver damage in laboratory data) can be misdiagnosed in patients with the overlapping leakage of transaminases due to various etiologies . Clinicians should keep in mind that abnormal liver function test results may be linked to a muscular problem . Clinicians should be aware of the possibility of systemic infection - associated myolysis / rhabdomyolysis and closely observe immunocompromised patients . This is the first report of lethal rhabdomyolysis caused by m. morganii as a complication in a non - hiv patient . Short description: fatal rhabdomyolysis caused by m. morganii can occur in a multiple myeloma patient without hiv.
Sarcomas are mesenchymal neoplasms with various lines of differentiation, i.e., fibrocytic, myogenic, neurogenic, vascular, chondro - osseus, or undefined . It is a spindle - cell tumor composed of collagen - rich and myxoid parts . Lgfs prefers subfascial soft tissue layers on the trunk and proximal extremities in younger adults but can also develop in internal organs . Pediatric cases have also been reported. [13] herein, we present a 48-year - old female patient who developed a lgfs on the lower leg . The clinical presentation, histopathology, surgical treatment, and follow - up are discussed . A 48-year - old woman was referred to our department because she had developed a slow - growing plaque below the left knee for 2 years . On examination we found an otherwise healthy, slim, woman with a symptomless firm subcutaneous plaque of about 2 cm size on the anterior aspect of the left lower leg figure 1.there was some bluish discoloration and circumscribed ulceration but no erythema or warmth . (a) overview and (b) the ulcerated honeycomb - like plaque routine laboratory tests were unremarkable . Thoracic dual - energy x - ray and abdominal and lymph node ultrasound excluded a metastatic spread . Histology revealed a spindle - cell tumor, with mild atypia and fibroblast - like morphology [figure 2]. Cells were arranged in a whorled or plexifiorm pattern, with alternating collagenous stroma and myxoid zones . The tumor was surgically removed with a wide safety margin (> 3 cm). The defect was covered by a mesh graft transplant covered by vacuum - assisted closure (vac; kci international) [figure 3]. Compression stockings were prescribed to protect the transplant and prevent leg edema . A regular follow - up for at least 5 years (a) overview (h&e, 4) and (b) detail (h&e, 40) surgical procedure . (a) wide excision of suprafascial soft tissue; (b) mesh graft transplantation; and (c) placement of microporous white sponge for vacuum - assisted closure above the transplant lgfs is a very rare and distinctive type of fibrosarcoma that was first described by evans in 1987 . There is a discrepancy between the bland histologic features with sparse mitotic figures and absent or mild nuclear and cellular pleomorphism and the anaplasia . The hyalinizing spindle - cell tumor with giant rosettes is considered a subtype of lgfs. [13] the tumor is further characterized by t(7;16)(q34;p11) translocation and fusion of fus and creb3l1 genes . Tumor cell phenotype is positive for vimentin, ema, cd99, and bcl-2, but negative for cd34, sma, s-100, desmin, keratins, neuron - specific enolase, and cd177 . The differential diagnosis includes other types of sarcomas, myxoma, neurofibroma, peripheral sheath tumor, histocytoma, desmoid tumor, and others . The diagnosis often is delayed - mainly because the patients do not seek treatment early . (2000) reported that in 15% of patients a histologic diagnosis was delayed by> 5 years . In our case although the histopathologic features suggest a low - grade malignancy, local recurrence is seen in more than 50% of patients and metastasis occurs in 6% of patients . Tumor cell dormancy is responsible for very late metastasis in some patients, with 45 years being the longest period observed between primary surgery and metastasis . Complete surgery with wide margins (> 3 cm) is the most important procedure . We used a combination of mesh graft transplantation and vacuum - assisted closure to cover the large defect . It has been demonstrated recently that such a combination, with microporous sponge for vacuum - assisted closure, results in a significantly improved take rate.
Only a minority of individuals living in the obesigenic environment currently encountered in developed nations are able to maintain a healthy body weight . For example, in the usa, evidence from the latest national health and nutrition examination survey (collected in 20072008) indicates that 68% of adults are overweight or obese . Given the health risks associated with obesity (heart disease, diabetes, hypertension, cancer), this has important implications for global health, and in light of the additional health - care costs associated with obesity, the global economy . Genome - wide association studies have identified numerous loci associated with obesity; however, their contribution to variation in body mass index and weight between individuals is estimated to be less than 2%, suggesting that environmental influences, such as increased availability of energy - dense food and an increasingly sedentary lifestyle, play a crucial role . Accumulating evidence from epidemiologic studies and animal models indicate that maternal health and nutritional state during pregnancy and lactation play a critical role in programming the neural circuitry that regulates energy balance and behavior in offspring, having a sustained influence on their physiology and behavior . Metabolic imprinting refers to the programming of an offspring's future metabolic responses by a stimulus occurring during a critical developmental period . As the type and amount of available nutrition are crucial determinants of survival and reproductive success, the evolutionary purpose of metabolic imprinting is to enhance offspring survival by programming energy balance regulation so that available metabolic fuels are most efficiently utilized . Mammals are exposed to two environments during development, the intrauterine and early postnatal environments, both of which are heavily impacted by maternal diet and adiposity . Perinatal nutrition has enduring effects on many aspects of physiology and behavior including the regulation of energy balance, susceptibility to metabolic disorders, programming of body weight set point, stress response and mental health - related behaviors . In addition to perinatal nutrition, a number of factors associated with maternal consumption of a high - fat diet (hfd), including maternal adiposity, hyperlipidemia, lipotoxicity, glucose levels and insulin resistance, also have a long - term impact on the developing offspring and are associated with increased risk of obesity, metabolic disorders, and mental health disorders . As maternal obesity is associated with consumption of a hfd in humans and a hfd is used to promote maternal obesity in most animal models, a major challenge faced by the field is the ability to disassociate the effects of the hfd from the maternal metabolic phenotype . The most common perturbation of maternal nutrition is nutritional excess and maternal obesity . In 2008, over 64% of women of child - bearing age in the usa were overweight or obese and the majority consume an excess amount of calories and fat . Epidemiological studies clearly indicate that maternal obesity is associated with increased incidence of obesity and metabolic syndrome in children . Although this review focuses on the impact of maternal hfd consumption, it is important to note that maternal undernutrition during gestation also increases the incidence of offspring obesity; thus, the relationship between birth weight and adult adiposity is thought of as a u-shaped curve with both a very high and a very low birth weight increasing the risk of obesity in adulthood . The relationship between maternal obesity and the development of offspring obesity appears to be independent of gestational diabetes as women who are obese, but able to maintain normal glycemia, also have heavier offspring with increased adiposity . Although epidemiological studies implicate the intrauterine environment, including maternal diet and energy status, in programming offspring obesity, in these studies it is not possible to directly link maternal diet and energy status with the offspring's metabolic profile, as several other factors could contribute to the association between maternal and offspring obesity, including genetics and shared environment factors such as access to energy - dense foods and a sedentary lifestyle . Also, there is relatively limited information on normal brain development in humans, making it challenging to examine the impact of maternal diet on offspring's brain development . Furthermore, it is difficult to accurately monitor and potentially unethical to manipulate the diet of pregnant women . Thus, it is critical to use animal models to directly examine the effects of maternal overnutrition on subsequent generations and to develop effective therapeutic intervention strategies . Providing animals with a hfd during pregnancy and lactation is a common method of inducing maternal obesity . However, the duration of exposure to the diet (acute vs. chronic), the percent of calories from fat, and diet composition are variable across studies, hindering comparisons . The diets commonly used to promote maternal obesity are either a purified hfd with fat in place of carbohydrates as an energy source or a cafeteria diet in which animals are provided with a selection of palatable food items that have a high fat content along with their regular diet . The cafeteria diet is most effective in promoting obesity; however, as several food items are provided, it is difficult to calculate the amount and composition of the diet consumed by each animal and the diet consumed by animals in the same group is variable . Several studies have used purified diets with different sources of fat to compare the impact of maternal consumption of saturated fat, polyunsaturated fat 3 or 6 fatty acids on offspring physiology and brain development . It is clear from these studies that the source of fat in the hfd matters . For example, a study in rodents determined that while a maternal diet high in saturated fat programs hyperphagia in offspring, exposure to a diet of equivalent percent of calories from fat as fish oil does not . Thus, it is essential that future studies determine which sources of fat in a mother's diet are beneficial and which are detrimental to the developing offspring in order for physicians and nutritionists to make appropriate recommendations to expecting mothers . Most rodent studies report that offspring exposed to a hfd during gestation and lactation have increased body weight and adiposity at weaning . However, several studies report that maternal hfd consumption results in lighter offspring which they speculate is due to hfd / obesity - induced impairments in the initiation and production of milk by obese mothers . The differences in offspring's body weight phenotypes across studies are likely due to differences in the duration of hfd consumption and fatty acid composition of the diets . Using nonhuman primates, our group is examining the impact of chronic maternal hfd consumption on offspring body weight regulation . Briefly, infant offspring from hfd mothers are underweight at birth and display rapid catch - up growth, so that by 6 months of age they are heavier and have increased adiposity . This offspring phenotype appears to be independent of whether the hfd - consuming mother is obese with insulin resistance or lean with normal sensitivity to insulin . These studies indicate that in primates, as in rodents, maternal hfd consumption, independent of maternal weight and metabolic status, predisposes offspring to increased risk of developing obesity and metabolic disorders early in life . Rodents that experience an early environment in which they are exposed to maternal hfd consumption or overnutrition are consistently reported to be hyperphagic as adults . A number of studies have found that maternal hfd consumption plays a critical role in programming hypothalamic pathways that regulate feeding . Early postnatal overfeeding increases the orexigenic peptides neuropeptide y and agouti - related peptide (agrp) in the arcuate nucleus of the hypothalamus (arh) of juvenile rats . Offspring of rat dams fed a hfd during the perinatal period also display a long - term upregulation in the expression of orexigenic peptides including galanin, enkephalin, and dynorphin in the paraventricular nucleus of the hypothalamus (pvh), and orexin and melanin - concentrating hormone in the perifornical lateral hypothalamus . Exposure to hfd during gestation stimulates the proliferation of neuronal and neuroepithelial cells of the embryonic third ventricle of the hypothalamus and increases their migration to hypothalamic regions resulting in an increase in the proportion of neurons expressing orexigenic peptides . Also, offspring from hfd mothers have reduced sensitivity to the anorectic effects of leptin . Thus, in rodents it is postulated that perinatal exposure to overnutrition or maternal hfd consumption results in disruption of the homeostatic feedback regulation and nutrient sensing capabilities of the hypothalamic feeding circuits leading to hyperphagia . Though rodent models have significant advantages, such as a relatively short period of gestation and the ability to manipulate genetics, the critical periods for the development of energy balance regulatory systems differ between rodents and humans . In rodents, the neural pathways regulating energy balance are immature at birth and are not completely developed until the third postnatal week (mice). In contrast, in humans, nonhuman primates, pigs, and sheep, the hypothalamic circuitry that regulates feeding develops primarily prenatally . Thus, models of maternal overnutrition in which the development of energy balance regulation occurs prenatally are particularly relevant . In the nonhuman primate model of maternal hfd - induced obesity, our group has determined that fetal offspring also display alterations in the development of the hypothalamic melanocortin system that may contribute to disrupted homeostatic signaling . In addition to hyperphagia, there is evidence that feeding behavior and food choice are also programmed by perinatal nutrition . Overweight children are reported to have increased preference for high - fat foods which is associated with increased parental adiposity . Also, children with one or two overweight parents consume a larger percentage of energy from fat than children who have two normal - weight parents . In these studies it is unclear if the children's increased fat preference is due to programming as a result of perinatal hfd exposure, genetics or increased availability of high - fat food during childhood . Animal models in which offspring are exposed to a hfd during the perinatal period provide further evidence for an influence of perinatal hfd exposure on food choice . For example, adult rat offspring exposed to junk food during either gestation or lactation displayed increased preference for fatty, sugary and salty foods . The source of fat in the perinatal diet influences the programming of food preference, as rat pups from mothers that consumed a hfd with lard as the main source of fat displayed increased preference for the hfd, whereas offspring exposed to maternal consumption of hfd with fish oil as the fat source do not . In rodents, there is evidence that maternal hfd consumption causes perturbation in the dopamine system of adult animals in areas associated with the rewarding value of food such as the nucleus accumbens and ventral tegmental area . Preliminary findings from our studies examining the impact of exposure to maternal hfd consumption on the food preference of nonhuman primate offspring also indicate that offspring display increased preference for diets with a high sugar and fat content [sullivan and grove, unpubl . Together, these studies provide compelling evidence that perinatal nutrition has a long - term influence on dietary preference and feeding behavior and may be an important contributing factor to the development of obesity . As differences in food preference and diet consumption will have a long - term impact on stress response and depressive behaviors of adult offspring, future studies examining the impact of perinatal hfd exposure on food preference should include the examination of stress response and behavioral disorders . Although many studies have examined the impact of perinatal nutrition on food intake and food intake regulation, the number of studies examining the other component of energy balance, energy expenditure, are limited . Offspring from mothers undernourished during gestation offspring exposed to maternal overnutrition have also been reported to be hypoactive or to have no difference in physical activity as compared to offspring exposed to a control diet . The effect of maternal diet on physical activity level depends on the type of fat in the diet . Rat pups from dams fed a diet rich in polyunsaturated fat displayed increased locomotor activity when compared to offspring from dams fed a saturated fat or standard laboratory diet . This study also reported increased locomotor response to stimulants in offspring from dams that consumed a saturated fat diet . Currently, the impact of early exposure to hfd on metabolic rate has not been examined . Thus, a major gap in the knowledge of the field of metabolic programming is the impact of perinatal hfd exposure on the regulation of energy expenditure . Future studies are needed which examine not only the effects of perinatal exposure to hfd on average basal energy expenditure, but its impact on compensatory changes in energy expenditure in response to metabolic challenges, such as fasting, dieting and chronic consumption of a hfd . In addition to being associated with metabolic disorders, epidemiological data indicate that obesity is associated with increased risk of behavioral / mental health disorders, such as depression, anxiety, and attention deficit hyperactivity disorder . Moreover, anxiety and depression influence the feeding behavior, food preference and physical activity level . Depression and anxiety are associated with increased craving for palatable food items and decreased physical activity level . Variations in mood also alter food choice, with increased preference for high - fat / high - sugar foods being reported during negative emotions . Maternal nutrition has long - term implications for the offspring's risk of developing mental health disorders . Perinatal exposure to a hfd may contribute to this association by altering the development of key pathways implicated in regulating mood and behavior such as the serotonin system . Recently, maternal hfd consumption has been associated with increased anxiety in rodent and nonhuman primate offspring . In a rat model, male adult offspring from mothers exposed to either a diet high in saturated or trans fat during gestation and lactation displayed increased anxiety and deficits in spatial learning . Using a nonhuman primate model, our group recently demonstrated that perinatal exposure to a hfd causes a decrease in serotonergic tone perturbations in the serotonin system, which predisposes female offspring to increased anxiety . The finding that female nonhuman primate offspring exposed to maternal hfd consumption are more sensitive to developing anxiety than male offspring is consistent with evidence in humans which suggests that females are more prone to anxiety than males and that the association between obesity and anxiety is stronger in women than men . Thus, nonhuman primates appear to be an ideal model in which to examine the impact of maternal hfd consumption on the development of mental health disorders such as anxiety and depression . Although there is clear evidence from studies in both rodents and nonhuman primates that maternal hfd consumption leads to increased risk of obesity and metabolic diseases, the mechanisms responsible are largely unknown . We have very limited information on the impact of maternal hfd consumption on the brain and the complex neural circuitry that regulates physiology and behavior . Recent evidence indicates that circulating factors such as hormones (leptin, insulin), nutrients (fatty acids, triglycerides and glucose) and inflammatory cytokines play important roles (fig . 1). Maternal obesity and diabetes result in maternal hyperglycemia, and as glucose can readily pass through the blood - placenta barrier, it is transferred to the fetus . However, the elevated insulin levels associated with maternal obesity do not cross the placenta; thus, the fetal pancreas must secrete increased levels of insulin to respond to the maternal hyperglycemia . This fetal hyperinsulinemia is postulated to be involved in the programming of obesity and diabetes in the developing offspring . Administering insulin to rats during the last term of gestation produces obesity in the offspring and administering insulin to the hypothalamus of rat pups during the time that projects from the arh to the pvh results in elevations in body weight, insulin level, impaired glucose tolerance, and increased vulnerability to diabetes . Insulin is an important growth factor in the central nervous system; thus, it is postulated that early exposure to hyperinsulinemia alters the development of the brain circuitry regulating energy balance and behavior . The hyperleptinemia that offspring from obese mothers experience during development is also implicated in metabolic imprinting . There is substantial evidence in rodents that postnatal leptin is a critical factor in the development of neural pathways in the hypothalamus . Human studies report that leptin is elevated in obese and diabetic mothers and lower in infants that experienced intrauterine growth restriction at term . However, in human and nonhuman primate gestation, circulating leptin levels do not rise until after hypothalamic development is mostly complete . Though critical for brain development in rodents, there is limited evidence for leptin's role in the development of primate brains . Increased adiposity is associated with elevations in peripheral markers of inflammation, such as c - reactive protein, interleukin-6, interleukin-1, and tumor necrosis factor- . These inflammatory markers are associated with increased risk of cardiovascular disease, heart disease, insulin resistance, type 2 diabetes mellitus and hypertension . The association between obesity and increased inflammatory cytokines has been confirmed in pregnant women such that obese pregnant women have increased levels of inflammatory cytokines which lead to endothelial dysfunction . Exposure of the developing fetus to increased circulating cytokines has been proposed as a potential mechanism by which maternal hfd consumption impacts brain development . The development of many neural systems that are critical in regulating energy balance, such as the melanocortinergic system, the serotonergic system, and the dopaminergic system, is sensitive to circulating cytokine levels . Also, rodent studies report that agrp and pro - opiomelanocortin neurons in the arh are directly impacted by cytokines . A recent study by bilbo and tsang reported that offspring from mothers consuming a saturated fat diet had increased microglial activation in the hippocampus at birth that persisted into adulthood . We have recently observed that fetuses from nonhuman primates consuming a hfd have increased circulating and hypothalamic cytokines . We postulate that exposure to increased inflammatory cytokines leads to the perturbations in the melanocortin and serotonin system observed in fetal offspring . Given the large number of neurotransmitter systems that are influenced by inflammation, future research is needed to examine the impact of maternal hfd - induced inflammation on each critical regulator of physiology and behavior . In summary, many common brain regions and neurotransmitters regulate energy balance, stress response and mental health disorders including the melanocortinergic system, serotoninergic system and dopaminergic system (fig . Thus, it is not surprising that maternal hfd consumption has a long - term impact on metabolic and behavioral regulation . As maternal hfd consumption and obesity are commonplace and rapidly increasing, we speculate that future generations will be at increased risk for both metabolic and mental health disorders . Given the prevalence of maternal obesity, future studies need to identify therapeutic strategies that are effective at preventing maternal hfd - induced malprogramming.
Dermatophytosis, one of the most common infectious diseases in the world, can be caused by dermatophyte fungal species of the genera trichophyton, microsporum and epidermophyton . Onychomycosis is a fungal infection of the nails, which may be caused by many types of fungi, other dermatophytes or yeast species (1). Treatment of onychomycosis is a challenge and infections are typically more severe and difficult to treat in toenails than in fingernails (1). Treatment requires long - term therapy with oral antifungal medication (with the cure rates ranging from 48% to 76%) which may have side effects, and/or topical agents such as ciclopirox (34% cure rate) (2, 3). Oral therapies (terbinafine, itraconazole) provide access to the nail bed and matrix and are also used to treat concomitant skin infections such as tinea pedis . To prevent side effects prior to and periodically during oral therapy, liver function the newer imidazoles may lead to a higher cure rate (4), but these drugs are considerably more expensive than routine treatments . Topical therapies are associated with low rates of adverse events and mild localized reactions at the application site, but due to inadequate penetration, especially when the nails are thick, their efficacy is limited . Uv radiation induces dna damage leading to inactivation of the pathogenic fungus commonly measured by viability count . Uv - a with long wavelength (315 - 400 nm) rays has a slow effect on the skin and is poorly absorbed by the ozone layer . This ray is 1,000 times more common than uv - b with the middle - range of 290 - 320 nm that causes visible damage, commonly observed as redness and blistering and are a major cause of sunburn and skin cancer (5). Uv - c with wavelengths less than 290 nm (100 - 280 nm) is the shortest and has the highest uv energy . It is filtered by the ozone layer; these wavelengths do not reach the earth surface and do not penetrate the atmosphere . Thus, people are exposed to large doses of uv - a throughout their life, but uv - b rays do not penetrate glass (5). High doses of uv - a and uv - b radiation have significant inhibitory effects on dermatophytes (6). The antibacterial activity of uv - c radiation reduces the number of bacteria on environmental surfaces and vegetative bacteria including mycobacterium tuberculosis, viruses and fungi (7, 8). The current study aimed to investigate the fungicidal effect of uv radiation on the growth of dermatophytes isolated from nails, considering the low efficacy of current treatment options and ease of exposing nails to uv light . The sample comprised all the patients referred to the dermatology clinic (shahid faghihi hospital, shiraz university of medical sciences, iran) with suspected onychomycosis . The inclusion criteria were clinical manifestations such as distal or proximal sub ungula onychomycosis, white superficial onychomycosis, pitting, paronychia, onycholysis and nail plate discoloration . All diagnoses were confirmed by the mycology laboratory of shahid faghihi hospital, shiraz university of medical sciences, iran . Since this procedure was part of the patients usual treatment protocol, informed consent for research purposes was not specifically sought . Fragments of nail plate and nail bed scrapes were inoculated onto sabouraud dextrose agar (sda) (merck, germany) with the antimicrobials cycloheximide (sigma - aldrich, germany) and chloramphenicol (merck, germany) (9), and incubated at 30c for 14 days . Isolated species were identified by macroscopic examination of colony morphology (size, color of the colonies from the top and bottom of the plate, and growth rate). Microscopic examinations were performed to identify dermatophyte species using lacto phenol - cotton blue and diagnostic methods such as growth on trichophyton agar media (quelab, uk) and rice, and hair perforation test were performed to identify (9). To determine the influence of different doses of uv light on the fungicidal effect, colony count and size, and growth rate of the isolated fungi were evaluated under laboratory conditions . Two strains of trichophyton mentagrophytes and two strains of t. rubrum were isolated from the patients and used in the current study . Trichophyton rubrum type 1 was white and had fluffy colony with pink to burgundy on reverse . Type 2 was granular with powdery - velvet, radially folded and pale brown in color and the reverse was dark tan . T. mentagrophyte type 1 was downy, floccose and white in color with the fade yellow on reverse . Finally, type 2 was coarsely granular and light buff in color with the buff - tan color on reverse . As in the skin samples from patients, only mycelium can be observed, in the current study mycelial colonies were harvested, suspended in sabouraud dextrose broth (sdb) (merck, germany) and dispersed to small part suspensions using a sonic dismembrator (dr . Hielscher gmbh, stahnsdorf, germany). Cell densities of the suspensions were counted in a neubauer hemocytometer (hausser scientific, horsham, pa, usa), and reached 10 pieces / ml . Fifty l of suspension was inoculated onto sda to observe macroscopic and microscopic colony characteristics . In each test, a 10 ml aliquot of the dermatophyte suspension was inoculated onto a petri dish . Uv - a and uv - b lights were supplied by a waldmann uv801kl apparatus (waldmann, villingen - schwenningen, germany) at 470 w, and uv - c light was supplied by an f30t8/gl apparatus (young deungpo - gu, seoul, south korea) at 30 w. five different doses of uv light were used: uv - a (315 - 400) 3, 6, 9, 12, 15 j / cm, uv - b (280 - 315) 0.5, 1, 1.5, 2, 2.5 j / cm and uv - c (100 - 280) 0.3, 0.6, 0.9, 1.2, 1.5 j / cm . One positive control without exposure to uv light was cultured in each round with the test samples . This distance is widely used in dental and cosmetic fields to sterilize tools (10). In the current study, for uv - a and uv - b radiation, the lamps were utilized in the centers for dermatological diseases in order to obtain some practical implications . Since radiation reduces the volume and raises the count because of evaporation, which may lead to errors, the specimen volumes were made up to the primary volume (10 ml) after irradiation, with sdb . For each irradiated specimen, the fungicidal effect of uv was evaluated by preparing five cultures to incubate at 30c under laboratory conditions; therefore, 75 cultures for each fungus were prepared . Colony size, morphology and count, and growth rate of all isolates were checked in the plates every two days and mean values were calculated . Since most colony growth occurs during the first 14 days, the results were graphed after this period . All statistical analyses were conducted using microsoft excel software.the current research conformed to the helsinki declaration and local legislation, and was approved by the local ethics committee (91 - 5) in clinical microbiology research center, shiraz university of medical sciences . In response to the uv treatments, compared with suspension control without irradiation, colony morphology for each dermatophyte remained unchanged, but colony count and size changed depending on the species and type of isolate . Of the two t. rubrum isolates (figure 1), type 1 was less sensitive to uv - a and uv - c, and more sensitive to uv - b . Continuous irradiation with the uv - a and uv - b doses used in the study did not completely suppress the growth of the isolates, but higher intensities of uv - c light inhibited the growth of t. rubrum . Mean colony counts at different intensities uv - a (315 - 400 nm), uv - b (290 - 320 nm) and uv - c (100 - 280 nm). Of the two t. mentagrophytes isolates (figure 2), colony count in type 1 decreased as the dose of all three types of uv radiation increased . However, colony count in t. mentagrophytes type 2 showed different patterns as the dose of uv radiation increased . Type 2 strain was slightly responsive to uv - abut compared to control cultures; there was no significant effect on colony count . Neither uv - b nor uv - c decreased the type 2 colony counts at low doses, but higher doses led to decreases in colony counts . Mean colony counts at different intensities of uv - a (315 - 400 nm), uv - b (290 - 320 nm) and uv - c (100 - 280 nm). According to table 1, uv - a irradiation led to a slight increase in colony size in the four strains except t. mentagrophytes type 1, in which colony size increased with the lowest dose of irradiation and significantly decreased with increasing the doses . Irradiation with uv - b increased colony size in t. mentagrophytes type 2, but in type 1 uv ray increased colony size at the lowest dose and decreased colony size with increasing doses . Uv - b irradiation decreased colony size in t. rubrum type 2 but had no significant effect on type 1 or either of the two strains of t. mentagrophytes . Irradiation with uv - c decreased colony size except for t. mentagrophytes type 2, in which higher doses increased colony size . Values are presented as mean sd . Values are presented as j / cm . According to obtained data, uv light can affect colony count and size of t. rubrum and t. mentagrophytes in the culture, the two fungal species frequently isolated from patients with onychomycosis . The estimated prevalence of onychomycosis caused by these species ranges from 10 to 21.3% in the general population in australia, the united kingdom and iran (11, 12). This prevalence rate may increase to 30% in specific populations such as people over 70 years old (13 - 15). It is challenging to determine the optimal treatment and circumventing drug resistance in pathogenic fungi, including dermatophytes . Since the nails and skin are being exposed to sunlight, many photodynamic treatments are developed in dermatologic studies . For example, uv light therapy is investigated in diseases such as scleroderma (16) and onychomycosis (17). The mechanism of uv radiation is the primary photochemical reaction, which damages dna and affects survival and rate of germination (18). This molecular change makes the dna unstable for essential biological processes such as transcription and replication . As a rule, the simpler the microorganism is in anatomical terms, the more easily it is inactivated by uv radiation . This is why viruses and prokaryotic cells such as bacteria are more easily destroyed than a complex of microorganisms such as eukaryotic cells, yeasts and vegetative fungi . In particular, fungal spores in which the dna is protected by a concentrated cytoplasm and pigmented cell wall some data suggest that solar uv radiation can inhibit the growth of fungi on the soil surface in the antarctic terrestrial environment (19), and has disinfectant effects that can vary depending on the type and dosage of irradiation, condition of the surface and exposure time . The effective exposure time was reported to be 45 minutes for yeast and 75 minutes for mold (20). Different doses of uv - b light show different effects on the growth of candida albicans . Increasing doses of uv - b strongly reduced hyphal growth in this fungus and triggered enhanced blastospore formation (21). The mechanism of resistance to uv radiation is not completely known, but intra hyphal growth and recolonization of old cells by new ones were observed in all the strains investigated by gorbushina et al . Uv - c radiation affected t. rubrum type 1: as the dose increased, colony count decreased . Irradiation with uv - a and uv - b also decreased the colony count of some strains tested in the current study which was proportional to the dose . This finding may be due to the synthesis of new hyphae through the old hyphae not exposed to irradiation, or may be related to the age of the exposed hyphae (i.e. Recolonization of older hyphae) in the suspension . Trichophyton rubrum is one of the most frequent causal agents in nail infections, and recurrence of the infection and resistance to therapy are the most important limitations to treatment success for this anthropophilic dermatophyte (22). This species was unaffected by infrared irradiation, responded with increased pigmentation to uv - a at13.5j / cm twice daily, and was inhibited by a single dose of uv - b at 15.1 j / cm (6). Growth rates, surface colony color and the texture of colony morphology are different in each strain . The current study compared two strains of t. mentagrophytes . According to figure 2, uv - a, uv - b and uv - c had similar effects on t. mentagrophytes type 1, but affecting type 2 strains differently . Increasing pigmentation in response to uv - a (13.5 j / cm twice daily) was observed in t. rubrum (6). Electron microscopy studies showed a marked thickening and blurred contours of the cell walls grown with a brightener effect related to interference by optical brighteners with the formation of normal chitin fibrils and thickening of the cell walls (23). In the present study, increasing doses of uv - a, uv - b and uv - c irradiation increased colony size in t. mentagrophytes type 2 . Uv - b irradiation of the skin is particularly well studied, and is accepted as the main cause of skin cancer . The depth of penetration of uv - c radiation into the human skin is very low; therefore, the risk of skin cancer associated with this type of treatment is low . There are studies which report the treatment of localized infections with multidrug - resistant microorganisms (24) and subungual onychomycosis (25) with uv - c light irradiation and favorable responses . As mentioned in these studies, the cases were limited and side effects should be taken into account more carefully . When uv irradiation is used to treat onychomycosis, it is important to minimize exposure by carefully masking the area with adhesive material . In addition, the dose of radiation must be minimized by careful dosimetery calculated with reference to the individual patient s nails, the etiologic agent, and optical properties . If there were enough samples, by using different types and doses of irradiation such as uv - a uv - b and uv - c for every single nail sample separately, the current study could directly examine the effects of radiation on them, but since the volume of samples was low, they were just cultured after the isolation of fungal strains and it was possible to evaluate different types and doses of irradiation in the isolated strains . Uv - a, uv - b and uv - c seem to be effective in decreasing colony growth in some prevalent fungi, which caused onychomycosis in the patients . Further studies are needed to determine the efficacy of this therapy, identify possible side effects and establish appropriate dosages for the antifungal action of uv radiation therapy.
A 39 year old female who was seven weeks pregnant presented to a community hospital emergency department with a first episode of chest pain . She had a twenty pack per year smoking history and a significant family history of coronary artery disease (cad) with her father developing cad in his thirties . She described the pain as a pressure sensation, retrosternal in location, with radiation down both arms . It was associated with intense nausea and vomiting, and she had some relief with aspirin . In the emergency department, she denied recreational drug use, and did not have any constitutional symptoms . Clinically there was no evidence of deep vein thrombosis (dvt), pulmonary embolism (pe), or pericarditis . On physical examination she was afebrile and hemodynamically stable, with a heart rate of 69 and regular, equal blood pressures in both arms of 95/65 mmhg, and an oxygen saturation of 97% on room air . There were no signs of congestive heart failure with no pedal edema, clear lungs, and no jugular venous distension . The precordial exam was normal with no heaves, thrills, normal s1 and s2 with normal physiological splitting and no extra heart sounds, rubs or murmurs . A 12-lead electrocardiogram (ecg) was completed that showed normal sinus rhythm at a rate of 60 bpm, normal axis, normal intervals with no evidence of chamber enlargement with 1 mm st segment depression in lead v4 and <1 mm depression in v5 (fig . 1). Initial blood work showed a significant elevation of the cardiac markers with a troponin t of 0.96 g / l and a creatinine kinase (ck) level of 718 u / l, all of which decreased on serial measurements . An echocardiogram demonstrated severe inferolateral wall hypokinesis with a preserved left ventricular systolic function and ejection fraction of 60% with no other abnormalities identified . Her past obstetrical history included three pregnancies with one full term delivery, one preterm delivery, and one therapeutic abortion . The patient was transferred from a community hospital to our tertiary center for further management . Pulmonary, gastrointestinal, psychiatric, neuromuskuloskeletal, along with non - ischemic cardiac causes of chest pain must be considered in these patients . The incidence of myocardial infarction in pregnancy has been estimated to be 6.2 per 100 000 deliveries with a mortality rate of 5.1%11% in recent reviews.1,2 prior estimates of mortality have reported it as substantially higher at 37% and estimates of incidence substantially lower at 2.8 per 100 000 deliveries.1,3 this incidence is approximately 34 times higher than the estimated age associated risk for non - pregnant women.2 the decreased mortality and increased incidence in the recent literature is likely due to use of more sensitive and specific serum cardiac markers, such as troponins, identifying more cases of subendocardial myocardial injury as well as the increasing cardiovascular risks, such as advancing maternal age.1 in a nationwide us population based study of acute myocardial infarction (ami) during pregnancy performed between 2000 and 2002, the anterior coronary circulation was found to be more commonly involved with 20% of reported infarctions occurring in this territory.1 although not elaborated on within the original articles, the preponderance of anterior circulation culprit vessels in ami may be due to the greater clinical presentation of these patients, while missing the smaller myocardial infarctions in the other vascular territories . Ladner et al found that in pregnant women with ami, 38% occurred in the antepartum, 21% occurred in the intrapartum, and 41% occurred in the 6-week postpartum period.4 within pregnancy, badui and colleagues identified that women in the third trimester had the highest risk of ami.5 the increased stroke volume and heart rate during pregnancy causes an increased myocardial oxygen demand, while the decreased diastolic blood pressure and related physiologic anemia result in decreased myocardial perfusion that may contribute to the ischemia when coronary blood flow is compromised . With labour, myocardial ischemia may be precipitated by a further increase in myocardial oxygen demand driven by pain, uterine contraction, and anxiety . After delivery, caval compression is relieved and blood flow is shifted from the uterus back to the systemic circulation resulting in further stress on the myocardium and likely contributing to the increased incidence of myocardial infarction in the puerperium . With a compromise in the coronary blood flow, the high demand physiological state of normal pregnancy would precipitate myocardial ischemia and potentially infarction.2 james et al reviewed the coronary anatomy through angiography and autopsy of pregnant women diagnosed with ami and found that 40% had evidence of atherosclerosis with or without thrombosis, 8% had thrombosis without atherosclerosis, 27% had coronary artery dissections and 13% had normal coronaries.1 in the general population, nearly all cases of acute myocardial infarction are due to coronary atherosclerotic disease and acute plaque rupture resulting in coronary artery occlusion . Rarely, vasculitic syndromes, hypercoagulable states, coronary artery spasm, increased myocardial demand, coronary emboli, congenital coronary anomalies, trauma and aneurysm may cause ami.6 the increased events associated with thrombosis without atherosclerosis, vasospasm and coronary artery dissection may be related to the physiological alterations associated with pregnancy . Pregnancy is a known hypercoagulable state.7 the association of thrombophilia with mi in pregnancy may be due to the increased testing for this in this particular population.1 in regards to vasospasm, the pregnant woman has more reactive vessels to norepinephrine and angiotensin ii, has associated endothelial dysfunction and has an increased renin secretion and angiotensin activity associated with uterine malperfusion with the supine position and the use of ergot derivatives to control post - partum hemorrhage all may contribute.8 this vasospasm may also be the precipitating mechanism for thrombosis in coronary vessels that have no evidence of atherosclerosis, with the spasm impeding blood flow and the physiologic hypercoagulable state resulting in a thrombosis.8 the risk factors for ami are also commonly seen in pregnancy, including diabetes mellitus, smoking, advanced maternal age, dyslipidemia, significant family history and hypertension.1,2 in addition novel risk factors such as black race, pre - eclamplsia, eclampsia, anemia, migraine headaches and thrombophilia have been identified.1,4 the associated risk with migraines may be due to overall disorder of the woman or due to heightened awareness of the physician for possible acs events in these patients.1 the association of pre - eclampsia and eclamplsia may be due to endothelial dysfunction that has been shown to persist up to one year post partum.9 the increased incidence of coronary dissection is thought to be due to the changes in progesterone resulting in several structural and biochemical changes within the vessel wall; however, other theories include changes in eosinophil activity and decreased prostacyclin activity have been postulated . It is these systemic changes in conjunction with the physiological changes of increased blood volume and cardiac output that likely result in increased shear forces that result in dissection occurring not only in single vessels, but frequently in multiple coronary arteries.2 the treatment of acs has been well established for the non - pregnant patient, but many uncertainties remain in the management of pregnant patient which may delay treatment . A classification scheme has been established to identify the associated risks with certain medications in pregnancy (table 1).6 nitroglycerine (class b) is widely used for ischemic pain, however there are concerns about maternal hypotension and uterine malperfusion.1,2 studies are required to fully elucidate the effect of nitrates in pregnancy.10 heparin (unfractionated heparin class c, low - molecular weight heparin lmwh class b) has been proven to be safe in pregnancy in numerous studies, however it is recommended to stop heparin prior to delivery and monitoring anti - xa levels if lmwh is used due to the pregnancy associated pharmacokinetic changes.11 beta - blockers (metoprolol class b, atenolol class c) have been used successfully; however there are anecdotal reports of fetal bradycardia, hypoglycemia, hyperbilirubinemia, and apnea.2 a cochrane review looking at oral beta blockers for use in treatment of mild to moderate hypertension in pregnant women found that there was a trend toward small for gestational age infants, but the results were skewed by a small outlier trial . There was insufficient data to comment on perinatal mortality or preterm delivery.12 atenolol has been linked to a possible increase in fetal growth restriction, especially when used in the first trimester.13 asa (class c) is debateable for use during pregnancy because animal studies have shown increased incidence of fissure of spine and skull, facial and eye defects, and malformations of the central nervous system (cns), viscera, and skeleton.2,10 chronic use of high dose asa during pregnancy should be avoided because of increased fetal hemorrhage, increased perinatal mortality, intrauterine growth restriction and teratogenic effects.2,6 a meta - analysis looking at antiplatelet agents found that low dose asa is safe in pregnancy.14 clopidogrel (class b) has very limited data for its use in pregnancy . It is recommended that clopidogrel be stopped 1 week prior to any regional anesthesia procedures.2 glycoprotein iib / iiia inhibitors (eptifibatide and tirofiban class b, abciximab class c) have not been studied in pregnant patients as all randomized trials of these agents excluded pregnant patients . These drugs cannot be recommended in pregnant patients, however if they are used a c - section delivery is recommended to decrease the potential for fetal intracranial hemorrhage . 2 angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) are contraindicated in pregnancy due to teratogenic side effects . Many animal and human studies have found that ace inhibitors and arbs cause multiple birth defects including renal dysgenesis, oligohydramnios, iugr, prematurity, bone malformations, limb contractures, death and multiple others.6,15 a recent retrospective analysis of fetuses exposed to ace inhibitors in the first trimester identified ace inhibitors as an independent risk factor for developing malformations of the cardiovascular and cns.16 statins (class x) are not recommended in pregnancy as information on use in pregnancy is limited . Although laboratory models show potential placental growth disruption and animal studies have shown skeletal abnormalities and increases in mortality, a recent systematic review found that most data of human teratogenicity were only case reports and that the overall risk is likely minimal . The authors stated that statin exposure did not warrant termination of pregnancy as a sole reason.2,17,18 a prospective cohort of 134 women inadvertently exposed to lovastatin and simvastatin found no difference in the incidence of adverse pregnancy outcomes.19 the use of invasive catheter procedures for management of ami in pregnancy is also not clearly identified . Numerous case studies have been published that describe results of both invasive and conservative management . In one report a patient was managed conservatively with asa and beta - blockers, while waiting for the post - partum period to undergo cardiac catheterization . Other reports have described treating pregnant women with early percutaneous coronary intervention (pci) and stent placement . Both reported favorable fetal and maternal outcomes.2022 bare metal stents have been used with success in the literature; however, there is limited data for the use of drug eluting stents and its necessary long - term clopidogrel treatment.2 the teratogenic effects of radiation were first reported in 1929 when goldstein and murphy observed a high rate of micorcephaly and reduced cranial circumference in women who had undergone radiation treatment for uterine cancer during pregnancy . While many studies have shown that a fetal dose of 5 rads is not related to teratogenicity at any period of gestation, the most vulnerable time for the fetus is 815 weeks of gestation.23 coronary angiography exposes patients to 2.55.0 msv (equivalent to 125250 chest x - rays), and percutaneous coronary intervention exposes patients to 5.015.0 msv (equivalent to 1151000 chest x - rays),24 which are both below the threshold for teratogenicity at any gestational age . The amount of radiation that reaches the fetus is a percentage of the total amount delivered to the patient and depends on the body parts being irradiated and the type of protection used . No necessary radiodiagnostic examination that is clinically justifiable should be avoided due to pregnancy, and protective measures for the mother and fetus should be taken . Other diagnostic procedures that are equally as effective but not as dangerous to the fetus should be preferentially used . A number of therapies ranging from multiple drugs to pci are available for the pregnant patient presenting with acs . It is important to weigh the risks and benefits of each potential therapy and tailor the management according to the clinical presentation . The patient was started on asa, beta - blockers, and intravenous unfractionated heparin . A quantitative -hcg and pelvic ultrasound were arranged to verify the viability of the pregnancy prior to starting medications with known teratogenic side effects and unclear risk profiles . Pci was discussed however not pursued as she had a normal left ventricular ejection fraction, no recurrent chest pain, no electrical or mechanical complications . A pelvic ultrasound showed an intrauterine pregnancy with no fetal heartbeat, consistent with fetal demise . Expectant management of the fetus was chosen and follow - up ultrasound was arranged with the obstetrics team . The cardiology team arranged follow - up regarding long term medications and planning for further risk stratification . Although acs in pregnancy has been historically uncommon, the increasing prevalence of atherosclerotic risk factors in women of child bearing age combined with the normal physiological changes of pregnancy will cause the incidence of this presentation to increase in clinical practice . It is important that physicians are familiar with the clinical presentation, risk factors, potential management options and their interactions with both the pregnant female and the fetus.
Clavicle fractures occur frequently, with the reported rates ranging between 8% and 15% of all paediatric fractures [13]. The vast majority of these injuries can be treated nonoperatively with excellent results [4, 5]. Reported indications for operative management include markedly displaced fractures with compromised skin integrity, open fractures, concomitant vascular injury requiring repair, and compromise of the brachial plexus [610]. More recently, there has been some support for operative management of middle third fractures with marked displacement or shortening [1113]. Some of these studies have specifically recommended fixation in children and adolescents [14, 15]. The aim of this study was to review the outcome of clavicle fractures in paediatric patients at our institute and to determine the number of such fractures that require operative management . We retrospectively reviewed all clavicle fractures in children treated at our institute over a two - year period . We used the agfa impax web1000 system to identify all radiographs of the shoulder region performed in children aged up to and including 15 years old . These radiographs were then reviewed to identify all clavicle fractures in the patient cohort group . Medical records and theatre records were then reviewed to establish the classification of each fracture, the treatment method used, the duration of radiographic followup, the duration of clinical followup, and the clinical outcome . Exclusion criteria were any patient aged 16 years or older, and any fracture as a result of birth trauma . We identified 487 clavicle fractures in 483 patients treated in our institute during the two - year period . Ten neonates were excluded because their injuries were related to birth trauma, and 283 patients were excluded because they were 16 years old or older . All fractures were classified using the system described by robinson [16, table 1]. One hundred and twenty - four fractures were undisplaced (65%) and 66 were displaced (35%). Immobilisation in the sling was continued until the patient was comfortable enough to mobilise without support . Further radiographs were only taken if the patient continued to have pain or limitation of function when reviewed . Initial review was within one week of injury, and second review was at three weeks after initial review . Further review appointments were arranged at the discretion of the clinician and were determined by clinical and/or radiographic assessment . The mean radiographic follow - up of the group as a whole was 35 days (5 weeks), and the mean clinical follow - up was 44 days (6.3 weeks). The mean radiological and clinical follow - up of the group when subdivided by age can be seen in figures 2 and 3 . Forty - four of 190 fractures (23%) had radiographic confirmation of fracture healing . The remaining 77% had radiographic examinations discontinued when clinical symptoms of pain and limitation of function had resolved . All fractures in this study had healed clinically when the child was discharged from follow - up . Clavicle fractures are common injuries in general, and, in this study, 39% of all fractures treated over a two - year period involved children of 15 years or younger . Despite this, there are surprisingly few published studies that specifically discuss paediatric clavicle fractures . Traditionally, clavicle fractures have been treated nonoperatively, particularly in children . This is largely due to the relatively low incidence of complications following non - operative management . Indications for operative management in the acute setting have included markedly displaced fractures with compromised skin integrity, open fractures, concomitant vascular injury requiring repair, and compromise of the brachial plexus [610]. Howard and shafer described fourteen clavicle fractures with associated neurovascular complications, but only one case occurred in a child, a ten - year old with a depressed clavicle fracture compressing the subclavian vein . Mital and aufranc also described a venous occlusion following a greenstick fracture of the clavicle . Keating and von ungern - sternberg recently published a case report entitled compression of the common carotid artery following clavicle fracture in a twelve - year - old but the report actually describes a clavicle dislocation . Fixation of a clavicle fracture associated with a dislocation [19, 20] and fixation of a fracture associated with a sternoclavicular physeal fracture have also been described in children . Operative management for nonunion of a clavicle fracture in a child has also been described . Reports of complications of clavicle fractures and operative management of clavicle fractures in paediatric patients are few . The examples cited above demonstrate that complications do occur, but these are extremely rare . In our group of 190 patients, none had significant associated injuries and none required operative management . Displaced midshaft fractures of the clavicle have received some attention recently, with some authors recommending operative management . It has been demonstrated that a periosteal hinge is important for fracture stability . In childhood, the periosteal sleeve is thick and protects the cortex, and the bone is softer and more pliable than in adults . In displaced fractures this periosteal sleeve and hinge has been mostly or completely disrupted (using robinson's classification, displaced fractures are those that are translated by 100% or more). Sixty - six patients (35%) in our group sustained displaced fractures of the midclavicle, and all of these healed clinically with non - operative management . This included five fractures that were comminuted segmental (robinson type 2b2), thereby having almost or complete disruption of the periosteal sleeve in at least one part of the bone . As figure 4 shows, displaced fractures occur more commonly in children as they get older . This can be explained by the more adult type bone and periosteum as the child grows and develops . Indeed, figures 3 and 4 demonstrate that the mean period of radiographic and clinical follow - up increased with increasing age of the children . This can in part be attributed to the larger percentage of displaced fractures being encountered with increasing age . In our institute, radiographic evaluation of clavicle fractures is discontinued when symptoms resolve . Some authors recommend that torus / buckle fractures do not require any radiographic review whatsoever, as the incidence of complications is so small [24, 25]. In our experience, clavicle fractures in children, whether displaced or undisplaced, heal clinically, as demonstrated by the absence of pain and the return of full function . This is achieved at a mean duration of six weeks for all fractures . For all age groups, clinical followup continued for at least as long as radiographic follow - up, and in all cases radiographs were only requested when clinically indicated . Some authors have recommended that paediatric patients with clavicle fractures require no follow - up at all . This is based on the fact that most paediatric clavicle fractures heal and is justified by detailed written instructions given to parents informing them of symptoms to be aware of and when to seek further review . The vast majority of patients reviewed in this study lived locally, and all patients are advised to seek further review if they develop symptoms after discharge . Patient records were reviewed at a mean of nineteen months after injury . Had any of these patients developed late complications within this time period, this would have been documented within their records in the form of referral back to the orthopaedic clinic by their family doctor or the local emergency department . We accept that we still may have overlooked complications in the small number of patients who did not live locally or those whose symptoms were not felt severe enough to warrant further orthopaedic consultation . However, radiographs take time, cost money, and expose patients to radiation [2729]. We firmly believe that radiographs should only be performed when they are likely to alter the management of the patient . Our radiographic follow - up in this study was a mean of five weeks, but we do recognise that only 23% of our patients had fracture union confirmed by radiographs . Some studies have suggested that radiographs are not required at all in the assessment of clavicle fractures [31, 32], but we feel that an initial radiograph to confirm and classify the injury is appropriate even where the fracture is obvious clinically . There is evidence in the literature that highlights the aetiology and risk factors for some of these complications . Most published articles report these complications in adult patients, and there is a relative paucity of the literature available that is specifically reporting upon clavicle fractures in children . Despite this, there are a small number of reports of complications in this group . In our experience, all paediatric clavicle fractures can be treated with simple immobilisation and analgesia, without development of complications . Radiographic review of paediatric clavicle fractures is unnecessary in the absence of clinical findings suggestive of delayed union.
The objective of this study is to quantitatively estimate the effect of medicare supplementation on use of health services by medicare beneficiaries . However, the extent of supplementary health insurance coverage is only one of many factors influencing the use of health services by medicare beneficiaries . Previous studies of health services utilization have found such factors as age, sex, race, income, and health status to be capable of exerting independent influences on the use of health services (leopold and langwell, 1978). In order to isolate the influence of supplementary health insurance coverage, it is necessary to control for the variation in utilization arising from these other factors . Accordingly, our empirical results are based upon a multivariate model that relates an individual's use of either physician or hospital services to a variety of underlying determinants . In particular, the empirical specification for the model is: where the subscript i denotes the ith medicare beneficiary; ui measures the person's annual use of health services (two alternative models are estimated: one for annual number of physician visits and one for annual number of hospital days); privi is a binary variable indicating whether the ith beneficiary supplements medicare with some type of private health insurance; caidi is a binary variable indicating whether the ith beneficiary received medicaid benefits during the past 12 months; x3i through xni compose a set of variables to control for other factors influencing the ith person's utilization; and ei is a stochastic error term, assumed to have a truncated - normal distribution . The set of control variables in this model is designed to capture the influence of family income, race, sex, region of residence, education, family size, health status, age, marital status, labor - force status, veteran status, and the availability of medical resources . The primary data source is the 1976 health interview survey (usdhew, 1977). Observations were excluded for persons for whom family income or education was unknown or not reported, reducing the sample for the analysis of hospital utilization to 8,325 . The elimination of observations pertaining to persons who failed to report the number of physician visits during the preceding 12 months further reduced the sample for analyzing physician utilization to 8,239 . For each of the dependent variables about 30 percent of the sample reported no use of physician services during the preceding 12 months, and approximately 80 percent did not use inpatient hospital services . Accordingly, the tobit estimation technique (tobin, 1958) was used for this study . Covariance analyses revealed that our investigation of physician utilization should be conducted with data stratified according to the presence or absence of chronic health problems . Thus, results from the analysis of physician utilization are reported separately for (1) elderly medicare beneficiaries who have no chronic health problems and (2) those who have one or more such conditions . In contrast, covariance tests for hospital utilization indicated that it was appropriate to pool over all health conditions . These tables highlight the influence of supplementary coverage on the utilization of health services by elderly medicare beneficiaries . Table 1 reports the average utilization of health services among medicare beneficiaries by type of supplementation, both in absolute terms and relative to the average utilization by medicare beneficiaries who do not supplement (shown in parentheses in the table). These average rates were tabulated directly from the health interview survey (his) data, and thus are unadjusted for other determinants of utilization . The differentials between these averages should not be interpreted as reliable indicators of the influence of supplementation on utilization . Since we shall compare these unadjusted averages with utilization rates adjusted for other determinants, a brief examination of the apparent implications of the results in table 1 is merited . These unadjusted averages reveal that beneficiaries who supplement medicare with private health insurance generally make greater use of health services than those who do not supplement . The largest differential between those with private supplementation and those with only medicare arises in connection with physician visits by beneficiaries with no chronic health problems: those who have private supplementation in this group report 39 percent more visits to physicians than their counterparts who rely solely on medicare . Private supplementation is also associated with somewhat greater use of hospital services (2.79 days per year), relative to the average utilization among those with no supplementation (2.51 days). In contrast, those with one or more chronic health problems and private supplementation tend to have fewer physician visits compared to those with no supplementary coverage . While the differential is small (6.23 visits versus 6.72 visits), it is nevertheless paradoxical . Public supplementation of medicare is associated with relatively high rates of utilization of both physician and hospital services . For example, medicare beneficiaries with medicaid supplementation spent an average of 76 percent more days in the hospital than those with no supplementation and 58 percent more than those with private supplementation . Based upon this type of evidence, one might be tempted to conclude that public supplementation of medicare has greatly stimulated the use of health services . However, while such a conclusion may be correct, it does not necessarily follow from the evidence in table 1 . The high utilization rates in the medicaid category may simply reflect the relatively poor health status of persons in this group . Table 2 contains the key findings of this study, namely, the predicted utilization rates by type of supplementation, adjusted for other determinants . These estimates are derived from a multivariate model that controls for a variety of determinants of utilization and thus isolates the influence of supplementation on utilization from the influence of other factors such as health status . The values reported in table 2 represent predicted utilization rates for typical medicare beneficiaries; that is, they are derived under the assumption that all of the determinants of utilization (except the supplementation variables) equal their mean values . What do these predicted utilization rates imply about the influence of supplementation on the use of health services by elderly medicare beneficiaries? First, other things equal, supplementation always stimulates use of health services, generally by statistically significant amounts . In particular, the paradox of relatively low demand for ambulatory care by those chronically ill beneficiaries with private supplementation disappears once the utilization rates are adjusted for other determinants . Second, public supplementation always stimulates more utilization of health services than does private supplementation . However, in no instance is the differential in utilization rates between those with private supplementation and those qualifying for medicaid statistically significant . We are justified in concluding only that, other things equal, medicaid supplementation of medicare permits elderly beneficiaries to use physician and hospital services at least as often as those who purchase private supplementary health insurance . This conclusion contrasts sharply with the one implied by the unadjusted average utilization rates in table 1: those estimates suggest that the influence of public supplementation on the demand for health services far outweighs the influence of private supplementation . A final important implication of the estimates reported in table 2 pertains to the differential effectiveness of supplementary coverage at stimulating demand for health services . Supplementation greatly increases the use of both hospital services and physician services among elderly persons with no chronic health problems . However, among those elderly beneficiaries with one or more chronic health problems (about 78 percent of the beneficiary population), persons with some type of supplementation have only slightly more physician visits than those with no additional coverage . Apparently the deductibles and coinsurance provisions of medicare's part b medical insurance do not represent an important barrier to ambulatory medical care for those beneficiaries who suffer from chronic health problems . For these individuals, supplementation particularly through medicaid serves mainly to redistribute income to the chronically ill medicare beneficiaries . Conclusion that public supplementation was very effective at stimulating demand for ambulatory care among those with chronic illnesses . The estimates presented in table 3 provide an additional perspective on supplementation's role in determining utilization rates among elderly medicare beneficiaries . Mean utilization for a group, u, can be defined as: where p is the probability that a person with mean characteristics will use a particular health service and u is the mean utilization rate among those who actually use the health service . Thus, any change in a group's utilization rate (u) can be divided along the following lines: this relationship can be restated in percentage terms as table 3 partitions the percentage increase in utilization arising from supplementation into the three components identified in equation 3: the percentage increase in the probability of using a particular health service, the percentage increase in the utilization rate among those that make use of the health service, and the interaction between these two factors . The estimates in table 3 reveal that supplementation raises a group's mean utilization rate (adjusted for other determinants) largely by increasing the proportion of the group that uses medical services . When compared to no supplementation for example, medicaid supplementation raises the use of hospital services by 47 percent . Most of this gain (29 percent out of the 47 percent) arises from the hospitalization of persons who, in the absence of medicaid supplementation, would not have received treatment on an inpatient basis . Private supplementation has a similar influence on the utilization of hospital services: most of the gain comes from more people being admitted to hospitals rather than from an increase in average length of stay . These results suggest that the part a deductible (approximately equal to the average charge for an inpatient hospital day) represents a significant barrier to the utilization of hospital services by the elderly . Supplementation increases utilization by persons in this group mainly by permitting a larger fraction of the group to visit a physician than would otherwise be the case . The part b deductible ($60) thus appears to also serve as an important barrier to the use of ambulatory medical services by those with no chronic health problems . In addition, the part b coinsurance rate (20 percent of covered services) apparently serves as an important deterrent to physician utilization by those elderly persons with no chronic conditions . This conclusion follows from the relatively large positive effect of supplementation on utilization among those who, in the absence of supplementary coverage, would have still made some use of physician services . To illustrate, private supplementation leads to a 42 percent increase in physician visits by persons in this health - status group . Of this increase, 16 percent is due directly to greater utilization among those who would have seen a physician even without supplementary coverage . As we noted earlier, supplementation stimulates physician utilization only to a small degree among those elderly with chronic health problems . Table 3 reveals the two underlying components of mean utilization to be about equally responsible for this small gain in the use of physician services . Apparently neither the deductible nor the coinsurance provisions of part b represent important barriers to physician utilization among those elderly beneficiaries with chronic health problems . Our principal finding is that beneficiaries' utilization of health care services rises when medicare is supplemented by private insurance coverage or by medicaid, though in varying amounts depending on individuals' health status . To turn the conclusion around, we find that medicare cost - sharing (when not vitiated by private or medicaid supplementation) leads to significantly lower levels of hospital and physician utilization than would have prevailed in the absence of the program's deductibles and coinsurance . The estimates we report allow for an illustrative calculation of the magnitude of the cost savings owing to medicare cost - sharing . Cost - sharing under parts a and b is an effective economic incentive for the 29 percent of the 24 million elderly beneficiaries (about 7 million) who have neither private nor public supplementary health insurance . The predicted hospital utilization rates in table 2 imply a utilization reduction of between 630 and 890 days per thousand beneficiaries per year when cost - sharing is effective (compared to private supplementation and medicaid, respectively). Therefore, the part a cost - sharing provisions result in 4.4 million to 6.2 million fewer days of hospital care for the elderly . Assuming a cost per day of $160 (the part a hospital inpatient deductible for calendar year 1979), medicare cost - sharing results in a reduction in total hospital expenditures by the elderly of between $700 million and $1 billion . Of course, only part of these savings accrue to the medicare program since some of the reduced utilization would have been subject to the part a deductible . However, cost control is not the sole goal of the medicare program . It has been argued that medicare cost - sharing is particularly perverse because it largely reduces the health services utilization of certain disadvantaged groups the medicaid - ineligible low income and nonwhite populations for whom medicare was designed to equalize access to medical care (gornick, 1976; davis and schoen, 1978). Thus it is conceivable that the advantage of cost - sharing in controlling health expenditures might be outweighed by the disproportionate reduction in utilization among certain population groups . To test the hypothesis that the availability of private health insurance supplementation leads to inequitable utilization advantages for whites and higher income groups, we re - estimated the above - described model after omitting the private insurance variable . Examination of the changes in the coefficients on the race and income variables, respectively, will reveal unequal access afforded by the private market availability of supplementary insurance, if any . In all three regressions physician visits for chronics and non - chronics and hospital days the coefficients for southern and non - southern blacks changed insignificantly . Similarly, as reported in link, long, and settle (1980), the variables representing income classes did not change significantly . Whatever inequitable access to services exists for medicare beneficiaries is apparently caused by factors other than the availability of private supplementary insurance . In summary, we conclude that medicare cost - sharing, in the absence of private or public supplementation, reduces medical care utilization and, therefore, costs to the program . Moreover, it appears that the burden of this cost - sharing is not so concentrated among race and income groups as to cause uniformly inequitable access to medical care . However, fine - tuning among these competing objectives remains in the province of policymakers, not researchers.
Parkinson s disease (pd) is one of the most common neurodegenerative disorders, with an estimated prevalence of approximately 1 to 2% among people 65 years of age or older . Clinically, pd patients show motor (e.g., tremor and rigidity) and nonmotor (e.g., dementia and depression) symptoms . It is believed that motor symptoms are largely attributable to the loss of dopaminergic neurons in the substantia nigra, whereas nonmotor symptoms involve many other brain regions . Effective treatment of pd has been hampered partially due to the fact that even newly diagnosed patients are already at relatively advanced stages pathologically . The other difficulty is lack of objective assessment of disease progression, which has led to extensive research on biomarkers that can be employed for early diagnoses of pd or assessing its progression . As of today, the best pd protein biomarkers are those found in cerebrospinal fluid (csf), the body fluid that is in direct contact with brain and spinal cord . Discovery of peripheral biomarkers reflecting motor components of pd is highly desirable because csf - based tests depend on the comparatively more invasive process of lumbar puncture that requires more specialized training to collect samples and are therefore not acceptable by some subjects, particularly those without apparent clinical symptoms . However, peripheral biomarker discovery has been largely unsuccessful, due primarily to factors intrinsic to blood; specifically, the plasma or serum proteome is quite complex, proteins are present across a wide dynamic range, and target proteins are often of extraordinarily low abundance . Thus, accurately detecting and quantifying brain - derived or disease - specific proteins in blood is challenging for current technologies . That said, a few blood biomarkers discovered recently, for example, egf and apoa1, appear to be related to cognitive impairment or disease onset in pd . Additionally, it has been suggested recently that -synuclein pathology, a key component of pd pathogenesis, exists in peripheral nerves, leading to a report demonstrating abnormal autonomic nerve staining in skin biopsies in pd . To facilitate peripheral biomarker discovery, in the current investigation, we began with proteins identified in our previous proteomic investigations using human brain tissues obtained at autopsy and csf obtained from living patients, with a focus on glycosylated proteins, which are highly enriched in body fluids, including plasma . We hypothesized that some of the brain or csf -derived proteins will reach plasma via mechanisms yet to be defined and that a subset of these proteins or peptides will be detectable in plasma using sufficiently sensitive measurements . To study the presumably low - abundance brain - derived proteins in plasma, we turned to a current quantitative mass spectrometry (ms) technique, selected reaction monitoring (srm), which has emerged as an alternative to immunoaffinity - based measurements of defined protein sets . Srm has the benefit of fast and cost - efficient assay development, and protein quantification by srm in complex samples using predefined assay coordinates is reproducible across different laboratories and instrument platforms . However, the main advantage of srm is the capacity to quantify multiple proteins in parallel at a low limit of detection and high accuracy . It has been reported that srm has the ability to detect plasma proteins at g / ml levels without any sample enrichment or fractionation, which suggests that a further enrichment would be necessary to detect central nervous system (cns)-derived proteins at lower levels . In recent years, a multitude of enrichment approaches have been developed . Among them, immune affinity depletion can be used to remove the most abundant proteins, improving detection of low - abundance proteins . However, one risk associated with this kind of predepletion is the proteins of interest might also be partially removed . In contrast, specific peptides of interest can be enriched after digestion (immuno - srm), dramatically increasing their relative concentration; however, such techniques are limited by the need for highly specific and high - affinity antibodies, which are either not available for all targets or expensive to generate . We chose to use the n - glycocapture technique, which is an antibody - free, hydrazide - based approach to selectively enrich n - glycopeptides . Additionally, glycosylation is known to be important in pd, and glycoproteins, which are prevalent in extracellular surface proteins and secreted proteins, are ideal sources of biomarkers . Therefore, to test our hypothesis in this study, n - glycoproteins were captured from plasma of a larger cohort of pd patients, along with healthy and diseased controls, by a hydrazide - based solid - phase capturing approach, followed by quantification of peptides that are uniquely associated with pd diagnosis or severity based on our previous experimentations . A total of 282 subjects, including patients with pd or alzheimer s disease (ad) and age - matched controls, recruited at the veterans affairs puget sound health care system / university of washington school of medicine, the oregon health and science university, and the university of california at san diego, were included in the investigation . The subjects consisted of two subcohorts: those collected prior to 2011 (75 pd, 15 ad, and 30 controls) and those collected more recently (98 pd, 15 ad, and 49 controls). All plasma samples were obtained after informed consent from patients, and all patients underwent medical history evaluation, physical, and neurological examinations, laboratory tests, and neuropsychological assessments . All pd subjects met the uk pd society brain bank clinical diagnostic criteria for pd, while ad cases were diagnosed according to nia criteria . Pd patient samples were further categorized based on updrs part iii on - state motor scores to approximate disease stage . Patients with updrs scores <15 were defined as early - stage pd, those with scores ranging from 1530 were classified as midstage pd, while those with scores> 30 were classified as late - stage pd patients . Control subjects were community volunteers in good health and had no signs or symptoms of cognitive impairment or neurological disease; all control subjects had a mini mental status examination (mmse) score between 28 and 30, a clinical dementia rating (cdr) score of 0, and new york university paragraph recall scores (immediate and delayed) of> 6 . All samples were collected and processed following standard clinical protocols and quality - control procedures at all participating sites, as defined previously . Isolation of n - linked glycopeptides (n - glycopeptides) from plasma was performed as previously described . In brief, starting with aliquots of 25 l individual or pooled plasma, samples were diluted 10-fold with ammonium bicarbonate (100 mm), then denatured with 50% tfe (2,2,2-trifluoroethanol, j.t . Baker, philipsburg, nj) and digested with mass - spectrometry - grade trypsin (promega, madison, wi). The peptides were desalted using c18 cartridges (waters, milford, ma) and were then oxidized with 10 mm naio4 . The resulting oxidized glycopeptides were coupled to hydrazide resin (affi - prep, bio - rad, hercules, ca) by incubation in coupling buffer (100 mm sodium acetate and 1.5 m sodium chloride, ph 4.5) overnight at room temperature with bottom - over - head rotation . The unbound nonglycosylated peptides were removed by several washes of sodium chloride (1.5 m), 80% acn, and ammonium bicarbonate (100 mm), respectively . N - glycopeptides were finally eluted from the resin by the addition of pngase f in 50 mm ammonium bicarbonate, ph 7.5, and incubation overnight at 37 c . An mcx (mixed - mode cation exchange) desalting step using an oasis elusion plate (waters, milford, ma) was performed before lc glycoproteomes of brain tissue and csf samples from pd and ad patients and age - matched controls were previously investigated . All n - glycopeptides with quantitative alterations in brain or csf were selected in the initial candidate library . To evaluate the utility of these glycoproteins as diagnostic markers for pd, we tested the 133 srm assay feasible glycopeptides (derived from 73 n - linked glycoproteins) in the initial pilot study using a few pooled plasma samples from pd and controls . Peptide selection criteria include: (1) length of 820 amino acid residues; (2) no chemically unstable residues (e.g., ng, dg, qg, n - terminal n, and n - terminal q); (3) fully tryptic; (4) avoiding cysteine residue if possible; and (5) sequence specific for the target protein (i.e., proteotypic peptides). All peptides used in this study were evaluated using blat (http://genome.ucsc.edu) and protein blast (http://blast.ncbi.nlm.nih.gov/blast.cgi) searches to ensure uniqueness of the target proteins at both proteomic and genomic levels . Finally, an srm theoretical collision calculator tool (http://proteomicsresource.washington.edu/cgi-bin/srmcalc.cgi) was applied to confirm the uniqueness of every q1/q3 pair for the target peptides . All srm analyses were performed on a tsq vantage triple quadrupole (qqq) mass spectrometer (thermo scientific) at the university of washington proteomics resource . The mass spectrometer was coupled to a nanoelectrospray ionization source and a waters nanoacquity uplc system . Glycopeptides from 1 l of original plasma (1 g peptides) were loaded onto a c18 trap column (20 mm long, 75 m i d) and separated by a 150 mm c18 column (75 m i d) over a 60 min 235% linear acetonitrile gradient . Spray voltage was set at 1700 v. scheduled srm was performed with 5 min retention time windows for most of the peptides and an instrument cycle time of 2000 500 ms . Dwell times were varied depending on the number of concurrent transitions; in all cases they were at least 10 ms . Both unpurified (thermo - fisher scientific, germany) and purified (aqua - grade, sigma - aldrich, usa) peptide standards that correspond to natural counterparts (light peptides) were synthesized with heavy isotopic lysine (c6n2) or arginine (c6n4) at the c - termini (heavy peptides). Collision energies (ces) were determined using the default formula from thermo (0.034 precursor mass m / z + 3.3140) and then optimized with four additional ce steps (5 v, 10 v). The top four abundant transition (q1/q3) pairs, including 4 light and 4 heavy determine the limits of detection (lod) and quantification (loq) for each target glycopeptide, we titrated heavy peptides at seven concentration points in a reference glycocaptured plasma matrix . A linear regression algorithm was used for fitting the seven serial dilution data points for each curve . The endogenous peptides were also monitored in these assays to help determine the amount of each peptide standard to spike - in . All raw srm data were processed using the skyline targeted proteomics environment (v1.3) (mccoss lab, university of washington) software developed for srm data sets . Settings including 0.055 th match tolerance m / z, default peak integration, and savitzky peptides with signal - to - noise ratio of at least 3 were considered detectable . Information including peak area and area ratio of light / heavy peptide pair were output from skyline to a text - delimited format worksheet . It should also be noted that the glycopeptide capturing method used in the current study has been well - optimized and widely employed, with cvs typically controlled in 1520% range . To further control this variable, in this study, we included one to two reference plasma samples in every batch of sample preparations as inter- and intrabatch controls . These samples showed an average of 16.5 and 14% for preanalytical (capture) and analytical (srm) variation (cv), respectively; that is, the variations associated with the capture and srm stages were reasonable in our investigation . The key challenge for the present analysis is a high dimension of the feature vector (large number of potentially predictive proteins) versus size of samples within the data set . Missing data were handled using k - nearest neighbor imputation algorithms (k = 10). Repeated (duplicate) measurements for the same protein all other analyses were generated in prism 6.0 (graphpad software, la jolla, ca) or spss 18.0 (ibm, chicago, il). One - way analysis of variance (anova), followed by the tukey s hsd post hoc test, was used to compare differences between groups . Receiver operating characteristic (roc) curves were used to calculate the relationship between sensitivity and specificity for pd versus the healthy control group and hence to evaluate the diagnostic performance of the analytes, either individually or in combinations . Logistic regression was used to determine the best linear combination of peptide analytes for predicting disease status (versus healthy controls), followed by roc analysis on the linear combination . Cutoff value from a roc curve is determined when the sum of sensitivity and specificity is maximal . Additionally, relationships between the analytes and the unified parkinson s disease rating scale (updrs) were analyzed with bivariate correlation using pearson s correlation coefficients . Stepwise multiple linear regression analysis was used to screen for the best predictors (linear combination of peptide analytes) that correlate the disease severity (updrs) to enrich secreted proteins in body fluids, in the past few years, we profiled n - linked glycoproteins in the brain and csf of patients with pd as compared with age - matched healthy and diseased controls (patients with ad). A subset of proteins has been previously published . Drawing from our previous work, we identified candidate peptides with quantitative alterations (defined by 50% over control cases; full list not shown) and selected a total of 133 n - glycopeptides, representing 73 n - linked glycoproteins based on peptide characteristics, especially amino acid sequence, suitable for srm assays, as defined in the methods section . With the aid of synthetic heavy - isotopic labeled peptides, srm conditions including q1/q3 transitions, collision energy (ce), retention time (rt), and the spiked - in amount of heavy peptide, were first optimized with a set of pooled samples obtained from healthy controls and pd patients . On the basis of the detectability of target peptides in the pooled samples (either control or pd), 50 formerly n - glycosylated peptides (deamidation happens during glycocapture, the n - glycopeptides monitored in srm are not glycosylated anymore) derived from 40 glycoproteins (supplemental table 1 in the supporting information, along with reported biological functions) were selected as srm targets for further analysis, with spiked synthetic corresponding heavy peptides in the initial stage of the validation to be discussed later . Brief workflow outlining the pipeline for screening plasma pd glycoprotein biomarkers by srm . In the pilot study, a primary synthetic peptide library containing 133 unpurified, deamidated n - glycopeptides was used to generate srm assays and test the detectability of these formerly n - glycosylated peptides in glycocaptured plasma . Refined srm assays with optimized settings including retention time (rt) were applied in next stages in the pipeline . In initial validation, 50 n - glycopeptides that could be detected in the pilot study were measured in a cohort including 15 pd, ad, and control samples pooled from 75 pd, 15 ad, and 30 control individuals, respectively . The final srm assay library containing 12 purified, deamidated n - glycopeptides was then used to detect the candidate biomarkers in an independent validation cohort for roc calculation and assessment of disease severity association (n = 162). Having identified peptides that are reliably detectable in plasma, we then aimed to narrow down the panel of potential biomarker candidates to those showing the largest alterations under the disease conditions . A total of 120 cases from pd, ad, and age - matched controls were included in this study, with the samples pooled based on disease status . To allow approximation of disease stage correlations, we further split plasma from pd subjects into three subgroups according to updrs - defined disease severity . To reduce the within - pool heterogeneity and facilitate statistical analysis, we combined samples in each group into three small pools (control: n = 3, early pd [updrs <15]: n = 3, intermediate pd [updrs 1530]: n = 3, late pd [updrs> 30]: n = 3, and ad: n = 3). Each pooled sample consisted of 10 individuals with age and gender evenly distributed, except the ad and early pd groups, where plasma from only five individuals was used because of limited samples in these subcohorts (table 1). Quantitative assessment of 15 pooled samples revealed clear differences among different diagnostic groups or between different stages of the disease (supplemental table 2 in the supporting information). Figure 2 shows the hierarchical clustering analysis, demonstrating a clear separation of pd with updrs> 15 (right side, pd2 _ * and pd3 _ * columns) from the three controls (left side columns). Interestingly, this phenomenon is missing in the three ad pools as well, suggesting that these elevations are unique in pd with updrs> 15 . The three updrs <15 samples (pd1_1, pd1_2, and pd1_3) did not present obvious changes compared with the controls, indicating that the current set of glycoproteins may lack the ability to distinguish pd in the early stage from controls, at least in these pooled samples . Overall, a total of 12 n - glycopeptides derived from 11 glycoproteins were detectable, with more than 50% difference between pd versus controls or between the stages of disease . Consequently, these 12 n - glycopeptides were selected to be the targets in the next stage of validation experiments . Heat map of hierarchical clustering analysis of the 50 n - glycopeptides that were detected in the initial validation cohort containing 15 pooled control, ad, and pd plasma samples . The analysis was based on srm measured peptide relative abundance against average of three pooled controls and conducted using average linkage and euclidean distance . Pd1, pd2, and pd3 represent pd at early, middle, and late stages of the disease, respectively, based on updrs . (also see table 1 .) Pooled samples, even with multiple pools as performed in our initial validation previously discussed, do not provide precise sensitivity and specificity information . Accordingly, in the final validation stage (figure 1), a total of 162 individual plasma samples from an independent cohort of subjects recruited from the same medical center were investigated . Additionally, to achieve higher - level accuracy, we used aqua level purified heavy isotopic - labeled peptides, with srm running conditions further optimized for the new peptide standards (supplemental table 3 in the supporting information). Peptide sequences, estimated plasma concentrations, and limits of detection and quantification of these 12 peptides are presented in table 2 . Of note, calibration curves were generated in a reference glycocaptured plasma matrix to fully characterize the performances of srm assays . Calibration curves for each aqua peptide are plotted on linear scales in supplemental figure 1 in the supporting information . Representative srm chromatograms of the 12 glycopeptides (both endogenous and spiked - in standard) are shown in supplemental figure 2 in the supporting information . With the exception of peptide sema4d - aadytsslnlpdk, the remaining 11 peptides were detected in more than 50% of the individuals (supplemental table 4 in the supporting information). P values and posthoc comparisons between each diagnostic group and age - matched controls were calculated for each of the 12 srm peptides using one - way anova, followed by tukey s hsd post hoc tests (table 3). Three of the 12 n - glycopeptides, derived from glycoproteins prnp (prion protein), golm1 (golgi membrane protein 1), and ncam1 (neural cell adhesion molecule 1), were significantly altered between pd versus age - matched controls (age 50; pd, n = 96; control, n = 34; p <0.05). Among them, the plasma concentration of peptide ncam1 was also significantly increased in ad patients compared with age - matched controls (p <0.05), suggesting its change may be related to neurodegeneration but not specific to pd . Furthermore, levels of 5 n - glycopeptides (chl1_iip, chl1_isg, golm1, hspg2, and tnc) were lower in younger controls (age <50), indicating that the blood level of these glycoproteins was likely associated with aging processes . Notes: n, deamidated asparagine residues . The limit of detection (lod) of each peptide was obtained from the average of lowest concentration point at which all three or four transitions were confidently detected . The limit of quantification (loq) was obtained from the average of lowest concentration point at which the intensity of the most abundant transition is on the linear scale along with the calibration curves . Settings in tukey s hsd post hoc tests: alpha = 0.05; n = average sample numbers in the two comparison groups . Proteins and peptides in bold are those with statistical alterations unique to pd (vs age - matched controls and ad). Significance level: p <0.01 . Significance level: p <0.05 . On the basis of the performance of each individual peptide on the group difference, a logistic regression - based multivariate analysis was performed to evaluate the sensitivity and specificity of the three pd unique peptides, alone or in combination with the rest of the 12 peptides for discriminating of pd from healthy controls (age 50) (table 4). As shown in supplemental figure 3 in the supporting information, the best individual performing peptide was prnp (area under curve (auc): 0.648; sensitivity: 76.8%; and specificity: 47.1%). The performance of roc became progressively better when more peptides were added to the panel; a combination of four peptides (prnp, hspg2, megf8, and ncam1) improved auc to 0.753, sensitivity to 90.4%, and specificity to 50.0% (figure 3a). Notably, however, adding more peptides only enhanced roc performance slightly; for example, a panel of 10 peptides (prnp, hspg2, megf8, ncam1, icam1, tnc, golm1, chl1_iip, chl1_isg, and lsamp) achieved the following values: auc, 0.840; sensitivity, 71.7%; and specificity, 83.3% . Also, as expected, there were more cases having missing values (i.e., peptides not detectable) when more peptides were included; specifically, the number of missing values for prnp, 4-peptide combination, and 10-peptide combination were 1, 4, and 53, respectively . Logistic regression of a panel of four n - glycopeptides and correlation with pd progression . (a) receiver operating characteristic (roc) curve and corresponding area under the curve (auc) of the classifier in distinguishing pd from controls are presented . This classifier includes four formerly n - linked glycopeptides (prnp, hspg2, megf8, and ncam1). (b) combination of two n - glycopeptides (icam 1 and megf8) correlates with pd progression . . Significant differences among pd subgroups at different disease stages (updrs <15, updrs 1530, updrs> 30) and age - matched controls were also observed (table 3). Next, the correlation of pd severity (as determined by updrs) with plasma levels of single peptide or a combination of peptides was evaluated by pearson s correlation . A stepwise multiple linear regression analysis was used to screen for the best predictors (linear combination of peptide analytes) for disease severity . Although none of the single peptides significantly correlated with updrs, when peptides were considered together, a combination of megf8 and icam1 was identified to correlate significantly with updrs (figure 3b; r = 0.293, p = 0.004). It has been exceptionally challenging to detect peripheral markers unique to cns diseases, including pd, largely because human blood is extremely complex, with proteins contributed from many organ systems, and cns - derived proteins are exceedingly low in concentration . In this study, we took a targeted approach, using previously identified cns - related proteins with changes specific to pd as the starting point . Several objectives are achieved in this study, including (1) detection of 11 cns - related glycoproteins (related to pd diagnosis or severity in previous proteomics profiling) in plasma, a much less invasive sample source (than brain, csf, or skin biopsy), (2) a combination of several peptides, that is, prnp, hspg2, megf8, and ncam1, provided good diagnostic sensitivity (90.4%) and specificity (50.0%) between pd and controls, and (3) a combination of two peptides, megf8 and icam1, significantly correlated with pd severity, as measured by updrs . All 12 of the n - glycopeptides studied in the final validation study are either relatively specific to the cns or have functions potentially important to pd pathogenesis or cns diseases in general . However, those that provided good sensitivity and specificity, alone and in combination, warrant further discussion . Prnp (prion protein or prp) is most predominantly expressed in the nervous system but occurs in many other tissues throughout the body . Brain barrier (bbb) and, when aggregated, becomes a major contributor to a variety of cognitive deficiencies and neurodegenerative diseases, especially creutzfeldt jakob disease . Prp has been implicated as a receptor and binding partner for a oligomers in ad, and its discovery as a protein altered in pd suggests interesting potential for a role in pd pathogenesis as well . Neural cell adhesion molecule 1 (ncam1) plays important roles in the inflammatory mechanisms associated with neurodegeneration and participates in the neuroprotective response in neurodegeneration . Its up - regulation in pd cases could be a compensatory mechanism during the disease process . Similarly, heparan sulfate proteoglycan 2 (hspg2) is an extracellular matrix protein that is primarily synthesized by vascular endothelial and smooth muscle cells, with several proposed functions, including maintenance of the integrity of the bbb, which has been reported to be compromised in pd . Finally, very little is known about megf8 (multiple epidermal growth factor - like domains 8) and its role in either pd or any other neurodegenerative disorder . Interestingly, a recent study has suggested that this protein could potentially be involved in phagocytic function of astrocytes . It should be stressed that while the four - peptide panel probably does not reach sufficient (80%) sensitivity / specificity to be used as a sole diagnostic criterion, it performed similarly to the best biomarkers discovered thus far . For example, in a previous study of -synuclein and dj-1 in csf of pd patients, sensitivity and specificity for distinguishing control from pd were 94 and 50% for dj-1 and 93 and 39% for -synuclein, respectively . This result is especially notable when considering that the previous results were obtained in csf, a biological fluid in direct contact with the extracellular space in the brain, while the current study used a peripheral fluid . Thus, while the current test performed similarly, it provides at least a promising lead to use a less invasive sample by sensitive detection of cns - related proteins in blood samples . That said, even a test without ideal sensitivity or specificity could be useful in clinical and research settings (e.g., selecting patients for clinical trials or screening for preclinical / premotor patients), where they may be useful for screening subjects or patients whose diagnosis would then be confirmed by more expensive or more invasive tests . Of note is also the observation that another glycoprotein, golm1 (golgi membrane protein, also known as golgi phosphoprotein 2 or golgi membrane protein gp73), was significantly changed between pd and controls but did not perform well in logistic regression screening for roc performance . However, this does not necessarily mean that it is not important to pd pathogenesis or development . Indeed, the golgi complex, including gp73, plays a key role in the sorting and modification of proteins exported from the endoplasmic reticulum, which has been demonstrated to be dysfunctional in pd or pd models . To date, biomarkers that could robustly correlate with pd severity or progression (assessed in longitudinal collected samples) are quite rare, if there are any . In fact, we are unaware of any such pd candidate markers (or ad for that matter) that have been validated by independent studies . To this end, our candidate markers, although vetted through discovery and validation phases, require yet another round of validation by independent groups, particularly for the identified models / combinations . That said, two candidate peptides, megf8 and icam1, were associated with pd severity in this study . As previously indicated, there is no clear evidence that megf8 is involved in cns disease at this point, and its potential role should be investigated, particularly if these findings are validated, as it may be a novel pd - related protein . Intercellular adhesion molecule 1 (icam1) is a cell - surface glycoprotein that is typically expressed on endothelial cells and cells of the immune system . One of the proposed cns functions of icam1 is involvement in vasoconstriction associated with subarachnoid hemorrhage, indicating its role in endothelial dysfunction . In addition to regulating endothelial functions, icam1 is also a mediator of cellular inflammation and reported to be regulated by dj-1 and -synuclein, two proteins intimately involved in pd, during neurodegeneration . The increase in circulating icam1 as a function of increasing pd severity is in line with the argument that there is persistent inflammation during pd development and progression . It remains to be determined, though, whether plasma icam1 is originated from the cns or the other way around . The two - peptide panel that correlated with disease severity may also be useful in developing an assay to track the progression of pd . However, as in all biomarker studies, the biomarkers studied here must be further validated in future studies . Moreover, because of the unbiased nature of the discovery stage of this study, some of the peptides discovered here (including the pair correlating with updrs) have not previously been associated with pd and are therefore also new putative candidates for future studies of pd pathogenesis . Interestingly, most of these peptides showed a pattern of greatest increase in the midstage pd group, followed by a decrease in the late pd group . The reasons for this pattern are not yet clear, but it should be emphasized that updrs does not reflect a linear pattern of degeneration in specific brain regions but rather a combination of the neurodegenerative and compensatory processes occurring throughout the brain . Moreover, pd processes are highly heterogeneous, and individual disease courses vary substantially . Thus, biomarker patterns may similarly be nonlinear and require interpretation depending on disease stage (e.g., screening tests at early stages vs tests for following progression at more advanced stages). It is also notable that although several peptides showed significant differences between control and pd subjects no single peptide correlated with pd severity . This result resembles those observed for -synuclein, which is reduced in the csf of pd patients but does not correlate with disease severity . For example, some biomarkers show floor / ceiling effects, in which the maximal change occurs early in the disease and therefore no correlation is observed with further progression . Furthermore, proteins involved in the disease process may undergo nonlinear changes, such as early compensatory up - regulation, followed by decreases accompanying more severe neurodegeneration . Moreover, it must be considered that the current results were obtained in plasma, while the proteins measured may have originated in the cns . Therefore, alterations in peripheral clearance or mechanisms of transfer across the bbb may change during the course of the disease as well, so plasma levels reflect a complex combination of disease processes . In the original selection of candidate peptides, we included peptides that changed 50% compared with controls . While this criterion is lenient, the low threshold could be justified in the preliminary steps, where the goal was choosing a pool of candidates for further analysis . Because further elimination of candidates would occur in following steps, to narrow a large number of detectable peptides to a feasible number of promising candidates for srm, failing to identify a candidate was more problematic than including a peptide that may not really change . An additional limitation was the pooling of subjects used for the first test set . In the preliminary validation step, the primary goal was to identify which of the candidate peptides were most altered in the disease conditions . We followed our previous strategy of combining samples into small pools by disease status . This represents a compromise, limiting costs by decreasing the samples to be processed and analyzed but also allowing statistical analyses . This strategy does have statistical costs, most notably dramatic reduction of the power to detect subtle changes (due to the reduction from the number of subjects to the number of pools) and masking the true variability of individual subjects . However, this limitation can be justified in the context of a preliminary study, in which the goal was to rank peptide candidates by the magnitude of their changes . That is, while we may not have detected subtle differences between groups in some peptides, these differences may not be the changes of the greatest relevance . Finally, an important aspect of the current study is the increase in efficiency of the use of large proteomics data sets . In biomarker research, unbiased studies can produce very large pools of candidate biomarkers, but validation of these candidates is hampered by limitations in assay development for individual proteins . Thus, this strategy of prioritizing peptides that change in the cns under the disease condition, while moving to peripheral fluids and using sensitive, relatively high - throughput assays such as srm, may allow improved use of these data in the future . In conclusion, using a relatively large cohort totaling 282 subjects, several cns - related protein markers, implicating potential novel mechanisms in pd pathogenesis were readily detected in human plasma . Several of them provided good diagnostic sensitivity / specificity for pd or correlated with pd severity . These results, if validated in independent investigations, could potentially help with establishing cns specific markers in blood for early disease diagnosis, monitoring disease progression or assessment of treatment effects.
Left - sided inferior vena cava (ivc) is an unusual abnormality that may be clinically significant during renal surgery . We report the unique case of a patient with a centrally located left renal mass who underwent laparoscopic radical nephrectomy . During the hilar dissection the patient was noted to have a left - sided inferior vena cava with multiple renal veins and anomalous tributaries . The embryology of a left - sided inferior vena cava is reviewed, and the safety and feasibility of a laparoscopic approach is discussed . Unexpected vascular anomalies may increase the risk of hemorrhage and necessitate conversion to open nephrectomy . We report the case of a major inferior vena cava (ivc) anomaly encountered during laparoscopic nephrectomy and discuss its embryogenesis and management . Computed tomography revealed a 4-cm enhancing lesion located centrally within the left kidney (figure 1). Laparoscopic left radical nephrectomy was planned and upon hilar dissection, the ivc was encountered without visualization of the aorta, confirming its left - sided orientation . In addition, 3 renal veins were noted, as was a left gonadal vein that drained directly into the ivc (figure 2). An aberrant vein was exposed that bridged the gonadal vein and the most caudal of the supernumerary renal veins . Prior to dissection of the renal artery, the caudal renal vein, gonadal vein, and bridging anomalous vein were ligated and transected . Abdominal ct scan illustrates the ivc as it courses from the normal anatomic position to the left - sided position: (a) ivc in right - sided anatomical position (thick arrow); (b) ivc crossing anterior to the aorta (thick arrow); (c) ivc crossing anterior to the aorta (thick arrow) and main left renal vein (thin arrow). Left renal mass is noted in a central location; (d) ivc in left - sided position (thick arrow) and second left renal vein (thin arrow). View of cephalad (left) and caudad (right) aspect of ivc; kidney (out of frame) is located towards top of image . Left gonadal vein drains into the left - sided ivc . Two of the 3 left renal veins are noted . Bridging vein between the main renal vein and gonadal vein is noted . The laparoscopic approach has become an accepted standard for extirpative renal surgery, with less postoperative pain, decreased hospital stay, and shorter convalescence compared with that of open surgery . Colombo et al reported that hemorrhage continues to be the most common intraoperative complication of laparoscopic surgery, occurring at a rate of 2.3% and requiring open conversion in 0.9% of cases . Preoperative knowledge of pertinent vascular anatomy is essential during laparoscopic renal surgery to avoid potential complications . To our knowledge, this is among the initial reported cases of a left - sided inferior vena cava encountered during laparoscopic radical nephrectomy . Left - sided ivc is an abnormality that occurs at a prevalence of 0.2% to 0.5% and is usually asymptomatic . Although commonly an incidental finding, it becomes clinically significant when it is mistaken for paraaortic lymphadenopathy or the main renal vein . The embryonic development of ivc involves the emergence and selective regression of 3 pairs of embryonic veins: the posterior cardinal, subcardinal, and supracardinal veins . Anomalies of the postrenal segment of the ivc include retrocaval ureter, duplicated ivc, and left - sided ivc . A left - sided ivc is caused by persistence of the left supracardinal vein with regression of the right supracardinal vein . The left subrenal ivc then joins the left renal vein and crosses anterior to the aorta where it unites with the right renal vein . As a result, renal venous development results from anastomosis of the subcardinal and supracardinal veins . During normal development, multiple persisting renal veins is the most common anomaly of the venous developmental system with a prevalence ranging from 9% to 20% . The main clinical significance of this finding is the risk of iatrogenic injury during renal surgery . When a left - sided ivc is encountered, the surgeon must be suspicious of other anomalies . In our patient, 3 renal veins, careful examination of preoperative radiographic imaging is mandatory in the diagnosis of a left - sided ivc . Case reports have described this phenomenon during laparoscopic live donor nephrectomy without adverse effects . In our experience, this anomaly may be more readily apparent when evaluating patients with higher resolution imaging . In accordance with other reports, our case demonstrates that laparoscopic radical nephrectomy can be successfully performed without complications in the presence of a left - sided ivc.
Langerhans' cell histiocytosis (lch) is a proliferative disease characterized by monoclonal proliferation and the infiltration of organs with langerhans' cells . Several organ systems may be involved in lch including the lungs, bone, skin, pituitary gland, liver, lymph nodes, and thyroid . Localized forms of lch in bone have been referred to as eosinophilic granuloma since lichtenstein first described them in 1940 . The term " pulmonary langerhans' cell histiocytosis (plch) " was first coined by farinacci in 1951 and refers to disease in adults that affects the lungs, either in isolation or in addition to other organ systems . Multi - systemic variants of this disease are known by a variety of names, including systemic histiocytosis x, letterer - siwe disease, and hand - schuller - christian disease . To avoid confusion, the histiocyte society has established a simplified classification system . According to this system, plch is a disease in adults that affects the lungs, either in isolation or in addition to other organ systems . The most common findings on high - resolution computed tomography (hrct) of the chest are multiple nodular and cystic changes, which occur predominantly in the middle and upper lobes . Nodular lesions are predominant in the early stage of plch and progress to cystic lesions in later stages of the disease . In korea, multiple cystic lesions are the main radiological findings of plch, and no cases with multiple nodular lesions without cysts have been reported . Here, we report a case of plch with multiple nodules without cysts throughout the lungs . A 31-year - old male was admitted to our hospital for a cough and exertional dyspnea, which had been present for 2 months . He had no hypertension, tuberculosis, or diabetes, and no history of surgery, medication, or travel . His vital signs were blood pressure 120/80 mmhg, pulse 84/minites, respiration rate 20/minites, and body temperature 37.2. physical examination of the head and neck revealed no palpable cervical lymph nodes or masses and no neck vein engorgement . Auscultation of the chest revealed a regular heart beat with no murmurs and clear breath sounds with no crackles or wheezing . Laboratory examination indicated a white blood count of 10,310/mm, hemoglobin concentration of 16.0 g / dl, and platelet count of 202,000/l, and the chemistry was unremarkable . Arterial blood gas analysis revealed a ph of 7.455, pao2 99.1 mmhg, paco2 38.2 mmhg, hco3 27 mmol / l, and 98% o2 saturation . The results of the pulmonary function test were forced vital capacity (fvc) 5.46 l / minites (105% of predicted), forced expiratory volume in 1 second (fev1) 4.59 l / minites (110% of predicted), and fev1/fvc 84% . We performed fiber optic bronchoscopy for bronchoalveolar lavage and observed no abnormal findings in the gram stain or culture, acid - fast bacillus stain or culture, tuberculosis - pcr, or cytology . The chest ct revealed variable - sized nodules with peribronchiolar or centrilobular distribution, some of which revealed thick - walled cavitary change (fig . The differential diagnosis based on the chest ct findings was that the pulmonary nodules represented hematogenous metastasis . Histologically, multiple nodules showed an infiltration of eosinophils, lymphocytes, and langerhans' cells (fig . Treatment consisted only of smoking cessation, and a chest radiograph 4 months later indicated that the nodules had decreased in size . After 15 months, we observed that the symptoms had improved further, and a follow - up chest radiograph and hrct indicated that the multiple nodules had disappeared (fig . This case involved a young male who currently smoked and had multiple pulmonary nodules, which were suspicious of metastasis . Plch is a rare disorder found in only 5% of biopsy - confirmed interstitial lung disease . Reviewed 102 cases with histopathologically confirmed plch drawn from a group with a mean age of 40.3 years and a 1: 1.5 ratio of males to females and found that 95% of the cases involved smokers . The only consistent epidemiologic association is cigarette smoking, which is involved in the overwhelming majority (> 90%) of cases . Several hypotheses have been proposed to explain the association between cigarette smoking and plch . According to one, cigarette smoke induces the secretion of bombesin - like peptides from neuroendocrine cells in the lungs . These peptides may have an important role in mediating lung injury and consequently induce lung fibrosis . Other components of cigarette smoke, such as tobacco glycoprotein, have also been implicated in the pathogenesis of plch . Nevertheless, plch occurs in a very small percentage of smokers, so genetic or environmental factors likely contribute to the development of this disease . A young male who currently smoked complained of a cough and exertional dyspnea, common clinical features of plch; however, the radiological findings did not support a diagnosis of plch . Hrct of the chest is a useful, sensitive tool in the diagnosis of plch . The combination of diffuse, irregularly shaped cystic spaces with small peribronchiolar nodular opacities, predominantly in the middle and upper lobes, is highly suggestive of plch [6 - 8,13]. When these features are present on hrct, they allow the clinician to make a diagnosis of plch without a lung biopsy . The lung cysts of plch are often less than 20 mm in diameter and typically have thin walls (<1 mm) [6 - 8]. The frequency of cystic change reflects the timing of imaging during the course of the disease [1,6 - 8]. In the early stages, the most common finding is nodular change, whereas in the later stages, cystic change and fibrosis predominate [6 - 8]. Based on the radiological staging, this case represents an early stage of plch comprised of nodular lesions only without cystic change . This finding is rare; only one other case of plch with multiple cavitating pulmonary nodules has been reported, although several cases of plch presenting as a solitary pulmonary nodule have been reported worldwide . None of these cases progressed to a more severe stage, such as cystic change or reduced pulmonary function . Brauner et al . Reported two cases of plch in which only nodules were present: one progressed to cystic change and the nodular lesion disappeared in the other . It is thought that predominantly nodular lesions in plch appear early in the reversible state of the disease . In korea, a number of cases of plch have been reported based on variable radiological findings, including multiple cysts and a combination of nodules and cysts . However, no case involving only nodular lesions has been reported . Atypical radiological findings as in this case need to be followed to determine whether sarcoidosis, pneumoconiosis, military tuberculosis, hypersensitivity pneumonitis, or metastatic lung nodules are present . The confirmative diagnostic tool is a pathological examination of a lung biopsy . In this case, an open lung biopsy was performed, which resulted in the diagnosis of plch based on positive staining for s-100 protein . The only treatment was smoking cessation, and clinical and radiological improvement was observed 15 months later . Here, we report a case of early stage plch in a young male that had atypical radiological features, which consisted of variable - sized nodules only without cystic changes.
Traumatic brain injury (tbi) is a nondegenerative, noncongenital insult to the brain from an external mechanical force causing temporary or permanent neurological dysfunction . It is one of the major causes of morbidity and mortality in both the developed as well as the developing countries . The prevalence of hypopituitarism after tbi is at least 25% among long - term survivors with significant impact on morbidities and mortalities . In india the main aim of the study was to determine the prevalence of pituitary hormone deficiencies in the acute phase of tbi . The secondary objectives were to correlate the severity of trauma with basal hormone levels and to determine whether initial hormone deficiencies predict mortality . In this cross - sectional, convenient sample study, 49 consecutive patients with tbi (41 men, 8 women; age range 1678 years) who were admitted to the trauma care center of tertiary care center in mumbai were included . The study was approved by our institutional ethical committee and conducted between 2013 and 2014 . The level of consciousness of the patients was evaluated according to the glasgow coma scale (gcs) as soon as the patient was admitted to the trauma care center . A score of 1315 was considered as mild, 912 moderate, and 8 as severe tbi . Patients who had suffered a prolonged hypotensive episode (systolic pressure <90 mmhg for more than 30 min), had a history of any known pituitary disorder or on any drug affecting hypothalamo - pituitary function, and pregnant women were excluded from the study . All patients underwent basal hormonal evaluation within the first 24 h of the admission before receiving glucocorticoids and dopamine . The samples of thyroid - stimulating hormone (tsh), free triiodothyronine (ft3), free thyroxine (ft4), prolactin (prl), cortisol, adrenocorticotropic hormone (acth), follicle stimulating hormone (fsh), luteinizing hormone (lh), insulin - like growth factor-1 (igf - i), growth hormone (gh), and total testosterone were collected and estimated on chemiluminescence immunoassay (advia centaur cp). The hormonal deficiency was defined as described in table 1 and the analytic methods to measure hormones with inter- and intra - assay coefficient of variance has been described in table 2 . Criterion to define deficiency of various hormones analytic methods to measure hormones in this study statistical analysis was performed using the statistical package for the social sciences 17.0 (chicago: spss inc) program . In addition, we used pearson's correlation analysis to determine whether significant correlations existed between chosen variables . A multifactorial logistic regression model was used to determine which variables independently predicted the development of mortality; p <0.05 was considered statistically significant . The main aim of the study was to determine the prevalence of pituitary hormone deficiencies in the acute phase of tbi . The secondary objectives were to correlate the severity of trauma with basal hormone levels and to determine whether initial hormone deficiencies predict mortality . In this cross - sectional, convenient sample study, 49 consecutive patients with tbi (41 men, 8 women; age range 1678 years) who were admitted to the trauma care center of tertiary care center in mumbai were included . The study was approved by our institutional ethical committee and conducted between 2013 and 2014 . The level of consciousness of the patients was evaluated according to the glasgow coma scale (gcs) as soon as the patient was admitted to the trauma care center . A score of 1315 was considered as mild, 912 moderate, and 8 as severe tbi . Patients who had suffered a prolonged hypotensive episode (systolic pressure <90 mmhg for more than 30 min), had a history of any known pituitary disorder or on any drug affecting hypothalamo - pituitary function, and pregnant women were excluded from the study . All patients underwent basal hormonal evaluation within the first 24 h of the admission before receiving glucocorticoids and dopamine . The samples of thyroid - stimulating hormone (tsh), free triiodothyronine (ft3), free thyroxine (ft4), prolactin (prl), cortisol, adrenocorticotropic hormone (acth), follicle stimulating hormone (fsh), luteinizing hormone (lh), insulin - like growth factor-1 (igf - i), growth hormone (gh), and total testosterone were collected and estimated on chemiluminescence immunoassay (advia centaur cp). The hormonal deficiency was defined as described in table 1 and the analytic methods to measure hormones with inter- and intra - assay coefficient of variance has been described in table 2 . Criterion to define deficiency of various hormones analytic methods to measure hormones in this study statistical analysis was performed using the statistical package for the social sciences 17.0 (chicago: spss inc) program . In addition, we used pearson's correlation analysis to determine whether significant correlations existed between chosen variables . A multifactorial logistic regression model was used to determine which variables independently predicted the development of mortality; p <0.05 was considered statistically significant . The most common mode of injury was a road traffic accident (55%) followed by a fall (34%), railway accident (6.1%), and assault (4.08%). Twenty - nine (59.18%) patients had mild, 4 (8.16%) had moderate, and 16 (32.65%) had severe tbi . Comparison between mild, moderate, and severe tbi groups in acute phase is described in table 3 . Ten of 49 patients died during the acute phase, 7 of them had severe tbi . Comparison between mild, moderate, and severe traumatic brain injury groups in acute phase based on the criterion defined in table 1, 63.5% patient had gonadotropin deficiency, 46.9% had low igf-1 level, 12.24% had cortisol level <7 mcg / dl, 6.1% had low ft4 level, and prl level was high in 4% of patients . Cortisol levels were significantly higher in patients with low gcs (r = 0.42; p = 0.0027) and cortisol levels were positively correlated with acth levels (r = 0.43, p = 0.002) suggestive of hypothalamo pituitary adrenal axis activation . Igf-1 did not correlated with severity of injury while gh is higher in patients with severe compared to mild tbi (p = 0 . The ft4 and ft3 positively correlated with gcs (r = 0.31; p = 0.041, r = 0.49; p = 0.0003, respectively). In our male cohort, there was a positive correlation between total testosterone and gcs in male, but the correlation was not significant (r = 0 . Significant correlations glasgow coma scale and serum cortisol level (a), serum adrenocorticotropic hormone and serum cortisol levels (b), between glasgow coma scale and serum prolactin level (c) mean age, gcs, and basal hormone levels of the patients who died after tbi no survivors (ns), 10 patients, were compared with the living patients survivors (s), 39 patients [table 4]. The ft3 level (ns: 2.05 1.6, s: 1.54 30.47; p = 0.0027) were significantly lower in nsg . There was no significant difference in mean ft4, tsh, prl cortisol, acth, fsh, lh, igf - i, gh, and total testosterone between the ns and s. logistic regression analysis revealed that mortality after tbi was unrelated to the basal pituitary hormone levels except low t3 level, which was found related to mortality . Comparison between survivors and nonsurvivors of traumatic brain injury tbi in acute phase can lead to pituitary dysfunction, results in a temporary increase or decrease in pituitary hormonal levels . In our study, gonadal axis was most common which was concordant with other studies . According to the previous studies, acute phase of tbi associated with low or high gh with low igf-1 . We found low igf-1 in 46.9% of patients, but it was not correlating with severity which is also demonstrated by tanriverdi et al . Gh was high in patients with severe tbi compared to mild tbi (p = 0.02). Gonadotroph and somatotroph cells are located in the vascular territory of the long hypophyseal system which can be easily affected by tbi, which explains most common involvement of these axes . As described in various studies cortisol level was found to increase immediately following tbi, and it was concordant with acth level suggestive of activation of hypothalamic pituitary adrenal axis . Low cortisol (<7 mcg / dl) was found in 12.24% of patients, suggestive of posttraumatic damage at the hypothalamic - pituitary level . Prl level correlated positively with the severity of the tbi, which may represent stress response to tbi . We found ft3, ft4 were significantly lower in patient with increasing severity of tbi . Increased level of prl and low level of ft3, ft4, and cortisol correlated significantly with the severity of tbi . During acute phase of tbi at least assessment of cortisol our future plan is to follow this cohort of patients for the assessment of long - term hormonal deficiency which could be factor for improvement in quality of life.
Systemic thrombolytic therapy with recombinant tissue plasminogen activator (rt - pa), intra - arterial thrombolytic therapy, and mechanical revascularization are therapeutic options in acute ischemic stroke (ais) in adults [1, 2, 3]. Published experience in ais in childhood a previously healthy 14-year - old boy was admitted with acute onset of severe left hemiparesis with a national institute of health stroke scale (nihss) score of 12 . There was no prior history of trauma, infection or family history of migraine or other cerebrovascular disease, but the boy was complaining of frontal headache over the last 2 weeks . Intravenous rt - pa infusion (0.9 mg / kg) was started 3 h after onset of symptoms but was discontinued after 2/3 of dosage because of serious neurological deterioration . We immediately performed an mri which revealed acute ischemic lesions in both territories of the anterior cerebral arteries (aca) and the left middle cerebral artery (mca). Mr angiography (mra) showed the right aca without flow signal, the left internal carotid artery (ica) with an intracranial stenosis, and several mca branches with lack of flow (fig . Several differential diagnoses were considered: dissection, isolated cerebral vasculitis, arterial spasms, infection, multiple cardiac embolism, and intoxication . Screening for heroin, amphetamine, ecstasy, ethanol, and cocaine in blood and urine was negative . A previously healthy 10-year - old boy was admitted with acute severe left hemiparesis (nihss 16) and headache after jumping on a trampoline . There was no history of infection or family history of migraine or other cerebrovascular disease . Mri revealed an acute ischemic lesion in the right mca territory, occlusion of the right ica, and a thrombus in the right mca (fig . 2). Intravenous rt - pa infusion (0.9 mg / kg) was started within 3 h after onset of symptoms . As no improvement was observed at the end of the rt - pa infusion, mechanical revascularization was performed . A goose neck snare was passed through the thrombus which was partially fragmented and finally dissolved . Flow was established in the right mca and the branches 5 h after the onset of stroke (fig . The potential trauma (bouncing trampoline) and thrombus formation in the ica made an intima dissection most likely, but could not be confirmed by angiography . No stent was used and platelet inhibition was started the next day . Transesophageal echocardiography and routine blood samples, including extensive coagulation tests, were all normal . The boy improved (nihss 13 the next day) and was transferred to the department of pediatric neurorehabilitation on day 3 . At 3 month, he was back to school, but was still suffering a slight hemiparesis (modified rankin scale (mrs) 2). A previously healthy 14-year - old boy was admitted with acute onset of severe left hemiparesis with a national institute of health stroke scale (nihss) score of 12 . There was no prior history of trauma, infection or family history of migraine or other cerebrovascular disease, but the boy was complaining of frontal headache over the last 2 weeks . Intravenous rt - pa infusion (0.9 mg / kg) was started 3 h after onset of symptoms but was discontinued after 2/3 of dosage because of serious neurological deterioration . We immediately performed an mri which revealed acute ischemic lesions in both territories of the anterior cerebral arteries (aca) and the left middle cerebral artery (mca). Mr angiography (mra) showed the right aca without flow signal, the left internal carotid artery (ica) with an intracranial stenosis, and several mca branches with lack of flow (fig . Several differential diagnoses were considered: dissection, isolated cerebral vasculitis, arterial spasms, infection, multiple cardiac embolism, and intoxication . Screening for heroin, amphetamine, ecstasy, ethanol, and cocaine in blood and urine was negative . A previously healthy 10-year - old boy was admitted with acute severe left hemiparesis (nihss 16) and headache after jumping on a trampoline . There was no history of infection or family history of migraine or other cerebrovascular disease . Mri revealed an acute ischemic lesion in the right mca territory, occlusion of the right ica, and a thrombus in the right mca (fig . 2). Intravenous rt - pa infusion (0.9 mg / kg) was started within 3 h after onset of symptoms . As no improvement was observed at the end of the rt - pa infusion, mechanical revascularization was performed . A goose neck snare was passed through the thrombus which was partially fragmented and finally dissolved . Flow was established in the right mca and the branches 5 h after the onset of stroke (fig . The potential trauma (bouncing trampoline) and thrombus formation in the ica made an intima dissection most likely, but could not be confirmed by angiography . Transesophageal echocardiography and routine blood samples, including extensive coagulation tests, were all normal . The boy improved (nihss 13 the next day) and was transferred to the department of pediatric neurorehabilitation on day 3 . At 3 month, he was back to school, but was still suffering a slight hemiparesis (modified rankin scale (mrs) 2). The current american heart association pediatric stroke guidelines advocate thrombolytic therapy for childhood ais only in the setting of clinical trials . In contrast, the european stroke organization guidelines suggest that intravenous rt - pa may be administered in selected patients under the age of 18 beyond the current european recommendations . Experience with systemic thrombolytic therapy and intra - arterial revascularization in ais in children is limited . The nationwide inpatient sample in the us recorded data of 2,904 children with stroke . (687 children), 2% of children received thrombolytic therapy, 9% received intravenous and 6% intra - arterial therapy . Both studies showed increased in - hospital mortality and worse outcome in children treated with thrombolytic therapy compared to conservative treatment, probably reflecting a treatment selection bias towards more severe stroke . Furthermore, the thrombolytic therapy often deviated from the current adult stroke guidelines by delayed initiation . In contrast, a review of 17 cases published in the literature, which were treated with systemic thrombolytic therapy or intra - arterial revascularization, probably reflects a publication bias towards positive results with good outcome and low mortality . Our two children were treated according to the stroke treatment guidelines for adults, and no rt - pa related intracranial hemorrhage or other side effects were seen . Lower concentration of tissue plasminogen activator and higher concentration of plasminogen activator inhibitor type 1 in children might influence the dose - response relation of rt - pa . Systemic thrombolytic therapy is approved in ais in adults, in whom atherothrombosis is the most frequent cause of ischemic stroke . In contrast, in children, cardiac disease, prothrombotic or hematologic disorders, dissection, moyamoya disease and vasculitis are common etiologies . However, our children were both previously healthy . In the first case, endovascular therapy was not available in our center at that time . The history of headache over the last 2 weeks was suspicious for vasculitis, but now signs of inflammation were found and the later organ transplantations caused no systemic vasculitis . There were no other medical conditions that might have indicated that the changes on mra were associated with the moyamoya syndrome . Another consideration was arterial spasm due to intoxication, but the screening for toxins was negative . Autopsy was not permitted by the parents, and the stroke etiology remained unclear . In the second case, the history of a potential trauma and thrombus formation in the distal ica made an intima dissection most likely, but could not be confirmed on angiography or mri after successful mechanical revascularization . The clinical outcome after 3 month was good (mrs 2) and full recovery was seen after 5 month . In conclusion, treatment of ais in childhood without being part of a randomized controlled trial needs a careful individual approach . Differences in the fibrinolytic system and the different etiology of ais in childhood may limit a simple extrapolation of the adult guidelines for systemic thrombolytic therapy . Acute multimodal imaging with mra, ct angiography, or angiography to clarify the etiology of ais might help to select the most appropriate treatment modality.
Healthcare is provided by public and private hospitals, and there are 5 major burns units . In january 2005, pustular lesions were observed on the foot of a patient in 1 of the burns units, and similar lesions subsequently appeared on other patients . Local health authorities were informed of the outbreak, and an investigatory team confirmed reports of similar infections in the city s other burns units, with retrospective identification of at least 19 probable cases occurring over a 5-month period . Most patients had a fever (39.0c40.5c) for 23 days, followed by the appearance of an eruption(s). Lesions were typically small, rounded, umbilicated, and nodular in appearance with an erythematous base (figure 1a). They contained cheesy pustular material and increased in size (from 12 mm to> 1 cm in diameter) and severity over 78 days . The lesions involved burn wounds and intact skin surrounding them, with the unhealed margins of the wounds being covered by a layer of thick yellow secretion . Some patients had a sparse rash; others had closely spaced lesions that produced a cobblestone appearance . A single lesion developed on a paramedical staff member s finger, and lesions developed around 1 patient s insertion site for an intravenous line (figure 1b). In all instances, the disease was self limiting, and patients recovered in 34 weeks . C) orthopoxvirus particles detected by electron microscopy (em) examination of negatively stained grids prepared from pustular material (magnification 73,000). D) transmission em examination of ultrathin sections of infected vero cell cultures showing classic intracytoplasmic orthopoxvirus factories and maturing virus particles (magnification 21,000). Results of bacteriologic and mycologic examination of biopsy samples, impression smears, and swab samples from lesions were negative . Histopathologic examination showed extensive ulceration and granulation, with epidermal necrosis and subepidermal edema plus acute and chronic inflammatory cell infiltration . Impression smears and biopsy tissues were sent to the special pathogens unit, national institute for communicable diseases (nicd), sandringham, south africa, and to the health protection agency (hpa), centre for emergency preparedness and response, porton down, salisbury, united kingdom . Electron microscopy of negative - stained grids prepared at hpa and ncid laboratories from pustular material showed orthopoxvirus particles, and examination of ultrathin sections prepared from infected vero cell cultures at hpa found classic orthopox intracytoplasmic virus factories and particle maturation sites (figure 1c, d). Pcr was performed on nucleic acid extracted from the samples, using primers specific for regions of the orthopoxvirus hemagglutinin gene (at nicd) and b5r membrane protein gene (at hpa). After nucleotide sequences were determined for the pcr products, phylogenetic analyses were conducted in relation to corresponding orthopoxvirus sequences obtained from genbank, using methods described elsewhere (14,15). The causative agent was found to cluster with buffalopoxvirus isolates within the vaccinia subgroup of orthopoxviruses (figure 2), and 3 patients from 2 separate burns units were shown to be infected with the identical virus, which was distinct from other known buffalopoxvirus isolates . To investigate the possibility of a shared source of infection, 17 samples of saline, antimicrobial drug ointments, petroleum jelly, cotton dressings, and swabs in common use were obtained from the 5 burns units and tested by pcr at nicd; no results were positive . Inquiries led to the suggestion that the outbreak was probably propagated by transfer of infected patients between burns units . This hypothesis was confirmed when a policy to isolate all new admissions, including referrals from other burn centers, for their first 2 weeks in a unit successfully controlled transmission of new endogenous cases . Maximum likelihood phylogenetic tree based on a 955-nt alignment of the karachi isolate and 33 orthopoxvirus sequences of the b5r gene from genbank constructed with clustalw (www.ebi.ac.uk/clustalw/index.html) and tree - puzzle (http://bioweb.pasteur.fr/seqanal/interfaces/puzzle.html); figures at nodes represent puzzle support values . The orthopoxvirus types are indicated to the right . Control measures included education of staff, single - room or cohort isolation of patients infected or suspected of being infected with buffalopoxvirus, and reinforcement of infection - control practices, such as hand disinfection after contact with any patient . To reduce virus load in the environment, the measures proved effective in reducing transmission within burns units, but they did not prevent the sporadic arrival of newly infected patients . Buffalopoxvirus outbreaks reported to date have been geographically restricted, and human cases have been limited to persons with direct exposure to infected animals, usually in rural communities (111). This reported outbreak uniquely involved nosocomial infections in 5 widely separated burns units in karachi, pakistan . However, buffaloes are the most common dairy animal in pakistan, even within the city limits of karachi, and buffalo fat, particularly in the form of butter or ghee, sometimes is used at home as a dressing for burns . Thus, burn patients newly infected with buffalopoxvirus due to disparity in the sophistication and cost of the care provided at the burns units in karachi, patients are often transferred or move themselves between units, thus facilitating the possible spread of infection . In this outbreak, 6 of the 19 patients with putative cases of buffalopoxvirus infection are known to have transferred between burns units during treatment . The infection was of low virulence for humans . Delay in recognizing and investigating the outbreak is cause for concern and can be ascribed to poor awareness and lack of resources.
In high - income countries, growing numbers of men as well as women are choosing to postpone parenthood . On average, if sustained, this demographic shift may have manifold implications for the health of successive generations . We discuss here the evidence for a beneficial effect of increasing paternal age at conception (pac) on the health of descendants . Basic genetics offers a strong foundation for hypothesizing that increased pac creates an increased risk for a host of disorders in offspring . In utero, the female germ cells undergo an estimated 22 cell divisions before meiosis and two divisions during meiosis . However, as only one chromosome replication takes place during meiosis, the female germ cells undergo a total of 23 chromosome replications . Postnatally, the meiotic process is arrested at the first meiosis and this persists until puberty . Thus, between the mother's birth and the conception of her offspring, her germ cells undergo no chromosome replication and only one cell division (regardless of her age at conception). For instance, the estimated cumulative numbers of germ - line stem cell (gsc) replications in men by the ages of 20 and 40 years are 150 and 610, respectively . It has long been recognised that rare conditions such as achondroplasia (the prevalence of which is one per twenty thousand) and marfan syndrome (which has a prevalence of one per five thousand) may arise from mutations in the male gscs . However, in the past decade, a large number of studies have linked increased pac to severe conditions that are not so rare in offspring, including autism, schizophrenia, and other neurodevelopmental disorders . It is often assumed, but not proven, that this is also due to mutations in the male germ line . Perhaps because of the gravity of these diseases, studies now suggest, however, that older pac may also confer benefits on the health of offspring . If increased pac may have both adverse and beneficial effects, understanding the balance of its risks and benefits will require the consideration of a broad scope of relationships between increased pac and offspring health . Yet we have only just begun to explore the potential benefits of older pac, and the evidence for them is still not widely understood . We therefore integrated work done across disciplines to articulate the case for a potentially major benefit of older pac on the basis of an intriguing finding in recent studies that leukocyte telomere length (ltl) is on average longer in the offspring of older fathers . This association of pac with offspring ltl has no threshold, as it has been observed for increasing pac from the age of 20 up to 60 years . As a longer ltl predicts reduced atherosclerotic risk and longer survival in the elderly, it is possible that older fathers, by endowing their offspring with a longer ltl, may also confer on them resistance to atherosclerosis and an advantage for increased longevity . That older pac is related to longer offspring ltl, it might even seem counter - intuitive, given the widespread awareness of genetic abnormalities related to both increased maternal age at conception (e.g., chromosomal aneuploidy) and pac (e.g., de novo mutations). The pac effect on offspring ltl is also perplexing on a deeper level, however, because of what it implies about age - related changes in the male germ line and how they are transmitted to offspring . Yet the outcome of these age - related changes, namely, longer telomere length (tl), is probably transmitted in mendelian fashion . We discuss below how this enigma could be resolved . For understanding of the following discussion, it is important to recognize four major aspects of tl in general and ltl in particular . First, telomeres are the ttaggg tandem repeats at both ends of each of the mammalian chromosomes, and together with telomere - binding proteins they cap the chromosomes . This capping stabilizes the telomere and prevents the chromosomal ends from being recognized by the dna repair processes in cells as dna break points and potential sites of chromosomal fusions . Second, as somatic cells replicate, their telomeres undergo progressive attrition because dna polymerase cannot completely replicate the 3' end of linear duplex dna . Once telomeres become very short, they often cause cells to exit from the replicative cycle and become senescent . Third, ltl is a complex human genetic trait in that it is determined by many genes, and its dynamics (birth ltl and age - dependent telomere attrition thereafter) reflect telomere dynamics in hematopoietic stem cells . Fourth, because the hematopoietic system is probably the most proliferative system among somatic tissues, ltl, and by inference tl in hematopoietic stem cells, can become critically short during the long human life course, thereby imposing a limit on the longevity of some individuals . The mechanisms underlying the association of pac with the ltl of offspring are not yet understood . Genome - wide association studies (gwas) of ltl have deciphered a number of genes and genetic loci associated with ltl in the general population . However, it is very unlikely that the pac effect on offspring ltl is mediated through increased mutation load with age in the paternal germ line . Such mutations are too rare to explain the pac effect on the offspring ltl, for the reason that the effect of older pac is manifested as a shift to a longer average ltl of the offspring in the population . Because there is no corresponding increase in variance of the offspring ltl, this population shift does not merely reflect an increase in the small subset of persons with extremely long ltl . An important clue to the causal mechanism of pac on ltl is that while telomeres undergo age - dependent shortening in replicating somatic cells, tl is longer in sperm samples donated by older men than in those donated by young ones . Thus, postulated mechanisms for the pac effect on ltl must ultimately explain the reason for why, on average, sperm cells of older men have longer tls than sperm cells of younger men . Each sperm is a distinct genetic package, ensuring genetic diversity among a father s offspring . Age might exert selection pressure at the level of the male gscs such that surviving gscs are those with relatively long telomeres . Indeed, there is some evidence for the overrepresentation of sperm with longer telomeres in older men . Another explanation for the effect of pac on ltl, not mutually exclusive with gsc selection, relates to the age - dependent elongation of telomeres in male gscs . This could be due to the difference in telomerase activity between somatic cells and male germ - line cells . Telomerase activity is repressed after birth in most human somatic cells, including hematopoietic stem cells . By contrast, telomerase displays robust activity in embryonic stem cells and in the testes of humans and other mammals, presumably because the enzyme is active in male gscs . The activity of telomerase is usually fine - tuned to maintain the length of telomeres constant in telomerase - positive cells . It seems, however, that a small increase in tl (only a few base pairs) occurs with each replication of gsc in males, suggesting that in these cells telomerase overshoots its mark . Because of the high number of replications of male gscs, a small elongation would result in considerable lengthening of sperm - cell tl over many replications . Although sperm have a long tl, the tl of mammalian oocytes is relatively short and is evidently however, regardless of mechanisms that affect tl in the embryo and fetus, human tl is evidently inherited in an allele - specific manner . Accordingly, ordinary mendelian principles would seem to suggest that since a child receives roughly half of its dna from its father, the slope of its ltl vs. pac should be approximately one half of the slope of tl in sperm vs. the ages of the sperm donors . The magnitude of the pac effect on offspring ltl is large, at 1520 base pairs of a longer ltl in the offspring for each year of pac . This is close to the average rate of age - dependent ltl attrition in adulthood, of 2030 base pairs per year . Moreover, the pac effect on offspring ltl appears to be cumulative across successive generations . Given the considerable magnitude of the pac effect on offspring ltl and its additive nature across successive generations, current demographic trends of an upward shift in paternal age might affect tl in future humans . From this standpoint, the pac effect on ltl is also directly relevant to the biological limit of human longevity . Short human telomeres could, in theory, impose a limit on human longevity, but the pac effect suggests that human tl is malleable . It is therefore essential to factor in the pac effect on tl dynamics in the offspring when considering the question of whether life expectancy is approaching its ultimate ceiling in modern humans . The large magnitude of the pac effect on offspring ltl also suggests that it could be worthwhile to use this knowledge to explore possibilities for improving population health . Before doing that, however, it is necessary to answer some basic questions about pac and ltl in life - course and cross - generational epidemiological studies . The main determinants of ltl at any age are ltl at birth and the magnitude of its attrition during growth . Yet the hypothesis of mendelian transmission of a longer tl from the paternal germ line to the offspring predicts that offspring of older fathers would be conceived with a longer tl . By the time of conception, the tl of the paternal germ line would already have been lengthened by gsc selection or age - related elongation or both, and this longer tl would be inherited by the offspring . But this prediction of mendelian inheritance of epigenetic changes has not yet been tested, leaving open the question of whether there might be some more complex process by which pac exerts a latent effect on ltl that becomes evident in utero or during the first two decades of life . We have noted above that increased pac is associated with rare mutations and related diseases . If increased pac also has beneficial effects on offspring health via increased tl, then we need to pose the following question: might the pac effect be understood from the perspective of natural forces that shape human biology with respect not only to disease but also to evolutionary fitness? Evolutionary theories suggest that delayed reproduction leads to increased longevity because of larger investment in maintenance and repair . This feature has been displayed in model organisms . For instance, selection based on delayed reproduction causes a pronounced increase in the lifespan of the fruit fly drosophila melanogaster . Because increased pac reflects delayed reproduction in humans, and longer ltl might predict increased lifespan, the pac effect may be a manifestation in humans of the same phenomenon as seen in the fruit fly . Moreover, in aging male fruit flies, the number of gscs is much smaller than that in younger male flies . Such a phenomenon may be the result of a stochastic depletion of gscs or may reflect the ability of surviving gscs to withstand the accruing burden of aging - related stress, primarily in the form of oxidative stress . The variant genetic and more likely epigenetic constitution that distinguishes the surviving gscs from the non - surviving ones might provide a potential mechanism for transmitting increased fitness and longevity from fathers to offspring . Studies across a variety of species suggest that tl and the expression of telomerase activity in somatic tissues have been fashioned by evolutionary forces . Consider, for instance, the body sizes of terrestrial mammals (among which humans are viewed as moderately sized mammals) and their life spans . Increased mammalian body size is associated with repression of telomerase, whereas increase in life span is associated with shorter telomeres . Humans, the longest - living terrestrial mammals, have short telomeres and repressed telomerase activity in their somatic cells during extra - uterine life . In principle, tl might hence curtail lifespan in humans to a greater extent than in other mammals . A postulated evolutionary explanation for repressed telomerase activity with increased body size and diminished tl with increased life span is that these features protect against cancer through the reproductive phase of the life span . Because somatic cells from relatively large and long - living mammals tend to undergo many more replications for growth and maintenance than do those of small, short - living mammals, they should be subject to a greater risk of cancer . Thus far, however, there is no empirical evidence for associations of cancer risk with body size or longevity among mammals . In this context, little is known about the effect among humans of inter - individual variation in tl with respect to the cost of having relatively short telomeres (perhaps less cancer risk early in life and more cardiovascular disease risk later in life) or long telomeres (perhaps more cancer risk early in life and less cardiovascular disease risk later in life). The pac effect on the health of offspring and, more broadly, on public health, requires fundamental re - thinking and new directions in research . Insight into this phenomenon and its links to human telomere biology will significantly advance the understanding of aging - related diseases in modern humans . Overall, the evidence supports the view that in some contexts the male germ line might drive the evolution of human tl . If so, this would represent a substantial addition to haldane s conceptualization of the evolutionary force of male - biased mutations and the subsequent recognition that the evolution of dna sequences is largely driven through the numerous replications of the male germ line . Because tl at birth and its rapid attrition during early life strongly influence tl throughout the human life course, an understanding of the pac effect on offspring tl requires going straight to the source, namely, tl at birth and its attrition during childhood . Ongoing studies, exemplified by the avon longitudinal study of parents and children (alspac) in the uk and the norwegian mother and child cohort study (moba), might be used to achieve this goal in a cost - effective manner . Lastly, it is clear that the enigma of the father s age and the health of his offspring are bound to engage telomere researchers, evolutionary biologists, epidemiologists, and demographers for quite some time.
Frequently, the introduced air is limited to the subcutaneous space and leads to local swelling and crepitus in the absence of any signs of inflammation such as erythema, edema, tenderness or lymphadenopathy [1, 2]. However, there is a potential risk that the air disperses along the facial planes to the periorbital and mediastinal spaces . In the literature, the most common causes of subcutaneous emphysema are the use of air turbine handpieces [2, 3] and air syringes [2, 3]. In addition, subcutaneous emphysema can be caused by coughing, blowing, smoking, vomiting and using straws after a dental treatment . Gilles de la tourette syndrome (gts) is a disorder that is characterized by multiple motor tics, especially affecting cranial and upper limb muscles and vocal tics . Although several gts patients can temporarily suppress their tics to some extent, tic emergence is rather random and intrusive . Therefore, it has been suggested that tics in gts patients are associated with an insufficient inhibitor motor control [6, 7]. To the best of our knowledge, the development of a subcutaneous emphysema in the context of the presence of gts has not been described yet . In this case report, a patient with gts who developed a subcutaneous emphysema following surgical wisdom tooth extraction is presented . A 30-year - old man was referred to the department of oral and maxillofacial surgery because of a soft tissue swelling in the facial area . This diffuse facial swelling had developed after bilateral surgical wisdom tooth extraction in the mandible in local anesthesia on the same day in a private dental office . Gts is characterized by repeated, quick movements or sounds, which cannot be controlled by the patients . This patient permanently repeated valsalva maneuvers and pressed air against his closed lips . At admission, the patient was hemodynamically stable, afebrile and the function of the facial and trigeminal nerves were normal . In addition, no general malaise was existing, and the c - reactive protein (crp) level was within normal ranges . Clinically, an extensive diffuse swelling in the whole facial area was visible, and the patient had difficulty in opening his eyes (figs 1 and 2). Mouth opening was slightly limited (cutting edge inter - incisor distance: 36 mm). On palpation, there was a manifest crepitus of the periorbital region, the cheek and the supraclavicular fossa . The floor of the mouth and the pharyngeal region were unaffected, and the patient was able to swallow but showed commencing respiratory distress . Figure 1:clinical appearance of the patient with distinct swelling in the periorbital, cheek and supraclavicular region after bilateral wisdom tooth removal . Clinical appearance of the patient with distinct swelling in the periorbital, cheek and supraclavicular region after bilateral wisdom tooth removal . The patient had difficulty to open his eyes . Computed tomography (ct) of the head and neck region revealed a bilaterally located subcutaneous air collection in the area of the periorbital, temporal, paramandibular and supraclavicular region (figs 35). There were no signs or symptoms of severe complications such as pneumothorax or pneumomediastinum . Figure 3:ct scan (coronal section in the first premolar region) showing multiple air inclusions within the facial soft tissue . Figure 4:the frontal ct scan of the head and neck region (section in the retromolar region) illustrates bilaterally trapped air into the soft tissue reaching from the temporal fascia through to the supraclavicular region . Figure 5:three - dimensional reconstruction of the ct scan showing the trapped air in the head and neck region . Ct scan (coronal section in the first premolar region) showing multiple air inclusions within the facial soft tissue . The frontal ct scan of the head and neck region (section in the retromolar region) illustrates bilaterally trapped air into the soft tissue reaching from the temporal fascia through to the supraclavicular region . Three - dimensional reconstruction of the ct scan showing the trapped air in the head and neck region . Because of the extensive emphysema, the patient was admitted for an inpatient monitoring . In order to avoid an infection, 2x / d for 2 days, pfizer ag, zrich, switzerland) were prescribed . In order to prevent the tic - induced powerful injection of air into the soft tissue, caused by the repetitive valsalva maneuvers, a 10-cm - long small tube (redon drainage tube cut in shape) was given to the patient, and the correct handling was instructed . By applying this tube, the building - up air pressure within the patient's oral cavity figure 6:the patient after 3 days with tube in place, which served as a valve to avoid air pressure within the oral cavity . The patient after 3 days with tube in place, which served as a valve to avoid air pressure within the oral cavity . After 3 days, the facial swelling was reduced approximately by half, and the subcutaneous crepitus was almost undetectable, and so the patient could leave the hospital . Oral antibiotic prophylaxis was continued for 7 days (amoxicillin / clavulanic acid, 875/125 mg tid; augmentin). At that time this case report was submitted and approved by the ethics committee of northwest and central switzerland (eknz). Subcutaneous emphysema following dental procedures, albeit rare, might cause severe complications including pneumomediastinum and mediastinitis . Spreading air in subcutaneous tissue increases the risk of subsequent connective tissue infections . Due to the communication of the facial spaces with the mediastinum, there is an increased risk for spreading of bacteria and the development of life - threatening infections of the retropharyngeal space and the mediastinum [2, 8, 9]. In principle, two factors are prerequisite for the formation of a subcutaneous emphysema: (i) air forced under pressure and (ii) a communication between the oral cavity and the subcutaneous tissue . In this case, the combination of bilateral surgical wisdom tooth extraction in the mandible and the presence of the motor tic to press air against the resistance of the closed lips caused by gts led to the distinct subcutaneous emphysema . The formation mechanism in this specific case is comparable with the first published case of a subcutaneous emphysema more than 100 years ago: a horn blower, who played his instrument immediately after a tooth extraction . As motor tics of gts patients can hardly be controlled, the usage of a redon drainage was instructed in order to prevent the patient from elevating pressure within the oral cavity . The diagnostic work - up of a patient with suspected subcutaneous emphysema includes a clinical examination, laboratory analysis (crp levels) and a ct of the affected region . Following the exclusion of potentially life - threatening complications, antibiotic prophylaxis to avoid infections concluding, a sudden swelling occurring after tooth extraction might be due to a subcutaneous emphysema . A thorough clinical examination has to be performed, and the patient should be referred to a specialized maxillofacial clinic.
A vast evidence base demonstrates that dysregulation of the human immune system occurs immediately following spaceflight.1,2 specific post - flight findings include: altered cytokine production patterns;37 diminished natural killer (nk) cell function;8 altered peripheral leukocyte distribution;9,10 diminished monocyte function;11 diminished granulocyte function;10,12 dysregulated t - cell intracellular signaling;1315 altered neuroendocrine responses;16,17 and inhibited leukocyte proliferation following activation.18,19 latent herpesviruses have been found to be reactivated in crew members during or following spaceflight.2022 humoral immunity may also be affected by spaceflight, as animal studies have indicated that the somatic hypermutation process responsible for diversification of antibody binding sites was reduced during spaceflight.23 recent data collected on board the space shuttle and international space station (iss) confirms that human immune dysregulation is indeed an in - flight phenomenon, and not merely a post - flight stress response to landing and readaptation following deconditioning.24,25 immune dysfunction seems to coincide with, and is likely the mechanistic cause for, the documented reactivation of various latent herpesviruses during short - duration flight.20,21,26 additional studies currently ongoing on board the iss will ascertain if other uninvestigated aspects of immune dysregulation persist for the duration of a 6-month orbital spaceflight . The effect of physiological stress on immunity is well established.27 stress hormone levels have been found to be elevated following flight, heavily dependent on mission duration.17,28 furthermore, other endogenous stress response systems, such as the endocannabinoid system in humans, have also been found to be dysregulated during spaceflight.29 persistent in - flight immune dysregulation could result in specific clinical risks to crew members participating in exploration class deep space missions.30 clinical events potentially related to immune system dysregulation may include a variety of bacterial or viral infections (skin, upper respiratory, urinary tract, etc . ), clinical viral reactivation, documented hypersensitivities, or increased incidence of allergies . During prolonged missions (mars, asteroids, etc . ), more prolonged - development diseases such as autoimmunity or malignancies may become possible . During exploration missions, the risk for malignancies may be directly related to immune dysregulation (diminished nk cell function) synergizing with radiation exposure . Basic understanding of the incidence of clinical symptoms during orbital spaceflight is important for anticipation of potential clinical risks during exploration class deep space missions . Immune dysregulation during orbital flight, documented by research data as described above, may be perceived to only result in subclinical changes . Crews are generally not thought, with some well - publicized exceptions, to suffer significant illness during flight . Whereas research findings may in a non - identifiable fashion be published, adverse medical events are known only to the crew member and his / her flight surgeon . Although it should be possible to examine national aeronautics and space administration (nasa) medical records and report incidence in an anonymous fashion, literature evidence detailing in - flight clinical incidence is lacking . These include a pre - flight quarantine period, defined as the health stabilization program (hsp). This program was implemented after an in - flight infectious disease event occurred during an apollo moon mission . Other in - place mitigation strategies include microbial monitoring of food, water, vehicles, and cargo . Reports of adverse clinical events on board the iss may appear in a variety of records or information sources . The primary source would normally be the crew electronic medical record (emr), in which the flight surgeon may document on - orbit illness of his / her particular crew member . However, notations of on - orbit illness may also be recorded in secondary sources of information, within both medical operations and biomedical research . The goal of our activity was to tabulate incidence of adverse clinical events during iss missions via reported symptoms by surveying the emr and the secondary information sources . In most cases, a confirmatory diagnosis is simply not possible, given the paucity of medical laboratory equipment on board the iss . A limitation of this activity is that it is not a standard epidemiological survey, but rather represents a simple tabulation of all available recorded occurrence of symptoms during flight . For some crew members, not all the various information sources were populated with information . As it is logical to assume that at least some events may not be recorded, based on the purview of the crew member and/or flight surgeon, the information reported here therefore may represent a baseline minimum compilation of known in - flight clinical incidence . The aim of this activity was to retrospectively analyze existing astronaut medical records, to derive the most accurate assessment of in - flight clinical incidence technically possible within existing operational constraints . However, to perform the survey, it was necessary to replace the actual confirmed incidence with reported symptomatology, as in - flight crews rarely have the ability to confirm diagnoses, and treatment is usually based on presentation of symptoms . Per current nasa policy, an attributability review of the information provided via this article was conducted by the epidemiology division / lifetime surveillance of astronaut health at the johnson space center, and approval to publish was granted . Further, per nasa policy, research based on extant de - identified data is exempt from institutional review board (irb) review . However, the current chair of the nasa irb has reviewed and approved release of the data contained within this manuscript . The nasa irb did not require written informed consent be obtained from the participants, as this was a retrospective surveillance activity, and all data was de - identified . For this study, we examined medical records from 46 long - duration us astronaut missions on board the iss . Of the 46 us crew members completing iss missions as of the date of this manuscript, all were included in this study . No symptoms or immunological events were reported by 8 crew members . During the analysis period, 5 astronauts actually repeated long - duration space missions on board the iss, for which each long - duration mission was counted as a separate flight / mission . Therefore, the records from 46 long - duration iss crew members were examined, which actually consisted of 41 flown us astronauts . Among the 46 us astronauts, there were 34 male and 12 female astronauts, with an average age of 47.51 years at time of flight . The average mission duration was 162.95 days; the sum total for the 46 crew members (representing 38 missions) was 7058 flight days or 20.57 flight years . For this report, an effort was made to review and categorize in - flight incidence, and tabulate those findings in a non - attributable fashion . The primary data review, consisting of iss emrs, was performed using the evaluation categories as described below . The iss data set consists of 46 flown space station crew members (repeat flyers were counted as separate crew member missions), resulting in a total of 20.57 crew flight years . Data regarding the incidence of clinical symptoms was obtained by reviewing the records of crew private medical conferences . A breakdown of all reported events was derived by mission month for the 6-month orbital spaceflights . The nasa integrated medical model (imm) is an operational nasa tool that helps to align science, technology, and operational activities . The imm is intended for use in optimizing crew health and safety and to ensure mission success by quantifying the probability and consequences of medical risks.31 the imm has identified 83 medical conditions that have occurred, or have the potential to occur, in - flight . From this list, we identified 13 imm conditions that could be associated with dysregulated immunity during spaceflight . For purposes of this review, otitis media and otitis externa were combined into a single analysis category of ear - related symptoms: pain, congestion, and itchiness . Additionally, the condition of upper respiratory infections was removed and the related symptoms, prolonged congestion, rhinitis, and sneezing were added instead . The imm category identified as two other conditions not contained within the imm, malignancy and autoimmunity, were also evaluated during this activity . Infections, other was created to account for those infectious disease conditions that occurred during spaceflight but were not associated with any specific imm condition (table 1). Skin rash and hypersensitivities were categorized, yet in some cases where rash was accompanied by symptoms of infection, it was classified under skin infection instead . For all such instances, the epidemiologist and immunologist evaluators worked to select the most appropriate category based on the event clinical symptoms reported . Observable symptom events thought not to be related to immune dysregulation, such as brief episodes of loose stools, or injury - related events, were not included in this evaluation . An attempt was made to particularly tabulate events that have the potential to relate to immune dysregulation . Where possible, if an event was reported as a recurrence of a previously noted event, we did not count twice . In addition to the 70 events reported and included in this tabulation, an additional 28 events were excluded, as they were deemed to not be associated with dysregulated immunity . Such events included intestinal complaints due to food or nutrition, abrasions, eye irritations and/or tearing . Events such as congestion, nausea, diarrhea, or constipation, which were reported and resolved within the first 30 days of flight, were excluded and classified as space adaptation symptoms . Since additional sources of information exist regarding medical events on board the iss, and due to operational constraints no single source would be complete for an individual crew member, we reviewed all available additional sources of clinical incidence information . These secondary sources included: medical requirements reports, private medical conference notes (non - emr), research clinical surveys, post - flight experiment debriefings, and space medicine operations team notes . Essentially, anywhere a notation was possible of an in - flight medical anomaly, clinical symptoms, or treatment, the records were reviewed and events tabulated . It is important to note that this deviates from a standard epidemiological investigation, since by definition, the recording techniques may differ among the 46 iss astronauts, and confirmatory laboratory diagnoses were not possible . Therefore, this information represents a tabulation of reported clinical symptoms . Notable event consisted of an event of: 1) prolonged duration of> 1 week, with some lasting through mission end; 2) repeated and/or intensified incidence; or 3) non - responsive to, or unresolved following, medication treatment during flight . To generally characterize the rashes occurring on board the iss from a clinical perspective, a more detailed evaluation of crew medical records documenting in - flight rash occurrence was performed by a nasa flight surgeon . For each subject, all available information (which could vary between subjects) was used for the evaluation . Rash appearance and location were recorded . In some cases, photographs of the rashes were available . Per current nasa policy, an attributability review of the information provided via this article was conducted by the epidemiology division / lifetime surveillance of astronaut health at the johnson space center, and approval to publish was granted . Further, per nasa policy, research based on extant de - identified data is exempt from institutional review board (irb) review . However, the current chair of the nasa irb has reviewed and approved release of the data contained within this manuscript . The nasa irb did not require written informed consent be obtained from the participants, as this was a retrospective surveillance activity, and all data was de - identified . For this study, we examined medical records from 46 long - duration us astronaut missions on board the iss . Of the 46 us crew members completing iss missions as of the date of this manuscript, all were included in this study . No symptoms or immunological events were reported by 8 crew members . During the analysis period, 5 astronauts actually repeated long - duration space missions on board the iss, for which each long - duration mission was counted as a separate flight / mission . Therefore, the records from 46 long - duration iss crew members were examined, which actually consisted of 41 flown us astronauts . Among the 46 us astronauts, there were 34 male and 12 female astronauts, with an average age of 47.51 years at time of flight . The average mission duration was 162.95 days; the sum total for the 46 crew members (representing 38 missions) was 7058 flight days or 20.57 flight years . For this report, an effort was made to review and categorize in - flight incidence, and tabulate those findings in a non - attributable fashion . The primary data review, consisting of iss emrs, was performed using the evaluation categories as described below . The iss data set consists of 46 flown space station crew members (repeat flyers were counted as separate crew member missions), resulting in a total of 20.57 crew flight years . Data regarding the incidence of clinical symptoms was obtained by reviewing the records of crew private medical conferences . A breakdown of all reported events was derived by mission month for the 6-month orbital spaceflights . The nasa integrated medical model (imm) is an operational nasa tool that helps to align science, technology, and operational activities . The imm is intended for use in optimizing crew health and safety and to ensure mission success by quantifying the probability and consequences of medical risks.31 the imm has identified 83 medical conditions that have occurred, or have the potential to occur, in - flight . From this list, we identified 13 imm conditions that could be associated with dysregulated immunity during spaceflight . For purposes of this review, otitis media and otitis externa were combined into a single analysis category of ear - related symptoms: pain, congestion, and itchiness . Additionally, the condition of upper respiratory infections was removed and the related symptoms, prolonged congestion, rhinitis, and sneezing were added instead . The imm category identified as two other conditions not contained within the imm, malignancy and autoimmunity, were also evaluated during this activity . Finally, one additional category identified as infections, other was created to account for those infectious disease conditions that occurred during spaceflight but were not associated with any specific imm condition (table 1). Skin rash and hypersensitivities were categorized, yet in some cases where rash was accompanied by symptoms of infection, it was classified under skin infection instead . For all such instances, the epidemiologist and immunologist evaluators worked to select the most appropriate category based on the event clinical symptoms reported . Observable symptom events thought not to be related to immune dysregulation, such as brief episodes of loose stools, or injury - related events, were not included in this evaluation . An attempt was made to particularly tabulate events that have the potential to relate to immune dysregulation . Where possible, if an event was reported as a recurrence of a previously noted event, we did not count twice . In addition to the 70 events reported and included in this tabulation, an additional 28 events were excluded, as they were deemed to not be associated with dysregulated immunity . Such events included intestinal complaints due to food or nutrition, abrasions, eye irritations and/or tearing . Events such as congestion, nausea, diarrhea, or constipation, which were reported and resolved within the first 30 days of flight, were excluded and classified as space adaptation symptoms . Since additional sources of information exist regarding medical events on board the iss, and due to operational constraints no single source would be complete for an individual crew member, we reviewed all available additional sources of clinical incidence information . These secondary sources included: medical requirements reports, private medical conference notes (non - emr), research clinical surveys, post - flight experiment debriefings, and space medicine operations team notes . Essentially, anywhere a notation was possible of an in - flight medical anomaly, clinical symptoms, or treatment, the records were reviewed and events tabulated . It is important to note that this deviates from a standard epidemiological investigation, since by definition, the recording techniques may differ among the 46 iss astronauts, and confirmatory laboratory diagnoses were not possible . For this review, the definition of a notable event consisted of an event of: 1) prolonged duration of> 1 week, with some lasting through mission end; 2) repeated and/or intensified incidence; or 3) non - responsive to, or unresolved following, medication treatment during flight . To generally characterize the rashes occurring on board the iss from a clinical perspective, a more detailed evaluation of crew medical records documenting in - flight rash occurrence was performed by a nasa flight surgeon . This represented a 16-astronaut subset of the overall 46 participating crew members . For each subject, all available information (which could vary between subjects) was used for the evaluation . Specific incidence data for reported symptoms from 46 long - duration missions onboard iss is summarized in table 1 . Spanning all event categories, 70 reports of symptoms potentially related to immune dysregulation the accumulated incidence rate for these events was found to be 3.40 events per flight year, or averaging approximately 1.7 events per 6-month iss expedition crew member . By far, the majority of the adverse events observed on board the iss were skin rashes (23 events), followed by upper respiratory symptoms, including congestion, rhinitis and/or sneezing (20 events). Tabulating the various types of infectious disease observed during spaceflight (including pharyngitis, skin infection, etc .) Additional categories included in the review, but for which no on - orbit reports were observed, included: malignancies, autoimmunity, sepsis, and anaphylaxis . Note that these immunologically related imm categories do consist of confirmed - diagnosis disease states; however, for completeness, we report that no incidence of relevant symptoms for these diseases was observed for the evaluated crew members . A more detailed evaluation of the 70 reported medical events further classified 42 events as notable events included such characteristics as prolonged duration, repeated or recurring and/or, being unresponsive to treatment . These 42 notable events were observed in 46% of the crew members, whereas the remaining 54% either experienced no notable events or only events that were not classified as the most likely diagnosis breakdown of these 42 noteworthy events, based on the reported symptoms, is presented in figure 2 . Almost one - half were found to be prolonged rashes, followed by infectious disease, atypical allergies, and cold sores . An evaluation of the 42 notable events in the context of mission kinetics (month) is presented in table 2 . The highest concentration of notable adverse medical events occurred during the first month of flight (18 events); however, beyond the first month, there was a generally even spread of events between months 2 and 6 of flight . An evaluation of the distribution of the 42 notable medical events among the 46 crew members is presented in figure 3 . Eleven crew members experienced a single notable in - flight medical event, whereas 10 individual crew members experienced 2 or more events ., rashes were observed to occur in the following locations: scalp, face, neck, chest, back, trunk, abdomen, arms, and hands . A representative photograph from a persistent rash incident on board the iss is presented in figure 4 . The appearance of the rashes generally consisted of bumps / nodules and/or small brown scaly patches, with or without petechiae, redness / hyperemia, and itching . Specific incidence data for reported symptoms from 46 long - duration missions onboard iss is summarized in table 1 . Spanning all event categories, 70 reports of symptoms potentially related to immune dysregulation the accumulated incidence rate for these events was found to be 3.40 events per flight year, or averaging approximately 1.7 events per 6-month iss expedition crew member . By far, the majority of the adverse events observed on board the iss were skin rashes (23 events), followed by upper respiratory symptoms, including congestion, rhinitis and/or sneezing (20 events). Tabulating the various types of infectious disease observed during spaceflight (including pharyngitis, skin infection, etc .) Additional categories included in the review, but for which no on - orbit reports were observed, included: malignancies, autoimmunity, sepsis, and anaphylaxis . Note that these immunologically related imm categories do consist of confirmed - diagnosis disease states; however, for completeness, we report that no incidence of relevant symptoms for these diseases was observed for the evaluated crew members . A more detailed evaluation of the 70 reported medical events further classified 42 events as notable events included such characteristics as prolonged duration, repeated or recurring and/or, being unresponsive to treatment . These 42 notable events were observed in 46% of the crew members, whereas the remaining 54% either experienced no notable events or only events that were not classified as notable the most likely diagnosis breakdown of these 42 noteworthy events, based on the reported symptoms, is presented in figure 2 . Almost one - half were found to be prolonged rashes, followed by infectious disease, atypical allergies, and cold sores . An evaluation of the 42 notable events in the context of mission kinetics (month) is presented in table 2 . The highest concentration of notable adverse medical events occurred during the first month of flight (18 events); however, beyond the first month, there was a generally even spread of events between months 2 and 6 of flight . An evaluation of the distribution of the 42 notable medical events among the 46 crew members is presented in figure 3 . Eleven crew members experienced a single notable in - flight medical event, whereas 10 individual crew members experienced 2 or more events . During spaceflight, rashes were observed to occur in the following locations: scalp, face, neck, chest, back, trunk, abdomen, arms, and hands . A representative photograph from a persistent rash incident on board the iss is presented in figure 4 . The appearance of the rashes generally consisted of bumps / nodules and/or small brown scaly patches, with or without petechiae, redness / hyperemia, and itching . Early (mercury, gemini, apollo) astronauts reported illness, including a noteworthy urinary tract infection on apollo 13, which eventually resulted in the nasa hsp.32 the hsp places astronauts in a 7-day period of restricted access before a spaceflight, in an attempt to mitigate crew infectious disease on - orbit . The hsp is not a pure isolation, as astronauts still have contact with family and staff . Rather, the hsp limits contact to individuals who have been medically screened and trained to minimize crew exposure to infectious disease . This program has been successful in reducing the rate of on - orbit communicable infectious disease;32 however, illness during spaceflight remains a possibility for astronauts . Breaks in the hsp quarantine are possible, and astronauts may become infected before flight and carry a pathogen to orbit . Certain pathogens, such as latent herpesviruses, cannot be screened out by a quarantine period . Although studies are ongoing to characterize immune dysregulation during flight, increased microbial virulence during space flight may also contribute to elevated risk for infectious disease . Recent data has demonstrated that some microbes actually increase their virulence during spaceflight.33 mermel recently summarized the infectious disease risk in the context of immunity, microbial virulence changes, and environmental conditions such as air handling, recycling, and hygiene.34 in conjunction with research data regarding human physiology during flight, it is important to know in - flight incidence rates for the iss program . The incidence data may provide the most important tool for assessing astronaut risk during exploration class missions . Unfortunately, detailed incidence rates for the space shuttle and iss programs have historically been lacking, due to astronaut medical privacy concerns . Although an extremely large number of us astronauts were able to fly short - duration space missions onboard the shuttle, incidence (or clinical symptoms) data from the space shuttle era is likely to be artificially elevated due to the presence of early space - adaptation symptoms during a 2-week orbital spaceflight . This is almost certainly true for stuffiness and congestion, which may certainly resemble allergic symptoms . Short - duration spaceflight is extremely busy and stressful, as most waking time is accounted for to meet mission objectives . Shuttle missions, with their short duration and very stressful timelines, are therefore likely to not be representative of the expected incidence for exploration class spaceflight . Iss missions consist of long - duration exposure to the microgravity and radiation environment of low earth orbit spaceflight . Iss crew members work on a somewhat more reasonable schedule compared to shuttle flights and generally adapt to the space environment by approximately 24 weeks on - orbit . Furthermore, crews are isolated from daily contact, so opportunity for communicable infectious disease is dramatically reduced . Iss incidence data therefore likely represents a more accurate reflection of in - flight clinical incidence, as human physiology would equilibrate during long - duration spaceflight . Generating an appropriate terrestrial comparison group for this dataset is particularly challenging . The relevant space agencies fly extremely healthy and fit individuals, screen their health frequently, put them through a pre - flight quarantine, and then deploy them in an extreme and physiologically disruptive (and entirely unique) environment . We suggest that the goal of the spaceflight medical program is that the incidence numbers should therefore be zero . However, as rashes were the most reported event on board the iss and are of clinical concern to flight surgeons, we will briefly discuss rash presentation and terrestrial incidence for this particular symptom . The prevalence of persistent rashes in the us population is about 0.044 cases / year,35 and our observed incidence of rashes on board the iss was elevated in comparison at 1.12 events per flight year . The incidence for rashes on board the iss is therefore 25-fold higher than terrestrial incidence . The body locations for rash occurrence on board the iss seemed to vary; however, the appearance was generally consistent, manifesting as bumps, nodules, small scaly patches, and itchy redness . Presumed diagnoses for such rashes could include eczema, contact dermatitis, psoriasis, allergic cholinergic urticaria, and/or simple acne . Ilscus et al speculated that contributing factors to on - orbit rashes could include constraints of hygiene, infection, atypical herpesvirus infection, photo dermatitis, or cutaneous decompression sickness.36 hygienic contributing factors may include soaps and no - rinse shampoo use with suboptimal ability to completely rinse the body areas after cleansing . Irritation from operational equipment, such as extravehicular (eva spacewalk) suits, electrocardiography electrodes, or oxygen masks could be other contributing factors, however the timing and locations of the rash events suggest that they were not related to eva activity . In addition, the environment onboard iss may be conducive to rashes, consisting of low humidity, prolonged contact with moisture from sweat or wet clothing, and constrained airflow . It is also possible, given that the majority of the notable in - flight events occurred in the first month of spaceflight, that early space adaptation influences these incidence numbers (figure 3). However, we speculate that the persistent dysregulation of the immune system, including some data indicating a th2 shift, may be a significant contributing factor to the incidence of on - orbit rashes . Recent research data confirms persistent immunological dysregulation during spaceflight.24,37 rashes and hypersensitivities would suggest immune dysregulation of a hypersensitive nature . There is other anecdotal commentary from recent iss astronauts during post - flight experiment debriefing sessions that supports the possibility for some aspects of th2 immunity to be sensitized . These include statements from multiple astronauts regarding upper respiratory symptoms (stuffiness, etc .) Which persisted for 6 months and were treated with, and reported responsive to, antihistamines (data not shown). Ilscus et al further suggested that possible mitigations for on - orbit rashes might include allergy / patch testing of crew members and various crew - use and/or food products, and avoidance of any defined irritants.36 suggested laboratory assays to further investigate rashes in astronauts could include immunoglobulin e, complement (c3a, 4a, 5a), cytokines (interleukin 5 and 13), mast / baso / eosinophils levels, prostaglandins (d2), stress hormones, and leukotrienes . Medication usage is not routinely tracked onboard iss; however, a recent nasa pharmacology survey of astronaut archival health records indicated that antihistamines were the most prescribed medication during flight for chronic conditions that persisted> 7 days.38 for acute (<7-day duration) issues, antihistamines were second only to sleep medications . Suggested treatments for persistent allergic / hypersensitivity symptoms in astronauts may include oral antihistamines, topical steroids, antibiotics (if an infectious agents is suspected), or oral steroids . The nasa human research program is currently characterizing the nature of immune system dysregulation in astronauts . This research may result in a monitoring strategy, which may be used to validate potential immune countermeasures . The clinical significance of these numbers is open to interpretation . Among the incidents reported here, certainly some category - specific clinical issues appear to be elevated compared to the general terrestrial population, and almost certainly appear elevated based on expectations for extremely healthy individuals in a putatively isolated environment . The hypersensitivity findings are seemingly discordant with various research data that indicates other aspects of adaptive immunity are depressed during spaceflight.2,24 it is established that several variables influence adaptive / cellular immune parameters in a negative fashion (microgravity, stress, radiation, etc . ), although this phenomenon had been believed to be largely subclinical (diminished function, altered cytokine profiles, latent viral reactivation, etc . ). For exploration missions, chronic immune dysregulation, coupled with increased radiation exposure and no return option, clinical conditions, such as tabulated here, are already being observed on - orbit . In fact, these incidence rates fit perfectly the model of spaceflight - associated immune alterations reported in the literature, which include diminished t - cell function (relates to infectious disease) and a th2 cytokine shift (relates to hypersensitivities).1 a linkage was recently reported between alterations in cytokine profiles in astronauts and the reactivation of latent herpesviruses,37 which supports the postulate that specific types of immune bias shifts will increase risks for specific types of disease . Although subclinical reactivation of these viruses has been previously reported during spaceflight, the incidence data confirms that 6 instances of oral herpesvirus reactivation have already occurred on board the iss . Herpesvirus reactivation resulting in fever blisters has also been reported by astronauts to occur during spaceflights . It is also interesting that the highest concentration of reported events occurred early within the 6-month mission durations . While the slope of immune alterations during spaceflight is still largely unknown, the literature to date generally finds that alterations persist for the duration of a 6-month orbital spaceflight.25 we anticipate upcoming investigations on board the iss will shed further light on the complexities of immune adaptation during flight, and planned 1-year mission durations will improve our understanding regarding the kinetics of these alterations . The authors acknowledge that this is a subjective report; however, great rigor was exercised to achieve a repeatable readout on iss incidence (based on reported symptoms), given the criteria that were used . Where possible, if an event was reported as a recurrence of a previously noted event, we did not count twice . If there was no indication from the flight surgeon notes or any other source documents that the event was a recurrence, we did not infer recurrence and as such, for the purposes of this tabulation, we counted it as a new event . An evaluation of the same records using different criteria would likely generate different results . To ensure as accurate and unbiased reporting as possible, the immunologists had no exposure to the record evaluation process . Records evaluation was strictly performed by the epidemiology staff, after the evaluation criteria were devised in consultation with the immunology discipline team at the johnson space center . On the iss, factors associated with spaceflight, including confinement, nutritional limits, and disrupted circadian rhythms, are likely to be similar to those anticipated for exploration class missions . Although a quick - return option exists for orbital flight, in - flight clinical care capability is likely similar to that experienced on exploration class missions . Therefore, we anticipate the incidence of adverse clinical events during an iss mission to represent the best available evidence from which to extrapolate incidence rates for exploration class missions . The incidence for missions beyond earth orbital spaceflight is likely to be higher than the rate observed during iss missions . Further studies on board the iss will complete our understanding of immune status during spaceflight . We suggest that a thorough investigation of th2 immunity on board the iss must be coupled with precise capture of crew clinical information and medication usage . Operational influences (crew equipment, hygiene, sleep, and work schedules), as well as deployment of unique allergens into the iss environment (such as a permanent murine habitat), must be thoroughly evaluated to not exacerbate an already - existing clinical issue . Finally, we note that assessments of astronauts during flight may provide unique insight into similar for example, it is well understood that stress adversely affects immunity, which may increase susceptibility to disease.27 aged individuals are known to present with increased infectious disease or latent viral reactivation, which may be related to immune senescence with decreased t - cell function.39 also, terrestrial space analog conditions exist; locations where remote deployment, prolonged isolation, and extreme environment recreate some of the stress effects of spaceflight.40 examples include undersea deployment or antarctica winter - over missions . It has been established that crew members participating in such terrestrial missions also shed latent herpesviruses.41 similarly, better controlled laboratory simulations of spaceflight confinement, such as a recent russian 500-day mars simulation, have also been found to re - create immune dysregulation, which shares some aspects of flight - associated dysregulation.42 we suggest monitoring strategies and countermeasures developed to assess and prevent incidence in astronauts may facilitate the reduction of illness on earth.
Asymptomatic carotid plaques are very commonly encountered in daily clinical practice, with prevalence ranging from 0.1% to 7.5% in the general population . Asymptomatic carotid stenosis refers to narrowing of the carotid artery caused by atherosclerosis in patients who have not experienced a stroke or transient ischemic attack in the territory of that artery . The pathogenesis of carotid plaque from asymptomatic to cerebrovascular events is a complex process which is still not completely understood . Although many systemic risk factors predispose to development of atherosclerosis, plaque is not uniformly distributed in the carotid arteries and it preferentially affects certain regions of circulation (e.g., the inner curve, close to side branches, and in the bulbs of the carotid artery [46]), suggesting that local mechanical factors might determine plaque development and growth . Plaque morphology and composition are evidently related to blood flow, which therefore influences the local predisposition to atherosclerosis progression, plaque rupture, and thrombosis . Furthermore, there is ample evidence that low wall shear stress is involved in plaque initiation and progression through various molecular mechanisms that influence endothelial cell morphology and function . Atherosclerosis is considered to be a disease caused by a variety of clinical environmental factors and genetic factors . In recent years, numerous polymorphisms of genes related to atherosclerosis have been found by genome - wide association study (gwas) and meta - analysis in large - scale studies [913]. On the other hands, other hemodynamic parameters related to atherosclerosis include wall pressure, shear strain rates, and flow velocity . Cfd is mechanical engineering field which is widely used in mechanical engineering to solve complex problems by analyzing fluid flow, heat transfer, and associated phenomena by using computer simulations . Examples are aerodynamics and hydrodynamics of vehicles, power plants and turbines, electronic engineering, chemical engineering, external and internal environmental architectural design, marine and environmental engineering, hydrology, meteorology, and biomedical engineering . In recent years, cfd biomedical research is more accessible because high - performance hardware and software are easily available with advances in computer science . Few studies have investigated the relationship between carotid plaque hemodynamic characteristics and risk of stroke in asymptomatic patients . Therefore, in this study, we aimed to investigate the relationship between hemodynamic parameters of asymptomatic carotid plaques by using cfd and magnetic resonance angiography (mra), including flow velocity across the lesion, wall shear stress (wss), wall pressure (wp), and the risk of stroke . This prospective study was performed at the chinese pla general hospital, beijing, china from march 2011 to june 2013 . For the purpose of our study, asymptomatic participants with carotid plaque (thickness 2 mm) in one of the carotid arteries were enrolled . The exclusion criteria included: 1) images were not suitable for cfd analysis: the stenosis degree greater than 95% or less than 10%; and 2) contraindications for mra: any electrically, magnetically, or mechanically activated implant, intracranial aneurysm clips (unless made of titanium), ferromagnetic surgical clips or staples, metallic foreign body in the eye, or pregnancy . Demographics, histories of common diseases (previously diagnosed or taking corresponding medications at baseline), and results of laboratory tests at baseline were collected from computerized medical records and ultrasound reports . The primary endpoint was defined as a stroke based on typical clinical symptoms and confirmed by follow - up brain ct or mra . Secondary endpoints included any ischemic stroke or tia, and all - cause death . A flow chart explaining the final data used for analysis is shown in figure 1 . The participants were divided into a stroke group and a non - stroke group according to follow - up data . Written informed consent the study was approved by the ethics committee for medical research of the chinese pla general hospital . Carotid plaques mras were obtained by a signal 1.5 t mr systems (general - electric healthcare, milwaukee, wi) using a bilateral phased - array surface coil with a slice thickness of 1.2 mm . Mra scan range was from the aortic arch to the skull base, and we selected the 4 cm within the scope of the original image of the carotid bifurcation (i.e., within the 4-cm scope of the cca, ica, and eca). After the scan was completed, the digital imaging and communications in medicine (dicom) format images were derived . All images were imported to the post - processing workstation adw4.5 - 1 (general - electric healthcare, milwaukee, wi) for image evaluation and analysis . Luminal area (la) was defined as the area encompassed by the inner boundary of the intimal surface in plaque slices . Total vessel area (tva) was defined by the outer boundary of the vessel . Wall area (wa) was calculated by the software, subtracting tva from la (wa = tvala). Geometric modeling was performed by importing the dicom format mra data into reverse engineering 3d reconstruction software mimics (materialise inc ., belgium), by threshold segmentation (figure 2a), region growing, and use of mask film editing (figure 2b) tools to get a sense of vessels in the region of interest (roi) mask by mimics the 3-dimensional (3d) transport calculation functions, connection of the extracted two - dimensional (2d) section, and generating a 3d carotid arteries geometric model (figure 2c). We then imported the 3d models into the forward engineering software 3-matic to remesh the surface of the arteries (figure 2d) and improve the quality of the grid . We imported stl format images into the icem - cfd software to identify the 3d space structure, and defined the 1-inlet, 2-outlets, the vessel wall, and other parts of arteries . Then, we set the parameters required for the tetrahedral mesh according to the size of the arteries . To guarantee the accuracy of the boundary of the target artery, we set up a 3-layer boundary calculate condition, and the growth factor was increased by 1.2 times . We imported tetrahedral mesh files into a fluent model with ansys software and set the following boundary conditions: (1) vascular wall characteristics: vascular walls were set to smooth, no penetration of rigid; (2) blood characteristics: the blood flow was assumed laminar, incompressible, and newtonian, with density of 1060 kg / m and viscosity of 0.0035pas; (3) the inlet velocity was measured by mra and the blood flow of internal and external carotid arteries were 55% and 45%, respectively; and (4) the calculation was based on continuity equation and navier - storks equation . The workflow of cfd analysis is shown in figure 4 ux+vy+wz=0 (u)t+div(uu)=div(gradu)-px+su (v)t+div(vu)=div(gradv)-py+sv (w)t+div(wu)=div(gradw)-pz+sw all continuous variables are presented as means sd . The normal distribution test for continuous variables was conducted using the kolmogorov - smirnov test . Statistical analysis of normal distribution data was performed using an unpaired t test between 2 groups . Non - normal distribution data were analyzed using the mann - whitney u test for continuous variables and the x test for discrete variables . Receiver operator characteristic curve (roc) and c - statistic testing were utilized to assess the performance of the constructed model in comparison with a previous published model . Differences were considered statistically significant at a two - tailed p value of less than 0.05 . This prospective study was performed at the chinese pla general hospital, beijing, china from march 2011 to june 2013 . For the purpose of our study, asymptomatic participants with carotid plaque (thickness 2 mm) in one of the carotid arteries were enrolled . The exclusion criteria included: 1) images were not suitable for cfd analysis: the stenosis degree greater than 95% or less than 10%; and 2) contraindications for mra: any electrically, magnetically, or mechanically activated implant, intracranial aneurysm clips (unless made of titanium), ferromagnetic surgical clips or staples, metallic foreign body in the eye, or pregnancy . Demographics, histories of common diseases (previously diagnosed or taking corresponding medications at baseline), and results of laboratory tests at baseline were collected from computerized medical records and ultrasound reports . The primary endpoint was defined as a stroke based on typical clinical symptoms and confirmed by follow - up brain ct or mra . Secondary endpoints included any ischemic stroke or tia, and all - cause death . A flow chart explaining the final data used for analysis is shown in figure 1 . The participants were divided into a stroke group and a non - stroke group according to follow - up data . Written informed consent the study was approved by the ethics committee for medical research of the chinese pla general hospital . Carotid plaques mras were obtained by a signal 1.5 t mr systems (general - electric healthcare, milwaukee, wi) using a bilateral phased - array surface coil with a slice thickness of 1.2 mm . Mra scan range was from the aortic arch to the skull base, and we selected the 4 cm within the scope of the original image of the carotid bifurcation (i.e., within the 4-cm scope of the cca, ica, and eca). After the scan was completed, the digital imaging and communications in medicine (dicom) format images were derived . All images were imported to the post - processing workstation adw4.5 - 1 (general - electric healthcare, milwaukee, wi) for image evaluation and analysis . Luminal area (la) was defined as the area encompassed by the inner boundary of the intimal surface in plaque slices . Total vessel area (tva) was defined by the outer boundary of the vessel . Wall area (wa) was calculated by the software, subtracting tva from la (wa = tvala). Geometric modeling was performed by importing the dicom format mra data into reverse engineering 3d reconstruction software mimics (materialise inc ., belgium), by threshold segmentation (figure 2a), region growing, and use of mask film editing (figure 2b) tools to get a sense of vessels in the region of interest (roi) mask by mimics the 3-dimensional (3d) transport calculation functions, connection of the extracted two - dimensional (2d) section, and generating a 3d carotid arteries geometric model (figure 2c). We then imported the 3d models into the forward engineering software 3-matic to remesh the surface of the arteries (figure 2d) and improve the quality of the grid . We imported stl format images into the icem - cfd software to identify the 3d space structure, and defined the 1-inlet, 2-outlets, the vessel wall, and other parts of arteries . Then, we set the parameters required for the tetrahedral mesh according to the size of the arteries . To guarantee the accuracy of the boundary of the target artery, we set up a 3-layer boundary calculate condition, and the growth factor was increased by 1.2 times . We imported tetrahedral mesh files into a fluent model with ansys software and set the following boundary conditions: (1) vascular wall characteristics: vascular walls were set to smooth, no penetration of rigid; (2) blood characteristics: the blood flow was assumed laminar, incompressible, and newtonian, with density of 1060 kg / m and viscosity of 0.0035pas; (3) the inlet velocity was measured by mra and the blood flow of internal and external carotid arteries were 55% and 45%, respectively; and (4) the calculation was based on continuity equation and navier - storks equation . The workflow of cfd analysis is shown in figure 4 ux+vy+wz=0 (u)t+div(uu)=div(gradu)-px+su (v)t+div(vu)=div(gradv)-py+sv (w)t+div(wu)=div(gradw)-pz+sw the normal distribution test for continuous variables was conducted using the kolmogorov - smirnov test . Statistical analysis of normal distribution data was performed using an unpaired t test between 2 groups . Non - normal distribution data were analyzed using the mann - whitney u test for continuous variables and the x test for discrete variables . Receiver operator characteristic curve (roc) and c - statistic testing were utilized to assess the performance of the constructed model in comparison with a previous published model . All tests were carried out using spss version 17.0 (spss inc ., chicago, il, usa) statistical software . Differences were considered statistically significant at a two - tailed p value of less than 0.05 . The study population included 120 men (52.63%) and 108 women (47.37%), the mean age was 59.218.48 years, and the mean follow - up duration was 1147.56224.84 days . The follow - up results showed 16 of 228 patients with asymptomatic carotid plaques had experienced a stroke . The participants were divided into a stroke group and a non - stroke group according to whether stroke had occurred . Detail information on demographics, medical history, physical examination, and results of laboratory tests of the 2 groups are shown in table 1 . There were no significant differences between the 2 groups in terms of age, sex, history of smoking, medical history, physical examination, or laboratory test results . Figure 5a is a streamlined picture of the flow, showing turbulent regions with obvious velocity slowing above and below the plaques . Local amplification of the velocity vector (figure 5b) shows the blood flow velocity was accelerated in the stenosis region . Contours of wp (figure 5c) show the wall pressure was gradually decreased from the inlet to the outlets of the carotid artery . However, the wp of the bifurcation site of the common carotid artery was abnormally high, and atherosclerotic lesions showed decreased wp . On the other hand, contours of wss the stroke group and non - stroke group were similar with respect to la, wa, and tva . The nwi of the stroke group was higher than in the non - stroke group (0.540.15 vs. 0.520.19, p=0.043). Qualitative assessment of hemodynamic parameters showed no significant difference between the stroke group and non - stroke group in wp; however, wss of the stroke group was higher than in the non - stroke group (7.871.34 vs. 5.862.14, p=0.013). Logistics regression analysis indicated that nwi and wss were associated with the development of carotid plaques (table 3). Carotid artery with higher other hemodynamic and mra characteristics of carotid artery did not show significant associations with the risk of stroke . To assess the predictive utility of wss, nwi, and wss+nwi, we compared the roc curves of these 3 parameters . The area under the roc curve values for wss, nwi, and wss+nwi were 0.772, 0.798, and 0.903, respectively (table 4). Wss+nwi predicted the onset of stroke better than wss and nwi (p<0.05), with no statistically significant differences between wss and nwi (figure 6). The study population included 120 men (52.63%) and 108 women (47.37%), the mean age was 59.218.48 years, and the mean follow - up duration was 1147.56224.84 days . The follow - up results showed 16 of 228 patients with asymptomatic carotid plaques had experienced a stroke . The participants were divided into a stroke group and a non - stroke group according to whether stroke had occurred . Detail information on demographics, medical history, physical examination, and results of laboratory tests of the 2 groups are shown in table 1 . There were no significant differences between the 2 groups in terms of age, sex, history of smoking, medical history, physical examination, or laboratory test results . Figure 5a is a streamlined picture of the flow, showing turbulent regions with obvious velocity slowing above and below the plaques . Local amplification of the velocity vector (figure 5b) shows the blood flow velocity was accelerated in the stenosis region . Contours of wp (figure 5c) show the wall pressure was gradually decreased from the inlet to the outlets of the carotid artery . However, the wp of the bifurcation site of the common carotid artery was abnormally high, and atherosclerotic lesions showed decreased wp . On the other hand, contours of wss (figure 5d) showed the uneven distribution of wss of the carotid artery . The stroke group and non - stroke group were similar with respect to la, wa, and tva . The nwi of the stroke group was higher than in the non - stroke group (0.540.15 vs. 0.520.19, p=0.043). Qualitative assessment of hemodynamic parameters showed no significant difference between the stroke group and non - stroke group in wp; however, wss of the stroke group was higher than in the non - stroke group (7.871.34 vs. 5.862.14, p=0.013). Logistics regression analysis indicated that nwi and wss were associated with the development of carotid plaques (table 3). Carotid artery with higher high wss increased the chance of the stroke by 6.974 times . Other hemodynamic and mra characteristics of carotid artery did not show significant associations with the risk of stroke . To assess the predictive utility of wss, nwi, and wss+nwi, we compared the roc curves of these 3 parameters . The area under the roc curve values for wss, nwi, and wss+nwi were 0.772, 0.798, and 0.903, respectively (table 4). Wss+nwi predicted the onset of stroke better than wss and nwi (p<0.05), with no statistically significant differences between wss and nwi (figure 6). The present study used morphologic data derived from mra and geometric parameter data obtained by cfd to examine the relationship between carotid plaques and the risk of stroke in patients with asymptomatic carotid plaques . Of the 228 participants with asymptomatic carotid plaques, 16 (7.02%) had stroke in the territory of the carotid artery, which was comparable to kakkos s and chung s studies . Additionally, the probability of stroke was much lower than in sadat s study . The difference may be that sadat s cohort had suffered a stroke before and were not asymptomatic as such . For the cfd analysis, most of the previous studies used standard flow velocity . Instead of this, we used person - specific inlet flow velocity measured by mra, which allowed us to get more accurate parameters of fluid dynamics . Several studies have investigated the association between carotid plaques progression and risk of cerebrovascular events in patients with asymptomatic carotid stenosis [1,22,2428]. These risk factors including age, sex, severity of stenosis, increased plaque area, carotid artery end - diastolic velocity, systolic blood pressure, and increased serum creatinine . Our analysis suggests that plaque morphology characteristics nwi and hemodynamics parameter wss are independent risk factors for the progression from asymptomatic to stroke . Logistics regression analysis and roc curve suggest that the combination of nwi and wss is better at predicting the risk of stroke . During the early stages of atherosclerotic plaque growth, the vessel may remodel and accommodate plaque growth without compromising luminal size . There are few plaque morphology characteristics, such as wa, used to assess plaques . Clinical trials have shown that in human carotid arteries, luminal stenosis has limited value as an indicator of atherosclerotic plaque vulnerability, enabling prediction of only 1 of 10 strokes in asymptomatic patients . Plaque burden, which is represented by plaque volume and maximum wall area (mwa), as a direct measure of the lesion itself, may have substantial usefulness in the assessment of atherosclerosis . Studies have shown nwi is a sensitive factor for early detection of carotid atherosclerosis plaques, and it can reflect the progression of plaque more accurately than other morphology characteristics such as la and wa . Calculated as wa / tva, nwi contains information about lumen stenosis and thickening of the wall, which make nwi is the most effective indicator for evaluating the severity of atherosclerosis . Furthermore, it can avoid the difference in the wall area caused by different vascular thicknesses . Therefore, nwi may also be used as a dynamic index to measure the dynamic changes of plaques . Even though morphology characteristics of plaques were thought to relate to the progression of local lesion, as mentioned above, plaque rupture is not solely dependent on plaque morphology, and other local factors are probably involved . As a consequence of improvements in imaging of atherosclerosis and in computational power, there is increasing evidence that hemodynamic factors are important in the atherogenic process and in the development of unstable plaque and thromboembolic stroke . Disturbances of hemodynamics are thought to have a role in several aspects of this process, particularly in the causation of plaque rupture and in the formation of surface thrombus . The follow - up radiographic results confirmed that the culprit sites were consistent with the regions of high wss . Wss is the parallel frictional force exerted by blood flow on the endoluminal surface of the arterial wall . The magnitude of wss is influenced by changes in luminal geometry, blood flow velocity, and plasma viscosity . Although studies suggested that a certain degree of high wss has anti - atherosclerosis effect, the increase of wss is not necessarily a protective factor for atherosclerosis . Lovett showed that plaque ruptures more frequently in the upper side of the lumen, which is affected by the high wss and leads to the production of vulnerable plaque with a thin fibrous cap . Studies of clinical pathological autopsies also showed that plaque rupture mainly occurred in the upper side of the lesion, consistent with the area of high wss . Although the absolute value of the wss is not enough to directly damage the structure of the fiber cap, the increased wss in the upper side of the lesion is the primary factor causing rupture of the plaque . Studies have confirmed that wss is associated with regulation of many vascular functions, such as maintenance of acute vessel tone, vascular permeability, adhesion of leukocytes, development of blood vessels, and secretion of pro - thrombotic and antithrombotic signaling molecules . Activated ecs produce chemokines, cytokines, and adhesion molecules that interact with leukocytes and platelets, and target inflammation to specific tissues as a host defense mechanism . Balaguru s team developed a versatile model based on cfd simulation to explore the shear stress - associated changes of biological function in endothelial cells (ecs). Cell morphology, cytoskeletal arrangement, cell death, reactive oxygen species profile, nitric oxide production, and disturbed flow markers under certain wss condition were assessed . They observed a 2~4-fold increase in vegfr2 expression in high - wss regions, but the increase in expression was not observed in low - wss areas . Mcp-1 involved in the recruitment of leukocytes plays a significant role in the development of atherosclerotic plaque formation . There was a 2-fold increase in expression of hif1- in high - wss regions, and a less than 2-fold increase in low - wss areas . Plaques that contain a large lipid - rich necrotic core, intraplaque hemorrhage, inflammation, and/or are covered by a thin fibrous cap are considered the most vulnerable to rupture . In animal studies and small case studies, an association between wss and plaque composition was observed . Although low wss may induce plaque initiation, it has been hypothesized that plaque destabilization can be caused by high shear stress on the plaque . Tuenter s study evaluated the association between shear stress and plaque components in asymptomatic persons . We found that the combination of plaque morphology characteristics nwi and hemodynamics parameter wss can improve the ability to identify patients at highest risk of rapid disease progression and may predict the risk of stroke in patients with asymptomatic carotid plaques . More prospective studies with larger sample sizes are necessary to validate the findings of this study and to discover new risk factors for imaging and fluid mechanics.
Diphtheritic polyneuropathy (dp) is recognized as one of the most severe complications of diphtheria, caused by exotoxin of corynebacterium diphtheriae . Among 20,000 cases of diphtheria reported by who during 20072011, 17,926 (89.6%) cases were from india alone . Scarce reports of dp in the indian literature may result from under - recognition and perhaps, under - reporting of this entity . In this paper, we present a case series of 13 children with dp from south india admitted in our hospital from july 2013 to december 2013 . During the same period, 19 children admitted with membranous tonsillitis in our hospital were identified as probable or confirmed cases of diphtheria with culture positivity in 13 children . They were treated with anti - diphtheritic serum (ads) and antibiotics . Among 19 children, the simultaneous resurgence of clinical diphtheria in our area helped us probe into the history of those children who presented with neuropathy and throw light on this latent entity which is almost forgotten by the present day physicians and neurologists . In india, apart from the case series from delhi, there is no other case series reported so far in the recent past . Investigations such as cerebrospinal fluid (csf) analysis, nerve conduction velocity (ncv) studies, magnetic resonance imaging (mri) brain were carried out . The results were tabulated and analyzed . Among 13 children, 10 presented with neuropathy whose past histories revealed membranous tonsillitis . Three children presented to us with respiratory diphtheria and developed neuropathy during follow - up . All the children had history of membranous tonsillitis with a latency period of 1540 days between the onset of tonsillitis and neurological symptoms . One child had unilateral lower motor neuron facial palsy and 6 children developed quadriparesis after palatal palsy, which was descending and symmetric in nature [table 1]. Details of children with dp supportive treatment with ryle's tube feeding was given to all children for 14 weeks . Children with isolated bulbar palsy recovered within 24 weeks while children with quadriparesis recovered within 56 weeks . Dp is seen in 20% and 75% of patients with mild and severe infection, respectively . The period between the appearance of first symptom of diphtheria and the development of dp is termed latency, which varies from 10 days to 3 months . The first indication of neuropathy is paralysis of the soft palate and posterior pharyngeal wall . Oculomotor and ciliary paralyses seen after 3 weeks are common and distinctive features of dp . Peripheral neuritis develops later, from 10 days to 3 months after the onset of oropharyngeal disease . In some patients, there may be a secondary worsening of the bulbar symptoms along with the occurrence of peripheral neuropathy . Biphasic course of disease was noted only in 2 children in our study which is contradictory to other studies . Various studies on major epidemic outbreaks from russia and europe in the past gave us valuable insights on pathophysiology and progression of dp . Diptheritic toxin penetrates into schwann cells and it inhibits the synthesis of myelin proteolipid and basic protein . It was observed in vitro that it binds to schwann cells as early as 1-hr postinjection, inducing the latent development of polyneuropathy . Local toxic effects occur by direct spread of toxin and result in the early bulbar problems while the ensuing generalized demyelinating neuropathy arises from hematogenous dissemination . The reason behind isolated palatal palsy in few children with no systemic involvement is probably nondissemination of the toxin . Dp has to be distinguished from other neuropathies especially guillain barre syndrome (gbs) which is more common in children . The clinical features differentiating dp from gbs are high prevalence of bulbar palsy, slower evolution of the neuropathy for more than 4 weeks, descending nature, and simultaneous involvement of other organ systems . Logina and donaghy reported an attenuated form of neuropathy in the immunized population of latvia . This was attributed to the protective effects of the vaccine, which attenuated the adverse effects of exotoxin . Compared to the previous epidemic in 1999, the incidence and severity were lower in same area due to early diagnosis and administration of ads within first 3 days of disease . With this review of literature and supportive evidence from our study, we stress upon the recognition of epidemic in its incipient stage and prompt administration of ads . Features of dp in various studies studies from russia showed a significant trend of decreasing immunity with increasing age, resulting in lack of protection, particularly for adults aged 30 to 50 years . As vaccine induced immunity wanes over time, periodic boosters are recommended every 10 years . Thus the susceptibility of adults to diphtheria is a new phenomenon of the vaccine era . In india, mohanta and mild respiratory muscle involvement was seen in one child in our study in contrast to the study by sandeep kumar et al. (2010) from delhi where respiratory muscles were involved in 85.4% cases and 60.4% required mechanical ventilation . In 2013, mateen et al . Reported a case series of 15 children with dp from nine states and union territories, detected through acute flaccid paralysis (afp) surveillance [20022008]. Pediatricians / neurophysicians should have a high index of suspicion to recognize dp in the wake of recent resurgence of diphtheria in some parts of india . Any child diagnosed with probable diphtheria should be followed for 36 months for neurological complications . As seen in our case series, dp carries good prognosis hence timely diagnosis and differentiation from other neuropathies is a prerequisite for rational management and contact tracing . Continued occurrence of diphtheria emphasizes the need for public health measures such as: strengthening of routine immunization and mandatory booster vaccination at school entry and td booster at 10 years intervals thereaftervaccination of susceptible contacts and prophylactic antibiotics to contactsensuring availability of ads for timely administration and thereby preventing complicationsafp surveillance system can be utilized to identify dp and thus areas of resurgence of diphtheria and strengthen immunization services in those pockets . Strengthening of routine immunization and mandatory booster vaccination at school entry and td booster at 10 years intervals thereafter vaccination of susceptible contacts and prophylactic antibiotics to contacts ensuring availability of ads for timely administration and thereby preventing complications afp surveillance system can be utilized to identify dp and thus areas of resurgence of diphtheria and strengthen immunization services in those pockets.
Traumatic cervical artery dissection (tcad) represents a rare but under certain circumstances life - threatening injury . Due to improvements in the diagnostic algorithms in the emergency room, blunt the most common injury mechanisms are combined movements of distraction and extension, distraction and flexion, and lateral flexion . The formation of a thrombus at the site of the intimal lesion can lead to an ischemic neurologic deficit if cerebral blood flow is compromised or embolization to the brain occurs . Even injuries of the vertebral arteries alone without any lesion of the carotid arteries can lead to symptomatic cerebral lesions . Interestingly, there seems to be no correlation between the size of the occlusion and the neurologic outcome.1 recent studies have shown tcad in 1.0 to 3.7% of all blunt trauma admissions.2 3 in patients at risk for tcad, aggressive screening has revealed an incidence of 13 to 39% of those serious vascular injuries.2 4 neurologic deficits were present in more than a quarter of patients suffering from tcad.4 cases of traumatic quadruple lesions of the cervical arteries have exhibited pronounced neurologic deficits.5 6 the following case report describes tcad of all four cervical arteries after a high - energy horse - riding injury of a woman with no resulting neurologic deficit . A 45-year - old woman was admitted to the emergency department after falling off a galloping horse without wearing a helmet . The patient was treated according to advanced trauma life support guidelines . At a glasgow coma scale of 15 points, the patient initially complained of right - sided chest pain and exhibited progressive respiratory insufficiency . After successful treatment of hematothorax with a thoracic drain, the patient was intubated and put under controlled ventilation . Diagnostics revealed right - sided pulmonary contusion, fracture of the sixth through tenth ribs, and fracture of the scapula as well as a compression fracture of the second lumbar vertebra, accompanied by extensive soft tissue contusions of the right side of the neck and shoulder . Following extubation and neurologic examination, the patient demonstrated paresis of abduction and anteversion of the right arm that was clinically allocated to an upper brachial plexus injury . Magnetic resonance imaging (mri) of the right shoulder revealed a diffuse accumulation of fluid around the upper right brachial plexus . A secondary finding was an elongated dissection of both vertebral arteries in the v2 segment (fig . Immediate mri examination of head and neck showed no signs of thromboembolic lesions and cerebral ischemia . However, imaging did show stenosis due to intimal flaps of both carotid arteries between the carotid bifurcation and the base of the skull without signs of thrombosis (fig . The diagnosis of quadruple cervical arterial injury was confirmed by computed tomography angiography (cta; fig . After 1 week, anticoagulation with heparin was stopped and antiplatelet therapy was initiated . Images from 1.5-t avanto (siemens, munich, germany) magnetic resonance imaging (mri). Flow voids in both common carotid arteries (arrows), indicating arteries are perfused normally . Thin flow voids in both vertebral arteries (va), indicating reduction of arterial lumen (open arrows). Flow voids in both vas (open arrows) but only partial flow voids in both internal carotid arteries (arrows), indicating reduced perfusion . Computed tomography angiography on 16-slice sensation (siemens, munich, germany). (a) normally perfused common carotid arteries (white arrows) but filiform - perfused vertebral arteries (va) (black arrows) at the level of the larynx . (b) filiform - perfused internal carotid arteries (ica) and normally perfused vas at the base of the skull . (c) regular perfusion of basilar artery and both icas at level of carotid canal . At the follow - up examination after 3 months mri after 3 months showed that all arterial lesions had regressed in size (fig . (a) both common carotid arteries and vertebral arteries (va) open with residual reduction of lumen of right va . (b) at the base of the skull, all four arteries are perfused; however, both internal carotid arteries still show reduced lumen . As far as we know, there has to date been no other case of an asymptomatic dissection of all four brain - supplying arteries originating from high - energy trauma to the neck . This case is highly interesting due to three facts: (1) asymptomatic tcad may be easily missed . (2) cta should be routinely performed in the emergency diagnostic procedures according to the patient's history . Of all diagnostic modalities, digital subtraction angiography (dsa) reaches the highest level of sensitivity and specificity in the detection of vascular lesions and represents the gold standard . The diagnostic accuracy of cta varies between institutions depending on the quality of the ct as well as the quality of image interpretation by the radiologist.7 in a recent prospective study, only 68 of 112 patients with cta results suggestive of tcad were confirmed by dsa.8 meta - analysis of the diagnostic accuracy of cta and dsa in the detection of traumatic cervical vascular injuries showed that sensitivity of cta (66%) was inferior to dsa, yet cta specificity (97%) was high . It was suggested that patients with cervical artery injury detected by cta, who suffered accompanying injuries indicating high risk of tcad, may receive treatment without confirmation of diagnosis by dsa.7 in clinical practice, cta is faster, less prone to complications, and more useful for diagnosing secondary injuries.9 in the face of these advantages on top of the increasing sensitivity of modern ct instruments, cta has become more and more important for the diagnosis of traumatic arterial dissections.10 with regard to our trauma protocol in the emergency room, cta has been included into the secondary trauma survey in the presence of a patient history indicating high risk of tcad . In contrast to dsa, not only does mri angiography allow screening of tcad, but it is also capable of detecting thromboembolic cerebral lesions.11 in addition, many patients with multiple injuries do not tolerate further invasive and sometimes long - lasting diagnostic procedures such as dsa . Because diagnosis of quadruple arterial dissection was positive in both cta and mri angiography, we did not perform dsa as a third diagnostic procedure after risk - benefit evaluation . In our case, surgical therapy of the vascular injuries was never considered due to the fact that neurologic deficit, signs of acute hemorrhage, or ischemia was not detected . In fact, tcad may be more benign than previously thought and therefore only requires aggressive surgical treatment in very rare cases.12 finally, any surgical procedure to be performed in a patient with multiple injuries has to be carefully evaluated . This latter point clearly makes the difference between a patient with tcad and a patient suffering from chronic stenosis of the carotid arteries . Most authors agree that some form of antithrombotic therapy is necessary after diagnosis of those injuries, although the overall incidence of ischemic stroke among patients with tcad is low.8 however, the type of antithrombotic therapy is controversial . The use of heparin is more popular than antiplatelet therapy, but the risk of hemorrhagic complications is high.2 13 14 15 16 nevertheless, we decided to use heparin in our patient with multiple injuries because of its capability to maintain continuous and immediate control of anticoagulation . After 1 week, during which the patient remained asymptomatic, her vital functions had stabilized, and no surgical procedures had been necessary, the treatment was changed to antiplatelet therapy . The latter have been found to be equivalent or even superior to heparin therapy with regard to thromboembolic complications, but with a more favorable profile of potential side effects . 17 18 19 this is in agreement with a recent study by cothren and coworkers.17 the authors looked at the therapeutic options after blunt tcad and clearly demonstrated that the type of treatment, heparin versus antiplatelet agents, does not appear to significantly affect either the risk of stroke or the injury healing rates.17 we conclude from the experience with our case and the recent literature that anticoagulation in the presence of tcad is necessary but may be different from patients with chronic atherosclerosis . We recommend heparin at the beginning because of its excellent capability of dose and effect regulation.
Atrial fibrillation (af) is the most common sustained cardiac arrhythmia and a major cause of substantial mortality and morbidity from stroke, thromboembolism, and heart failure, leading to an impaired quality of life.1 - 5) with the increase of elderly population in the developed world, the prevalence of af is increasing, resulting in a major public health problem.6) maintenance of sinus rhythm is the main therapeutic goal in patients with af . Radiofrequency catheter ablation (rfca) for af has been proposed as an effective therapeutic option for af that is resistant to pharmacologic rhythm or rate control, with successful long - term maintenance of sinus rhythm in the absence of treatment with anti - arrhythmic drugs reported in the majority of patients.7) however, the recurrence of af after rfca is not uncommon . The recurrence rate of af after rfca has been reported to be between 30 and 40%, depending on the ablation strategy and the type of af.7 - 10) some investigators have reported that the left atrial diameter / volume and hypertension are predictors of af recurrence after rfca.11)12) recent research has focused increasingly on atrial structural remodeling and electrical dysfunction, which underlie the development of af in different pathologic conditions.13 - 17) a particular interest has been generated in the role of renin - angiotensin system (ras) blockade in reversing the electrical and structural remodeling of diseased atria . Angiotensin - converting enzyme inhibitors (aceis) are known to suppress structural and functional remodeling and prevent the induction and promotion of af in chronic rapid atrial pacing dogs.18) in several animal models, it has been suggested that angiotension ii type 1 receptor blockers (arbs) might be useful for preventing af recurrence after the termination of sustained af by decreasing interstitial fibrosis.19)20) however, there is still uncertainty about the role of aceis and arbs for the reduction of af recurrence after rfca in clinical practice . This study was aimed to evaluate the effect of aceis and arbs on the recurrence of af after rfca . We retrospectively evaluated 152 patients (mean age, 5710 years; m: f=94: 58) who underwent af ablation due to drug - refractory paroxysmal (mean age, 5710 years; m: f=58: 43) or persistent af (mean age, 5610 years; m: f=36: 15). Among the 152 patients, 101 had paroxysmal af (paf) and 51 had persistent af (peaf). Paf was defined as the occurrence of two or more episodes of af during the previous 12 months, typically lasting <7 days and terminating spontaneously . Peaf was defined as the occurrence of af episodes lasting <7 days, and typically requiring cardioversion for restoration of normal sinus rhythm . The af ablation strategy was 3d mapping (carto)-guided circumferential ablation, pulmonary vein ostial ablation, and roof line, mitral valve isthmus, and right cavotricuspid isthmus block ., the patients were followed 1 - 2 weeks later, and then visited our hospital every 1 - 3 months during the first year after rfca . Whenever the patients visited our clinic, a history was taken and a physical examination was performed . Additionally, a 12-lead electrocardiography (ecg) and 24-hour ambulatory holter ecg monitoring (24 hours holter monitoring) were obtained . Thereafter, we immediately checked a 12-lead ecg and 24 hours holter monitoring if the patients complained of symptoms, such as palpitations that suggest af . Twenty - four hours holter monitoring was also performed on asymptomatic patients every 6 months . Recurrence was defined if the patients showed af on a 12-lead ecg and/or on 24 hours holter monitoring, or complained of typical episodes of palpitation sat the 3-month follow - up visit after rfca . We compared the recurrence rates between the paf and peaf groups that received aceis or arbs . Aceis or arbs were prescribed at least 1 month prior to ablation and for the duration of the follow - up . The statistical package for social sciences (spss) for windows, version 15.0 (chicago, il, usa) was used for all analyses . The continuous variables are presented as the mean valuesstandard deviations (sds) and were compared using paired or unpaired student's t - tests . The statistical package for social sciences (spss) for windows, version 15.0 (chicago, il, usa) was used for all analyses . The continuous variables are presented as the mean valuesstandard deviations (sds) and were compared using paired or unpaired student's t - tests . The paf and peaf groups, with or without aceis or arbs, were similar with respect to important baseline clinical characteristics (table 1 and 2). Furthermore, there was no difference in medications, including anti - arrhythmic drugs other than aceis / arbs for the paf and peaffgroups (table 3 and 4) in paf patients, no significant differences existed in the recurrence rates between the group with aceis or arbs and the group without aceis or arbs (24.2% vs. 22.9%, p=0.87) (fig . 1). However, in peaf patients, compared with the group not using aceis or arbs, the recurrence rate was significantly decreased in the group using aceis or arbs (12.1% vs. 61.1%, p<0.01) (fig . The left atrium dimension (44.28.4 mm vs. 44.35.8 mm, respectively, p=0.45) and the left ventricle ejection fraction (626.5% vs. 61.56.2%, respectively, p=0.28) were not significantly different . In multivariate analysis, the use of aceis or arbs was independently associated with recurrence after adjusting for the left atrium dimension and the left ventricle ejection fraction (or=0.078, 95% ci=0.02 - 0.35, p<0.01). The paroxysms of af are consistently initiated by a spontaneous firing focus arising from several sites, including the pulmonary veins,21 - 24) the vein of marshall,25) the coronary sinus,23)26) the crista terminalis,23)26) the superior vena cava, and the inferior vena cava . However, electrophysiologic remodeling is commonly observed and is probably an important factor contributing to the persistence of af . In addition to eletrophysiologic remodeling, structural remodeling, such as fibrosis (at both the subcellular and tissue levels) is also often present and contributes to the altered tissue substrate that promotes af maintenance.27) although there are many anti - arrhythmic drugs available to maintain sinus rhythm and reduce the ventricular rate, there is no drug which can control af effectively . Because rfca can effectively treat patients with drug - refractory af, rfca has emerged as a major treatment option for af . However, this procedure is limited by a significant rate of af recurrence and the effect of rfca on patients with peaf is not as effective as on paf . This phenomenon may be caused by structural and electrical remodeling of the atrium during the long period of af . Several experimental studies have demonstrated that atrial cells express all components of ras in a pig model of af,28) remodeling of the atrium correlated with a high level of angiotensin in atrial tissue, and blockade of ras through aceis or arbs can reverse atrial remodeling.13)18)29) nakashima et al.30) first demonstrated the role of aceis and arbs in preventing atrial electrical remodeling . They found that in dogs in which the atria were paced at 800 beats / min, candesartan and captopril prevented the atrial effective refractory period from shortening . In the current study, the overall recurrence rate after ablation therapy was 26% (n=39) the recurrence rate was significantly decreased in the peaf group that used aceis or arbs (12.1% vs. 61.1%, p<0.01). This result might be caused by the effect of aceis or arbs on alteration of left atrial remodeling and reduction of interstitial fibrosis through ras blockade . However, this difference in the recurrence rate was not observed in paf patients (24.2% vs. 22.9%, p=0.87). We presume that the reason for this outcome might be caused by difference of pathophysiologic mechanisms between paf and peaf . That is to say, paf is mainly triggered by electric foci, but electrophysiologic or structural remodeling underlie peaf . Therefore, aceis or arbs which can alter remodeling, played an important role in reducing the recurrence rate of peaf, but not paf . Aceis or arbs might be effective in preventing af recurrence after catheter ablation in peaf patients . Aceis or arbs might be effective in preventing af recurrence after catheter ablation in peaf patients.
Comorbidity of anxiety and depression is substantially high, about 55% to 76% of the individuals with a lifetime history of major depression and anxiety report anxiety and depression respectively (1). Furthermore, patients with comorbidity of anxiety and depression have earlier age of onset, greater likelihood of suicide, greater symptom severity, higher chronicity, greater impairment in functioning and poorer overall quality of life (2 - 5). Traditionally, cognitive - behavioral therapy focuses on specific disorder approach . Specific manuals are developed that account for the assessment and treatment protocols in specific disorders . Clinicians and therapists are trained according to these manuals, but the high comorbidity among emotional disorders, especially anxiety and unipolar mood disorders leads many researchers and clinicians to find out the reasons and explanations that illuminate this darkness (6). These struggles open the scientific gate to exploring shared processes common across multiple disorders including depression and anxiety, called transdiagnostic components . The transdiagnostic approach assumes an incremental value to understand these shared maintenance factors underlying psychological disorders . Many surveys are done regarding the role of some of the transdiagnostic components in many mental disorders especially depression and anxiety . Some of these studies try to answer what differences there are between patients with anxiety and those with depression in almost each of these common vulnerabilities . Most of the aforementioned studies found no differences (7 - 13), but some others revealed small or medium differences (14 - 16). In addition to the interrelationship between all shared vulnerabilities to anxiety and depression symptoms, there is evidence supporting the idea of correlations of some of these common factors . These two variables are highly correlated with each other; however remain as separate variables that predict depression and anxiety symptoms over time (17). Furthermore, there are significant relationships between neuroticism and rumination, worry and depression (17). Another study showed significant correlation between worry, rumination, intolerance of uncertainty and anxiety symptoms . Results showed that worry partially mediated the relationship between intolerance of uncertainty and anxiety and rumination fully mediated the relationship between intolerance of uncertainty and depression symptoms (18). In addition, rumination and worry are considered by different researchers and clinicians as different kinds of avoidance and emotion regulation strategies (19, 20). Suppression as one of the emotion regulation strategies is considered a kind of experiential avoidance (21). From a more specific perspective, some of these shared variables are mediators which connect other common components such as neuroticism and experiential avoidance to anxiety and depression . For example, emotional avoidance and non - acceptance of negative emotions are associated with anxiety and depressive related psychopathology (10, 12, 22). Furthermore, experiential avoidance predicts depression and anxiety such as posttraumatic stress disorder (ptsd) (23, 24). In addition, evidence shows that experiential avoidance is more than an emotional disorder phenomenon and may have a casual effect on the course of emotional disorder (23, 25). Individuals with anxiety and depressive symptoms use emotion regulation maladaptive strategies to avoid experiencing negative and unwanted emotions; therefore, emotion regulation has mediating effect in the relationship between some common factors such as neuroticism and experiential avoidance, and anxiety and depression symptoms, and their related psychopathology . The mediating role of emotion regulation is demonstrated in some researches (26) and has been the treatment target in several recent treatment approaches (27, 28). While difficulties in emotion regulation and maladaptive emotion regulation strategies contribute to emotional disorders, personality traits such as neuroticism may also play a pivotal role in etiology and maintenance of emotional disorders (29, 30). Moreover, there is a strong relationship between neuroticism and emotion regulation deficits (31, 32). The boundaries between transdiagnostic components including emotion regulation components (emotional clarity, non - acceptance of emotion, etc . ), worry (as an emotion regulation strategy), experiential avoidance and neuroticism are unclear (33, 34); hence, further study is needed to determine which of these variables have more casual role in relation to other variables for prediction anxiety and depression as emotional disorder symptoms . The current study aimed to assess the mediating roles of emotion regulation deficits and worry, as a maladaptive emotion regulation strategy, between experiential avoidance and neuroticism (as independent variables), and anxiety and depression symptoms (as dependent variables). Three hundred and thirty individuals at university of social welfare and rehabilitation sciences, tehran university of medical sciences and iran university of medical sciences volunteered to complete a series of questionnaires (through convenient sampling). Fourteen participants were dropped from the sample due to missing data and deleting outliers after data cleaning, resulting in a sample size of 316 individuals . To assess experiential avoidance and neuroticism as independent variables, worry and emotion regulation as mediator variables, and anxiety and depression as dependent variables, authors employed acceptance and action questionnaire - ii (aaq - ii), the neuroticism scale of shortened and revised form of eysenck personality questionnaire scale (epq - rs), penn state worry questionnaire (pswq), difficulties in emotion regulation scale (ders), beck anxiety questionnaire (bai) and beck depression questionnaire (bdi - ii), respectively . The acceptance and action questionnaire (aaq - ii) was first developed in 2004 (35). To reduce defects of the former version and make it more applicable, the second version of aaq - ii was then developed (36). It is a 10-item scale and assesses the tendency to evaluate unwanted thoughts and feelings negatively, not accept and try to alter or escape from them (37). In total, it refers to a construct meaning acceptance, experiential avoidance and psychological flexibility . Responses range from 1 (never true) to 7 (always true) with higher scores reflecting greater psychological flexibility . The mean alpha coefficient is reported 0.84 (0.78 - 0.88) and the three- and twelve - month test - retest reliability values were 0.81 and 0.79 respectively (36). The internal consistency of the persian version of the aaq - ii in general and clinical population is high, = 0.71 - 0.84 . Moreover, avoidance of emotional experiences significantly correlated with depressive and anxiety symptoms as well as poor mental health (38). It consists of four subscales: introversion (stability /emotionality) extraversion (extraversion / introversion), psychoticism and a lie subscale to reveal falsehood . The neuroticism subscale is a 12-item subscale with dichotomous (yes or no) answers . It is used for the age range 16 - 70 years . The internal consistency () for the neuroticism subscales for male and female were = 0.84 and = 0.80, respectively (39). Psychometric properties of iranian version of epq - rs are reported acceptable with high internal consistency, r = 0.74 (40). Each statement is scored on a 5-point likert scale ranging from 1 to 5 yielding a total score ranging from 16 to 80, with higher scores indicating greater worry levels (41). The scale has very good internal consistency (= of 0.93) and high test - retest reliability (r = 0.74 - 0.93) (41). The pswq has also quite favorable internal consistency in patients with generalized anxiety disorder (gad), different anxiety disorder groups (0.86 to 0.95) and the control group (0.90) (42). Psychometric properties of persian version of the scale had high internal consistency (0.88) and test - retest reliability (0.79). In addition, the significant correlation between pswq and two variables of trait anxiety (0.68) and depression (0.49) indicated good validity of pswq (43). The ders is a 36-item scale (44) and assesses individuals typical tendencies toward emotion regulation . It consists of several facets such as: 1- non - acceptance of emotional responses (nonacceptance); 2- difficulties engaging in goal directed behaviors (goals); 3- impulse control difficulties (impulse); 4- lack of emotional awareness (awareness); 5- limited access to emotion regulation strategies (strategies) and 6- lack of emotional clarity (clarity). The ders has high internal consistency (= higher than 0.80 for each subscale) and good test - retest reliability (r = 0.88) furthermore the correlation between ders and zuckerman - kuhlman personality questionnaire was significant, showing acceptable validity of the questionnaire (46). The bai consists of 21 items in a 4-point likert scale that assesses cognitive and somatic symptoms of anxiety . Scores range from 0 to 63, with higher scores indicating higher levels of anxiety (47). Bai has high internal consistency (= 0.92) and test - retest reliability over a week (r = 0.75). In addition, the correlation between bai and the revised hamilton anxiety rating scale is moderate (0.51) (48). Good and acceptable psychometric properties are reported by other studies in clinical and non - clinical samples (49, 50). The persian version of bai has a good reliability (0.72), a very good validity (0.83) and an excellent internal consistency (alpha = 0.92) (51). Each item is scored on a likert scale from 0 to 3, with higher scores indicating greater depressive symptoms . Total scores are obtained by summing all items and range from 0 to 63 (52). Bdi mean coefficient alpha is reported 0.86 in psychiatric populations and 0.81 in non - psychiatric populations . Its mean test - retest reliability is reported 0.86 (47). In iran, in a study on 354 patients with recovered depressed, coefficient alpha, test - retest over a week and convergent validity (with bdi) were reported 0.91, 0.81, and 0.61, respectively (53). Participants were recruited from university campuses and dormitories . After signing informed consent and answering any of their questions about the research, they were given a packet of questionnaires . The current cross - sectional study used path analyze via regression by spss ver . 20 to analyze the data . The recommended procedure and evaluative criteria of baron and kenny (54) experiential avoidance and neuroticism were independent variables, emotion regulation and worry (as a kind of emotion regulation strategy) were mediator variables, and anxiety and depression were dependent variables . Three hundred and thirty individuals at university of social welfare and rehabilitation sciences, tehran university of medical sciences and iran university of medical sciences volunteered to complete a series of questionnaires (through convenient sampling). Fourteen participants were dropped from the sample due to missing data and deleting outliers after data cleaning, resulting in a sample size of 316 individuals . To assess experiential avoidance and neuroticism as independent variables, worry and emotion regulation as mediator variables, and anxiety and depression as dependent variables, authors employed acceptance and action questionnaire - ii (aaq - ii), the neuroticism scale of shortened and revised form of eysenck personality questionnaire scale (epq - rs), penn state worry questionnaire (pswq), difficulties in emotion regulation scale (ders), beck anxiety questionnaire (bai) and beck depression questionnaire (bdi - ii), respectively . The acceptance and action questionnaire (aaq - ii) was first developed in 2004 (35). To reduce defects of the former version and make it more applicable, the second version of aaq - ii was then developed (36). It is a 10-item scale and assesses the tendency to evaluate unwanted thoughts and feelings negatively, not accept and try to alter or escape from them (37). In total responses range from 1 (never true) to 7 (always true) with higher scores reflecting greater psychological flexibility . The mean alpha coefficient is reported 0.84 (0.78 - 0.88) and the three- and twelve - month test - retest reliability values were 0.81 and 0.79 respectively (36). The internal consistency of the persian version of the aaq - ii in general and clinical population is high, = 0.71 - 0.84 . Moreover, avoidance of emotional experiences significantly correlated with depressive and anxiety symptoms as well as poor mental health (38). It consists of four subscales: introversion (stability /emotionality) extraversion (extraversion / introversion), psychoticism and a lie subscale to reveal falsehood . The neuroticism subscale is a 12-item subscale with dichotomous (yes or no) answers . It is used for the age range 16 - 70 years . The internal consistency () for the neuroticism subscales for male and female were = 0.84 and = 0.80, respectively (39). Psychometric properties of iranian version of epq - rs are reported acceptable with high internal consistency, r = 0.74 (40). Each statement is scored on a 5-point likert scale ranging from 1 to 5 yielding a total score ranging from 16 to 80, with higher scores indicating greater worry levels (41). The scale has very good internal consistency (= of 0.93) and high test - retest reliability (r = 0.74 - 0.93) (41). The pswq has also quite favorable internal consistency in patients with generalized anxiety disorder (gad), different anxiety disorder groups (0.86 to 0.95) and the control group (0.90) (42). Psychometric properties of persian version of the scale had high internal consistency (0.88) and test - retest reliability (0.79). In addition, the significant correlation between pswq and two variables of trait anxiety (0.68) and depression (0.49) indicated good validity of pswq (43). The ders is a 36-item scale (44) and assesses individuals typical tendencies toward emotion regulation . It consists of several facets such as: 1- non - acceptance of emotional responses (nonacceptance); 2- difficulties engaging in goal directed behaviors (goals); 3- impulse control difficulties (impulse); 4- lack of emotional awareness (awareness); 5- limited access to emotion regulation strategies (strategies) and 6- lack of emotional clarity (clarity). The ders has high internal consistency (= higher than 0.80 for each subscale) and good test - retest reliability (r = 0.88). Furthermore the correlation between ders and zuckerman - kuhlman personality questionnaire was significant, showing acceptable validity of the questionnaire (46). The bai consists of 21 items in a 4-point likert scale that assesses cognitive and somatic symptoms of anxiety . Scores range from 0 to 63, with higher scores indicating higher levels of anxiety (47). Bai has high internal consistency (= 0.92) and test - retest reliability over a week (r = 0.75). In addition, the correlation between bai and the revised hamilton anxiety rating scale is moderate (0.51) (48). Good and acceptable psychometric properties are reported by other studies in clinical and non - clinical samples (49, 50). The persian version of bai has a good reliability (0.72), a very good validity (0.83) and an excellent internal consistency (alpha = 0.92) (51). Each item is scored on a likert scale from 0 to 3, with higher scores indicating greater depressive symptoms . Total scores are obtained by summing all items and range from 0 to 63 (52). Bdi mean coefficient alpha is reported 0.86 in psychiatric populations and 0.81 in non - psychiatric populations . Its mean test - retest reliability is reported 0.86 (47). In iran, in a study on 354 patients with recovered depressed, coefficient alpha, test - retest over a week and convergent validity (with bdi) were reported 0.91, 0.81, and 0.61, respectively (53). The acceptance and action questionnaire (aaq - ii) was first developed in 2004 (35). To reduce defects of the former version and make it more applicable, the second version of aaq - ii was then developed (36). It is a 10-item scale and assesses the tendency to evaluate unwanted thoughts and feelings negatively, not accept and try to alter or escape from them (37). In total, it refers to a construct meaning acceptance, experiential avoidance and psychological flexibility . Responses range from 1 (never true) to 7 (always true) with higher scores reflecting greater psychological flexibility . The mean alpha coefficient is reported 0.84 (0.78 - 0.88) and the three- and twelve - month test - retest reliability values were 0.81 and 0.79 respectively (36). The internal consistency of the persian version of the aaq - ii in general and clinical population is high, = 0.71 - 0.84 . Moreover, avoidance of emotional experiences significantly correlated with depressive and anxiety symptoms as well as poor mental health (38). It consists of four subscales: introversion (stability /emotionality) extraversion (extraversion / introversion), psychoticism and a lie subscale to reveal falsehood . The neuroticism subscale is a 12-item subscale with dichotomous (yes or no) answers . It is used for the age range 16 - 70 years . The internal consistency () for the neuroticism subscales for male and female were = 0.84 and = 0.80, respectively (39). Psychometric properties of iranian version of epq - rs are reported acceptable with high internal consistency, r = 0.74 (40). Each statement is scored on a 5-point likert scale ranging from 1 to 5 yielding a total score ranging from 16 to 80, with higher scores indicating greater worry levels (41). The scale has very good internal consistency (= of 0.93) and high test - retest reliability (r = 0.74 - 0.93) (41). The pswq has also quite favorable internal consistency in patients with generalized anxiety disorder (gad), different anxiety disorder groups (0.86 to 0.95) and the control group (0.90) (42). Psychometric properties of persian version of the scale had high internal consistency (0.88) and test - retest reliability (0.79). In addition, the significant correlation between pswq and two variables of trait anxiety (0.68) and depression (0.49) indicated good validity of pswq (43). The ders is a 36-item scale (44) and assesses individuals typical tendencies toward emotion regulation . It consists of several facets such as: 1- non - acceptance of emotional responses (nonacceptance); 2- difficulties engaging in goal directed behaviors (goals); 3- impulse control difficulties (impulse); 4- lack of emotional awareness (awareness); 5- limited access to emotion regulation strategies (strategies) and 6- lack of emotional clarity (clarity). The ders has high internal consistency (= higher than 0.80 for each subscale) and good test - retest reliability (r = 0.88). Furthermore the correlation between ders and zuckerman - kuhlman personality questionnaire was significant, showing acceptable validity of the questionnaire (46). The bai consists of 21 items in a 4-point likert scale that assesses cognitive and somatic symptoms of anxiety . Scores range from 0 to 63, with higher scores indicating higher levels of anxiety (47). Bai has high internal consistency (= 0.92) and test - retest reliability over a week (r = 0.75). In addition, the correlation between bai and the revised hamilton anxiety rating scale is moderate (0.51) (48). Good and acceptable psychometric properties are reported by other studies in clinical and non - clinical samples (49, 50). The persian version of bai has a good reliability (0.72), a very good validity (0.83) and an excellent internal consistency (alpha = 0.92) (51). Each item is scored on a likert scale from 0 to 3, with higher scores indicating greater depressive symptoms . Total scores are obtained by summing all items and range from 0 to 63 (52). Bdi mean coefficient alpha is reported 0.86 in psychiatric populations and 0.81 in non - psychiatric populations . Its mean test - retest reliability is reported 0.86 (47). In iran, in a study on 354 patients with recovered depressed, coefficient alpha, test - retest over a week and convergent validity (with bdi) were reported 0.91, 0.81, and 0.61, respectively (53). Participants were recruited from university campuses and dormitories . After signing informed consent and answering any of their questions about the research, they were given a packet of questionnaires . The current cross - sectional study used path analyze via regression by spss ver . 20 to analyze the data . The recommended procedure and evaluative criteria of baron and kenny (54) experiential avoidance and neuroticism were independent variables, emotion regulation and worry (as a kind of emotion regulation strategy) were mediator variables, and anxiety and depression were dependent variables . The sample consisted of 160 (50.6%) females and 156 (49.4%) males with the mean age of 22.3 (sd = 2.89), ranged from 18 to 35 years . In terms of marital status, 56.6% were undergraduate and 43.4% graduate students (master student = 33.5% and doctoral student = 9.8%). Raw mean scores, standard deviations and correlations among all main study variables are reported in table 1 . After making sure that multicollinearity was not a problem, a series of regression analysis was done to assess these four hypotheses: 1, there are significant relationships between experiential avoidance and anxiety, experiential avoidance and depression, neuroticism and anxiety, and neuroticism and depression; 2, there are significant relationships between experiential avoidance and emotion regulation, experiential avoidance and worry, neuroticism and emotion regulation and neuroticism and worry; 3, there are significant relationships between emotion regulation and anxiety, emotion regulation and depression, worry and anxiety, worry and depression and all of the aforementioned relations are met; 4, there are significant relationships between all independent variables (experiential avoidance, neuroticism, emotion regulation and worry) and dependent variables (anxiety and depression). The first regression analysis used aaq - ii and neuroticism as predictor variables and bai as dependent variable: experiential avoidance (beta = -0.115, t = -2.105, p <0.036) and neuroticism (beta = 0.440, t = -0.8.078, p <0.001) were significant predictors of anxiety, r = 0.502, f (2, 313) = 52.591, p <0.001 . The second regression analysis used aaq - ii and neuroticism as independent variables and ders as dependent variable: both aaq - ii (beta = -0.202, t = -4.224, p <0.001) and neuroticism (beta = 0.538, t = 11.271, p <0.001) were significant predictors of ders, r = 0.653, f(2, 313) = 116.055, p <0.001; for the second mediator, another analysis used aaq - ii and neuroticism as independent variables and pswq as dependent variable: both aaq - ii (beta = -0.171, t = -3.311, p <0.001) and neuroticism (beta = 0.474, t = 9.175, p <0.001) were significant predictors of pswq, r = 0.571, f (2, 313) = 75.728, p <0.001 . The third regression analysis used ders and pswq as independent variables and bai as dependent variable: both ders (beta = 0.328, t = 5.501, p <0.001) and pswq (beta = 0.243, t = 4.083, p <0.001) were significant predictors of bai, r = 0.508, f (2, 313) = 54.471, p <0.001 . The fourth regression analysis used all aaq - ii, neuroticism, ders and pwsq as independent variables and bai as dependent variable: all variables including aaq - ii (beta = -0.049, t = -0.9, p <0.369), neuroticism (beta = 0.263, t = 4.041, p <0.001), ders (beta = 0.19, t = 2.864, p <0.004), and pswq (beta = 0.16, t = 2.612, p <0.009) were significant predictors of bai, r = 0.549, f (4, 311) = 33.576, p <0.001 . In total, results showed that pswq especially ders were mediating the relationship between neuroticism and bai partially and the relationship between aaq - ii and bai fully . The path model of the results is presented in figure 1 . To predict depression, another series of regressions were done . The first regression analysis used aaq - ii and neuroticism as predictor variables and bdi as dependent variable: experiential avoidance (beta = -0.211, t = -4.2, p <0.001) and 0.001) were significant predictors of bdi, r = 0.605, f (2, 313) = 90.238, p <0.001 . The second regression analysis used aaq - ii and neuroticism as independent variables and ders as dependent variable: both aaq - ii (beta = -0.202, t = -4.224, p <0.001) and neuroticism (beta = -0.538, t = -11.271, p <r = 0.653, f (2, 313) = 116.055, p <0.001; for the second mediator, another analysis used aaq - ii and neuroticism as independent variables and pswq as dependent variable: both aaq - ii (beta = -0.171, t = -3.311, p <0.001) and neuroticism (beta = 0.474, t = 9.175, p <0.001) were significant predictors of pswq, r = 0.571, f (2, 313) = 75.728, p <0.001 . The third regression analysis used ders and pswq as independent variables and bdi as dependent variable: both ders (beta = 0.438, t= 9.067, p <0.001) and pswq (beta = 0.223, t = 4.196, p <0.001) were significant predictors of bdi, r = 0.638, f (2, 313) = 107.460, p <0.001 . The fourth regression analysis used all aaq - ii, neuroticism, ders and pwsq as independent variables and bdi as dependent variable: all variables including aaq - ii (beta = -0.121, t = -2.521, p <0.012), neuroticism (beta = 0.241, t = 4.205, p <0.001), ders (beta = 0.331, t = 5.682, p <0.001), and pswq (beta = 0.132, t = 2.457, p r = 0.677, f (4, 311) = 65.768, p <0.001 . In total, results showed that pswq especially ders were mediating the relationship between neuroticism and bdi and the relationship between aaq and bdi, partially . As hypothesized, present data support the notion that both neuroticism (negative affect) and experiential avoidance predict anxiety and depression symptoms independently; and also mediate the effects of difficulties in emotion regulation and worry . It appears that heightened neuroticism is associated with increase in difficulties in emotion regulation and worry which contribute to anxiety and depression symptoms . Since watson and clark proposed the tripartite model, the important role of neuroticism or negative affectivity is underscored in the etiology and maintenance of anxiety and depression as a common factor (55). In fact, individuals with high neuroticism may be more prone to anxiety and depression symptoms (56, 57). However, many individuals with heightened neuroticism may not exhibit high levels of anxiety and depressive symptomatology; therefore, there may be other factors which mediate or moderate the relationship between neuroticism and anxiety and depression symptoms such as emotion regulation and worry shown in the present study . The mediating effect of emotion regulation in the relationship between negative affect and anxiety and depression was demonstrated previously (58). A study using structural equation modeling tested the relationship between negative affect, emotion regulation and psychological distress in clinical and nonclinical samples; it suggested that negative effect leads people to control negative emotions via maladaptive emotion regulation strategies and these lead to psychopathology (59). Neuroticism or negative emotionality as a higher - order factor operates from a distinct motivational system, and the behavioral inhibition system (bis); therefore, the individuals with high levels of neuroticism exhibit higher levels of bis sensitivity which is somehow underlying factor of neuroticism and may explain casualty better and more precisely (61). The aversive motivational systems (bis system) are gaining attention of researchers (62) and the most recent psychological treatments that target emotion regulation strategies and emotion regulation therapy, use the motivation and behavioral approach system (bas) and inhibition system in conceptualizing and targeting it in corresponded treatment via some emotional and cognitive techniques (63). Therefore, it seems more reasonable and more effective to target some underlying factors such as neuroticism, besides emotion regulation . Experiential avoidance as an independent variable in the present study has significant relationship with anxiety and depression related psychopathology (22, 25), that is consistent with the results of the current study . Furthermore, people with lower psychological flexibility or higher experiential avoidance level rarely tolerate the negative emotion and to avoid or lessen the experience of negative emotion engage in series of emotion regulation strategies . This will lead to temporary relief, and this negative reinforcement is the cause of repeating maladaptive behavior (34). Additionally, experiential avoidance has robust relationship with emotion regulation and some theorists suggest that the experiential avoidance is a kind of emotion regulation such as suppression (64). The pivotal role of experiential avoidance in the etiology and maintenance of emotional disorders were indicated in some recent and evidence based treatments, such as acceptance and commitment therapy (65), dialectical behavior therapy (66)), mindfulness - base cognitive therapy (67), emotion - focused therapy (68) and the emotion regulation therapy (63). As mentioned, many researchers tried to find different pathways that connect emotional disorders and common etiological and maintenance factors, but it was not enough . Assessment and psychotherapy areas need more subtle investigation to find out the route to more straight, economical and effective instrument to assess and cue emotions and comorbid disorders via affecting fundamental factors . First, the design of the present study was cross - sectional which limited establishment of the casual links . Furthermore, the analysis method was path analysis via regression with some limitations, path analysis or structural equation modeling using amose and other more advanced methods with fewer errors would lead to more valid and reliable results . Second, there was no consistent definition of emotion regulation concept and different approaches yield distinct conceptualization and related instruments to assess the concepts; therefore, the measure of emotion regulation used in the present study was just one of many instruments designed to assess emotion regulation, and the results of the present study should be interpreted for other emotion regulation concepts cautiously . Third, the current study used some limited variables as mediators and there were more transdiagnostic variables that may be mediating the relationships between other common underlying factors and emotional disorders . Hence, future studies are needed to examine the mediating or moderating roles of variables . Finally, the present study sample was nonclinical; therefore, future studies should be done on clinical samples.
Nine participants (6 women; 3 men, 1950 years) with no history of neurological injury participated . Two participants were subsequently excluded due to excessive head movement (> 5 mm). Previous studies have shown that n = 7 is sufficient to reveal significant gamma - band differences across conditions (adjamian, hadjipapas, barnes, hillebrand, & holliday, 2008). Experimental procedures were in accordance with the declaration of helsinki and received approval from the university of aston's ethical committee . Angry, fearful, and neutral faces were selected from the nimstim face set (tottenham et al ., 2009). Eight actors (four men) posing three different expressions (angry, fearful, and neutral) were selected (figure 1a). In all cases, actors depicted in the stimuli displayed a direct gaze and were photographed straight on . All stimuli were gray - scaled, matched for size, and fitted to an oval - shaped layer of dimensions 2 3 (this layer obscured nonfacial features, e.g., hair, earrings). An in - house matlab script was then used to standardize the faces for luminance and contrast (the mathworks, inc ., uk). Data were collected with a 275-channel, whole - head neuromagnetometer scanner at a sampling rate of 625 hz, using a third - order gradiometer configuration with an antialiasing filter cutoff of 200 hz . Participants were seated in an upright position, and three electromagnetic coils were fastened to the participant's nasion and auricular points respectively, to determine head position within the meg helmet . The display monitor was positioned outside the shielded room and viewed through a window in the room, using a front - silvered mirror . The stimuli were viewed binocularly at an optical viewing distance of 2 m. during the recording session, each subject's head was stabilized within the helmet with an inflatable head - cuff . (a) angry versus neutral facial expressions: a group snpm comparison for the time window 50250 ms demonstrates a power difference in ba 18, bilateral lingual gyrus (p <.05, 23 significant voxels; 6, 87, 12). The threshold was set at p <.10, and significant voxels (i.e., p <.05) are white in color . (b) a time - frequency plot for a significant voxel in ba 18 averaged across participants for the angry face condition demonstrates a decrease in power at approximately 100 ms within the 5080 hz range for the angry face stimuli . This power decrease was absent for the neutral facial expressions (results not shown). On each trial, a single face was presented for 200 ms with a 1.31.6 s interstimulus interval (isi). Stimulus eccentricity corresponded to a visual angle of 1.5 (i.e., central vision). Simultaneously, a stream of mirror - reversed letters (one every 350 ms, no isi) was presented at central fixation (i.e., letters appeared non - reversed). By a button - press response device, participants were required to respond to the letter x, which appeared on 10% of trials . This task design (1) rendered the faces task - irrelevant and (2) helped maintain an alert attentive state in the participants . Data from trials displaying an x or those where a button - press response was recorded were not analyzed . For each stimulus type, data from approximately 100 trials were recorded over two experimental runs of 8 min duration . Immediately after data acquisition, a polhemus isotrak 3d digitizer was used to map the surface shape of each participant's head and localize the electromagnetic head coils (medsim, usa). These surface points were then coregistered with the individual participant's anatomical mri by a surface - matching procedure (huppertz et al ., 1998). Data were bandpass filtered between 0.5 and 100 hz and dc corrected according to a pre - stimulus baseline, in addition to a 50 hz powerline filter . Moreover, all data were visually inspected, enabling the removal of trials with eye - blink (e.g., trials in which an extreme dipolar frontal pattern was observed to emerge within the recorded epoch) and/or movement artifacts (e.g., trials in which noise was observed for a period of 25% or more of the recorded epoch). Synthetic aperture magnetometry (sam) was then used to spatially map task - related power changes in oscillatory brain activity across participants . This method uses the same principle of fixed array - weighted channels as found in contemporary radar systems to scan the brain for the sources of magnetic signals recorded at the scalp (hillebrand, singh, holliday, furlong, & barnes, 2005). Therefore, sam requires no a priori assumptions as to the number of sources activated and is ideally suited for the analysis of neuronal activity not strictly time - locked to stimulus onset? I.e ., induced activity . In brief, when using sam, for each voxel in a predefined source space, an optimal spatial filter is constructed that links activity in that voxel to the meg system's sensor array . In the present study, each individual's mri was divided into voxels of 5 5 5 mm (5 mm grids). The filter output for each voxel was calculated independently as the weighted sum of the sensor signals, yielding a measure of current density as a function of time (hillebrand et al ., 2005). This procedure is conceptually similar to the use of an invasive electrode at the neural source location; therefore, the spatial filter is often termed a virtual electrode (barnes & hillebrand, 2003). The sam beamformer technique also actively suppresses any undetected noise or artifact sources that may have occurred in spatially removed locations such as around the eyes (e.g., kinsey, anderson, hadjipapas, & holliday, 2011). For the time windows 50250 ms (early differences), 250450 ms (later differences), and 100600 ms (sustained differences) post - stimulus onset, statistical maps (t maps) of the difference between the facial expressions were calculated for gamma - band oscillations . These analyses involved assessing the difference between spectral power estimates for every voxel with a pseudo - t statistic (robinson & vrba, 1999), enabling a 3d image of activity to be generated for every participant and time window . Data were then normalized and nonparametric permutation analyses (singh, barnes, & hillebrand, 2003) performed with the snpm toolbox (www.fil.ion.ucl.ac.uk/spm/snpm/) to assess significant group effects for voxel - level inferences . Such analyses, unlike random and fixed - effects models, are suited for the robust analysis of data with low degrees of freedom (singh et al ., 2003). Additionally, as the snpm procedure employed included the use of a probability distribution map, generated by the largest t values in the volume rather than the t values at each voxel, the problem of multiple comparisons was circumvented (nichols & holmes, 2002). Probability maps for significant group effects (p <.05, corrected) were visualized by using mri3dx (http://www.cubric.cf.ac.uk/documentation/mri3dx/), and regions - of - interest (rois) were determined from the significant voxel clusters . To examine the specific time course of any changes in oscillatory activity within these rois, time - frequency plots were calculated for all individuals, using a morlet wavelet transform . These plots were created from single - trial activation waveforms for a given roi, and from these an average time - frequency plot was created . This revealed percent change in energy per time - frequency bin relative to the pre - stimulus phase . Nine participants (6 women; 3 men, 1950 years) with no history of neurological injury participated . Two participants were subsequently excluded due to excessive head movement (> 5 mm). Previous studies have shown that n = 7 is sufficient to reveal significant gamma - band differences across conditions (adjamian, hadjipapas, barnes, hillebrand, & holliday, 2008). Experimental procedures were in accordance with the declaration of helsinki and received approval from the university of aston's ethical committee . Angry, fearful, and neutral faces were selected from the nimstim face set (tottenham et al ., 2009). Eight actors (four men) posing three different expressions (angry, fearful, and neutral) were selected (figure 1a). In all cases, actors depicted in the stimuli displayed a direct gaze and were photographed straight on . All stimuli were gray - scaled, matched for size, and fitted to an oval - shaped layer of dimensions 2 3 (this layer obscured nonfacial features, e.g., hair, earrings). An in - house matlab script was then used to standardize the faces for luminance and contrast (the mathworks, inc ., uk). Data were collected with a 275-channel, whole - head neuromagnetometer scanner at a sampling rate of 625 hz, using a third - order gradiometer configuration with an antialiasing filter cutoff of 200 hz . Participants were seated in an upright position, and three electromagnetic coils were fastened to the participant's nasion and auricular points respectively, to determine head position within the meg helmet . The display monitor was positioned outside the shielded room and viewed through a window in the room, using a front - silvered mirror . The stimuli were viewed binocularly at an optical viewing distance of 2 m. during the recording session, each subject's head was stabilized within the helmet with an inflatable head - cuff . (a) angry versus neutral facial expressions: a group snpm comparison for the time window 50250 ms demonstrates a power difference in ba 18, bilateral lingual gyrus (p <.05, 23 significant voxels; 6, 87, 12). The threshold was set at p <.10, and significant voxels (i.e., p <.05) are white in color . (b) a time - frequency plot for a significant voxel in ba 18 averaged across participants for the angry face condition demonstrates a decrease in power at approximately 100 ms within the 5080 hz range for the angry face stimuli . This power decrease was absent for the neutral facial expressions (results not shown). On each trial, a single face was presented for 200 ms with a 1.31.6 s interstimulus interval (isi). Stimulus eccentricity corresponded to a visual angle of 1.5 (i.e., central vision). Simultaneously, a stream of mirror - reversed letters (one every 350 ms, no isi) was presented at central fixation (i.e., letters appeared non - reversed). By a button - press response device, participants were required to respond to the letter x, which appeared on 10% of trials . This task design (1) rendered the faces task - irrelevant and (2) helped maintain an alert attentive state in the participants . Data from trials displaying an x or those where a button - press response was recorded were not analyzed . For each stimulus type, data from approximately 100 trials were recorded over two experimental runs of 8 min duration . Immediately after data acquisition, a polhemus isotrak 3d digitizer was used to map the surface shape of each participant's head and localize the electromagnetic head coils (medsim, usa). These surface points were then coregistered with the individual participant's anatomical mri by a surface - matching procedure (huppertz et al ., 1998). Data were bandpass filtered between 0.5 and 100 hz and dc corrected according to a pre - stimulus baseline, in addition to a 50 hz powerline filter . Moreover, all data were visually inspected, enabling the removal of trials with eye - blink (e.g., trials in which an extreme dipolar frontal pattern was observed to emerge within the recorded epoch) and/or movement artifacts (e.g., trials in which noise was observed for a period of 25% or more of the recorded epoch). Synthetic aperture magnetometry (sam) was then used to spatially map task - related power changes in oscillatory brain activity across participants . This method uses the same principle of fixed array - weighted channels as found in contemporary radar systems to scan the brain for the sources of magnetic signals recorded at the scalp (hillebrand, singh, holliday, furlong, & barnes, 2005). Therefore, sam requires no a priori assumptions as to the number of sources activated and is ideally suited for the analysis of neuronal activity not strictly time - locked to stimulus onset? I.e ., induced activity . In brief, when using sam, for each voxel in a predefined source space, an optimal spatial filter is constructed that links activity in that voxel to the meg system's sensor array . In the present study, each individual's mri was divided into voxels of 5 5 5 mm (5 mm grids). The filter output for each voxel was calculated independently as the weighted sum of the sensor signals, yielding a measure of current density as a function of time (hillebrand et al ., 2005). This procedure is conceptually similar to the use of an invasive electrode at the neural source location; therefore, the spatial filter is often termed a virtual electrode (barnes & hillebrand, 2003). The sam beamformer technique also actively suppresses any undetected noise or artifact sources that may have occurred in spatially removed locations such as around the eyes (e.g., kinsey, anderson, hadjipapas, & holliday, 2011). For the time windows 50250 ms (early differences), 250450 ms (later differences), and 100600 ms (sustained differences) post - stimulus onset, statistical maps (t maps) of the difference between the facial expressions were calculated for gamma - band oscillations . These analyses involved assessing the difference between spectral power estimates for every voxel with a pseudo - t statistic (robinson & vrba, 1999), enabling a 3d image of activity to be generated for every participant and time window . Data were then normalized and nonparametric permutation analyses (singh, barnes, & hillebrand, 2003) performed with the snpm toolbox (www.fil.ion.ucl.ac.uk/spm/snpm/) to assess significant group effects for voxel - level inferences . Such analyses, unlike random and fixed - effects models, are suited for the robust analysis of data with low degrees of freedom (singh et al ., 2003). Additionally, as the snpm procedure employed included the use of a probability distribution map, generated by the largest t values in the volume rather than the t values at each voxel, the problem of multiple comparisons was circumvented (nichols & holmes, 2002). Probability maps for significant group effects (p <.05, corrected) were visualized by using mri3dx (http://www.cubric.cf.ac.uk/documentation/mri3dx/), and regions - of - interest (rois) were determined from the significant voxel clusters . To examine the specific time course of any changes in oscillatory activity within these rois, time - frequency plots were calculated for all individuals, using a morlet wavelet transform . These plots were created from single - trial activation waveforms for a given roi, and from these an average time - frequency plot was created . This revealed percent change in energy per time - frequency bin relative to the pre - stimulus phase . In comparing the facial displays of anger with the neutral facial expressions, snpm analyses revealed a significant difference in the gamma frequency band within the 50250 ms time window only . This difference took the form of a significant power decrease (p <.05, corrected for multiple comparisons) in extra - striate visual cortex (ba 18, including lingual gyrus; figure 1a). By using the morlet wavelet transform to examine the exact time course of this difference in gamma activity, group - averaged time - frequency plots for the 200 to 400 ms time period were calculated . These time - frequency plots revealed that the difference in power reflected a decrease in the lower (4080 hz) gamma frequency range for angry facial expressions at approximately 100 ms post - stimulus onset (figure 1b), an effect that was absent for neutral facial expressions . For all other comparisons and time windows, the analyses revealed no significant differences . While the above analyses demonstrate greater power decreases for anger, the extent to which these changes represent modulation of pre - stimulus versus post - stimulus gamma activity is unclear . Thus, gamma - band activity for angry and neutral faces relative to baseline was examined further . In each of the seven subjects, guided by our snpm rois, power changes in gamma - band activity pre - stimulus (250 to 50 ms) compared with post - stimulus (50250 ms) onset were investigated for two voxels in ba 18 . To avoid confounds associated with using normalized mri brain templates (woods, 1996), voxels within this roi table 1a subject - wise overview of frequency fluctuations in the gamma band in the extrastriate cortex . Subject - specific voxel coordinates (top line; bold) show the foci of gamma modulation and the direction of that modulation (pd = power decrease, pi = power increase, nc = no change) for both the right and left hemispheres . For reference, nearest talaraich coordinates are provided (second line)leftrightsubjectxyzangerneutralxyzangerneutralm1113208127pdnc138202134pdpi14.198.418.011.092.425.0f1110210127pdpi134214129pdpi17.1100.418.08.0100.420.0f2121202110pdnc139202109pdpd6.091.41.012.092.40.0m2116206110pdpi145206111pipd11.096.41.018.196.42.0f3108214107pdpd143213107pipi19.1104.42.016.1103.42.0f4115202115pdpi140202115pdpi12.092.46.013.192.46.0f5115191132pdpi142186120pdpi16.181.323.015.176.311.0 a subject - wise overview of frequency fluctuations in the gamma band in the extrastriate cortex . Subject - specific voxel coordinates (top line; bold) show the foci of gamma modulation and the direction of that modulation (pd = power decrease, pi = power increase, nc = no change) for both the right and left hemispheres . For reference, nearest talaraich coordinates are provided (second line) these single - subject roi analyses revealed gamma - band power decreases in left extrastriate visual cortex (ba 18) for the angry expressions in all participants (as compared to only one participant for the neutral expressions) and a bilateral reduction for the angry faces in this region for five participants (as compared to none for the neutral expressions; see table 1). For comparison, figure 2 shows time - frequency plots for one subject for the period 100 to 300 ms . For the angry expressions (figure 2a), a reduction in power (5080 hz) however, a similar reduction in power was absent for the neutral expressions (figure 2b). Given that the group analyses revealed no differences for the fearful faces, these data were not analyzed further . Time - frequency plots for a significant voxel in ba 18 for a representative individual demonstrate: (a) a power decrease at approximately 80150 ms within the 5080 hz frequency range for the angry face stimuli; (b) the absence of such an effect in the neutral face stimuli time - frequency plot; and (c) the absence of such an effect in the participant's grand - averaged time - frequency plot for the angry stimuli . This demonstrates that the power decrease observed for the angry face stimuli reflect an induced (not evoked) response . In each plot, the stimulus appeared on screen at time zero, and the red / blue colors represent percent change in power . It was hypothesized that differential modulation of gamma - band activity by threat - related faces as compared to neutral faces would be evident in primary areas of occipital cortex, and that, if observed, these differences would occur within 250 ms of stimulus onset . The findings of this study support the hypotheses with (1) a significant power decrease for facial displays of anger compared to the neutral expressions in extrastriate cortex, including lingual gyrus, and (2) this decrease occurring approximately 100 ms post - stimulus onset (i.e., within the 50250 ms post - stimulus time window). Moreover, as shown by the individual subject - level analyses, the power decrease in the gamma band for the angry stimuli was observed in ba 18 post - stimulus onset in all subjects . The current results reveal that perception of facial expressions of anger is associated with modulation of gamma - band activity in the occipital cortex as early as 100 ms post - stimulus onset, a finding consistent with recent meg research by luo et al . Observed power increases, we observed power decreases, a result that is consistent with the methodological differences of our tasks; most notably, in the present research, the stimuli were task - irrelevant i.e ., parallels can be drawn with recent findings where manipulating the attentional focus away from facial displays of threat resulted in the suppression of amygdala and fusiform cortex activity (brassen, gamer, rose, & buchel, 2010) 2010) report task - related decreases in gamma - band activity, with strong suppressions of power in areas not related to immediate task demands . Measures of cortical deactivation, then, can be used to identify activity in task - irrelevant areas that needs to be disengaged for appropriate attentional focus to occur . Our data suggest that where threat - related, task - irrelevant emotional stimuli are involved, initial rapid engagement of emotional processing areas requires active suppression as indexed by the gamma - band decreases . As this did not occur during identical presentation of neutral stimuli, it is possible that the threat valence of the stimuli initiated early processing that required subsequent suppression . Moreover, our results fit well with (1) research implicating the lingual gyrus and extrastriate cortex in threat detection networks; (2) the temporal model of emotional face recognition proposed by vuilleumier and pourtois (2007); and (3) the idea that it is periods of both synchrony and desynchrony that enable the rapid detection and processing of significant information (jensen et al . 2010). As the lingual gyrus has afferent connectivity with the amygdala (amaral & price, 1984), our results are also in keeping with the idea that threat circuitry includes regions of primary occipital cortex (anderson et al ., 2003). In accordance with this, convergent evidence indicates that early gamma activity observed within visual cortices may reflect mechanisms associated with the fast perceptual processing of aversive stimuli and the initial stage of perceptual processes responsible for recognizing facial expressions of threat (keil et al ., 2001; vuilleumier & pourtois, 2007). Thus, it may be that early oscillatory activity in the gamma frequency range (i.e., that within the first 250 ms of stimulus onset) serves as the mechanism by which selective perceptual processing of threatening facial expressions occurs . The gamma power decrease may reflect a suppression of visual processing resources that have initially been focused away from task - relevant information (in this instance, the central stream of letters) toward task - irrelevant direct threat cues (angry faces). This account would seem consistent with well - documented findings that threatening faces, especially angry faces, receive prioritized processing that, behaviorally, is manifest in many ways (e.g., rapid reorienting of attention, efficient threat detection) (maratos, mogg, & bradley, 2008). It has also been suggested that gamma power decrease reflects an active suppression (or dampening down) of neuronal activity in response to negative information (vidal et al ., therefore, given that the angry faces were task - irrelevant, the power decreases could reflect inhibition of the hypothesized threat - detection system (in regions of early visual cortex) to enable an individual to focus upon the task at hand . Alternatively, it is possible that our results represent a suppression of gamma synchrony during the central letter task when the angry faces were present ., reprioritization of processing resources from the central letter task to the task - irrelevant (angry) faces . The absence of an observable gamma response to fearful faces may stem from either qualitative or quantitative differences between the different types of threat cues . Fearful and angry faces are indirect and direct signals of threat, respectively, as fearful faces provide less information about the source of danger (whalen et al ., 2001). Fearful and angry faces may also differ in emotional intensity or arousal, with angry faces being more emotionally potent . Thus, there may be a threshold effect for changes in gamma activity, which could be investigated in future studies, as by using morphed angry and fearful facial expressions which manipulate emotional intensity (mogg, garner, & bradley, 2007). Another stimulus dimension which would be useful to investigate further is the perceptibility of the stimuli . For example, when the face stimuli used here were blurred (by low - pass filtering), meg results indicated modulation of theta activity by threat - related expressions (maratos, mogg, bradley, rippon, & senior, 2009), suggesting an association between theta activity and processing of more ambiguous threat cues . Finally, the present work may also be extended by manipulating task relevance within a single study; this was not practical in the present investigation . The present data reveal that task - irrelevant threat (facial expressions of anger) is associated with a power decrease in the gamma - band within 250 ms of stimulus onset in the occipital cortex (in keeping with neural theories of visual processing of threat e.g ., davis & whalen, 2001). Taken together with recent research into the role of gamma in information processing, our research reveals, for angry faces at least, that gamma desynchrony, in addition to gamma synchrony, is key when considering neural networks associated with the perceptual processing of this threat cue.
Renal ischemia - reperfusion (ir) injury is major cause of acute kidney injury, a common clinical problem associated with an increasing prevalence and high mortality rate (1). The severity of the injury depends on the duration of ischemia and subsequent reperfusion phase . Reperfusion causes damage through generation of reactive oxygen species and inflammation rather than restoration of normal function (2). Therefore, the need for additional therapeutic modalities to prevent renal ir injury is quite urgent . Erythropoietin is a glycoprotein hormone that was originally identified as the humoral factor, which control production of erythroid precursor cells . However, many evidence suggests that epo has several functions independent of its effects on red blood cells production . Recently, studies in vitro and vivo have shown that epo attenuates cell damage (3). The proper effects of the epo - related changes are not fully clearly, even though it has anti - apoptotic, antioxidative and anti - inflammatory properties . Its pro - angiogenic potential seems to be related to epo - mediated protective effect . Ischemic preconditioning (ipc), defined as brief sublethal periods of ischemia followed by reperfusion before master ischemia, is a way to minimize subsequent events of ir injury (4, 5). Researches show that ipc regimen can protect the target organs or distant parts of organs and tissues (6, 7). Protective effects of ipc on ir injury have been frequently demonstrated in other organs such as skeletal muscle (9). Antioxidants have been presented to be protective against ir interposed oxidative damage in different animal models (8). Lipid peroxidation refers to the oxidative deterioration of lipids and is one of the most important sources of oxidative stress . Several experimental studies have shown that lipid peroxidation occurred in renal ir insult (10). Therefore, ros has been shown to contribute to the cellular damage induced by ir . The aim of this study was to compare the effects of recombinant human epo and ipc on renal ischemia / reperfusion injury and their effects on the production of ros . Male wistar albino rats, weighing 220 - 270 g, were used . Until the beginning of the research, rats were kept at room temperature (23c - 25c) and 40% - 60% relative humidity on a 12/12 hour light / dark cycle and were fed with standard pellet diet and water ad libitum . All procedures described below were performed under approval of the animal ethics committee of tehran university of medical sciences . Rats were anaesthetized by an intraperitoneal administration mixture of ketamine (50 mg / kg) and xylazine (10 mg / kg). Systolic blood pressure and heart rate were recorded by a tail - cuff connected to a pulse transducer device (mlt125/r; ad instruments, castle hill, nsw, australia). The transducer was linked to a powerlab/4sp data - acquisition system (chart, version 5; ad instruments). After satisfactory surgical anesthesia had been achieved, a midline laparotomy was done, in which the abdominal cavity was fully exposed . Bilateral renal pedicles were isolated carefully and clamped by non - crushing microvascular clamp to effect complete cessation of renal blood flow . After 50 minutes, the clamps were removed to allow return of blood flow to the kidneys, and then kidneys were undergone to reperfusion for 24 hours . The edges of the abdominal incision were joined to each other and covered by a piece of gauze soaked with warm isotonic saline (37c) to prevent undue loss of body fluids . The abdomen was irrigated with warm isotonic saline, and then the abdominal wound was closed in two layers by continuous stitches . The ischemic preconditioning (ipc) was performed by three cycles of alternating 3 minutes of bilateral renal pedicles ischemia and 3 minutes reperfusion . Rats were allocated randomly into four experimental groups (n = 6): sham group, after laparotomy, rats were subjected to surgical manipulation without intervention; ir group, rats were subjected to bilaterally renal ischemia for 50 minutes followed by 24 hours reperfusion; ir + epo, rats were subjected to ir (as in ir group), a single dose of epo (5000 u / kg) was injected intraperitoneally thirty minutes before the onset of ischemia; ir + ipc, rats were subjected to ischemic preconditioning (ipc) regimen before the onset of ischemia (figure 1). At the end of the surgery, after 24 hours reperfusion, they were anaesthetized by mixture of ketamine (50 mg / kg) and xylazine (10 mg / kg) and blood samples were collected from the inferior vena cava . Rats were sacrificed and their kidneys were harvested before being washed and dissected in cold normal saline . Part of the left kidney was immediately snap - frozen in liquid nitrogen and stored at 70c for renal oxidative stress status assay . Blood samples were centrifuged at 1,000 g for 15 minutes within 1 hour after collection . Plasma samples were analyzed for blood urea nitrogen (bun) and plasma creatinine with commercial kits by an autoanalyzer . Kidney (50 mg) tissue samples were homogenized with trichloroacetic acid (tca). Thiobarbituric acid (tba) solution was added to the supernatant and boiled for 60 minutes . After samples were cooled down, the optical density of supernatant was measured at 532 nm . To measure sod activity, 50 mg of kidney tissue samples were homogenized in buffer phosphate . A spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation kidney tissue samples (50 mg) were homogenized in ethylenediaminetetraacetic acid (edta)-buffer phosphate and tca solution . The assay for gsh is based on the reaction of gsh with 5, 5-dithiobis (2-nitrobenzoic acid), which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm (13). Male wistar albino rats, weighing 220 - 270 g, were used . Until the beginning of the research, rats were kept at room temperature (23c - 25c) and 40% - 60% relative humidity on a 12/12 hour light / dark cycle and were fed with standard pellet diet and water ad libitum . All procedures described below were performed under approval of the animal ethics committee of tehran university of medical sciences . Rats were anaesthetized by an intraperitoneal administration mixture of ketamine (50 mg / kg) and xylazine (10 mg / kg). Systolic blood pressure and heart rate were recorded by a tail - cuff connected to a pulse transducer device (mlt125/r; ad instruments, castle hill, nsw, australia). The transducer was linked to a powerlab/4sp data - acquisition system (chart, version 5; ad instruments). After satisfactory surgical anesthesia had been achieved, a midline laparotomy was done, in which the abdominal cavity was fully exposed . Bilateral renal pedicles were isolated carefully and clamped by non - crushing microvascular clamp to effect complete cessation of renal blood flow . After 50 minutes, the clamps were removed to allow return of blood flow to the kidneys, and then kidneys were undergone to reperfusion for 24 hours . The edges of the abdominal incision were joined to each other and covered by a piece of gauze soaked with warm isotonic saline (37c) to prevent undue loss of body fluids . The abdomen was irrigated with warm isotonic saline, and then the abdominal wound was closed in two layers by continuous stitches . The ischemic preconditioning (ipc) was performed by three cycles of alternating 3 minutes of bilateral renal pedicles ischemia and 3 minutes reperfusion . Rats were allocated randomly into four experimental groups (n = 6): sham group, after laparotomy, rats were subjected to surgical manipulation without intervention; ir group, rats were subjected to bilaterally renal ischemia for 50 minutes followed by 24 hours reperfusion; ir + epo, rats were subjected to ir (as in ir group), a single dose of epo (5000 u / kg) was injected intraperitoneally thirty minutes before the onset of ischemia; ir + ipc, rats were subjected to ischemic preconditioning (ipc) regimen before the onset of ischemia (figure 1). At the end of the surgery, after 24 hours reperfusion, they were anaesthetized by mixture of ketamine (50 mg / kg) and xylazine (10 mg / kg) and blood samples were collected from the inferior vena cava . Rats were sacrificed and their kidneys were harvested before being washed and dissected in cold normal saline . Part of the left kidney was immediately snap - frozen in liquid nitrogen and stored at 70c for renal oxidative stress status assay . Blood samples were centrifuged at 1,000 g for 15 minutes within 1 hour after collection . Plasma samples were analyzed for blood urea nitrogen (bun) and plasma creatinine with commercial kits by an autoanalyzer . Kidney (50 mg) tissue samples were homogenized with trichloroacetic acid (tca). Thiobarbituric acid (tba) solution was added to the supernatant and boiled for 60 minutes . After samples were cooled down, the optical density of supernatant was measured at 532 nm . To measure sod activity, a spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation . The optical density of supernatant was measured at 340 nm (12). Reduced glutathione (gsh) levels in kidney tissue were determined by spectrophotometery . Kidney tissue samples (50 mg) were homogenized in ethylenediaminetetraacetic acid (edta)-buffer phosphate and tca solution . The assay for gsh is based on the reaction of gsh with 5, 5-dithiobis (2-nitrobenzoic acid), which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm (13). Kidney (50 mg) tissue samples were homogenized with trichloroacetic acid (tca). Thiobarbituric acid (tba) solution was added to the supernatant and boiled for 60 minutes . After samples were cooled down, the optical density of supernatant was measured at 532 nm . To measure sod activity, 50 mg of kidney tissue samples were homogenized in buffer phosphate . A spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation . Kidney tissue samples (50 mg) were homogenized in ethylenediaminetetraacetic acid (edta)-buffer phosphate and tca solution . The assay for gsh is based on the reaction of gsh with 5, 5-dithiobis (2-nitrobenzoic acid), which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm (13). Bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in a significant increase in plasma bun compared with that in the sham group (22.50 3.18 vs 133.33 5.48 mg / dl respectively; p <0.05) and an increase in plasma creatinine (0.29 0.08 vs 2.75 0.40 mg / dl, respectively; p <0.05). Furthermore, epo significantly decreased plasma bun and creatinine (64.33 8.75 mg / dl and 1.26 0.36 mg / dl; respectively; both p <0.05) compared to that in the ir group . Ischemic preconditioning also significantly decreased plasma bun and creatinine (68.60 5.87 mg / dl and 1.28 0.14 mg / dl; respectively; both p <0.05) compared to that in the ir group (figure 2). The sham group did not undergo any further interventions; the ischemia / reperfusion (ir) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . A single dose of epo (5000 u / kg, i.p) was administered 30 minutes prior to ischemia . Ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia, followed by 24 hours reperfusion . Data are the mean sem (n = 6 to 9). * p <0.05 compared to the sham group; #p <0.05 compared to the ir group (one - way anova followed by tukey s test). Bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in significant increase in mda content compared to the sham group (1.20 0.09 vs 2.13 0.14 mol/100 mg tissue, respectively; p <0.05). However, epo significantly decreased mda content (1.56 0.1 mol/100 mg tissue; p <0.05) compared to the ir group . Ischemic preconditioning also significantly decreased mda content (1.54 0.07 mol/100 mg tissue p <0.05) compared to the ir group (figure 3a). The sham group did not undergo any further interventions; the ischemia / reperfusion (ir) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . A single dose of epo (5000 u / kg, i.p) was administered 30 minutes prior to ischemia . Ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia, followed by 24 hours reperfusion . * p <0.05 compared to the sham group; #p <0.05 compared to the ir group (one - way anova followed by tukey s test). In addition, bilateral renal ischemia (50 minutes) and 24 hours reperfusion significantly decreased sod activity compared to the sham group (29.84 2.78 vs 9.04 1.44 u / g tissue, respectively; p <0.05). The sod activity in the epo group (22.37 2.34 u / g tissue; p <ischemic preconditioning also significantly increased the sod activity (21.48 3.22 u / g tissue; p <0.05) compared to the ir group (figure 3b). Compared to the sham group, bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in a significant decrease in gsh levels (79.63 3.5 vs 26.02 4.36 mol / g tissue, respectively; p <0.05). The gsh level was increased in the epo group (67.69 4.48 mol / g tissue p <0.05) compared to that in the ir group . The gsh levels were also increased in the ipc group (64.74 3.42 mol / g tissue; p <0.05) compared to that in the ir group (figure 3c). Abbreviations: ir, ischemia / reperfusion; epo, erythropoietin; ipc, ischemic preconditioning; bun, blood urea nitrogen; scr, serum creatinine; mda, malondialdehyde; sod, superoxide dismutase; gsh, glutathione . Bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in a significant increase in plasma bun compared with that in the sham group (22.50 3.18 vs 133.33 5.48 mg / dl respectively; p <0.05) and an increase in plasma creatinine (0.29 0.08 vs 2.75 0.40 mg / dl, respectively; p <0.05). Furthermore, epo significantly decreased plasma bun and creatinine (64.33 8.75 mg / dl and 1.26 0.36 mg / dl; respectively; both p <0.05) compared to that in the ir group . Ischemic preconditioning also significantly decreased plasma bun and creatinine (68.60 5.87 mg / dl and 1.28 0.14 mg / dl; respectively; both p <0.05) compared to that in the ir group (figure 2). The sham group did not undergo any further interventions; the ischemia / reperfusion (ir) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . A single dose of epo (5000 u / kg, i.p) was administered 30 minutes prior to ischemia . Ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia, followed by 24 hours reperfusion . Data are the mean sem (n = 6 to 9). * p <0.05 compared to the sham group; #p <0.05 compared to the ir group (one - way anova followed by tukey s test). Bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in significant increase in mda content compared to the sham group (1.20 0.09 vs 2.13 0.14 mol/100 mg tissue, respectively; p <0.05). However, epo significantly decreased mda content (1.56 0.1 mol/100 mg tissue; p <0.05) compared to the ir group . Ischemic preconditioning also significantly decreased mda content (1.54 0.07 mol/100 mg tissue p <0.05) compared to the ir group (figure 3a). The sham group did not undergo any further interventions; the ischemia / reperfusion (ir) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . A single dose of epo (5000 u / kg, i.p) was administered 30 minutes prior to ischemia . Ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia, followed by 24 hours reperfusion . Data are the mean sem (n = 6 to 9). * p <0.05 compared to the sham group; #p <0.05 compared to the ir group (one - way anova followed by tukey s test). In addition, bilateral renal ischemia (50 minutes) and 24 hours reperfusion significantly decreased sod activity compared to the sham group (29.84 2.78 vs 9.04 1.44 u / g tissue, respectively; p <0.05). The sod activity in the epo group (22.37 2.34 u / g tissue; p <ischemic preconditioning also significantly increased the sod activity (21.48 3.22 u / g tissue; p <0.05) compared to the ir group (figure 3b). Compared to the sham group, bilateral renal ischemia (50 minutes) and 24 hours reperfusion resulted in a significant decrease in gsh levels (79.63 3.5 vs 26.02 4.36 mol / g tissue, respectively; p <0.05). The gsh level was increased in the epo group (67.69 4.48 mol / g tissue p <0.05) compared to that in the ir group . The gsh levels were also increased in the ipc group (64.74 3.42 mol / g tissue; p <0.05) compared to that in the ir group (figure 3c). Abbreviations: ir, ischemia / reperfusion; epo, erythropoietin; ipc, ischemic preconditioning; bun, blood urea nitrogen; scr, serum creatinine; mda, malondialdehyde; sod, superoxide dismutase; gsh, glutathione . Renal ir injury is a complicated process in which the kidney is subjected to morphological and functional damage during the ischemic phase and undergoes further insult during reperfusion . In the present study, we used a rat model of renal ir injury (50 minutes) bilaterally . The effect of pretreatment with epo or ipc on renal function, as well as on mda (a marker of lipid peroxidation) and sod and gsh (markers of antioxidants) were investigated . In this study, we present the finding that epo pretreatment and application of ipc protect the kidney against ir injury induced by 50 minutes bilateral ischemia followed by 24 hours reperfusion . Our study show that epo pretreatment and application of ipc reduce renal dysfunction (assessed by bun and creatinine), oxidative stress (assessed by sod and gsh), and lipid peroxidation (assessed by mda). The present study demonstrated that 50 minutes bilateral renal ir injury caused significant increases in plasma levels of bun and creatinine . These findings confirm that ir injury of the kidney causes both glomerular and tubular dysfunctions and are in agreement with those reported by gobe et al . The sequential events of renal ir injury include both cellular damage caused by ischemic insult and generation of reactive oxygen species which results in activated vascular endothelial cells after reperfusion (15) that cause renal cell injury . To dismiss toxic reactive oxygen species, cells have many natural defense enzyme mechanisms, including the enzymes sod and cat and the anti - oxidant molecule gsh . An increase in free radical causes overproduction of ros during ir which may lead to the consumption and depletion of these endogenous scavenger antioxidants . In the present study, these observations are in accordance with previous studies that reported that renal iri is associated with decreases in sod activity (16). Our results demonstrated that, pretreatment with epo as a single dose or application of ipc, significantly increased sod activity and gsh content . In agreement with these findings baranano and snyder (18) reported that epo increased the production of radical scavengers, and akisu et al . (19) showed that epo inhibited the iron - catalyzed reactions for generating free oxygen radicals . The data of another study showed that epo pretreatment improved the cellular antioxidant defense system following renal ir injury (20). Previous studies have demonstrated that, mda tissue content is also commonly used as a marker of oxidative stress in renal ir injury (21). In the present study, we demonstrated that ir resulted in increased mda content in renal tissues and was associated with impaired kidney function . These data are in good agreement with those of jiang et al . Who found high lipid peroxidation after renal ir injury (22). Our results demonstrated that pretreatment with epo significantly decreased the level of mda, indicating lower level of oxidative stress and subsequently less lipid peroxidation . (23) demonstrated that epo decreased the level of mda after ir injury in a rat model . Our results showed that application of ipc caused a significant reduction in mda production, indicating a reduction in lipid peroxidation and cellular damage . (24) demonstrated that, ipc significantly reversed the increase in lipid hydroperoxide levels to a considerable extent . Renal ir damage leads to extensive oxidative stress . Reducing the excessive production of reactive oxygen species minimizes the secondary destruction after renal injury (25). Erythropoietin may exert its anti - oxidative effects by stimulating endothelial nitric oxide synthase and inducing intracellular anti - oxidative mechanisms . Plentiful of reactive oxygen species during the first stage of reperfusion has been presented out as the good evidence for the pathogenesis of the tissue injury . Reactive oxygen species generation increases sharply at the onset of reperfusion, although such substances are detectable in further moments too (26). The ipc acts in this phase in a way that has not been completely clear, probably attenuating the oxidative stress through increased production of nitric oxide . A limitation of the current study is the lack of long - term evaluation of the kidney function of the experimental groups . Another limitation is the lack of some groups to measure the parameters in the middle of the reperfusion period . In conclusion, pretreatment with epo and application of ipc significantly ameliorated the renal injury induced by bilateral renal ir.
It is my pleasure to review progress in numerical relativity based upon characteristic evolution . In the spirit of living reviews in relativity, we are now in an era in which einstein s equations can effectively be considered solved at the local level . Several groups, as reported here and in other living reviews in relativity, have developed 3d codes which are stable and accurate in some sufficiently local setting . Since my last review, the pioneering works (based upon a harmonic formulation) and [66, 19] (based upon bssn formulations [218, 32]) have initiated dramatic progress in the ability of cauchy codes to simulate the inspiral and merger of binary black holes, the premier problem in classical relativity . In particular, none of the existing codes are capable of computing the waveform of the gravitational radiation emanating from a binary inspiral with only the error introduced by the numerical approximation . The best that these cauchy codes by themselves can provide is an extraction of the waveform based upon the analytical approximation that the outer boundary is at infinity . Just as several coordinate patches are necessary to describe a spacetime with nontrivial topology, the most effective attack on the binary black hole waveform might involve a global solution patched together from pieces of spacetime handled by a combination of different codes and techniques . Most of the effort in numerical relativity has centered about the cauchy {3 + 1} formalism, with the gravitational radiation extracted by perturbative methods based upon introducing an artificial schwarzschild background in the exterior region [1, 4, 2, 3, 208, 204, 177]. A different approach which is specifically tailored to study radiation is based upon the characteristic initial value problem . In the 1960s, bondi [53, 54] and penrose pioneered the use of null hypersurfaces to describe gravitational waves . It led to the first unambiguous description of gravitational radiation in a fully nonlinear context . By formulating asymptotic flatness in terms of characteristic hypersurfaces extending to infinity, they were able to reconstruct, in a nonlinear geometric setting, the basic properties of gravitational waves which had been developed in linearized theory on a minkowski background . The major new nonlinear features were the bondi mass and news function, and the mass loss formula relating them . The bondi news function is an invariantly defined complex radiation amplitude n = n+ in, whose real and imaginary parts correspond to the time derivatives th and th of the plus and cross polarization modes of the strain h incident on a gravitational wave antenna . The corresponding waveforms are important both for the design of detection templates for a binary black hole inspiral and merger and for the determination of the resulting recoil velocity . The recent success of cauchy evolutions in simulating binary black holes emphasizes the need of applying these global techniques to accurate waveform extraction . The successful cauchy simulation of binary black holes has been based upon adaptive mesh techniques and upon fourth order finite difference approximations . One of the prime factors affecting the accuracy of any characteristic code is the introduction of a smooth coordinate system covering the sphere, which labels the null directions on the outgoing light cones . This is also an underlying problem in meteorology and oceanography . In a pioneering paper on large - scale numerical weather prediction, phillips put forward a list of desirable features for a mapping of the sphere to be useful for global forecasting . This led to two distinct choices which had been developed earlier in purely geometrical studies: stereographic coordinates (two coordinate patches) and cubed - sphere coordinates (six patches). Both coordinate systems have been rediscovered in the context of numerical relativity (see section 4.1). The cubed - sphere method has stimulated two new attempts at improved codes for characteristic evolution (see section 4.2.3). Another issue affecting code accuracy is the choice between a second vs first differential order reduction of the evolution system . Historically, the predominant importance of computational fluid dynamics has favored first order systems, in particular the reduction to symmetric hyperbolic form . However, in acoustics and elasticity theory, where the natural treatment is in terms of second order wave equations, an effective argument for the second order form has been made . In general relativity, the question of whether first or second order formulations are more natural depends on how einstein s equations are reduced to a hyperbolic system by some choice of coordinates and variables . The second order form is more natural in the harmonic formulation, where the einstein equations reduce to quasilinear wave equations . The first order form is more natural in the friedrich - nagy formulation, which includes the weyl tensor among the evolution variables, and was used in the first demonstration of a well - posed initial - boundary value problem for einstein s equations . Recent investigations of first order formulations of the characteristic initial value problem are discussed in section 4.2.2 . The major drawback of a stand - alone characteristic approach arises from the formation of caustics in the light rays generating the null hypersurfaces . In the most ambitious scheme proposed at the theoretical level such caustics only a few structural stable caustics can arise in numerical evolution, and their geometrical properties are well enough understood to model their singular behavior numerically, although a computational implementation has not yet been attempted . In the typical setting for the characteristic initial value problem, the domain of dependence of a single smooth null hypersurface is empty . In order to obtain a nontrivial evolution problem, the null hypersurface must either be completed to a caustic - crossover region where it pinches off, or an additional inner boundary must be introduced . So far, the only caustics that have been successfully evolved numerically in general relativity are pure point caustics (the complete null cone problem). When spherical symmetry is not present, the stability conditions near the vertex of a light cone place a strong restriction on the allowed time step nevertheless, point caustics in general relativity have been successfully handled for axisymmetric vacuum spacetimes . Progress toward extending these results to realistic astrophysical sources has been made in two ph.d . Florian siebel s thesis work, at the technische universitt mnchen, integrated an axisymmetric characteristic gravitational - hydro code with a high resolution shock capturing code for the relativistic hydrodynamics . This coupled general relativistic code has been thoroughly tested and has yielded state - of - the - art results for the gravitational waves produced by the oscillation and collapse of a relativistic star (see sections 7.1 and 7.2). Nevertheless, computational demands to extend these results to 3d evolution would be prohibitive using current generation supercomputers, due to the small timestep required at the vertex of the null cone (see section 3.3). This is unfortunate because, away from the caustics, characteristic evolution offers myriad computational and geometrical advantages . Vacuum simulations of black hole spacetimes, where the inner boundary can be taken to be the white hole horizon, offer a scenario where both the timestep and caustic problems can be avoided and three - dimensional simulations are practical . In yosef zlochower s thesis work, at the university of pittsburgh, the gravitational waves generated from the post - merger phase of a binary black black hole were computed using a fully nonlinear three - dimensional characteristic code (see section 4.5). He showed how the characteristic code could be employed to investigate the nonlinear mode coupling in the response of a black hole to the infall of gravitational waves . At least in the near future, fully three - dimensional computational applications of characteristic evolution are likely to be restricted to some mixed form, in which data is prescribed on a non - singular but incomplete initial null hypersurface n and on a second inner boundary b, which together with the initial null hypersurface determine a nontrivial domain of dependence . The hypersurface b may be either (i) null, (ii) timelike or (iii) spacelike, as schematically depicted in figure 1 . The first two possibilities give rise to (i) the double null problem and (ii) the nullcone - worldtube problem . Possibility (iii) it may be regarded as a cauchy initial - boundary value problem where the outer boundary is null . An alternative interpretation is the cauchy - characteristic matching (ccm) problem, in which the cauchy and characteristic evolutions are matched transparently across a worldtube w, as indicated in figure 1 . Figure 1the three applications of characteristic evolution with data given on an initial null hypersurface n and boundary b. the shaded regions indicate the corresponding domains of dependence . The three applications of characteristic evolution with data given on an initial null hypersurface n and boundary b. the shaded regions indicate the corresponding domains of dependence . In ccm, it is possible to choose the matching interface between the cauchy and characteristic regions to be a null hypersurface, but it is more practical to match across a timelike worldtube . Ccm combines the advantages of characteristic evolution in treating the outer radiation zone in spherical coordinates which are naturally adapted to the topology of the worldtube with the advantages of cauchy evolution in treating the inner region in cartesian coordinates, where spherical coordinates would break down . In this review, we trace the development of characteristic algorithms from model 1d problems to a 2d axisymmetric code which computes the gravitational radiation from the oscillation and gravitational collapse of a relativistic star and to a 3d code designed to calculate the waveform emitted in the merger to ringdown phase of a binary black hole . And we trace the development of ccm from early feasibility studies to successful implementation in the linear regime and through current attempts to treat the binary black hole problem . A major achievement has been the successful application of ccm to the linearized matching problem between a 3d characteristic code and a 3d cauchy code based upon harmonic coordinates (see section 5.8). Here the linearized cauchy code satisfies a well - posed initial - boundary value problem, which seems to be a critical missing ingredient in previous attempts at ccm in general relativity . Recently a well - posed initial - boundary value problem has been established for fully nonlinear harmonic evolution (see section 5.3), which should facilitate the extension of ccm to the nonlinear case . In previous reviews, i tried to include material on the treatment of boundaries in the computational mathematics and fluid dynamics literature because of its relevance to the ccm problem . The fertile growth of this subject warrants a separate living review in relativity on boundary conditions, which is presently under consideration . In anticipation of this, i will not attempt to keep this subject up to date except for material of direct relevance to ccm . See for an independent review of boundary conditions that have been used in numerical relativity . The problem of computing the evolution of a neutron star, in close orbit about a black hole is of clear importance to the new gravitational wave detectors . The interaction with the black hole could be strong enough to produce a drastic change in the emitted waves, say by tidally disrupting the star, so that a perturbative calculation would be inadequate . The understanding of such nonlinear phenomena requires well behaved numerical simulations of hydrodynamic systems satisfying einstein s equations . Several numerical relativity codes for treating the problem of a neutron star near a black hole have been developed, as described in the living review in relativity on numerical hydrodynamics in general relativity by font . Although most of these efforts concentrate on cauchy evolution, the characteristic approach has shown remarkable robustness in dealing with a single black hole or relativistic star . In this vein, state - of - the - art axisymmetric studies of the oscillation and gravitational collapse of relativistic stars have been achieved (see section 7.2) and progress has been made in the 3d simulation of a body in close orbit about a schwarzschild black hole (see sections 4.6 and 7.3). Characteristics have traditionally played an important role in the analysis of hyperbolic partial differential equations . However, the use of characteristic hypersurfaces to supply the foliation underlying an evolution scheme has been mainly restricted to relativity . This is perhaps natural because in curved spacetime there is no longer a preferred cauchy foliation provided by the euclidean 3-spaces allowed in galilean or special relativity . The method of shooting along characteristics is a standard technique in many areas of computational physics, but evolution based upon characteristic hypersurfaces is quite uniquely limited to relativity . Bondi s initial use of null coordinates to describe radiation fields was followed by a rapid development of other null formalisms . These were distinguished either as metric based approaches, as developed for axisymmetry by bondi, metzner and van der burg and generalized to 3 dimensions by sachs, or as null tetrad approaches in which the bianchi identities appear as part of the system of equations, as developed by newman and penrose . At the outset, null formalisms were applied to construct asymptotic solutions at null infinity by means of 1/r expansions . Soon afterward, penrose devised the conformal compactification of null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal i}$\end{document} (scri), thereby reducing to geometry the asymptotic quantities describing the physical properties of the radiation zone, most notably the bondi mass and news function . The characteristic initial value problem rapidly became an important tool for the clarification of fundamental conceptual issues regarding gravitational radiation and its energy content . It laid bare and geometrised the gravitational far field . The initial focus on asymptotic solutions clarified the kinematic properties of radiation fields but could not supply the dynamical properties relating the waveform to a specific source . It was soon realized that instead of carrying out a 1/r expansion, one could reformulate the approach in terms of the integration of ordinary differential equations along the characteristics (null rays). The integration constants supplied on some inner boundary then played the role of sources in determining the specific waveforms obtained at infinity . In the double - null initial value problem of sachs, the integration constants are supplied at the intersection of outgoing and ingoing null hypersurfaces . In the worldtube - nullcone formalism, the sources were represented by integration constants on a timelike worldtube . These early formalisms have gone through much subsequent revamping . Some have been reformulated to fit the changing styles of modern differential geometry . Rather than including a review of the extensive literature on characteristic formalisms in general relativity, i concentrate here on those approaches which have been implemented as computational evolution schemes . The well - posedness of the associated initial - boundary value problems, which is essential for the success of numerical simulations, is treated in a separate living review in relativity on theorems on existence and global dynamics for the einstein equations by rendall . The fundamental ingredient is a foliation by null hypersurfaces u = const . Which are generated by a two - dimensional set of null rays, labeled by coordinates x, with a coordinate varying along the rays . In (u,, x) null coordinates, the main set of einstein equations take the schematic form 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f_{,\lambda}} = {h_f}[f, g]$$\end{document} and 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${g_{,u\lambda}} = {h_g}[f, g,{g_{,u}}].$$\end{document} here f represents a set of hypersurface variables, g a set of evolution variables, and hf and hg are nonlinear hypersurface operators, i.e. They operate locally on the values of f, g and g, u intrinsic to a single null hypersurface . In the bondi formalism, these hypersurface equations have a hierarchical structure in which the members of the set f can be integrated in turn in terms of the characteristic data for the evolution variables and the computed values of prior members of the hierarchy . In addition to these main einstein equations, there is a subset of four subsidiary einstein equations which are satisfied by virtue of the bianchi identities, provided that they are satisfied on a hypersurface transverse to the characteristics . But they are not elliptic since they may be intrinsic to null or timelike hypersurfaces, rather than spacelike cauchy hypersurfaces . Computational implementation of characteristic evolution may be based upon different versions of the formalism (i.e. Metric or tetrad) and different versions of the initial value problem (i.e. Double null or worldtube - nullcone). However, most characteristic evolution codes share certain common advantages: the characteristic initial data is free, i.e. There are no elliptic constraints on the data . This eliminates the need for time consuming iterative constraint solvers with their accompanying artificial boundary conditions . This flexibility and control in prescribing initial data has the trade - off of limited experience with prescribing physically realistic characteristic initial data.the coordinates are very rigid, i.e. There is very little remaining gauge freedom.the constraints satisfy ordinary differential equations along the characteristics which force any constraint violation to fall off asymptotically as 1/r.no second time derivatives appear so that the number of basic variables is at most half the number for the corresponding version of the cauchy problem.the main einstein equations form a system of coupled ordinary differential equations with respect to the parameter varying along the characteristics . This allows construction of an evolution algorithm in terms of a simple march along the characteristics.in problems with isolated sources, the radiation zone can be compactified into a finite grid boundary with the metric rescaled by 1/r as an implementation of penrose s conformal boundary at future null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Because \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is a null hypersurface, no extraneous outgoing radiation condition or other artificial boundary condition is required . The analogous treatment in the cauchy problem requires the use of hyperboloidal spacelike hypersurfaces asymptoting to null infinity . For reviews of the hyperboloidal approach and its status in treating the associated three - dimensional computational problem, see [148, 92].the grid domain is exactly the region in which waves propagate, which is ideally efficient for radiation studies . Since each null hypersurface of the foliation extends to infinity, the radiation is calculated immediately (in retarded time).in black hole spacetimes, a large redshift at null infinity relative to internal sources is an indication of the formation of an event horizon and can be used to limit the evolution to the exterior region of spacetime . While this can be disadvantageous for late time accuracy, it allows the possibility of identifying the event horizon on the fly, as opposed to cauchy evolution where the event horizon can only be located after the evolution has been completed . Perhaps most important from a practical view, characteristic evolution codes have shown remarkably robust stability and were the first to carry out long term evolutions of moving black holes . The characteristic initial data is free, i.e. There are no elliptic constraints on the data . This eliminates the need for time consuming iterative constraint solvers with their accompanying artificial boundary conditions . This flexibility and control in prescribing initial data has the trade - off of limited experience with prescribing physically realistic characteristic initial data . The coordinates are very rigid, i.e. There is very little remaining gauge freedom . The constraints satisfy ordinary differential equations along the characteristics which force any constraint violation to fall off asymptotically as 1/r . No second time derivatives appear so that the number of basic variables is at most half the number for the corresponding version of the cauchy problem . The main einstein equations form a system of coupled ordinary differential equations with respect to the parameter varying along the characteristics . This allows construction of an evolution algorithm in terms of a simple march along the characteristics . In problems with isolated sources, the radiation zone can be compactified into a finite grid boundary with the metric rescaled by 1/r as an implementation of penrose s conformal boundary at future null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Because \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is a null hypersurface, no extraneous outgoing radiation condition or other artificial boundary condition is required . The analogous treatment in the cauchy problem requires the use of hyperboloidal spacelike hypersurfaces asymptoting to null infinity . For reviews of the hyperboloidal approach and its status in treating the associated three - dimensional computational problem, the grid domain is exactly the region in which waves propagate, which is ideally efficient for radiation studies . Since each null hypersurface of the foliation extends to infinity, the radiation is calculated immediately (in retarded time). In black hole spacetimes, a large redshift at null infinity relative to internal sources is an indication of the formation of an event horizon and can be used to limit the evolution to the exterior region of spacetime . While this can be disadvantageous for late time accuracy, it allows the possibility of identifying the event horizon on the fly, as opposed to cauchy evolution where the event horizon can only be located after the evolution has been completed . Characteristic schemes also share as a common disadvantage the necessity either to deal with caustics or to avoid them altogether . The scheme to tackle the caustics head on by including their development and structure as part of the evolution is perhaps a great idea still ahead of its time but one that should not be forgotten . There are only a handful of structurally stable caustics, and they have well known algebraic properties . The structural stability of the singularities should in principle make this possible, and algorithms to evolve the elementary caustics have been proposed [79, 230]. In the axisymmetric case, cusps and folds are the only structurally stable caustics, and they have already been identified in the horizon formation occurring in simulations of head - on collisions of black holes and in the temporarily toroidal horizons occurring in collapse of rotating matter [172, 217]. In a generic binary black hole horizon, where axisymmetry is broken, there is a closed curve of cusps which bounds the two - dimensional region on the event horizon where the black holes initially form and merge [165, 150]. Limited computer power, as well as the instabilities arising from non - hyperbolic formulations of einstein s equations, necessitated that the early code development in general relativity be restricted to spacetimes with symmetry . The techniques for other special relativistic fields which propagate on null characteristics are similar to the gravitational case . We postpone treatment of relativistic fluids, whose characteristics are timelike, until section 7 . It is often said that the solution of the general ordinary differential equation is essentially known, in light of the success of computational algorithms and present day computing power . But, in this spirit, it is fair to say that the general system of hyperbolic partial differential equations in one spatial dimension seems to be a solved problem in general relativity . Codes have been successful in revealing important new phenomena underlying singularity formation in cosmology and in dealing with unstable spacetimes to discover critical phenomena . As described below one of the earliest characteristic evolution codes, constructed by corkill and stewart [79, 229], treated spacetimes with two killing vectors using a grid based upon double null coordinates, with the null hypersurfaces intersecting in the surfaces spanned by the killing vectors . They simulated colliding plane waves and evolved the khan - penrose collision of impulsive (-function curvature) plane waves to within a few numerical zones from the final singularity, with extremely close agreement with the analytic results . Their simulations of collisions with more general waveforms, for which exact solutions are not known, provided input to the understanding of singularity formation which was unforeseen in the analytic treatments of this problem . Many {1 + 1}-dimensional characteristic codes have been developed for spherically symmetric systems . Here initially the characteristic evolution of matter was restricted to simple cases, such as massless klein - gordon fields, which allowed simulation of gravitational collapse and radiation effects in the simple context of spherical symmetry . Its application to hydrodynamics has made significant contributions to general relativistic astrophysics, as reviewed in section 7 . The synergy between analytic and computational approaches has led to dramatic results in the massless klein - gordon case . On the analytic side, working in a characteristic initial value formulation based upon outgoing null cones, christodoulou made a penetrating study of the spherically symmetric problem [69, 70, 71, 72, 73, 74]. In a suitable function space, he showed the existence of an open ball about minkowski space data whose evolution is a complete regular spacetime; he showed that an evolution with a nonzero final bondi mass forms a black hole; he proved a version of cosmic censorship for generic data; and he established the existence of naked singularities for non - generic data . What this analytic tour - de - force did not reveal was the remarkable critical behavior in the transition to the black hole regime, which was discovered by choptuik [67, 68] in simulations using cauchy evolution . This phenomenon has now been understood in terms of the methods of renormalization group theory and intermediate asymptotics, and has spawned a new subfield in general relativity, which is covered in the living review in relativity on critical phenomena in gravitational collapse by gundlach . The characteristic evolution algorithm for the spherically symmetric einstein - klein - gordon problem provides a simple illustration of the techniques used in the general case . It centers about the evolution scheme for the scalar field, which constitutes the only dynamical field . Given the scalar field, all gravitational quantities can be determined by integration along the characteristics of the null foliation . This is a coupled problem, since the scalar wave equation involves the curved space metric . It illustrates how null algorithms lead to a hierarchy of equations which can be integrated along the characteristics to effectively decouple the hypersurface and dynamical variables . In a bondi coordinate system based upon outgoing null hypersurfaces u = const . And a surface area coordinate r, the metric is 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = - {e^{2\beta}}du\left({{v \over r}du + 2dr} \right) + {r^2}(d{\theta ^2} + {\sin ^2}\theta d{\phi ^2}).$$\end{document} smoothness at r = 0 allows imposition of the coordinate conditions 4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{v(u, r) = r + {\mathcal o}({r^3})}\\ {\beta (u, r) = {\mathcal o}({r^2}). }\\ \end{array}$$\end{document} the field equations consist of the curved space wave equation = 0 for the scalar field and two hypersurface equations for the metric functions: 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\beta _ {, r}} = 2\pi r{({\phi _ {, r}})^2},$$\end{document} 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${v_{,r}} = {e^{2\beta}}.$$\end{document} the wave equation can be expressed in the form 7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\square^{(2)}}g - {\left({{v \over r}} \right)_{,r}}{{{e^{- 2\beta}}g} \over r} = 0,$$\end{document} where g = r and is the dalembertian associated with the two - dimensional submanifold spanned by the ingoing and outgoing null geodesics . Initial null data for evolution consists of (u0, r) at the initial retarded time u0 . Because any two - dimensional geometry is conformally flat, the surface integral of g over a null parallelogram gives exactly the same result as in a flat 2-space, and leads to an integral identity upon which a simple evolution algorithm can be based . Let the vertices of the null parallelogram be labeled by n, e, s, and w corresponding, respectively, to their relative locations (north, east, south, and west) in the 2-space, as shown in figure 2 . Upon integration of equation (7), curvature introduces an integral correction to the flat space null parallelogram relation between the values of g at the vertices: 8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${g_n} - {g_w} - {g_e} + {g_s} = {1 \over 2}\int\nolimits_\sigma {du\;dr} {\left({{v \over r}} \right)_{,r}}{g \over r}.$$\end{document} figure 2the null parallelogram . After computing the field at point n, the algorithm marches the computation to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} by shifting the corners by n n, e e, s e, w n. the null parallelogram . After computing the field at point n, the algorithm marches the computation to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} by shifting the corners by n n, e e, s e, w n. this identity, in one form or another, lies behind all of the null evolution algorithms that have been applied to this system . The prime distinction between the different algorithms is whether they are based upon double null coordinates, or upon bondi coordinates as in equation (3). When a double null coordinate system is adopted, the points n, e, s, and w can be located in each computational cell at grid points, so that evaluation of the left hand side of equation (8) requires no interpolation . As a result, in flat space, where the right hand side of equation (8) vanishes, it is possible to formulate an exact evolution algorithm . In curved space, of course, there is a truncation error arising from the approximation of the integral, e.g., by evaluating the integrand at the center of . The identity (8) gives rise to the following explicit marching algorithm, indicated in figure 2 . Let the null parallelogram lie at some fixed and and span adjacent retarded time levels u0 and u0 + u . Imagine for now that the points n, e, s, and w lie on the spatial grid, with rn rw = re rs = r . If g has been determined on the entire initial cone u0, which contains the points e and s, and g has been determined radially outward from the origin to the point w on the next cone u0 + u, then equation (8) determines g at the next radial grid point n in terms of an integral over . The integrand can be approximated to second order, i.e. To \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}(\delta r\delta u)$\end{document}, by evaluating it at the center of . To this same accuracy, the value of g at the center equals its average between the points e and w, at which g has already been determined . Similarly, the value of (v / r),r at the center of can be approximated to second order in terms of values of v at points where it can be determined by integrating the hypersurface equations (5, 6) radially outward from r = 0 . After carrying out this procedure to evaluate g at the point n, the procedure can then be iterated to determine g at the next radially outward grid point on the u0 + u level, i.e. Point n in figure 2 . Upon completing this radial march to null infinity, in terms of a compactified radial coordinate such as x = r/(1 + r), the field g is then evaluated on the next null cone at u0 + 2u, beginning at the vertex where smoothness gives the startup condition that g(u, 0) = 0 . In the compactified bondi formalism, the vertices n, e, s, and w of the null parallelogram cannot be chosen to lie exactly on the grid because, even in minkowski space, the velocity of light in terms of a compactified radial coordinate x is not constant . As a consequence, the fields g,, and v at the vertices of are approximated to second order accuracy by interpolating between grid points . However, cancellations arise between these four interpolations so that equation (8) is satisfied to fourth order accuracy . The net result is that the finite difference version of equation (8) steps g radially outward one zone with an error of fourth order in grid size, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}({({\delta _ u})^2}{(\delta x)^2})$\end{document}. In addition, the smoothness conditions (4) can be incorporated into the startup for the numerical integrations for v and to insure no loss of accuracy in starting up the march at r = 0 . The resulting global error in g, after evolving a finite retarded time, is then \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}(\delta u\delta x)$\end{document}, after compounding errors from 1/(ux) number of zones . When implemented on a grid based upon the (u, r) coordinates, the stability of this algorithm is subject to a courant - friedrichs - lewy (cfl) condition requiring that the physical domain of dependence be contained in the numerical domain of dependence . In the spherically symmetric case, this condition requires that the ratio of the time step to radial step be limited by (v / r) u 2r, where r = [x/(1x)]. This condition can be built into the code using the value v / r = e, corresponding to the maximum of v / r at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The strongest restriction on the time step then arises just before the formation of a horizon, where v / r at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. This infinite redshift provides a mechanism for locating the true event horizon on the fly and restricting the evolution to the exterior spacetime . Points near \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} must be dropped in order to evolve across the horizon due to the lack of a nonsingular compactified version of future time infinity i. the situation is quite different in a double null coordinate system, in which the vertices of the null parallelogram can be placed exactly on grid points so that the cfl condition is automatically satisfied . A characteristic code based upon double null coordinates was developed by goldwirth and piran in a study of cosmic censorship based upon the spherically symmetric gravitational collapse of a massless scalar field . Their early study lacked the sensitivity of adaptive mesh refinement (amr) which later enabled choptuik to discover the critical phenomena appearing in this problem . Subsequent work by marsa and choptuik combined the use of the null related ingoing eddington - finklestein coordinates with unruh s strategy of singularity excision to construct a 1d code that runs forever . Later, garfinkle constructed an improved version of the goldwirth - piran double null code which was able to simulate critical phenomena without using adaptive mesh refinement . In this treatment, as the evolution proceeds on one outgoing null cone to the next, the grid points follow the ingoing null cones and must be dropped as they cross the origin at r = 0 . However, after half the grid points are lost they are then recycled at new positions midway between the remaining grid points . This technique is crucial for resolving the critical phenomena associated with an r 0 size horizon . An extension of the code was later used to verify that scalar field collapse in six dimensions continues to display critical phenomena . Hamad and stewart also applied a double null code to study critical phenomena . In order to obtain the accuracy necessary to confirm choptuik s results they developed the first example of a characteristic grid with amr . They did this with both the standard berger and oliger algorithm and their own simplified version, with both versions giving indistinguishable results . Their simulations of critical collapse of a massless scalar field agreed with choptuik s values for the universal parameters governing mass scaling and displayed the echoing associated with discrete self - similarity . Hamad, horne, and stewart extended this study to the spherical collapse of an axion / dilaton system and found in this case that self - similarity was a continuous symmetry of the critical solution . Brady, chambers, and gonalves used garfinkle s double null algorithm to investigate the effect of a massive scalar field on critical phenomena . The introduction of a mass term in the scalar wave equation introduces a scale to the problem, which suggests that the critical point behavior might differ from the massless case . They found that there are two different regimes depending on the ratio of the compton wavelength 1/m of the scalar mass to the radial size of the scalar pulse used to induce collapse . When m 1, the critical solution is the one found by choptuik in the m = 0 case, corresponding to a type ii phase transition . However, when m 1, the critical solution is an unstable soliton star (see), corresponding to a type i phase transition where black hole formation turns on at a finite mass . A code based upon bondi coordinates, developed by husa and his collaborators, has been successfully applied to spherically symmetric critical collapse of a nonlinear -model coupled to gravity . Critical phenomena cannot be resolved on a static grid based upon the bondi r - coordinate . Instead, the numerical techniques of garfinkle were adopted by using a dynamic grid following the ingoing null rays and by recycling radial grid points . They studied how coupling to gravity affects the critical behavior previously observed by bizo and others in the minkowski space version of the model . For a wide range of the coupling constant, they observe discrete self - similarity and typical mass scaling near the critical solution . The code is shown to be second order accurate and to give second order convergence for the value of the critical parameter . The first characteristic code in bondi coordinates for the self - gravitating scalar wave problem was constructed by gmez and winicour . They introduced a numerical compactification of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} for the purpose of studying effects of self - gravity on the scalar radiation, particularly in the high amplitude limit of the rescaling a. as a, the red shift creates an effective boundary layer at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} which causes the bondi mass mb and the scalar field monopole moment q to be related by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${m_{\rm{b}}} \sim \pi \vert q\vert/\sqrt 2$\end{document}, rather than the quadratic relation of the weak field limit . This could also be established analytically so that the high amplitude limit provided a check on the code s ability to handle strongly nonlinear fields . In the small amplitude case, this work incorrectly reported that the radiation tails from black hole formation had an exponential decay characteristic of quasinormal modes rather than the polynomial 1/t or 1/t falloff expected from price s work on perturbations of schwarzschild black holes . In hindsight, the error here was not having confidence to run the code sufficiently long to see the proper late time behavior . Gundlach, price, and pullin [129, 130] subsequently reexamined the issue of power law tails using a double null code similar to that developed by goldwirth and piran . Their numerical simulations verified the existence of power law tails in the full nonlinear case, thus establishing consistency with analytic perturbative theory . They also found normal mode ringing at intermediate time, which provided reassuring consistency with perturbation theory and showed that there is a region of spacetime where the results of linearized theory are remarkably reliable even though highly nonlinear behavior is taking place elsewhere . These results have led to a methodology that has application beyond the confines of spherically symmetric problems, most notably in the the study of the radiation tail decay of a scalar field was subsequently extended by gmez, schmidt, and winicour using a characteristic code . They showed that the newman - penrose constant for the scalar field determines the exponent of the power law (and not the static monopole moment as often stated). When this constant is non - zero, the tail decays as 1/t on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, as opposed to the 1/t decay for the vanishing case . (they also found t log t corrections, in addition to the exponentially decaying contributions of the quasinormal modes .) This code was also used to study the instability of a topological kink in the configuration of the scalar field . The kink instability provides the simplest example of the turning point instability [152, 226] which underlies gravitational collapse of static equilibria . Brady and smith have demonstrated that characteristic evolution is especially well adapted to explore properties of cauchy horizons . They examined the stability of the reissner - nordstrm cauchy horizon using an einstein - klein - gordon code based upon advanced bondi coordinates (,r) (where the hypersurfaces = const are ingoing null hypersurfaces). They studied the effect of a spherically symmetric scalar pulse on the spacetime structure as it propagates across the event horizon . Their numerical methods were patterned after the work of goldwirth and piran, with modifications of the radial grid structure that allow deep penetration inside the black hole . In accord with expectations from analytic studies, they found that the pulse first induces a weak null singularity on the cauchy horizon, which then leads to a crushing spacelike singularity as r 0 . The null singularity is weak in the sense that an infalling observer experiences a finite tidal force, although the newman - penrose weyl component 2 diverges, a phenomenon known as mass inflation . These results confirm the earlier result of gnedin and gnedin that a central spacelike singularity would be created by the interaction of a charged black hole with a scalar field, in accord with a physical argument by penrose that a small perturbation undergoes an infinite redshift as it approaches the cauchy horizon . Burko has confirmed and extended these results, using a code based upon double null coordinates which was developed with ori in a study of tail decay . He found that in the early stages the perturbation of the cauchy horizon is weak and in agreement with the behavior calculated by perturbation theory . Brady, chambers, krivan, and laguna have found interesting effects of a non - zero cosmological constant on tail decay by using a characteristic einstein - klein - gordon code to study the effect of a massless scalar pulse on schwarzschild - de sitter and reissner - nordstrm - de sitter spacetimes . First, by constructing a linearized scalar evolution code, they show that scalar test fields with 0 have exponentially decaying tails, in contrast to the standard power law tails for asymptotically flat spacetimes . Rather than decaying, the monopole mode asymptotes at late time to a constant, which scales linearly with, in contrast to the standard no - hair result . This unusual behavior for the = 0 case was then independently confirmed with a nonlinear spherical characteristic code . Using a combination of numerical and analytic techniques based upon null coordinates, hod and piran have made an extensive series of investigations of the spherically symmetric charged einstein - klein - gordon system dealing with the effect of charge on critical gravitational collapse and the late time tail decay of a charged scalar field on a reissner - nordstrm black hole [143, 146, 144, 145]. These studies culminated in a full nonlinear investigation of horizon formation by the collapse of a charged massless scalar pulse . They track the formation of an apparent horizon which is followed by a weakly singular cauchy horizon which then develops a strong spacelike singularity at r = 0 . This is in complete accord with prior perturbative results and nonlinear simulations involving a pre - existing black hole . Oren and piran increased the late time accuracy of this study by incorporating an adaptive grid for the retarded time coordinate u, with a refinement criterion to maintain r / r = const . The accuracy of this scheme is confirmed through convergence tests as well as charge and constraint conservation . They were able to observe the physical mechanism which prohibits black hole formation with charge to mass ration q / m> 1 . Electrostatic repulsion of the outer parts of the scalar pulse increases relative to the gravitational attraction and causes the outer portion of the charge to disperse to larger radii before the black hole is formed . Inside the black hole, they confirm the formation of a weakly singular cauchy horizon which turns into a strong spacelike singularity, in accord with other studies . Hod extended this combined numerical - analytical double null approach to investigate higher order corrections to the dominant power law tail, as well as corrections due to a general spherically symmetric scattering potential and due to a time dependent potential . He found (log t)/t modifications to the leading order tail behavior for a schwarzschild black hole, in accord with earlier results of gmez et al . . These modifications fall off at a slow rate so that a very long numerical evolution (t 3000 m)is necessary to cleanly identify the leading order power law decay . The foregoing numerical - analytical work based upon characteristic evolution has contributed to a very comprehensive classical treatment of spherically symmetric gravitational collapse . Sorkin and piran have investigated the question of quantum corrections due to pair creation on the gravitational collapse of a charged scalar field . For observers outside the black hole, several analytic studies have indicated that such pair - production can rapidly diminish the charge of the black hole . Sorkin and piran apply the same double - null characteristic code used in studying the classical problem to evolve across the event horizon and observe the quantum effects on the cauchy horizon . The quantum electrodynamic effects are modeled in a rudimentary way by a nonlinear dielectric constant that limits the electric field to the critical value necessary for pair creation . The back - reaction of the pairs on the stress - energy and the electric current are ignored . They found that quantum effects leave the classical picture of the cauchy horizon qualitatively intact but that they shorten its lifetime by hastening the conversion of the weak null singularity into a strong spacelike singularity . The southampton group has constructed a {1 + 1}-dimensional characteristic code for space - times with cylindrical symmetry [77, 87]. The original motivation was to use it as the exterior characteristic code in a test case of ccm (see section 5.5.1 for the application to matching). Subsequently, sperhake, sjdin, and vickers [223, 227] modified the code into a global characteristic version for the purpose of studying cosmic strings, represented by massive scalar and vector fields coupled to gravity . Using a geroch decomposition with respect to the translational killing vector, they reduced the global problem to a {2 + 1}-dimensional asymptotically flat spacetime, so that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} could be compactified and included in the numerical grid . Rather than the explicit scheme used in ccm, the new version employs an implicit, second order in space and time, crank - nicholson evolution scheme . The code showed long term stability and second order convergence in vacuum tests based upon exact weber - wheeler waves and xanthopoulos rotating solution, and in tests of wave scattering by a string . The results show damped ringing of the string after an incoming weber - wheeler pulse has excited it and then scattered to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The ringing frequencies are independent of the details of the pulse but are inversely proportional to the masses of the scalar and vector fields . Frittelli and gmez have cast the spherically symmetric einstein - klein - gordon problem in symmetric hyperbolic form, where in a bondi - sachs gauge the fundamental variables are the scalar field, lapse and shift . The bondi - sachs gauge conditions relate the usual adm variables (the 3-metric and extrinsic curvature) to the lapse and shift, which obey simpler evolution equations . The resulting cauchy problem is well - posed and the outer boundary condition is constraint preserving (although whether the resulting ibvp is well - posed is not addressed, i.e. Whether the boundary condition is dissipative). A numerical evolution algorithm based upon the system produces a stable simulation of a scalar pulse scattering off a black hole . The initial data for the pulse satisfies t = 0 so, as expected, it contains an ingoing part, which crosses the horizon, and an outgoing part, which leaves the grid at the outer boundary with a small amount of back reflection . The goal of computing waveforms from relativistic binaries, such as a neutron star or stellar mass back hole spiraling into a supermassive black hole, requires more than a stable convergent code . It is a delicate task to extract a waveform in a spacetime in which there are multiple length scales: the size of the supermassive black hole, the size of the star, the wavelength of the radiation . It is commonly agreed that some form of mesh refinement is essential to attack this problem . Mesh refinement was first applied in characteristic evolution to solve specific spherically symmetric problems regarding critical phenomena and singularity structure [104, 135, 60]. Pretorius and lehner have presented a general approach for amr to a generic characteristic code . Although the method is designed to treat 3d simulations, the implementation has so far been restricted to the einstein - klein - gordon system in spherical symmetry . The 3d approach is modeled after the berger and oliger amr algorithm for hyperbolic cauchy problems, which is reformulated in terms of null coordinates . The resulting characteristic amr algorithm can be applied to any unigrid characteristic code and is amenable to parallelization . They applied it to the problem of a spherically symmetric massive klein - gordon field propagating outward from a black hole . The non - zero rest mass restricts the klein - gordon field from propagating to infinity . Instead it diffuses into higher frequency components which pretorius and lehner show can be resolved using amr but not with a comparison unigrid code . One - dimensional characteristic codes enjoy a very special simplicity due to the two preferred sets (ingoing and outgoing) of characteristic null hypersurfaces . This eliminates a source of gauge freedom that otherwise exists in either two- or three - dimensional characteristic codes . However, the manner in which the characteristics of a hyperbolic system determine domains of dependence and lead to propagation equations for shock waves is the same as in the one - dimensional case . This makes it desirable for the purpose of numerical evolution to enforce propagation along characteristics as extensively as possible . In basing a cauchy algorithm upon shooting along characteristics, the infinity of characteristic rays (technically, bicharacteristics) at each point leads to an arbitrariness which, for a practical numerical scheme, makes it necessary either to average the propagation equations over the sphere of characteristic directions or to select out some preferred subset of propagation equations . The latter approach was successfully applied by butler to the cauchy evolution of two - dimensional fluid flow, but there seems to have been very little follow - up along these lines . The closest resemblance is the use of riemann solvers for high resolution shock capturing in hydrodynamic codes (see section 7.1). The formal ideas behind the construction of two- or three - dimensional characteristic codes are similar, although there are various technical options for treating the angular coordinates which label the null rays . Historically, most characteristic work graduated first from 1d to 2d because of the available computing power . The first characteristic code based upon the original bondi equations for a twist - free axisymmetric spacetime was constructed by j. welling in his phd thesis at pittsburgh . The spacetime was foliated by a family of null cones, complete with point vertices at which regularity conditions were imposed . This allowed studies of the bondi mass and radiation flux on the initial null cone, but it could not be used as a practical evolution code because of instabilities . These instabilities came as a rude shock and led to a retreat to the simpler problem of axisymmetric scalar waves propagating in minkowski space, with the metric 9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = - d{u^2} - 2du\;dr + {r^2}(d{\theta ^2} + {\sin ^2}\theta d{\phi ^2})$$\end{document} in outgoing null cone coordinates . A null cone code for this problem was constructed using an algorithm based upon equation (8), with the angular part of the flat space laplacian replacing the curvature terms in the integrand on the right hand side . This simple setting allowed one source of instability to be traced to a subtle violation of the cfl condition near the vertices of the cones . In terms of the grid spacing x, the cfl condition in this coordinate system takes the explicit form 10\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\delta u} \over {\delta r}} <- 1 + {[{k^2} + {(k - 1)^2} - 2k(k - 1)\cos \delta \theta] ^{1/2}},$$\end{document} where the coefficient k, which is of order 1, depends on the particular startup procedure adopted for the outward integration . Far from the vertex, the condition (10) on the time step u is quantitatively similar to the cfl condition for a standard cauchy evolution algorithm in spherical coordinates . But condition (10) is strongest near the vertex of the cone where (at the equator = /2) it implies that 11\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta u <k\delta r{(\delta \theta)^2}.$$\end{document} this is in contrast to the analogous requirement 12\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta u <k\delta r\delta \theta$$\end{document} for stable cauchy evolution near the origin of a spherical coordinate system . The extra power of is the price that must be paid near the vertex for the simplicity of a characteristic code . Nevertheless, the enforcement of this condition allowed efficient global simulation of axisymmetric scalar waves . Global studies of backscattering, radiative tail decay, and solitons were carried out for nonlinear axisymmetric waves, but three - dimensional simulations extending to the vertices of the cones were impractical at the time on existing machines . Aware now of the subtleties of the cfl condition near the vertices, the pittsburgh group returned to the bondi problem, i.e. To evolve the bondi metric 13\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = \left({{v \over r}{e^{2\beta}} - {u^2}{r^2}{e^{2\gamma}}} \right)d{u^2} + 2{e^{2\beta}}du \; dr + 2u{r^2}{e^{2\gamma}}du \; d\theta - {r^2}({e^{2\gamma}}d{\theta ^2} + {e^{- 2\gamma}}{\sin ^2}\theta d{\phi ^2}),$$\end{document} by means of the three hypersurface equations 14\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\beta _ {, r}} = {1 \over 2}r{({\gamma _ {, r}})^2},$$\end{document} 15\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\left [{{r^4}{e^{2(\gamma - \beta)}}{u_{,r}}} \right]_{,r}} = 2{r^2}\left [{{r^2}{{\left({{\beta \over {{r^2}}}} \right)}_{,r\theta}} - {{{{({{\sin}^2}\theta \gamma)}_{,r\theta}}} \over {{{\sin}^2}\theta}} + 2{\gamma _ {, r}}{\gamma _ {, \theta}}} \right],$$\end{document} 16\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{{v_{,r}} = - {1 \over 4}{r^4}{e^{2(\gamma - \beta)}}{{({u_{,r}})}^2} + {{{{({r^4}\sin \theta u)}_{,r\theta}}} \over {2{r^2}\sin \theta}}}\\{\quad \quad + {e^{2(\beta - \gamma)}}\left [{1 - {{{{(\sin \theta {\beta _ {, \theta}})}_{,\theta}}} \over {\sin \theta}} + {\gamma _ {, \theta \theta}} + 3\cot {\theta _ {\gamma, \theta}} - {{({\beta _ {, \theta}})}^2} - {2_{\gamma, \theta}}({\gamma _ {, \theta}} - {\beta _ {, \theta}})} \right],}\\\end{array}$$\end{document} and the evolution equation 17\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{4r{{(r\gamma)}_{,ur}} = {{\left\{{2r{\gamma _ {, r}}v - {r^2}\left [{2{\gamma _ {, \theta}}u + \sin \theta {{\left({{u \over {\sin \theta}}} \right)}_{,\theta}}} \right]} \right\}}_{,r}} - 2{r^2}{{{{({\gamma _ {, r}}u\sin \theta)}_{,\theta}}} \over {\sin \theta}}}\\{\quad \quad \quad \quad \quad + {1 \over 2}{r^4}{e^{2(\gamma - \beta)}}{{({u_{,r}})}^2} + 2{e^{2(\beta - \gamma)}}\left [{{{({\beta _ {, \theta}})}^2} + \sin \theta {{\left({{{{\beta _ {, \theta}}} \over {\sin \theta}}} \right)}_{,\theta}}} \right]. }\\\end{array}$$\end{document} the beauty of the bondi equations is that they form a clean hierarchy . Given on an initial null hypersurface, the equations can be integrated radially to determine, u, v, and,u on the hypersurface (in that order) in terms of integration constants determined by boundary conditions, or smoothness conditions if extended to the vertex of a null cone . The initial data is unconstrained except for smoothness conditions . Because represents an axisymmetric spin-2 field, it must be \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}({\sin ^2}\theta)$\end{document} near the poles of the spherical coordinates and must consist of l 2 spin-2 multipoles . In the computational implementation of this system by the pittsburgh group, the null hypersurfaces were chosen to be complete null cones with nonsingular vertices, which (for simplicity) trace out a geodesic worldline r = 0 . The vertices of the cones were not the chief source of difficulty . A null parallelogram marching algorithm, similar to that used in the scalar case, gave rise to another instability that sprang up throughout the grid . In order to reveal the source of this instability, physical considerations suggested looking at the linearized version of the bondi equations, where they can be related to the wave equation . If this relationship were sufficiently simple, then the scalar wave algorithm could be used as a guide in stabilizing the evolution of . A scheme for relating to solutions of the wave equation had been formulated in the original paper by bondi, metzner, and van der burgh . However, in that scheme, the relationship of the scalar wave to was nonlocal in the angular directions and was not useful for the stability analysis . A local relationship between and solutions of the wave equation was found . This provided a test bed for the null evolution algorithm similar to the cauchy test bed provided by teukolsky waves . More critically, it allowed a simple von neumann linear stability analysis of the finite difference equations, which revealed that the evolution would be unstable if the metric quantity u was evaluated on the grid . For a stable algorithm, the grid points for u must be staggered between the grid points for,, and v. this unexpected feature emphasizes the value of linear stability analysis in formulating stable finite difference approximations . It led to an axisymmetric code [186, 120] for the global bondi problem which ran stably, subject to a cfl condition, throughout the regime in which caustics and horizons did not form . Stability in this regime was verified experimentally by running arbitrary initial data until it radiated away to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Also, new exact solutions as well as the linearized null solutions were used to perform extensive convergence tests that established second order accuracy . The code generated a large complement of highly accurate numerical solutions for the class of asymptotically flat, axisymmetric vacuum spacetimes, a class for which no analytic solutions are known . All results of numerical evolutions in this regime were consistent with the theorem of christodoulou and klainerman that weak initial data evolve asymptotically to minkowski space at late time . An additional global check on accuracy was performed using bondi s formula relating mass loss to the time integral of the square of the news function . The bondi mass loss formula is not one of the equations used in the evolution algorithm but follows from those equations as a consequence of a global integration of the bianchi identities . Thus it not only furnishes a valuable tool for physical interpretation but it also provides a very important calibration of numerical accuracy and consistency . An interesting feature of the evolution arises in regard to compactification . By construction, the u - direction is timelike at the origin where it coincides with the worldline traced out by the vertices of the outgoing null cones . But even for weak fields, the u - direction generically becomes spacelike at large distances along an outgoing ray . Geometrically, this reflects the property that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is itself a null hypersurface so that all internal directions are spacelike, except for the null generator . For a flat space time, the u - direction picked out at the origin leads to a null evolution direction at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, but this direction becomes spacelike under a slight deviation from spherical symmetry . Thus the evolution generically becomes superluminal near \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Remarkably, this leads to no adverse numerical effects . This remarkable property apparently arises from the natural way that causality is built into the marching algorithm so that no additional resort to numerical techniques, such as causal differencing, is necessary . Stewart has implemented a characteristic evolution code which handles the bondi problem by a null tetrad, as opposed to metric, formalism . The geometrical algorithm underlying the evolution scheme, as outlined in [233, 99], is friedrich s conformal - null description of a compactified spacetime in terms of a first order system of partial differential equations . The variables include the metric, the connection, and the curvature, as in a newman - penrose formalism, but in addition the conformal factor (necessary for compactification of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal i}$\end{document}) and its gradient . Without assuming any symmetry, there are more than 7 times as many variables as in a metric based null scheme, and the corresponding equations do not decompose into as clean a hierarchy . This disadvantage, compared to the metric approach, is balanced by several advantages: the equations form a symmetric hyperbolic system so that standard theorems can be used to establish that the system is well-posed.standard evolution algorithms can be invoked to ensure numerical stability.the extra variables associated with the curvature tensor are not completely excess baggage, since they supply essential physical information.the regularization necessary to treat \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is built in as part of the formalism so that no special numerical regularization techniques are necessary as in the metric case . (this last advantage is somewhat offset by the necessity of having to locate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal i}$\end{document} by tracking the zeroes of the conformal factor .) The equations form a symmetric hyperbolic system so that standard theorems can be used to establish that the system is well - posed . The extra variables associated with the curvature tensor are not completely excess baggage, since they supply essential physical information . The regularization necessary to treat \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is built in as part of the formalism so that no special numerical regularization techniques are necessary as in the metric case . (this last advantage is somewhat offset by the necessity of having to locate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal i}$\end{document} by tracking the zeroes of the conformal factor .) The code was intended to study gravitational waves from an axisymmetric star . Since only the vacuum equations are evolved, the outgoing radiation from the star is represented by data (4 in newman - penrose notation) on an ingoing null cone forming the inner boundary of the evolved domain . This inner boundary data is supplemented by schwarzschild data on the initial outgoing null cone, which models an initially quiescent state of the star . But by choosing a scenario in which a black hole is formed, it is possible to evolve the entire region exterior to the horizon . An obvious test bed is the schwarzschild spacetime for which a numerically satisfactory evolution was achieved (although convergence tests were not reported). Physically interesting results were obtained by choosing data corresponding to an outgoing quadrupole pulse of radiation . By increasing the initial amplitude of the data 4, it was possible to evolve into a regime where the energy loss due to radiation was large enough to drive the total bondi mass negative . Although such data is too grossly exaggerated to be consistent with an astrophysically realistic source, the formation of a negative mass was an impressive test of the robustness of the code . Papadopoulos has carried out an illuminating study of mode mixing by computing the evolution of a pulse emanating outward from an initially schwarzschild white hole of mass m. the evolution proceeds along a family of ingoing null hypersurfaces with outer boundary at r = 60 m. the evolution is stopped before the pulse hits the outer boundary in order to avoid spurious effects from reflection and the radiation is inferred from data at r = 20 m. although gauge ambiguities arise in reading off the waveform at a finite radius, the work reveals interesting nonlinear effects: (i) modification of the light cone structure governing the principal part of the equations and hence the propagation of signals; (ii) modulation of the schwarzschild potential by the introduction of an angular dependent mass aspect; and (iii) quadratic and higher order terms in the evolution equations which couple the spherical harmonic modes . A compactified version of this study was later carried out with the 3d pitt code, which confirms these effects as well as new effects which are not present in the axisymmetric case (see section 4.5 for details). Oliveira and rodrigues have presented the first code based upon the galerkin spectral method to evolve the axisymmetic bondi problem . The spectral decomposition reduces the partial differential evolution system to a system of ordinary differential equations for the spectral coefficients . Several numerical tests were performed to verify stability and convergence, including linearized gravitational waves and the global energy momentum conservation law relating the bondi mass to the radiated energy flux . The main feature of the galerkin method is that each basis function is chosen to automatically satisfy the boundary conditions, in this case the regularity conditions on the bondi variables on the axes of symmetry and at the vertices of the outgoing null cones and the asymptotic flatness condition at infinity . Although \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is not explicitly compactified, the choice of radial basis functions allows verification of the asymptotic relations governing the coefficients of the leading gauge dependent terms of the metric quantities . It will be interesting to see whether the approach can be generalized to the full 3d case . The southampton group, as part of its goal of combining cauchy and characteristic evolution, has developed a code [85, 86, 194] which extends the bondi problem to full axisymmetry, as described by the general characteristic formalism of sachs . By dropping the requirement that the rotational killing vector be twist - free, they were able to include rotational effects, including radiation in the cross polarization mode (only the plus mode is allowed by twist - free axisymmetry). The null equations and variables were recast into a suitably regularized form to allow compactification of null infinity . Regularization at the vertices or caustics of the null hypersurfaces was not necessary, since they anticipated matching to an interior cauchy evolution across a finite worldtube . The code was designed to insure standard bondi coordinate conditions at infinity, so that the metric has the asymptotically minkowskian form corresponding to null - spherical coordinates . In order to achieve this, the hypersurface equation for the bondi metric variable must be integrated radially inward from infinity, where the integration constant is specified . This differs from the pittsburgh code in which all the equations are integrated radially outward, so that the coordinate conditions are determined at the inner boundary and the metric is asymptotically flat but not asymptotically minkowskian . The southampton scheme simplifies the formulae for the bondi news function and mass in terms of the metric . It is anticipated that the inward integration of causes no numerical problems because this is a gauge choice which does not propagate physical information . However, the code has not yet been subject to convergence and long term stability tests so that these issues cannot be properly assessed at the present time . The matching of the southampton axisymmetric code to a cauchy interior is discussed in section 5.6 . Numerical calculations of asymptotic quantities such as the bondi mass must pick off non - leading terms in an asymptotic expansion about infinity . This is similar to the experimental task of determining the mass of an object by measuring its far field . For example, in an asymptotically inertial bondi frame at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} (in which the metric takes an asymptotically minkowski form in null spherical coordinates)), the mass aspect \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m}(u, \theta, \phi)$\end{document} is picked off from the asymptotic expansion of bondi s metric quantity v (see equation (16)) of the form \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$v = r - 2{\mathcal m} + {\mathcal o}(1/r)$\end{document}. In gauges which incorporate some of the properties of an asymptotically inertial frame, such as the null quasi - spherical gauge in which the angular metric is conformal to the unit sphere metric, this can be a straightforward computational problem . However, the job can be more difficult if the gauge does not correspond to a standard bondi frame at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. One must then deal with an arbitrary coordinatization of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} which is determined by the details of the interior geometry . As a result, v has a more complicated asymptotic behavior, given in the axisymmetric case by 18\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{v - r = {{{r^2}(l\sin \theta)_{,\theta}} \over {\sin \theta}} + r{e^{2(h - k)}} \times}\\{\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad\quad \quad \quad\quad \quad \quad \left [{\left({1 - {e^{- 2(h - k)}}} \right) + {{2{{({h_{,\theta}}\sin \theta)}_{,\theta}}} \over {\sin \theta}} + {k_{,\theta \theta}} + 3{k_{,\theta}}\cot \theta + 4{{({h_{,\theta}})}^2} - 4{h_{,\theta}}{k_{,\theta}} - 2{{({k_{,\theta}})}^2}} \right]}\\{\quad \quad\quad \quad \quad - 2{e^{2h}}{\mathcal m} + {\mathcal o}({r^{- 1}}),}\\\end{array}$$\end{document} where l, h, and k are gauge dependent functions of (u,) which would vanish in an inertial bondi frame [237, 153]. The calculation of the bondi mass requires regularization of this expression by numerical techniques so that the coefficient \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal m}$\end{document} can be picked off . The task is now similar to the experimental determination of the mass of an object by using non - inertial instruments in a far zone which contains \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}(1/r)$\end{document} radiation fields . It was accomplished in stewart s code by re - expressing the formula for the bondi mass in terms of the well - behaved fields of the conformal formalism . In the pittsburgh code, it was accomplished by re - expressing the bondi mass in terms of renormalized metric variables which regularize all calculations at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} and made them second order accurate in grid size . The calculation of the bondi news function (which provides the waveforms of both polarization modes) is an easier numerical task than the bondi mass . It has also been implemented in both of these codes, thus allowing the important check of the bondi mass loss formula . An alternative approach to computing the bondi mass is to adopt a gauge which corresponds more closely to an inertial bondi frame at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} and simplifies the asymptotic limit . Such a choice is the null quasi - spherical gauge in which the angular part of the metric is proportional to the unit sphere metric, and as a result the gauge term k vanishes in equation (18). This gauge was adopted by bartnik and norton at canberra in their development of a 3d characteristic evolution code (see section 4 for further discussion). It allowed accurate computation of the bondi mass as a limit as r of of the hawking mass . Mainstream astrophysics is couched in newtonian concepts, some of which have no well defined extension to general relativity . In order to provide a sound basis for relativistic astrophysics, it is crucial to develop general relativistic concepts which have well defined and useful newtonian limits . The results of characteristic codes show that the energy of a radiating system can be evaluated rigorously and accurately according to the rules for asymptotically flat spacetimes, while avoiding the deficiencies that plagued the pre - numerical era of relativity: (i) the use of coordinate dependent concepts such as gravitational energy - momentum pseudotensors; (ii) a rather loose notion of asymptotic flatness, particularly for radiative spacetimes; (iii) the appearance of divergent integrals; and (iv) the use of approximation formalisms, such as weak field or slow motion expansions, whose errors have not been rigorously estimated . Characteristic codes have extended the role of the bondi mass from that of a geometrical construct in the theory of isolated systems to that of a highly accurate computational tool . The bondi mass loss formula provides an important global check on the preservation of the bianchi identities . The numerical results demonstrate that computational approaches, rigorously based upon the geometrical definition of mass in general relativity, can be used to calculate radiation losses in highly nonlinear processes where perturbation calculations would not be meaningful . Numerical calculation of the bondi mass has been used to explore both the newtonian and the strong field limits of general relativity . For a quasi - newtonian system of radiating dust, the numerical calculation joins smoothly on to a post - newtonian expansion of the energy in powers of 1/c, beginning with the newtonian mass and mechanical energy as the leading terms . This comparison with perturbation theory has been carried out to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}(1/{c^7})$\end{document}, at which stage the computed bondi mass peels away from the post - newtonian expansion . It remains strictly positive, in contrast to the truncated post - newtonian behavior which leads to negative values . A subtle feature of the bondi mass stems from its role as one component of the total energy - momentum 4-vector, whose calculation requires identification of the translation subgroup of the bondi - metzner - sachs group . Is tantamount to knowing the conformal transformation to an inertial bondi frame in which the time slices of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} have unit sphere geometry . Both stewart s code and the pittsburgh code adapt the coordinates to simplify the description of the interior sources . This results in a non - standard foliation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The determination of the conformal factor which relates the 2-metric hab of a slice of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} to the unit sphere metric is an elliptic problem equivalent to solving the second order partial differential equation for the conformal transformation of gaussian curvature . In the axisymmetric case, the pde reduces to an ode with respect to the angle, which is straightforward to solve . Stewart proposes an approach based upon the dyad decomposition 19\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h_{ab}}d{x^a}d{x^b} = {m_a}d{x^a}{\bar m_b}d{x^b}.$$\end{document} the desired conformal transformation is obtained by first relating hab conformally to the flat metric of the complex plane . Denoting the complex coordinate of the plane by can then be determined from the integrability condition 20\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${m_{\left [a \right. }}{\partial _ b \right]}}.$$\end{document} this is equivalent to the classic beltrami equation for finding isothermal coordinates . It would appear to be a more effective scheme than tackling the second order pde directly, but numerical implementation has not yet been carried out . The initial work on 3d characteristic evolution led to two independent codes, one developed at canberra and the other at pittsburgh (the pitt code), both with the capability to study gravitational waves in single black hole spacetimes at a level not yet mastered at the time by cauchy codes . The pittsburgh group established robust stability and second order accuracy of a fully nonlinear 3d code which was able to calculate the waveform at null infinity [49, 46] and to track a dynamical black hole and excise its internal singularity from the computational grid [119, 116]. The canberra group implemented an independent nonlinear 3d code which accurately evolved the exterior region of a schwarzschild black hole . Both codes pose data on an initial null hypersurface and on a worldtube boundary, and evolve the exterior spacetime out to a compactified version of null infinity, where the waveform is computed . However, there are essential differences in the underlying geometrical formalisms and numerical techniques used in the two codes and in their success in evolving generic black hole spacetimes . Recently two new codes have evolved from the pitt code by introducing a new choice of spherical coordinates [111, 201]. Any characteristic code extending to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} requires the ability to handle tensor fields and their derivatives on the sphere . Spherical coordinates and spherical harmonics are natural analytic tools for the description of radiation, but their implementation in computational work requires dealing with the impossibility of smoothly covering the sphere with a single coordinate grid . Polar coordinate singularities in axisymmetric systems can be regularized by standard tricks . In the absence of symmetry, these techniques do not generalize and would be especially prohibitive to develop for tensor fields . The development of grids smoothly covering the sphere has had a long history in computational meteorology that has led to two distinct approaches: (i) the stereographic approach in which the sphere is covered by two overlapping patches obtained by stereographic projection about the north and south poles; and (ii) the cubed - sphere approach in which the sphere is covered by the 6 patches obtained by a projection of the faces of a circumscribed cube . A discussion of the advantages of each of these methods and a comparison of their performance in a standard fluid testbed are given in . In numerical relativity, the stereographic method has been reinvented in the context of the characteristic evolution problem; and the cubed - sphere method has been reinvented in building an apparent horizon finder . The cubed sphere module, including the interpatch transformations, has been integrated into the cactus toolkit and later applied to black hole excision . Perhaps the most ingenious treatment of the sphere, based upon a toroidal map, was devised by the canberra group in building their characteristic code . Motivated by problems in meteorology, g. l. browning, j. j. hack and p. n. swartztrauber developed the first finite difference scheme based upon a composite mesh with two overlapping stereographic coordinate patches, each having a circular boundary centered about the north or south poles . Values for quantities required at ghost points beyond the boundary of one of the patches were interpolated from values in the other patch . Because a circular boundary does not fit regularly on a stereographic grid, dissipation was found necessary to remove the short wavelength error resulting from the inter - patch interpolations . They used the shallow water equations as a testbed to compare their approach to existing spectral approaches in terms of computer time, execution rate and accuracy . Such comparisons of different numerical methods can be difficult . Both the finite difference and spectral approaches gave good results and were competitive in terms of overall operation count and memory requirements for the particular initial data sets tested, the spectral approach had an advantage but not enough to give clear indication of the suitability of one method over another . The spectral method with m modes requires o(m) operations per time step compared with o(n) for a finite difference method on a n n grid . However, assuming that the solution is smooth, the accuracy of the spectral method is o(e) compared to, say, o(n) for a sixth order finite difference method . Hence, for comparable accuracy, m = o(ln n) which implies that the operation count for the spectral and finite difference methods are o[(ln n)] and o(n), respectively . Thus for sufficiently high accuracy, i.e. Large n, the spectral method requires fewer operations . The issue of spectral vs finite difference methods thus depends on the nature of the smoothness of the physical problem being addressed and the accuracy desired . The pitt null code was first developed using two stereographic patches with square boundaries, each overlapping the equator . This has recently been modified based upon the approach advocated in, which retains the original stereographic coordinates but shrinks the overlap region by masking a circular boundary near the equator . However, the corners of the square boundary, besides being a significant waste of economy, were a prime source of inaccuracy . The resolution at the corners is only 1/9th that at the poles due to the stretching of the stereographic map . Near the equator, the use of a circular boundary requires an angular version of numerical dissipation to control the resulting high frequency error (see section 4.2.1). A crucial ingredient of the pitt code is the - module which incorporates a computational version of the newman - penrose eth - formalism . The underlying method can be applied to any smooth coordinatization x of the sphere based upon several patches . The unit sphere metric qab, defined by these coordinates, is decomposed in each patch in terms of a complex basis vector qa, 21\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_{ab}} = q{(_a}{\bar q_b})$$\end{document} vector and tensor fields on the sphere, and their covariant derivatives, are then represented by their basis components . For example, the vector field u is represented by the complex spin - weight 1 field u = uqa . The covariant derivative d associated with qab is then expressed in terms of the operator according to 22\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_a}{q_b}{d^a}{u^b} = \eth u,\quad {\bar q_a}{q_b}{d^a}{u^b} = \bar \eth u.$$\end{document} the eth - calculus simplifies the underlying equations, avoids spurious coordinate singularities and allows accurate differentiation of tensor fields on the sphere in a computationally efficient and clean way . Its main weakness is the numerical noise introduced by interpolations between the patches . C. ronchi, r. iacono and p. s. paolucci, developed the cubed - sphere approach as a new gridding method for solving global meteorological problems . The method decomposes the sphere into the 6 identical regions obtained by projection of a cube circumscribed on its surface . This gives a variation of the composite mesh method in which the 6 domains butt up against each other along shared grid boundaries . As a result only 1-dimensional intergrid interpolations are necessary (as opposed to the 2-dimensional interpolations of the stereographic grid), which results in enhanced accuracy . The symmetry of the scheme, in which the six patches have the same geometric structure and grid, also allows efficient use of parallel computer architectures . Their tests of the cubed sphere method based upon the simulation of shallow water waves in spherical geometry show that the numerical solutions are as accurate as those with spectral methods, with substantial saving in execution time . Recently, the cubed - sphere method has also been developed for application to characteristic evolution in numerical relativity [201, 111]. The eth - calculus is used to treat tensor fields on the sphere in the same way as in the stereographic method except the interpatch transformations now involve 6, rather than 2, sets of basis vectors . The canberra group treats fields on the sphere by taking advantage of the existence of a smooth map from the torus to the sphere . The pullback of this map allows functions on the sphere to be expressed in terms of toroidal coordinates . The intrinsic topology of these toroidal coordinates allow them to take advantage of of fast - fourier transforms to implement a highly efficient pseudo - spectral treatment . The pitt code uses a standard bondi - sachs null coordinate system, 23\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = - \left({{e^{2\beta}}{v \over r} - {r^2}{h_{ab}}{u^a}{u^b}} \right)d{u^2} - 2{e^{2\beta}}du\;dr - 2{r^2}{h_{ab}}{u^b}du\;d{x^a} + {r^2}{h_{ab}}d{x^a}d{x^b},$$\end{document} where det(hab) = det(qab) for some standard choice qab of the unit sphere metric . This generalizes equation (13) to the three - dimensional case . The characteristic version of einstein s equations are the hypersurface equations derive from the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${g_\mu}^\nu {\nabla _ \nu}u$\end{document} components of the einstein tensor . They take the explicit form 24\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\beta _ {, r}} = {1 \over {16}}r\;{h^{ac}}{h^{bd}}{h_{ab, r}}{h_{cd, r}},$$\end{document} 25\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${({r^4}{e^{- 2\beta}}{h_{ab}}u_{,r}^b)_{,r}} = 2{r^4}{({r^{- 2}}{\beta _ {, a}})_{,r}} - {r^2}{h^{bc}}{d_c}({h_{ab, r}})$$\end{document} 26\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{2{e^{- 2\beta}}{v_{,r}} = {\mathcal r} - 2{d^a}{d_a}\beta - 2({d^a}\beta){d_a}\beta + {r^{- 2}}{e^{- 2\beta}}{d_a}\left({{{({r^4}{u^a})}_{,r}}} \right)}\\{\quad \quad \quad \quad - {1 \over 2}{r^4}{e^{- 4\beta}}{h_{ab}}u_{,r}^au_{,r}^b,}\\\end{array}$$\end{document} where da is the covariant derivative and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal r}$\end{document} the curvature scalar of the conformal 2-metric hab of the r = const . Surfaces, and capital latin indices are raised and lowered with hab . Given the null data hab on an outgoing null hypersurface, this hierarchy of equations can be integrated radially in order to determine, u and v on the hypersurface in terms of integration constants on an inner boundary . The evolution equations for the u - derivative of the null data derive from the trace - free part of the angular components of the einstein tensor, i.e. The components mmgab where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${h^{ab}} = 2{m^{(a}}{{\bar m}^b})$\end{document}. They take the explicit form 27\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}c}{{m^a}{m^b}\left({{{(r{h_{ab, u}})}_{,r}} - {1 \over {2r}}{{(rv{h_{ab, r}})}_{,r}} - {2 \over r}{e^\beta}{d_a}{d_b}{e^\beta} + r{h_{ac}}{d_b}(u_{,r}^c)} \right. }\\ {\quad \quad \quad \quad \quad \quad \;\; - {{{r^3}} \over 2}{e^{- 2\beta}}{h_{ac}}{h_{bd}}u_{,r}^cu_{,r}^d + 2{d_a}{u_b} + {r \over 2}{h_{ab, r}}{d_c}{u^c}}\\{\left . {\quad \quad \quad \quad \quad \quad \;\; + r{u^c}{d_c}({h_{ab, r}}) + r{h_{ad, r}}{h^{cd}}({d_b}{u_c} - {d_c}{u_b})} \right) = 0. }\\\end{array}$$\end{document} a compactified treatment of null infinity is achieved by introducing the radial coordinate x = r/(r + r), where r is a scale parameter adjusted to the size of the inner boundary . Thus x = 1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The canberra code employs a null quasi - spherical (nqs) gauge (not to be confused with the quasi - spherical approximation in which quadratically aspherical terms are ignored). The nqs gauge takes advantage of the possibility of mapping the angular part of the bondi metric conformally onto a unit sphere metric, so that hab qab . The required transformation x y (u, r, x) is in general dependent upon u and r so that the nqs angular coordinates y are not constant along the outgoing null rays, unlike the bondi - sachs angular coordinates . Instead the coordinates y display the analogue of a shift on the null hypersurfaces u = const . The radiation content of the metric is contained in a shear vector describing this shift . This results in the description of the radiation in terms of a spin - weight 1 field, rather than the spin - weight 2 field associated with hab in the bondi - sachs formalism . In both the bondi - sachs and nqs gauges, the independent gravitational data on a null hypersurface is the conformal part of its degenerate 3-metric . The bondi - sachs null data consist of hab, which determines the intrinsic conformal metric of the null hypersurface . In the nqs case, hab = qab and the shear vector comprises the only non - trivial part of the conformal 3-metric . Both the bondi - sachs and nqs gauges can be arranged to coincide in the special case of shear - free robinson - trautman metrics [82, 26]. The formulation of einstein s equations in the nqs gauge is presented in, and the associated gauge freedom arising from (u, r) dependent rotation and boosts of the unit sphere is discussed in . As in the pitt code, the main equations involve integrating a hierarchy of hypersurface equations along the radial null geodesics extending from the inner boundary to null infinity . In the nqs gauge the source terms for these radial odes are rather simple when the unknowns are chosen to be the connection coefficients . However, as a price to pay for this simplicity, after the radial integrations are performed on each null hypersurface a first order elliptic equation must be solved on each r = const . Cross - section to reconstruct the underlying metric . For a {3 + 1} evolution algorithm based upon a system of wave equations, or any other symmetric hyperbolic system, numerical dissipation can be added in the standard kreiss - oliger form . Dissipation cannot be added to the {2 + 1 + 1} format of characteristic evolution in this standard way for {3 + 1} cauchy evolution . In the original version of the pitt code, which used square stereographic patches with boundaries aligned with the grid, this was sufficient to establish numerical stability . In the new version of the code with circular stereographic patches, whose boundaries fit into the stereographic grid in an irregular way, angular dissipation is necessary to suppress the resulting high frequency error . Angular dissipation can be introduced in the following way . In terms of the spin - weight 2 variable j = qqhab, the evolution equation (27) takes the form 28\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ u}{\partial _ r}(rj) = s,$$\end{document} where s represents the right hand side terms . We add angular dissipation to the u - evolution through the modification 29\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ u}{\partial _ r}(rj) + {\epsilon _ u}{h^3}{\eth^2}{\bar \eth^2}{\partial_r}(rj) = s,$$\end{document} where h is the discretization size and u 0 is an adjustable parameter independent of h. this leads to 30\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ u}\left({\vert{\partial _ _ r}(r\bar j)} \right)s\}{. }$$\end{document} integration over the unit sphere with solid angle element d then gives 31\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ u}\oint {{{\left\vert {{\partial _ r}(rj)} \right\vert}^2}} d\omega + 2{\epsilon _ u}{h^3}\oint {{{\left\vert {{\eth^2}\left({{\partial _ r}(rj)} \right)} \right\vert}^2}d\omega = 2\re \oint {\left({{\partial _ r}(r\bar j)} \right)} sd\omega}.$$\end{document} thus the u - term has the effect of damping high frequency noise as measured by the l2 norm of r(rj) over the sphere . Similarly, dissipation can be introduced in the radial integration of (28) through the substitution 32\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ u}{\partial _ r}(rj) \rightarrow{\partial _ u}{\partial _ r}(rj) + {\epsilon_r}{h^3}{\eth^2}{\bar \eth^2}{\partial _ u}(rj),$$\end{document} with r 0 . Angular dissipation can also be introduced in the hypersurface equations, e.g. In (26) through the substitution 33\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\partial _ _ r}v + {\epsilon_v}{h^3}\bar \eth \eth \eth \bar \eth v.$$\end{document} the pitt code was originally formulated in the second differential form of equations (24, 25, 26, 27), which in the spin - weighted version leads to an economical number of 2 real and 2 complex variables . Subsequently, the variable 34\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_a} = {r^2}{e^{- 2\beta}}{h_{ab}}u_{,r}^b$$\end{document} was introduced to reduce equation (25) to two first order radial equations, which simplified the startup procedure at the boundary . Although the resulting code was verified to be stable and second order accurate, its application to problems involving strong fields and gradients led to numerical errors which made small scale effects of astrophysical importance difficult to measure . In particular, in initial attempts to simulate a white hole fission, gmez encountered an oscillatory error pattern in the angular directions near the time of fission . The origin of the problem was tracked to numerical error of an oscillatory nature introduced by terms in the hypersurface and evolution equations . Gmez s solution was to remove the offending second angular derivatives by introducing additional variables and reducing the system to first differential order in the angular directions . This suppressed the oscillatory mode and subsequently improved performance in the simulation of the white hole fission problem (see section 4.4.2). This success opens the issue of whether a completely first differential order code might perform even better, as has been proposed by gmez and frittelli . By the use of uhab as a fundamental variable, they cast the bondi system into duff s first order quasilinear canonical form . At the analytic level this provides standard uniqueness and existence theorems (extending previous work for the linearized case) and is a starting point for establishing the estimates required for well - posedness . At the numerical level, gmez and frittelli point out that this first order formulation provides a bridge between the characteristic and cauchy approaches which allows application of standard methods for constructing numerical algorithms, e.g. To take advantage of shock - capturing schemes . Although true shocks do not exist for vacuum gravitational fields, when coupled to hydro the resulting shocks couple back to form steep gradients which might not be captured by standard finite difference approximations . In particular, the second derivatives needed to compute gravitational radiation from stellar oscillations have been noted to be a troublesome source of inaccuracy in the characteristic treatment of hydrodynamics . The benefits of this completely first order approach are not simple to decide without code comparison . The part of the code in which the operator introduced the oscillatory error mode in was not identified, i.e. Whether it originated in the inner boundary treatment or in the interpolations between stereographic patches where second derivatives might be troublesome . There are other possible ways to remove the oscillatory angular modes, such as adding angular dissipation (see section 4.2.1). The finite difference algorithm in the original pitt code only introduced numerical dissipation in the radial direction . The economy of variables and other advantages of a second order scheme should not be abandoned without further tests and investigation . The pitt code is an explicit finite difference evolution algorithm based upon retarded time steps on a uniform three - dimensional null coordinate grid based upon the stereographic coordinates and a compactified radial coordinate . Most of these involve smoothing or filtering and have obvious advantage for removing short wavelength noise but would be unsuitable for modeling shocks . There have been two recent projects, to improve the performance of the pitt code by using the cubed - sphere method to coordinatize the sphere . They both include an adaptation of the eth - calculus to handle the transformation of spin - weighted variables between the six patches . In one of these projects, gmez, barreto and frittelli develop the cubed - sphere approach into an efficient, highly parallelized 3d code, the leo code, for the characteristic evolution of the coupled einstein - klein - gordon equations in the bondi - sachs formalism . This code was demonstrated to be convergent and its high accuracy in the linearized regime with a schwarzschild background was demonstrated by the simulation of the quasinormal ringdown of the scalar field and its energy - momentum conservation . Because the characteristic evolution scheme constitutes a radial integration carried out for each angle on the sphere of null directions, the natural way to parallelize the code is distribute the angular grid among processors . Thus given m m processors one can distribute the n n points in each spherical patch (cubed - sphere or stereographic), assigning to each processor equal square grids of extent n / m in each direction . To be effective this requires that the communication time between processors scales effectively . This depends upon the ghost point location necessary to supply nearest neighbor data and is facilitated in the cubed - sphere approach because the ghost points are aligned on 1-dimensional grid lines, whose pattern is invariant under grid size . In the stereographic approach, the ghost points are arranged in an irregular pattern which changes in an essentially random way under rescaling and requires a more complicated parallelization algorithm . Their goal is to develop the leo code for application to black hole - neutron star binaries in a close orbit regime where the absence of caustics make a pure characteristic evolution possible . Their first anticipated application is the simulation of a boson star orbiting a black hole, whose dynamics is described by the einstein - klein - gordon equations . They point out that characteristic evolution of such systems of astrophysical interest have been limited in the past by resolution due to the lack off the necessary computational power, parallel infrastructure and mesh refinement . Most characteristic code development has been geared toward single processor machines whereas the current computational platforms are designed toward performing high resolution simulations in reasonable times by parallel processing . At the same time also constructed a characteristic code for the bondi - sachs problem based upon the cubed - sphere infrastructure of thornburg [242, 241]. The canberra code handles fields on the sphere by means of a 3-fold representation: (i) as discretized functions on a spherical grid uniformly spaced in standard (,) coordinates, (ii) as fast - fourier transforms with respect to (,) (based upon the smooth map of the torus onto the sphere), and (iii) as a spectral decomposition of scalar, vector, and tensor fields in terms of spin - weighted spherical harmonics . The grid values are used in carrying out nonlinear algebraic operations; the fourier representation is used to calculate (,)-derivatives; and the spherical harmonic representation is used to solve global problems, such as the solution of the first order elliptic equation for the reconstruction of the metric, whose unique solution requires pinning down the = 1 gauge freedom . The sizes of the grid and of the fourier and spherical harmonic representations are coordinated . In practice, the spherical harmonic expansion is carried out to 15th order in, but the resulting coefficients must then be projected into the 10 subspace in order to avoid inconsistencies between the spherical harmonic, grid, and fourier representations . The canberra code solves the null hypersurface equations by combining an 8th order runge - kutta integration with a convolution spline to interpolate field values . The radial grid points are dynamically positioned to approximate ingoing null geodesics, a technique originally due to goldwirth and piran to avoid the problems with a uniform r - grid near a horizon which arise from the degeneracy of an areal coordinate on a stationary horizon . The time evolution uses the method of lines with a fourth order runge - kutta integrator, which introduces further high frequency filtering . Analytic stability analysis of the finite difference equations has been crucial in the development of a stable evolution algorithm, subject to the standard courant - friedrichs - lewy (cfl) condition for an explicit code . Linear stability analysis on minkowski and schwarzschild backgrounds showed that certain field variables must be represented on the half - grid [120, 49]. Nonlinear stability analysis was essential in revealing and curing a mode coupling instability that was not present in the original axisymmetric version of the code [46, 163]. This has led to a code whose stability persists even in the regime that the u - direction, along which the grid flows, becomes spacelike, such as outside the velocity of light cone in a rotating coordinate system . Robust stability was established by imposing random initial data on the initial characteristic hypersurface and random constraint violating boundary data on an inner worldtube . (robust stability was later adopted as one of the standardized tests for cauchy codes .) The pitt code was the first 3d general relativistic code to pass this robust stability test . The use of random data is only possible in sufficiently weak cases where terms quadratic in the field gradients are not dominant . . Runs for a time of 60, 000 m were carried out for a moving, distorted schwarzschild black hole (of mass m), with the marginally trapped surface at the inner boundary tracked and its interior excised from the computational grid [116, 117]. At the time, this was by far the longest simulation of a dynamic black hole . Furthermore, the scattering of a gravitational wave off a schwarzschild black hole was successfully carried out in the extreme nonlinear regime where the backscattered bondi news was as large as n = 400 (in dimensionless geometric units), showing that the code can cope with the enormous power output nc / g 10 w in conventional units . This exceeds the power that would be produced if, in 1 second, the entire galaxy were converted to gravitational radiation . The characteristic codes using the cubed - sphere grid [111, 201] are based upon the same variables and equations as the pitt code, with the same radial integration scheme . Thus it should be expected that stability be maintained since the main difference is that the interpatch interpolations are simpler, i.e. Only 1-dimensional . This appears to be the case in the reported tests, although robust stability was not directly confirmed . In particular, the leo code showed no sign of instability in long time, high resolution simulations of the quasinormal ringdown of a scalar field scattering off a schwarzschild black hole . Angular dissipation was not necessary analytic stability analysis of the underlying finite difference equations is impractical because of the extensive mix of spectral techniques, higher order methods, and splines . Although there is no clear - cut cfl limit on the code, stability tests show that there is a limit on the time step . The damping of high frequency modes due to the implicit filtering would be expected to suppress numerical instability, but the stability of the canberra code is nevertheless subject to two qualifications [28, 29, 30]: (i) at late times (less than 100 m), the evolution terminates as it approaches an event horizon, apparently because of a breakdown of the nqs gauge condition, although an analysis of how and why this should occur has not yet been given . (ii) numerical instabilities arise from dynamic inner boundary conditions and restrict the inner boundary to a fixed schwarzschild horizon . The designed second order accuracy has been verified in an extensive number of testbeds [49, 46, 116, 255, 256], including new exact solutions specifically constructed in null coordinates for the purpose of convergence tests: linearized waves on a minkowski background in null cone coordinates.boost and rotation symmetric solutions .schwarzschild in rotating coordinates.polarization symmetry of nonlinear twist - free axisymmetric waveforms.robinson-trautman waveforms from perturbed schwarzschild black holes.nonlinear robinson - trautman waveforms utilizing an independently computed solution of the robinson - trautman equation.perturbations of a schwarzschild black hole utilizing an independently computed solution of the teukolsky equation . In addition to these testbeds, a set of linearized solutions has recently been obtained in the bondi - sachs gauge for either schwarzschild or minkowski backgrounds . The solutions are generated by the introduction of a thin shell of matter whose density varies with time and angle . For a minkowski background, the solution is given in exact analytic form and, for a schwarzschild background, in terms of a power series . They supply a new and very useful testbed for the calibration and further development of characteristic evolution codes for einstein s equations, analogous to the role of the teukolsky waves in cauchy evolution . The pitt code showed clean second order convergence in both the l2 and l error norms in tests based upon waves in a minkowski background . Nonlinear robinson - trautman waveforms utilizing an independently computed solution of the robinson - trautman equation . Perturbations of a schwarzschild black hole utilizing an independently computed solution of the teukolsky equation . Convergence of the cubed - sphere code developed by reisswig et al was also checked using linearized waves in a minkowski background . The convergence rate for the l2 error norm was approximately second order accurate but in some cases there was significant degradation . They conjectured that the underlying source of error arises at the corners of the six patches . Comparison with the l error norm would discriminate such a localized source of error but such results were not reported . The designed convergence rate of the operator used in the leo code was verified for second, fourth and eighth order finite difference approximations, using the spin - weight 2 spherical harmonic 2y43 as a test . Similarly, the convergence of the integral relations governing the orthonormality of the spin - weighted harmonics was verified . Although convergence of the evolution code was not explicitly checked, high accuracy in the linearized regime with schwarzschild background was demonstrated in the simulation of quasinormal ringdown of the scalar field and in the energy - momentum conservation of the scalar field . The complexity of the algorithm and nqs gauge makes it problematic to establish accuracy by direct means . Exact solutions do not provide an effective convergence check, because the schwarzschild solution is trivial in the nqs gauge and other known solutions in this gauge require dynamic inner boundary conditions which destabilize the present version of the code . Instead indirect tests by means of geometric consistency and partial convergence tests are used to calibrate accuracy . The consistency tests were based on the constraint equations, which are not enforced during null evolution except at the inner boundary . The balance between mass loss and radiation flux through \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is a global consequence of these constraints . No appreciable growth of the constraints was noticeable until within 5 m of the final breakdown of the code . In weak field tests where angular resolution does not dominate the error, partial convergence tests based upon varying the radial grid size verify the 8th order convergence in the shear expected from the runge - kutta integration and splines . When the radial source of error is small, reduced error with smaller time step can also be discerned . In practical runs, the major source of inaccuracy is the spherical harmonic resolution, which was restricted to 15 by hardware limitations . Truncation of the spherical harmonic expansion has the effect of modifying the equations to a system for which the constraints are no longer satisfied . A natural physical application of a characteristic evolution code is the nonlinear version of the classic problem of scattering off a schwarzschild black hole, first solved perturbatively by price . Here the inner worldtube for the characteristic initial value problem consists of the ingoing branch of the r = 2 m hypersurface (the past horizon), where schwarzschild data are prescribed . The nonlinear problem of a gravitational wave scattering off a schwarzschild black hole is then posed in terms of data on an outgoing null cone which describe an incoming pulse with compact support . Part of the energy of this pulse falls into the black hole and part is backscattered to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. This problem has been investigated using both the pitt and canberra codes . The pittsburgh group studied the backscattered waveform (described by the bondi news function) as a function of incoming pulse amplitude . The computational eth - module smoothly handled the complicated time dependent transformation between the non - inertial computational frame at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} and the inertial (bondi) frame necessary to obtain the standard plus and cross polarization modes . In the perturbative regime, the news corresponds to the backscattering of the incoming pulse off the effective schwarzschild potential . When the energy of the pulse is no larger than the central schwarzschild mass, the backscattered waveform still depends roughly linearly on the amplitude of the incoming pulse . Its amplitude is greater than that predicted by linear scaling and its shape drastically changes and exhibits extra oscillations . In this very high amplitude case, the mass of the system is completely dominated by the incoming pulse, which essentially backscatters off itself in a nonlinear way . The canberra code was used to study the change in bondi mass due to the radiation . The hawking mass mh(u, r) was calculated as a function of radius and retarded time, with the bondi mass mb(u) then obtained by taking the limit r . The limit had good numerical behavior . For a strong initial pulse with = 4 angular dependence, in a run from u = 0 to u = 70 (in units where the interior schwarzschild mass is 1), the bondi mass dropped from 1.8 to 1.00002, showing that almost half of the initial energy of the system was backscattered and that a surprisingly negligible amount of energy fell into the black hole . A possible explanation is that the truncation of the spherical harmonic expansion cuts off wavelengths small enough to effectively penetrate the horizon . The bondi mass decreased monotonically in time, as necessary theoretically, but its rate of change exhibited an interesting pulsing behavior whose time scale could not be obviously explained in terms of quasinormal oscillations . The bondi mass loss formula was confirmed with relative error of less than 10 . This is impressive accuracy considering the potential sources of numerical error introduced by taking the limit of the hawking mass with limited resolution . The code was also used to study the appearance of logarithmic terms in the asymptotic expansion of the weyl tensor . In addition, the canberra group studied the effect of the initial pulse amplitude on the waveform of the backscattered radiation, but did not extend their study to the very high amplitude regime in which qualitatively interesting nonlinear effects occur . The pitt code has also been implemented to evolve along an advanced time foliation by ingoing null cones, with data given on a worldtube at their outer boundary and on the initial ingoing null cone . The code was used to evolve a black hole in the region interior to the worldtube by implementing a horizon finder to locate the marginally trapped surface (mts) on the ingoing cones and excising its singular interior . The code tracks the motion of the mts and measures its area during the evolution . Data at the outer worldtube was induced from a schwarzschild or kerr spacetime but the worldtube was allowed to move relative to the stationary trajectories; i.e. With respect to the grid the worldtube is fixed but the black hole moves inside it . The initial null data consisted of a pulse of radiation which subsequently travels outward to the worldtube where it reflects back toward the black hole . The approach of the system to equilibrium was monitored by the area of the mts, which also equals its hawking mass . When the worldtube is stationary (static or rotating in place), the distorted black hole inside evolved to equilibrium with the boundary . A boost or other motion of the worldtube with respect to the black hole did not affect this result . The marginally trapped surface always reached equilibrium with the outer boundary, confirming that the motion of the boundary was pure gauge . This was the first code that ran forever in a dynamic black hole simulation, even when the worldtube wobbled with respect to the black hole to produce artificial periodic time dependence . An initially distorted, wobbling black hole was evolved for a time of 60,000 m, longer by orders of magnitude than permitted by the stability of other existing 3d black hole codes at the time . This exceptional performance opens a promising new approach to handle the inner boundary condition for cauchy evolution of black holes by the matching methods reviewed in section 5 . Note that setting the pulse to zero is equivalent to prescribing shear free data on the initial null cone . Combined with schwarzschild boundary data on the outer worldtube however, the evolution of such shear free null data combined with kerr boundary data would have an initial transient phase before settling down to a kerr black hole . This is because the twist of the shear - free kerr null congruence implies that kerr data specified on a null hypersurface are not generally shear free . The event horizon is an exception but kerr null data on other null hypersurfaces have not been cast in explicit analytic form . (curiously, kerr data on a null hypersurface with a conical type singularity do take a simple analytic form, although unsuitable for numerical evolution .) Using some intermediate analytic results of israel and pretorius, venter and bishop have recently constructed a numerical algorithm for transforming the kerr solution into bondi coordinates and in that way provide the necessary null data numerically . The conformal metric of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document} is provided by the conformal horizon model for a binary black hole horizon [165, 150], which treats the horizon in stand - alone fashion as a three - dimensional manifold endowed with a degenerate metric ab and affine parameter t along its null rays . The metric is obtained from the conformal mapping \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\gamma _ {ab}} = {\omega ^2}{{\hat \gamma}_{ab}}$\end{document} of the intrinsic metric \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \gamma}_{ab}}$\end{document} of a flat space null hypersurface emanating from a convex surface \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_0}$\end{document} embedded at constant time in minkowski space . The horizon is identified with the null hypersurface formed by the inner branch of the boundary of the past of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_0}$\end{document}, and its extension into the future . The flat space null hypersurface expands forever as its affine parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat t}$\end{document} (minkowski time) increases, but the conformal factor is chosen to stop the expansion so that the cross - sectional area of the black hole approaches a finite limit in the future . At the same time, the raychaudhuri equation (which governs the growth of surface area) forces a nonlinear relation between the affine parameters t and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat t}$\end{document}. This is what produces the nontrivial topology of the affine t - slices of the black hole horizon . The relative distortion between the affine parameters t and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat t}$\end{document}, brought about by curved space focusing, gives rise to the trousers shape of a binary black hole horizon . An embedding diagram of the horizon for an axisymmetric head - on collision, obtained by choosing \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}_0}$\end{document} to be a prolate spheroid, is shown in figure 3 . The black hole event horizon associated with a triaxial ellipsoid reveals new features not seen in the degenerate case of the head - on collision, as depicted in figure 4 . If the degeneracy is slightly broken, the individual black holes form with spherical topology but as they approach, an effective tidal distortion produces two sharp pincers on each black hole just prior to merger . At merger, the inner hole of the torus subsequently closes up to produce first a peanut shaped black hole and finally a spherical black hole . No violation of topological censorship occurs because the hole in the torus closes up superluminally . Consequently, a causal curve passing through the torus at a given time can be slipped below the bottom of a trouser leg to yield a causal curve lying entirely outside the hole . In the degenerate axisymmetric limit, the pincers reduce to a point so that the individual holes have teardrop shape and they merge without a toroidal phase . Figure 4upper left: tidal distortion of approaching black holes upper right: formation of sharp pincers just prior to merger . Upper left: tidal distortion of approaching black holes upper right: formation of sharp pincers just prior to merger . The conformal horizon model determines the data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}^ +} $\end{document}. The remaining data necessary to evolve the exterior spacetime are given by the conformal geometry of j, which constitutes the outgoing radiation waveform . The determination of the merger - ringdown waveform proceeds in two stages . In the first stage, this outgoing waveform is set to zero and the spacetime is evolved backward in time to calculate the incoming radiation entering from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ -}$\end{document}. (this incoming radiation is eventually absorbed by the black hole .) From a time reversed point of view, this evolution describes the outgoing waveform emitted in the fission of a white hole, with the physically correct initial condition of no ingoing radiation . Preliminary calculations show that at late times the waveform is entirely quadrupolar (= 2) but that a strong octopole mode (= 4) exists just before fission . In the second stage of the calculation, this waveform could be used to generate the physically correct outgoing waveform for a black hole merger . The passage from the first stage to the second is the nonlinear equivalent of first determining an inhomogeneous solution to a linear problem and then adding the appropriate homogeneous solution to satisfy the boundary conditions . In this context, the first stage supplies an advanced solution and the second stage the homogeneous retarded minus advanced solution . When the evolution is carried out in the perturbative regime of a kerr or schwarzschild background, as in the close approximation, this superposition of solutions is simplified by the time reflection symmetry . The second stage has been carried out in the perturbative regime of the close approximation using a characteristic code which solves the teukolsky equation, as described in section 4.4 . More generally, beyond the perturbative regime, the merger - ringdown waveform must be obtained by a more complicated inverse scattering procedure, which has not yet been attempted . There is a complication in applying the pitt code to this double null evolution because a dynamic horizon does not lie precisely on r - grid points . As a result, the r - derivative of the null data, i.e. The ingoing shear of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal h}$\end{document}, the ingoing shear is part of the free data specified at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal s}^ +} $\end{document}. Its value on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal h}$\end{document} can be determined by integrating (backward in time) a sequence of propagation equations involving the horizon s twist and ingoing divergence . A horizon code which carries out these integrations has been tested to give accurate data even beyond the merger . The code has revealed new global properties of the head - on collision by studying a sequence of data for a family of colliding black holes which approaches a single schwarzschild black hole . The resulting perturbed schwarzschild horizon provides global insight into the close limit, in which the individual black holes have joined in the infinite past . A marginally anti - trapped surface divides the horizon into interior and exterior regions, analogous to the division of the schwarzschild horizon by the r = 2 m bifurcation sphere . In passing from the perturbative to the strongly nonlinear regime there is a rapid transition in which the individual black holes move into the exterior portion of the horizon . The data pave the way for the pitt code to calculate whether this dramatic time dependence of the horizon produces an equally dramatic waveform . See section 4.4.2 for first stage results . The nonlinear 3d pitt code has been calibrated in the regime of small perturbations of a schwarzschild spacetime [255, 256] by measuring convergence with respect to independent solutions of the teukolsky equation . By decomposition into spherical harmonics, the teukolsky equation reduces the problem of a perturbation of a stationary black hole to a 1d problem in the (t, r) subspace perturbations for a component of the weyl tensor . Campanelli, gmez, husa, winicour, and zlochower [65, 151] have reformulated the teukolsky formalism as a double - null characteristic evolution algorithm . The evolution proceeds on a family of outgoing null hypersurfaces with an ingoing null hypersurface as inner boundary and with the outer boundary compactified at future null infinity . It applies to either the weyl component 0 or 4, as classified in the newman - penrose formalism . The 0 component comprises constraint - free gravitational data on an outgoing null hypersurface and 4 comprises the corresponding data on an ingoing null hypersurface . In the study of perturbations of a schwarzschild black hole, 0 is prescribed on an outgoing null hypersurface \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document}, representing an early retarded time approximating past null infinity, and 4 is prescribed on the inner white hole horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document}. The physical setup is described in figure 5 . The outgoing null hypersurfaces extend to future null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} on a compactified numerical grid . Consequently, there is no need for either an artificial outer boundary condition or an interior extraction worldtube . The outgoing radiation is computed in the coordinates of an observer in an inertial frame at infinity, thus avoiding any gauge ambiguity in the waveform . A star of mass m has undergone spherically symmetric collapse to form a black hole . The ingoing null worldtube \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document} lies outside the collapsing matter . Inside \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document} (but outside the matter) there is a vacuum schwarzschild metric . Outside of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document}, data for an ingoing pulse is specified on the initial outgoing null hypersurface \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document}. As the pulse propagates to the black hole event horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document}, part of its energy is scattered to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The physical setup for the scattering problem . A star of mass m has undergone spherically symmetric collapse to form a black hole . The ingoing null worldtube \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document} lies outside the collapsing matter . Inside \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document} (but outside the matter) there is a vacuum schwarzschild metric . Outside of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal n}$\end{document}, data for an ingoing pulse is specified on the initial outgoing null hypersurface \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document}. As the pulse propagates to the black hole event horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document}, part of its energy is scattered to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The first calculations were carried out with nonzero data for 4 on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} and zero data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} (so that no ingoing radiation entered the system). The resulting simulations were highly accurate and tracked the quasi - normal ringdown of a perturbation consisting of a compact pulse through 10 orders of magnitude and tracked the final power law decay through an additional 6 orders of magnitude . The measured exponent of the power law decay varied from 5.8, at the beginning of the tail, to 5.9 near the end, in good agreement with the predicted value of 2 + 2 = 6 for a quadrupole wave . The accuracy of the perturbative solutions provide a virtual exact solution for carrying out convergence tests of the nonlinear pitt null code . In this way, the error in the bondi news function computed by the pitt code was calibrated for perturbative data consisting of either an outgoing pulse on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} or an ingoing pulse on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document}. For the outgoing pulse, clean second order convergence was confirmed until late times in the evolution, when small deviations from second order arise from accumulation of roundoff and truncation error . For the bondi news produced by the scattering of an ingoing pulse, clean second order convergence was again confirmed until late times when the pulse approached the r = 2 m black hole horizon . The late time error arises from loss of resolution of the pulse (in the radial direction) resulting from the properties of the compactified radial coordinate used in the code . This type of error could be eliminated by using characteristic the amr techniques under development . The characteristic teukolsky code has been used to study radiation from axisymmetric white holes and black holes in the close approximation . The radiation from an axisymmetric fissioning white hole was computed using the weyl data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} supplied by the conformal horizon model described in section 4.3, with the fission occurring along the axis of symmetry . The close approximation implies that the fission takes place far in the future, i.e. In the region of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} above the black hole horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document}. The data have a free parameter which controls the energy yielded by the white hole fission . The radiation waveform reveals an interesting dependence on the parameter . In the large limit, the waveform consists of a single pulse, followed by ringdown and tail decay . The amplitude of the pulse scales quadratically with and the width decreases with . As is reduced, the initial pulse broadens and develops more structure . In the small limit, the amplitude scales linearly with and the shape is independent of . Since there was no incoming radiation, the above model gave the physically appropriate boundary conditions for a white hole fission (in the close approximation). From a time reversed view point, the system corresponds to a black hole merger with no outgoing radiation at future null infinity, i.e. The analog of an advanced solution with only ingoing but no outgoing radiation . In the axisymmetric case studied, the merger corresponds to a head - on collision between two black holes . The physically appropriate boundary conditions for a black hole merger correspond to no ingoing radiation on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} and binary black hole data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document}. Because \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document} are disjoint, the corresponding data cannot be used directly to formulate a double null characteristic initial value problem . However, the ingoing radiation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} supplied by the advanced solution for the black hole merger could be used as stage i of a two stage approach to determine the corresponding retarded solution . In stage ii, this ingoing radiation is used to generate the analogue of an advanced minus retarded solution . A pure retarded solution (with no ingoing radiation but outgoing radiation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} can then be constructed by superposition . The time reflection symmetry of the schwarzschild background is key to carrying out this construction . This two stage strategy has been carried out by husa, zlochower, gmez, and winicour . The superposition of the stage i and ii solutions removes the ingoing radiation from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} while modifying the close approximation perturbation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ +} $\end{document}, essentially making it ring . The amplitude of the radiation waveform at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} has a linear dependence on the parameter, which in this black hole scenario governs the energy lost in the inelastic merger process . Unlike the fission waveforms, there is very little -dependence in their shape and the amplitude continues to scale linearly even for large . It is not surprising that the retarded waveforms from a black hole merger differs markedly from the retarded waveforms from a white hole merger . The white hole process is directly visible at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} whereas the merger waveform results indirectly from the black holes through the preceding collapse of matter or gravitational energy that formed them . This explains why the fission waveform is more sensitive to the parameter which controls the shape and timescale of the horizon data . However, the weakness of the dependence of the merger waveform on is surprising and has potential importance for enabling the design of an efficient template for extracting a gravitational wave signal from noise . In the purely vacuum approach to the binary black hole problem, the stars which collapse to form the black holes are replaced either by imploding gravitational waves or some past singularity as in the kruskal picture . The imploding waves either emanate from a past singularity, in which case the time - reversed application of cosmic censorship implies the existence of an anti - trapped surface; or they emanate from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ -}$\end{document}, which complicates the issue of gravitational radiation content in the initial data and its effect on the outgoing waveform . These complications are avoided in the two stage approach adopted in the close approximation studies described in section 4.4.1, where advanced and retarded solutions in a schwarzschild background can be rigorously identified and superimposed . Computational experiments have been carried out to study the applicability of this approach in the nonlinear regime . From a time reversed viewpoint, the first stage is equivalent to the determination of the outgoing radiation from a fission of a white hole in the absence of ingoing radiation, i.e. The physically appropriate retarded waveform from a white hole fission . This fission problem can be formulated in terms of data on the white hole horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} and data representing the absence of ingoing radiation on a null hypersurface j which emanates from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} at an early time . The data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document} is provided by the conformal horizon model for a fissioning white hole . This allows study of a range of models extending from the perturbative close approximation regime, in which the fission occurs inside a black hole event horizon, to the nonlinear regime of a fission visible from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The study concentrates on the axisymmetric spinless fission (corresponding in the time reversed view to the head - on collision of non - spinning black holes). In the perturbative regime, the news function agrees with the close approximation waveforms . In the highly nonlinear regime, a bare fission was found to produce a dramatically sharp radiation pulse, which then undergoes a damped oscillation . Because the black hole fission is visible from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, it is a more efficient source of gravitational waves than a black hole merger and can produce a higher fractional mass loss! The pitt code has been used to model the nonlinear generation of waveforms by scattering off a schwarzschild black hole [255, 256]. The physical setup is similar to the perturbative study in section 4.4 . A radially compact pulse is prescribed on an early time outgoing null hypersurface \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal j}^ -}$\end{document} and schwarzschild null data is given on the interior white hole horizon \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal h}^ -}$\end{document}, which is causally unaffected by the pulse . The input pulse is standardized to (= 2, m = 0) and (= 2, m = 2) quadrupole modes with amplitude a. the outgoing null hypersurfaces extend to future null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} on a compactified numerical grid . The evolution code then provides the news function at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, in the coordinates of an observer in an inertial frame at infinity, thus avoiding any gauge ambiguity in the waveform . This provides a simple setting for how the nonlinearities generated by high amplitudes affect the waveform . The study reveals several features of qualitative importance: the mode coupling amplitudes consistently scale as powers a of the input amplitude a corresponding to the nonlinear order of the terms in the evolution equations which produce the mode . This allows much economy in producing a waveform catalog: given the order n associated with a given mode generation, the response to any input amplitude a can be obtained from the response to a single reference amplitude.the frequency response has similar behavior but in a less consistent way . The dominant frequencies produced by mode coupling are in the approximate range of the quasinormal frequency of the input mode and the expected sums and difference frequencies generated by the order of nonlinearity.large phase shifts, ranging up 15% in a half cycle relative to the linearized waveform, are exhibited in the news function obtained by the superposition of all output modes, i.e. In the waveform of observational significance . These phase shifts, which are important for design of signal extraction templates, arise in an erratic way from superposing modes with different oscillation frequencies . This furnishes a strong argument for going beyond the linearized approximation in designing a waveform catalog for signal extraction.besides the nonlinear generation of harmonic modes absent in the initial data, there is also a stronger than linear generation of gravitational wave output . This provides a potential mechanism for enhancing the strength of the gravitational radiation produced during, say, the merger phase of a binary inspiral above the strength predicted in linearized theory.in the non - axisymmetric m = 2 case, there is also considerable generation of radiation in polarization states not present in the linearized approximation . In the simulations, input amplitudes in the range a = 0.1 to a = 0.36 lead to nonlinear generation of the polarization mode which is of the same order of magnitude as the mode (which would be the sole polarization in the linearized regime). As a result, significant nonlinear amplification and phase shifting of the waveform would be observed by a gravitational wave detector, depending on its orientation . The mode coupling amplitudes consistently scale as powers a of the input amplitude a corresponding to the nonlinear order of the terms in the evolution equations which produce the mode . This allows much economy in producing a waveform catalog: given the order n associated with a given mode generation, the response to any input amplitude a can be obtained from the response to a single reference amplitude . The dominant frequencies produced by mode coupling are in the approximate range of the quasinormal frequency of the input mode and the expected sums and difference frequencies generated by the order of nonlinearity . Large phase shifts, ranging up 15% in a half cycle relative to the linearized waveform, are exhibited in the news function obtained by the superposition of all output modes, i.e. In the waveform of observational significance . These phase shifts, which are important for design of signal extraction templates, arise in an erratic way from superposing modes with different oscillation frequencies . This furnishes a strong argument for going beyond the linearized approximation in designing a waveform catalog for signal extraction . Besides the nonlinear generation of harmonic modes absent in the initial data, there is also a stronger than linear generation of gravitational wave output . This provides a potential mechanism for enhancing the strength of the gravitational radiation produced during, say, the merger phase of a binary inspiral above the strength predicted in linearized theory . In the non - axisymmetric m = 2 case, there is also considerable generation of radiation in polarization states not present in the linearized approximation . In the simulations, input amplitudes in the range a = 0.1 to a = 0.36 lead to nonlinear generation of the polarization mode which is of the same order of magnitude as the mode (which would be the sole polarization in the linearized regime). As a result, significant nonlinear amplification and phase shifting of the waveform would be observed by a gravitational wave detector, depending on its orientation . These effects arise from the nonlinear modification of the schwarzschild geometry identified by papadopoulos in his prior work on axisymmetric mode coupling, reported in section 3.3.2 . Although papadopoulos studied nonlinear mode generation produced by an outgoing pulse, as opposed to the case of an ingoing pulse studied in [255, 256], the same nonlinear factors were in play and gave rise to several common features . In both cases, analogs of features 1, 2, 3, and 4 above are all apparent in papadopoulos s work . At the finite difference level, both codes respect the reflection symmetry inherent in einstein s equations and exhibit the corresponding selection rules arising from parity considerations . In the axisymmetric case considered by papadopoulos, this forbids the nonlinear generation of a mode from a mode, as described in feature 5 above . The evolution along ingoing null hypersurfaces in the axisymmetric work of papadopoulos has complementary numerical features with the evolution along outgoing null hypersurfaces in the 3d work . The grid based upon ingoing null hypersurfaces avoids the difficulty in resolving effects close to r = 2 m encountered with the grid based upon outgoing null hypersurfaces . The outgoing code would require amr in order to resolve the quasinormal ringdown for as many cycles as achieved by papadopoulos . However, the outgoing code avoids the late time caustic formation noted in papadopoulos work, as well as the complications of gauge ambiguity and backscattering introduced by a finite outer boundary . One attractive option would be to combine the best features of these approaches by matching an interior evolution based upon ingoing null hypersurfaces to an exterior evolution based upon outgoing null hypersurfaces, as implemented in for spherically symmetric einstein - klein - gordon waves . The waveform of relevance to gravitational wave astronomy is the superposition of modes with different frequency compositions and angular dependence . Although this waveform results from a complicated nonlinear processing of the input signal, which varies with choice of observation angle, the response of the individual modes to an input signal of arbitrary amplitude can be obtained by scaling the response to an input of standard reference amplitude . The einstein - klein - gordon (ekg) system can be used to simulate many interesting physical phenomena . In 1d, characteristic ekg codes have been used to simulate critical phenomena and the perturbation of black holes (see section 3.1), and a cauchy ekg code has been used to study boson star dynamics . Extending these codes to 3d would open up a new range of possibilities, e.g., the possibility to study radiation from a boson star orbiting a black hole . A first step in that direction has been achieved with the construction of a 3d characteristic code by incorporating a massless scalar field into the pitt code . Since the scalar and gravitational evolution equations have the same basic form, the same evolution algorithm could be utilized . It was applied to the fully nonlinear simulation of an asymmetric pulse of ingoing scalar radiation propagating toward a schwarzschild black hole . The resulting scalar radiation and gravitational news backscattered to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} was computed . The amplitudes of the scalar and gravitational radiation modes exhibited the expected power law scaling with respect to the initial pulse amplitude . In addition, the computed ringdown frequencies agreed with the results from perturbative quasinormal mode calculations . The leo code developed by gmez et al . Has been applied to the characteristic evolution of the coupled einstein - klein - gordon fields, using the cubed - sphere coordinates . The long term plan is to simulate a boson star orbiting a black hole . In simulations of a scalar pulse incident on a schwarzschild black hole, they find the interesting result that scalar energy flow into the black hole reaches a maximum at spherical harmonic index = 2, and then decreases for larger due to the centrifugal barrier preventing the harmonics from effective penetration . Characteristic evolution of such systems of astrophysical interest have been limited in the past by resolution . They note that at the finest resolution considered in, it would take 1.5 months on the fastest current (single) processor to track a star in close orbit around a black hole . This is so even though the grid in question is only 81 123 points, which is moderate by today s standards . Its main disadvantage is the existence of either a caustic, where neighboring characteristics focus, or a milder version consisting of a crossover between two distinct characteristics . The vertex of a light cone is a highly symmetric caustic which already strongly limits the time step for characteristic evolution because of the cfl condition (11). It does not appear possible for a single characteristic coordinate system to cover the entire exterior region of a binary black hole spacetime without developing very complicated caustics and crossovers . This limits the waveform determined by a purely characteristic evolution to the post merger period . Ccm is a way to avoid this limitation by combining the strong points of characteristic and cauchy evolution into a global evolution . One of the prime goals of computational relativity is the simulation of the inspiral and merger of binary black holes . Given the appropriate data on a worldtube surrounding a binary system, characteristic evolution can supply the exterior spacetime and the radiated waveform . The potential advantages of ccm over traditional boundary conditions are accurate waveform and polarization state at infinity, computational efficiency for radiation problems in terms of both the grid domain and the computational algorithm, elimination of an artificial outer boundary condition on the cauchy problem, which eliminates contamination from back - reflection and clarifies the global initial value problem, anda global picture of the spacetime exterior to the event horizon . Accurate waveform and polarization state at infinity, computational efficiency for radiation problems in terms of both the grid domain and the computational algorithm, elimination of an artificial outer boundary condition on the cauchy problem, which eliminates contamination from back - reflection and clarifies the global initial value problem, and a global picture of the spacetime exterior to the event horizon . These advantages have been realized in model tests (see sections 5.55.8), but ccm has not yet been achieved in fully nonlinear 3-dimensional general relativity . The early attempts to implement ccm in general relativity involved the arnowitt - deser - misner (adm) formulation for the cauchy evolution . The major problem was later traced to a pathology in the way boundary conditions have traditionally been applied in adm codes . Exponentially growing instabilities introduced at boundaries have emerged as a major problem common to all adm code development . Linearized studies [234, 235, 15] of adm evolution - boundary algorithms with prescribed values of lapse and shift show the following: on analytic grounds, those adm boundary algorithms which supply values for all components of the metric (or extrinsic curvature) are inconsistent.a consistent boundary algorithm allows free specification of the transverse - traceless components of the metric with respect to the boundary.using such a boundary algorithm, linearized adm evolution can be carried out in a bounded domain for thousands of crossing times without sign of an exponential growing instability but with error that grows secularly in time . The linearized evolution satisfied the original criterion for robust stability that there be no exponential growth when the initial cauchy data and free boundary data are prescribed as random numbers (in the linearized regime). However, it was subsequently shown that adm is only weakly hyperbolic so that in the linear regime there are instabilities which grow as a power law in time . In the nonlinear regime, it is symptomatic of weakly hyperbolic systems that such secular instabilities become exponential . This has led to refined criteria for robust stability as a standardized test . On analytic grounds, those adm boundary algorithms which supply values for all components of the metric (or extrinsic curvature) are inconsistent . A consistent boundary algorithm allows free specification of the transverse - traceless components of the metric with respect to the boundary . Using such a boundary algorithm, linearized adm evolution can be carried out in a bounded domain for thousands of crossing times without sign of an exponential growing instability but with error that grows secularly in time . This is necessarily so because interpolations continually introduce short wavelength noise into the neighborhood of the boundary . It has been demonstrated that the pitt characteristic code has a robustly stable boundary (see section 4.2.4), but robustness of the cauchy boundary has only recently been studied . Boundary conditions are both the most important and the most difficult part of a theoretical treatment of most physical systems . Usually, that s where all the physics is . And, in computational approaches, that s usually where all the agony is . Computational boundaries for hyperbolic systems pose special difficulties . Even with an analytic form of the correct physical boundary condition in hand, there are seemingly infinitely more unstable numerical implementations than stable ones . In general, a stable problem places more boundary requirements on the finite difference equations than on the corresponding partial differential equations . Furthermore, the methods of linear stability analysis are often more unwieldy to apply to the boundary than to the interior evolution algorithm . The von neumann stability analysis of the interior algorithm linearizes the equations, while assuming a uniform grid with periodic boundary conditions, and checks that the discrete fourier modes do not grow exponentially . The mode e, with k real, is not included in the von neumann analysis for periodic boundary conditions . However, for the half plane problem in the domain x 0, one can legitimately prescribe such a mode as initial data as long as k> 0 so that it has finite energy . Thus the stability of such boundary modes must be checked . In the case of an additional boundary, e.g. For a problem in the domain 1 x 1, the godunov - ryabenkii theory gives as a necessary condition for stability the combined von neumann stability of the interior and the stability of the allowed boundary modes . The correct physical formulation of any cauchy problem for an isolated system also involves asymptotic conditions at infinity . These conditions must ensure not only that the total energy and energy loss by radiation are both finite, but they must also ensure the proper 1/r asymptotic falloff of the radiation fields . However, when treating radiative systems computationally, an outer boundary is often established artificially at some large but finite distance in the wave zone, i.e. Many wavelengths from the source . Imposing an appropriate radiation boundary condition at a finite distance is a difficult task even in the case of a simple radiative system evolving on a fixed geometric background . Gustaffson and kreiss have shown in general that the construction of a nonreflecting boundary condition for an isolated system requires knowledge of the solution in a neighborhood of infinity . When the system is nonlinear and not amenable to an exact solution, a finite outer boundary condition must necessarily introduce spurious physical effects into a cauchy evolution . The domain of dependence of the initial cauchy data in the region spanned by the computational grid would shrink in time along ingoing characteristics unless data on a worldtube traced out by the outer grid boundary is included as part of the problem . In order to maintain a causally sensible evolution, this worldtube data must correctly substitute for the missing cauchy data which would have been supplied if the cauchy hypersurface had extended to infinity . In a scattering problem, this missing exterior cauchy data might, for instance, correspond to an incoming pulse initially outside the outer boundary . In a scalar wave problem with field where the initial radiation is confined to a compact region inside the boundary, the missing cauchy data outside the boundary would be =,t = 0 at the initial time t0 . However, the determination of cauchy data for general relativity is a global elliptic constraint problem so that there is no well defined scheme to confine it to a compact region . Furthermore, even in the scalar field case where =,t = 0 is appropriate cauchy data outside the boundary at t0, it would still be a non - trivial evolution problem to correctly assign the associated boundary data for t t0 . It is common practice in computational physics to impose an artificial boundary condition (abc), such as an outgoing radiation condition, in an attempt to approximate the proper data for the exterior region . This abc may cause partial reflection of an outgoing wave back into the system [168, 154, 138, 202], which contaminates the accuracy of the interior evolution and the calculation of the radiated waveform . Furthermore, nonlinear waves intrinsically backscatter, which makes it incorrect to try to entirely eliminate incoming radiation from the outer region . In general, a systematic reduction of this error can only be achieved by moving the computational boundary to larger and larger radii . A traditional abc for the wave equation is the sommerfeld condition . For a scalar field satisfying the minkowski space wave equation 35\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\eta ^{\alpha \beta}}{\partial _ \alpha}{\partial _ \beta}\phi = s,$$\end{document} with a smooth source s of compact support emitting outgoing radiation, the exterior retarded field has the form 36\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi = {{f(t - r, \theta, \phi)} \over r} + {{g(t - r,\theta, \phi)} \over {{r^2}}} + {{h(t, r,\theta, \phi)} \over {{r^3}}},$$\end{document} where f, g and h and their derivatives are smooth bounded functions . The simplest case is the monopole radiation 37\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi = {{f(t - r)} \over r},$$\end{document} which satisfies (t + r)(r) = 0 . This motivates the use of the sommerfeld condition 38\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${1 \over r}({\partial _ t} + {\partial _ r})(r\phi){\vert _ r} = q(t, r,\theta, \phi)$$\end{document} on a finite boundary r = r. a homogeneous sommerfeld condition, i.e. Q = 0, is exact only in the spherically symmetric case . The sommerfeld boundary data q in the general case (36) falls off as 1/r, so that a homogeneous sommerfeld condition introduces an error, which is small only for large r. as an example, for the dipole solution 39\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ {dipole}} = {\partial _ z}{{f(t - r)} \over r} = - \left({{{f{\prime}(t - r)} \over r} + {{f(t - r)} \over {{r^2}}}} \right)\cos \theta$$\end{document} we have 40\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q = {{f(t - r)\cos \theta} \over {{r^3}}}.$$\end{document} a homogeneous sommerfeld condition at r = r would lead to a solution \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\tilde \phi}_{dipole}}$\end{document} containing a reflected ingoing wave . For large r, 41\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tilde \phi _ {dipole}} \sim {\phi _ {dipole}} + \kappa {{f(t + r - 2r)\cos \theta} \over r},$$\end{document} where tf(t) = f(t) and the reflection coefficient has asymptotic behavior = o(1/r). More precisely, the fourier mode 42\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tilde \phi _ {dipole}}(\omega) = {\partial _ z}\left({{{{e^{i\omega (t - r)}}} \over r} + {\kappa _ \omega}{{{e^{i\omega (t + r - 2r)}}} \over r}} \right),$$\end{document} satisfies the homogeneous boundary condition \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({\partial _ t} + {\partial _ r})(r{{\tilde \phi}_{dipole}}(\omega){\vert _ r} = 0$\end{document} with reflection coefficient 43\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\kappa _ \omega} = {1 \over {2{\omega ^2}{r^2} + 2i\omega r - 1}} \sim {1 \over {2{\omega ^2}{r^2}}}.$$\end{document} much work has been done on formulating boundary conditions, both exact and approximate, for linear problems in situations that are not spherically symmetric . These boundary conditions are given various names in the literature, e.g., absorbing or non - reflecting . See the articles [107, 202, 245, 209, 43] for general discussions . Local abcs have been extensively applied to linear problems with varying success [168, 89, 35, 244, 138, 52, 155]. Some of these conditions are local approximations to exact integral representations of the solution in the exterior of the computational domain, while others are based on approximating the dispersion relation of the so - called one - way wave equations [168, 244]. Higdon showed that this last approach is essentially equivalent to specifying a finite number of angles of incidence for which the abcs yield perfect transmission . Local abcs have also been derived for the linear wave equation by considering the asymptotic behavior of outgoing solutions, thus generalizing the sommerfeld outgoing radiation condition . Although this type of abc is relatively simple to implement and has a low computational cost, the final accuracy is often limited because the assumptions made about the behavior of the waves are rarely met in practice [107, 245]. The disadvantages of local abcs have led some workers to consider exact nonlocal boundary conditions based on integral representations of the infinite domain problem [243, 107, 245]. Even for problems where the green s function is known and easily computed, such approaches were initially dismissed as impractical; however, the rapid increase in computer power has made it possible to implement exact nonlocal abcs for the linear wave equation and maxwell s equations in 3d [80, 126]. If properly implemented, this method can yield numerical solutions to a linear problem which converge to the exact infinite domain problem in the continuum limit, while keeping the artificial boundary at a fixed distance . However, due to nonlocality, the computational cost per time step usually grows at a higher power with grid size \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({\mathcal o}({n^4})$\end{document} per time step in three dimensions) than in a local approach [107, 80, 245]. The problem is normally treated by linearizing the region between the outer boundary and infinity, using either local or nonlocal linear abcs [245, 209]. The neglect of the nonlinear terms in this region introduces an unavoidable error at the analytic level . This is a subtle global problem because the correct boundary data must correspond to the continuity of fields and their normal derivatives when extended across the boundary into the linearized exterior . This is a clear requirement for any consistent boundary algorithm, since discontinuities in the field or its derivatives would otherwise act as a spurious sheet source on the boundary, which contaminates both the interior and the exterior evolutions . But the fields and their normal derivatives constitute an overdetermined set of data for the boundary problem . So it is necessary to solve a global linearized problem, not just an exterior one, in order to find the proper data . The designation exact abc is given to an abc for a nonlinear system whose only error is due to linearization of the exterior . An exact abc requires the use of global techniques, such as the difference potentials method, to eliminate back reflection at the boundary . Thompson generalized a previous nonlinear abc of hedstrom to treat 1d and 2d problems in gas dynamics . These boundary conditions performed poorly in some situations because of their difficulty in adequately modeling the field outside the computational domain [240, 107]. Hagstrom and hariharan have overcome these difficulties in 1d gas dynamics by a clever use of riemann invariants . They proposed a heuristic generalization of their local abc to 3d, but this approach has not yet been validated . In order to reduce the level of approximation at the analytic level, an artificial boundary for a nonlinear problem must be placed sufficiently far from the strong - field region . There is no numerical method which converges (as the discretization is refined) to the infinite domain exact solution of a strongly nonlinear wave problem in multi - dimensions, while keeping the artificial boundary fixed . Attempts to use compactified cauchy hypersurfaces which extend the domain to spatial infinity have failed because the phase of short wavelength radiation varies rapidly in spatial directions . Ccm is a strategy that eliminates this nonlinear source of error . In the simplest version of ccm, cauchy and characteristic evolution algorithms are pasted together in the neighborhood of a worldtube to form a global evolution algorithm . The characteristic algorithm provides an outer boundary condition for the interior cauchy evolution, while the cauchy algorithm supplies an inner boundary condition for the characteristic evolution . The characteristic evolution is based upon the outgoing null hypersurfaces emanating from these slices, with the evolution proceeding from one hypersurface to the next by the outward radial march described in section 3.1). There is no need to truncate spacetime at a finite distance from the source, since compactification of the radial null coordinate used in the characteristic evolution makes it possible to cover the infinite space with a finite computational grid . In this way, the true waveform may be computed up to discretization error by the finite difference algorithm . Ccm evolves a mixed spacelike - null initial value problem in which cauchy data is given in a spacelike hypersurface bounded by a spherical boundary \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal s}$\end{document} and characteristic data is given on a null hypersurface emanating from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal s}$\end{document}. The general idea is not entirely new . An early mathematical investigation combining spacelike and characteristic hypersurfaces appears in the work of duff . The three chief ingredients for computational implementation are: (i) a cauchy evolution module, (ii) a characteristic evolution module and, (iii) a module for matching the cauchy and characteristic regions across their interface . In the simplest scenario, the interface is the timelike worldtube which is traced out by the flow of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal s}$\end{document} along the worldlines of the cauchy evolution, as determined by the choice of lapse and shift . Matching provides the exchange of data across the worldtube to allow evolution without any further boundary conditions, as would be necessary in either a purely cauchy or purely characteristic evolution . The most important application of ccm is anticipated to be the waveform and momentum recoil in the binary black hole inspiral and merger . The 3d cauchy codes being applied to simulate this problem employ a single cartesian coordinate patch . In principle, the application of ccm to this problem might seem routine, tantamount to translating into finite difference form the textbook construction of an atlas consisting of overlapping coordinate patches . In practice, it is a complicated project . The underlying algorithm consists of the following main submodules: the boundary module which sets the grid structures . This defines masks identifying which points in the cauchy grid are to be evolved by the cauchy module and which points are to be interpolated from the characteristic grid, and vice versa . The reference structures for constructing the mask is the inner characteristic boundary, which in the cartesian cauchy coordinates is the spherical extraction worldtube \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${x^2} + {y^2} + {z^2} = r_e^2$\end{document}, and the outer cauchy boundary \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${x^2} + {y^2} + {z^2} = r_i^2$\end{document}, where the cauchy boundary data is injected . The choice of lapse and shift for the cauchy evolution governs the dynamical and geometrical properties of these worldtubes.the extraction module whose input is cauchy grid data in the neighborhood of the extraction worldtube at re and whose output is the inner boundary data for the exterior characteristic evolution . This module numerically implements the transformation from cartesian {3 + 1} coordinates to spherical null coordinates . The algorithm makes no perturbative assumptions and is based upon interpolations of the cauchy data to a set of prescribed grid points near re . The metric information is then used to solve for the null geodesics normal to the slices of the extraction worldtube . This provides the jacobian for the transformation to null coordinates in the neighborhood of the worldtube . The characteristic evolution module is then used to propagate the data from the worldtube to null infinity, where the waveform is calculated.the injection module which completes the interface by using the exterior characteristic evolution to inject the outer boundary data for the cauchy evolution at ri . This is the inverse of the extraction procedure but must be implemented with ri> re to allow for overlap between the cauchy and characteristic domains . The overlap region can be constructed either to have a fixed physical size or to shrink to zero in the continuum limit . In the latter case, the inverse jacobian describing the transformation from null to cauchy coordinates can be obtained to prescribed accuracy in terms of an affine parameter expansion along the null geodesics emanating from the worldtube . The above strategy provides a model of how cauchy and characteristic codes can be pieced together as modules to form a global evolution code . The boundary module which sets the grid structures . This defines masks identifying which points in the cauchy grid are to be evolved by the cauchy module and which points are to be interpolated from the characteristic grid, and vice versa . The reference structures for constructing the mask is the inner characteristic boundary, which in the cartesian cauchy coordinates is the spherical extraction worldtube \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${x^2} + {y^2} + {z^2} = r_e^2$\end{document}, and the outer cauchy boundary \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${x^2} + {y^2} + {z^2} = r_i^2$\end{document}, where the cauchy boundary data is injected . The choice of lapse and shift for the cauchy evolution governs the dynamical and geometrical properties of these worldtubes . The extraction module whose input is cauchy grid data in the neighborhood of the extraction worldtube at re and whose output is the inner boundary data for the exterior characteristic evolution . This module numerically implements the transformation from cartesian {3 + 1} coordinates to spherical null coordinates . The algorithm makes no perturbative assumptions and is based upon interpolations of the cauchy data to a set of prescribed grid points near re . The metric information is then used to solve for the null geodesics normal to the slices of the extraction worldtube . This provides the jacobian for the transformation to null coordinates in the neighborhood of the worldtube . The characteristic evolution module is then used to propagate the data from the worldtube to null infinity, where the waveform is calculated . The injection module which completes the interface by using the exterior characteristic evolution to inject the outer boundary data for the cauchy evolution at ri . This is the inverse of the extraction procedure but must be implemented with ri> re to allow for overlap between the cauchy and characteristic domains . The overlap region can be constructed either to have a fixed physical size or to shrink to zero in the continuum limit . In the latter case, the inverse jacobian describing the transformation from null to cauchy coordinates can be obtained to prescribed accuracy in terms of an affine parameter expansion along the null geodesics emanating from the worldtube . The full advantage of ccm lies in the numerical treatment of nonlinear systems where its error converges to zero in the continuum limit of infinite grid resolution [38, 39, 76]. For high accuracy, it has been shown that the relative amount of computation required for ccm (accm) compared to that required for a pure cauchy calculation (ac) goes to zero, accm / ac o as o [49, 45]. An important factor here is the use of a compactified characteristic evolution, so that the whole spacetime is represented on a finite grid . From a numerical point of view this means that the only error made in a calculation of the radiation waveform at infinity is the controlled error due to the finite discretization . Accuracy of a cauchy algorithm which uses an abc requires a large grid domain in order to avoid error from nonlinear effects in its exterior . The computational demands of ccm are small because the interface problem involves one less dimension than the evolution problem . Because characteristic evolution algorithms are more efficient than cauchy algorithms, the efficiency can be further enhanced by making the matching radius as small as possible consistent with the avoidance of caustics . At present, the computational strategy of ccm is mainly the tool of numerical relativists, who are used to dealing with dynamical coordinate systems . The first discussion of its potential was given in and its feasibility has been more fully explored in [76, 77, 87, 42, 236]. Recent work has been stimulated by the requirements of the binary black hole problem, where ccm is one of the strategies to provide boundary conditions and determine the radiation waveform . However, it also has inherent advantages in dealing with other hyperbolic systems in computational physics, particularly nonlinear 3-dimensional problems . A detailed study of the stability and accuracy of ccm for linear and nonlinear wave equations has been presented in, illustrating its potential for a wide range of problems . A special issue arising in general relativity is whether the boundary conditions on an outer world - tube preserve the constraints . It is typical of hyperbolic reductions of the einstein equations that the hamiltonian and momentum constraints propagate in a domain of dependence dictated by the characteristics . Unless the boundary conditions enforce these constraints, they will be violated outside the domain of dependence of the initial cauchy hypersurface . This issue of a constraint - preserving initial boundary value problem has only recently been addressed . The first fully nonlinear treatment of a well - posed constraint preserving formulation of the einstein initial - boundary value problem (ibvp) has subsequently been given by friedrich and nagy . Their treatment is based upon a frame formulation in which the evolution variables are the tetrad, connection coefficients and weyl curvature . Although this system has not yet been implemented computationally, it has spurred the investigation of simpler treatments of einstein equations which give rise to a constraint preserving ibvp under various restrictions [63, 236, 64, 100, 128, 207, 162]. Well - posedness of the ibvp, in addition to constraint preservation, is a necessary condition for computational success . This is particularly cogent for dealing with waveform extraction in the simulation of black holes by bssn or harmonic formulations . There is no well - posed outer boundary theory for the bssn formulation and the strategy is to place the boundary out far enough so that it does no harm . The harmonic formulation has a simpler mathematical structure as a system of coupled quasilinear wave equations which is more amenable to an analytic treatment . Standard harmonic coordinates satisfy the covariant wave equation 44\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\gamma ^\alpha} = - \square{x^\alpha} = - {1 \over {\sqrt {- g}}}{\partial _ \beta}{\gamma ^{\alpha \beta}} = 0,\quad {\gamma ^{\alpha \beta}} = {\sqrt {- gg} ^{\alpha \beta}}.$$\end{document} (this can easily be generalized to include gauge forcing, whereby = f(x, g). For simplicity of discussion, i will set = 0, although gauge forcing is an essential tool in simulating black holes .) When = 0, einstein s equations reduce to the 10 quasilinear wave equations 45\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${g^{\mu \nu}}{\partial _ \nu}{\gamma ^{\alpha \beta}} = {s^{\alpha \beta}},$$\end{document} where s does not enter the principle part and vanishes in the linearized approximation . Straightforward techniques can be applied to formulate a well - posed ibvp for the system (45). The catch is that einstein s equations are not necessarily satisfied unless the constraints are also satisfied . In the harmonic formalism, the constraints can be reduced to the harmonic coordinate conditions (44). For the resulting ibvp to be constraint preserving numerous early attempts to accomplish this failed because (44) contains derivatives tangent to the boundary, which do not fit into the standard methods for obtaining the necessary energy estimates . The use of pseudo - differential techniques developed for similar problems in elasticity theory has led to the first well - posed formulation for the general ibvp for the harmonic einstein equations . Subsequently, these results were also obtained using standard energy estimates by means of a novel, non - conventional choice of the energy for the harmonic system . Furthermore, the allowed boundary conditions include those of the sommerfeld type which are nonreflecting in the sense that the boundary data for g falls off as o(1/r) as the boundary radius r of a cauchy evolution code, the abigel code, has been based upon a discretized of this well - posed harmonic ibvp . The code was tested to be stable, convergent and constraint preserving in the nonlinear regime . A linearized version of the abigel code has been used to successfully carry out ccm (see section 5.8). In the present harmonic codes used to simulate the binary black holes, the best that can be done is to impose a constraint preserving boundary condition for which homogeneous boundary data, i.e. Zero boundary values, is a good approximation . One proposal of this type is a boundary condition that requires the newman - penrose weyl tensor component 0 to vanish . In the limit that the outer boundary goes to infinity this outer boundary condition becomes exact . In the present state of the art of black hole simulations, this approach comes closest to a satisfactory treatment of the outer boundary . In numerous analytic studies outside of general relativity, matching techniques have successfully cured pathologies in perturbative expansions . Matching is a strategy for obtaining a global solution by patching together solutions obtained using different coordinate systems for different regions . By adopting each coordinate system to a length scale appropriate to its domain, a globally convergent perturbation expansion is sometimes possible in cases where a single coordinate system would fail . In general relativity, burke showed that matching could be used to eliminate some of the divergences arising in perturbative calculations of gravitational radiation . Kates and kegles further showed that use of an exterior null coordinate system in the matching scheme could eliminate problems in the perturbative treatment of a scalar radiation field on a schwarzschild background . The schwarzschild light cones have drastically different asymptotic behavior from the artificial minkowski light cones used in perturbative expansions based upon a flat space green function . Use of the minkowski light cones leads to nonuniformities in the expansion of the radiation fields which are eliminated by the use of true null coordinates in the exterior . Kates, anderson, kegles, and madonna extended this work to the fully general relativistic case and reached the same conclusion . Anderson later applied this approach to the slow motion approximation of a binary system and obtained a derivation of the radiation reaction effect on the orbital period which avoided some objections to earlier approaches . The use of the true light cones was also essential in formulating as a mathematical theorem that the bondi news function satisfies the einstein quadrupole formula to leading order in a newtonian limit . Although questions of mathematical consistency still remain in the perturbative treatment of gravitational radiation, it is clear that the use of characteristic methods pushes these problems to a higher perturbative order . One of the first computational applications of characteristic matching was a hybrid numerical - analytical treatment by anderson and hobill of the test problem of nonlinear 1d scalar waves [7, 8, 9]. They matched an inner numerical solution to a far field solution which was obtained by a perturbation expansion . A key ingredient is that the far field is solved in retarded null coordinates (u, r). Because the transformation from null coordinates (u, r) to cauchy coordinates (t, r) is known analytically for this problem, the matching between the null and cauchy solutions is quite simple . Causality was enforced by requiring that the system be stationary prior to some fixed time . This eliminates extraneous incoming radiation in a physically correct way in a system which is stationary prior to a fixed time but it is nontrivial to generalize, say, to the problem of radiation from an orbiting binary . Later, a global, characteristic, numerical study of the self - gravitating version of this problem confirmed that the use of the true null cones is essential in getting the correct radiated waveform . For quasi - periodic radiation, the phase of the waveform is particular sensitive to the truncation of the outer region at a finite boundary . Although a perturbative estimate would indicate an \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal o}(m / r)$\end{document} error, this error accumulates over many cycles to produce an error of order in the phase . Anderson and hobill proposed that their method be extended to general relativity by matching a numerical solution to an analytic 1/r expansion in null coordinates . Most perturbative - numerical matching schemes that have been implemented in general relativity have been based upon perturbations of a schwarzschild background using the standard schwarzschild time slicing [1, 4, 2, 3, 208, 204, 177]. It would be interesting to compare results with an analytic - numeric matching scheme based upon the true null cones . Although the full proposal by anderson and hobill has not been carried out, characteristic techniques have been used [170, 65, 151] to study the radiation content of numerical solutions by treating the far field as a perturbation of a schwarzschild spacetime . Most metric based treatments of gravitational radiation are based upon perturbations of the schwarzschild metric and solve the underlying regge - wheeler and zerilli equations using traditional spacelike cauchy hypersurfaces . At one level, these approaches extract the radiation from a numerical solution in a region with outer boundary \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal b}$\end{document} by using data on an inner worldtube \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal w}$\end{document} to construct the perturbative solution . Ambiguities are avoided by use of moncrief s gauge invariant perturbation quantities . For this to work, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal w}$\end{document} must not only be located in the far field, i.e. Many wavelengths from the source, but, because of the lack of proper outer boundary data, it is necessary that the boundary \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal b}$\end{document} be sufficiently far outside \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal w}$\end{document} so that the extracted radiation is not contaminated by back - reflection for some significant window of time . This extraction strategy has also been carried out using characteristic evolution in the exterior of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal w}$\end{document} instead of a perturbative solution, i.e. Cauchy - characteristic extraction (see section 6). A study by babiuc, szilgyi, hawke, and zlochower carried out in the perturbative regime show that cce compares favorably with zerilli extraction and has advantages at small extraction radii . When the extraction worldtube is sufficiently large, e.g. R = 200, where is the characteristic wavelength of the radiation, the zerilli and cce methods both give excellent results . However, the accuracy of cce remains unchanged at small extraction radii, e.g. R = 10, whereas the zerilli approach shows error associated with near zone effects . The contamination of the extracted radiation by back - reflection can only be eliminated by matching to an exterior solution which injects the physically appropriate boundary data on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal w}$\end{document}. Cauchy - perturbative matching [208, 204] has been implemented using the same modular structure described for ccm in section 5.2 . At present, perturbative matching and ccm share the common problem of long term stability of the outer cauchy boundary in 3d applications . These model problems provided a controlled environment for the development of ccm, in which either exact solutions or independent numerical solutions were known . In the following studies ccm worked like a charm in a variety of 1d applications, i.e. The matched evolutions were essentially transparent to the presence of the interface . The southampton group chose cylindrically symmetric systems as their model problem for developing matching techniques . In preliminary work, they showed how ccm could be consistently carried out for a scalar wave evolving in minkowski spacetime but expressed in a nontrivial cylindrical coordinate system . Although the problem involves only one spatial dimension, there are two independent modes of polarization . The cauchy metric was treated in the jordan - ehlers - kompaneets canonical form, using coordinates (t, r,, z) adapted to the (, z) cylindrical symmetry . The advantage here is that u = t r is then a null coordinate which can be used for the characteristic evolution . They successfully recast the equations in a suitably regularized form for the compactification of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} in terms of the coordinate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$y = \sqrt {1/r}$\end{document}. The simple analytic relationship between cauchy coordinates (t, r) and characteristic coordinates (u, y) facilitated the translation between cauchy and characteristic variables on the matching worldtube, given by r = const . It is notable that they report no problems with instability, which have arisen in other attempts at unconstrained leapfrog evolution in general relativity . The characteristic evolution also used a leapfrog scheme for the evolution between retarded time levels u, while numerically integrating the hypersurface equations outward along the characteristics . The matching interface was located at points common to both the cauchy and characteristic grids . In order to update these points by cauchy evolution, these values were obtained by interpolation from characteristic grid points (lying on three levels of null hypersurfaces in order to ensure second order accuracy). Similarly, the boundary data for starting up the characteristic integration was obtained by interpolation from cauchy grid values inside the worldtube . The matching code was first tested using exact weber - wheeler cylindrical waves, which come in from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ -}$\end{document}, pass through the symmetry axis and expand out to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The numerical errors were oscillatory with low growth rate, and second order convergence was confirmed . Comparisons of ccm were made with cauchy evolutions using a standard outgoing radiation boundary condition . At high amplitudes the standard condition developed a large error very quickly and was competitive only for weak waves with a large outer boundary . In contrast, the matching code performed well even with a small matching radius . Some interesting simulations were presented in which an outgoing wave in one polarization mode collided with an incoming wave in the other mode, a problem studied earlier by pure cauchy evolution . The code was next tested using exact cylindrically symmetric solutions, due to piran, safier, and katz, which contain both degrees of freedom . These solutions are singular at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} so that the code had to be suitably modified . The convergence rate of the numerical solution starts off as second order but diminishes to first order after long time evolution . This performance could perhaps be improved by incorporating subsequent improvements in the characteristic code made by sperhake, sjdin, and vickers (see section 3.1). A joint collaboration between groups at pennsylvania state university and the university of pittsburgh applied ccm to the ekg system with spherical symmetry . Initial data were specified on the union of a spacelike hypersurface and a null hypersurface . The evolution used a 3-level cauchy scheme in the interior and a 2-level characteristic evolution in the compactified exterior . A constrained cauchy evolution was adopted because of its earlier success in accurately simulating scalar wave collapse . Characteristic evolution was based upon the null parallelogram algorithm (8). The matching between the cauchy and characteristic foliations was achieved by imposing continuity conditions on the metric, extrinsic curvature and scalar field variables, ensuring smoothness of fields and their derivatives across the matching interface . The extensive analytical and numerical studies of this system in recent years aided the development of ccm in this non - trivial geometrical setting by providing basic knowledge of the expected physical and geometrical behavior, in the absence of exact solutions . The ccm code accurately handled wave propagation and black hole formation for all values of m / r at the matching radius, with no symptoms of instability or back - reflection . Second order accuracy was established by checking energy conservation . In further developmental work on the ekg model, the pittsburgh group used ccm to formulate a new treatment of the inner cauchy boundary for a black hole spacetime . In the excision strategy, the inner boundary of the cauchy evolution is located at an apparent horizon, which must lie inside (or on) the event horizon, so that truncation of the interior spacetime at the apparent horizon cannot causally affect the gravitational waves radiated to infinity . However, instabilities reported in some early attempts at excision motivated an alternative treatment . In the ccm excision strategy, illustrated in figure 6, the interior black hole region is evolved using an ingoing null algorithm whose inner boundary is a marginally trapped surface, and whose outer boundary lies outside the black hole and forms the inner boundary of a region evolved by the cauchy algorithm . In turn, the outer boundary of the cauchy region is handled by matching to an outgoing null evolution extending to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Data are passed between the inner characteristic and central cauchy regions using a ccm procedure similar to that already described for an outer cauchy boundary . The main difference is that, whereas the outer cauchy boundary data is induced from the bondi metric on an outgoing null hypersurface, the inner cauchy boundary is now obtained from an ingoing null hypersurface which enters the event horizon and terminates at a marginally trapped surface . A cauchy evolution, with data at t0 is matched across worldtubes r0 and r1 to an ingoing null evolution, with data at 0, and an outgoing null evolution, with data at u0 . The ingoing null evolution extends to an inner trapped boundary q, and the outgoing null evolution extends to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Black hole excision by matching . A cauchy evolution, with data at t0 is matched across worldtubes r0 and r1 to an ingoing null evolution, with data at 0, and an outgoing null evolution, with data at u0 . The ingoing null evolution extends to an inner trapped boundary q, and the outgoing null evolution extends to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The translation from an outgoing to an incoming null evolution algorithm can be easily carried out . The substitution + i/2 in the 3d version of the bondi metric (3) provides a simple formal recipe for switching from an outgoing to an ingoing null formalism . In order to ensure that trapped surfaces exist on the ingoing null hypersurfaces, such data can be obtained from initial cauchy data for a black hole . However, rather than extending the cauchy hypersurface inward to an apparent horizon, it was truncated sufficiently far outside the apparent horizon to avoid computational problems with the cauchy evolution . The initial cauchy data were then extended into the black hole interior as initial null data until a marginally trapped surface was reached . First, the inner matching surface must be chosen to be convex, in the sense that its outward null normals uniformly diverge and its inner null normals uniformly converge . (this is trivial to satisfy in the spherically symmetric case .) Given any physically reasonable matter source, the focusing theorem guarantees that the null rays emanating inward from the matching sphere continue to converge until reaching a caustic . Second, the initial null data must lead to a trapped surface before such a caustic is encountered . This is a relatively easy requirement to satisfy because the initial null data can be posed freely, without any elliptic or algebraic constraints other than continuity with the cauchy data . Its performance showed that ccm provides as good a solution to the black hole excision problem in spherical symmetry as any previous treatment [214, 215, 171, 11]. Ccm is computationally more efficient than these pure cauchy approaches (fewer variables) and much easier to implement . Depending upon the cauchy formalism adopted, achieving stability with a pure cauchy scheme in the region of an apparent horizon can be quite tricky, involving much trial and error in choosing finite difference schemes . There were no complications with stability of the null evolution at the marginally trapped surface . The cauchy evolution was carried out in ingoing eddington - finklestein (ief) coordinates . The initial cauchy data consisted of a schwarzschild black hole with an ingoing gaussian pulse of scalar radiation . Since ief coordinates are based on ingoing null cones, it is possible to construct a simple transformation between the ief cauchy metric and the ingoing null metric . Initially there was no scalar field present on either the ingoing or outgoing null patches . The initial values for the bondi variables and v were determined by matching to the cauchy data at the matching surfaces and integrating the hypersurface equations (5, 6). As the evolution proceeds, the scalar field passes into the black hole, and the marginally trapped surface (mts) grows outward . The mts is easily located in the spherically symmetric case by an algebraic equation . In order to excise the singular region, the grid points inside the marginally trapped surface the backscattered radiation propagated cleanly across the outer matching surface to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The strategy worked smoothly, and second order accuracy of the approach was established by comparing it to an independent numerical solution obtained using a second order accurate, purely cauchy code . As discussed in section 5.9, this inside - outside application of ccm has potential application to the binary black hole problem . In a variant of this double ccm matching scheme, lehner has eliminated the middle cauchy region between r0 and r1 in figure 6 . He constructed a 1d code matching the ingoing and outgoing characteristic evolutions directly across a single timelike worldtube . In this way, he was able to simulate the global problem of a scalar wave falling into a black hole by purely characteristic methods . The southampton ccm project is being carried out for spacetimes with (twisting) axial symmetry . The formal basis for the matching scheme was developed by dinverno and vickers [85, 86]. Similar to the pittsburgh 3d strategy (see section 5.2), matching is based upon an extraction module, which supplies boundary data for the exterior characteristic evolution, and an injection module, which supplies boundary data for the interior cauchy evolution . However, their use of spherical coordinates for the cauchy evolution (as opposed to cartesian coordinates in the 3d strategy) allows use of a matching worldtube r = rm which lies simultaneously on cauchy and characteristic gridpoints . This tremendously simplifies the necessary interpolations between the cauchy and characteristic evolutions, at the expense of dealing with the r = 0 coordinate singularity in the cauchy evolution . The characteristic code (see section 3.3.4) is based upon a compactified bondi - sachs formalism . The use of a radial cauchy gauge, in which the cauchy coordinate r measures the surface area of spheres, simplifies the relation to the bondi - sachs coordinates . In the numerical scheme, the metric and its derivatives are passed between the cauchy and characteristic evolutions exactly at r = rm, thus eliminating the need of a matching interface encompassing a few grid zones, as in the 3d pittsburgh scheme . Preliminary results in the development of the southampton ccm code are described by pollney in his thesis . The cauchy code was based upon the axisymmetric adm code of stark and piran and reproduces their vacuum results for a short time period, after which an instability at the origin becomes manifest . The characteristic code has been tested to reproduce accurately the schwarzschild and boost - rotation symmetric solutions, with more thorough tests of stability and accuracy still to be carried out . Ccm has been successfully implemented in the fully 3d problem of nonlinear scalar waves evolving in a flat spacetime [43, 42]. This study demonstrated the feasibility of matching between cartesian cauchy coordinates and spherical null coordinates, the setup required to apply ccm to the binary black hole problem . Unlike spherically or cylindrically symmetric examples of matching, the cauchy and characteristic patches do not share a common coordinate which can be used to define the matching interface . This introduces a major complication into the matching procedure, resulting in extensive use of intergrid interpolation . The nonlinear waves were modeled by the equation 46\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c^{- 2}}\partial _ t^2\phi = {\nabla ^2}\phi + f(\phi) + s(t, x, y, z),$$\end{document} with self - coupling f() and external source s. the initial cauchy data (t0, x, y, z) and t(t0, x, y, z) are assigned in a spatial region bounded by a spherical matching surface of radius rm . The characteristic initial value problem (46) is expressed in standard spherical coordinates (r,,) and retarded time u = t r + rm: 47\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2{\partial _ u}{\partial _ r}g = \partial _ r^2 g - {{{l^2}g} \over {{r^2}}} + r(f + s),$$\end{document} where g = r and l is the angular momentum operator 48\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${l^2}g = - {{{\partial _ ^2 g} \over {{{\sin}^2}\theta}}.$$\end{document} the initial null data consist of g(r,,, u0) on the outgoing characteristic cone u0 = t0 emanating at the initial cauchy time from the matching worldtube at r = rm . Ccm was implemented so that, in the continuum limit, and its normal derivatives would be continuous across the matching interface . The use of a cartesian discretization in the interior and a spherical discretization in the exterior complicated the treatment of the interface . In particular, the stability of the matching algorithm required careful attention to the details of the intergrid matching . Nevertheless, there was a reasonably broad range of discretization parameters for which ccm was stable . One was based upon two overlapping stereographic grid patches and the other upon a multiquadric approximation using a quasi - regular triangulation of the sphere . Also, two separate tactics were used to implement matching, one based upon straightforward interpolations and the other upon maintaining continuity of derivatives in the outward null direction (a generalization of the sommerfeld condition). The solutions were second order accurate and the richardson extrapolation technique could be used to accelerate convergence . The performance of ccm was compared to traditional abcs . As expected, the nonlocal abcs yielded convergent results only in linear problems, and convergence was not observed for local abcs, whose restrictive assumptions were violated in all of the numerical experiments . The computational cost of ccm was much lower than that of current nonlocal boundary conditions . In strongly nonlinear problems, ccm appears to be the only available method which is able to produce numerical solutions which converge to the exact solution with a fixed boundary . Although the individual pieces of the ccm module have been calibrated to give a second order accurate interface between cauchy and characteristic evolution modules in 3d general relativity, its stability has not yet been established . However, a stable version of ccm for linearized gravitational theory has recently been demonstrated . The cauchy evolution is carried out using a harmonic formulation for which the reduced equations have a well - posed initial - boundary problem . Previous attempts at ccm were plagued by boundary induced instabilities of the cauchy code . Although stable behavior of the cauchy boundary is only a necessary and not a sufficient condition for ccm, the tests with the linearized harmonic code matched to a linearized characteristic code were successful . The harmonic conditions consist of wave equations for the coordinates which can be used to propagate the gauge as four scalar waves using characteristic evolution . This allows the extraction worldtube to be placed at a finite distance from the injection worldtube without introducing a gauge ambiguity . Furthermore, the harmonic gauge conditions are the only constraints on the cauchy formalism so that gauge propagation also insures constraint propagation . This allows the cauchy data to be supplied in numerically benign sommerfeld form, without introducing constraint violation . Using random initial data, robust stability of the ccm algorithm figure 7 shows a sequence of profiles of the metric component \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\gamma ^{xy}} = \sqrt {- g} {g^{xy}}$\end{document} linearized wave propagates cleanly through the spherical injection boundary and passes to the characteristic grid, where it is propagated to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Figure 7sequence of slices of the metric component, evolved with the linear matched cauchy - characteristic code . In the last snapshot, sequence of slices of the metric component, evolved with the linear matched cauchy - characteristic code . In the last snapshot, ccm also offers a new approach to singularity excision in the binary black hole problem in the manner described in section 5.5.3 for a single spherically symmetric black hole . In a binary system, there are computational advantages in posing the cauchy evolution in a frame which is co - rotating with the orbiting black holes . In this co - orbiting description, the cauchy evolution requires an inner boundary condition inside the black holes and also an outer boundary condition on a worldtube outside of which the grid rotation is likely to be superluminal . Thus, successful implementation of ccm could solve the exterior boundary problem for this co - orbiting description . Ccm also has the potential to handle the two black holes inside the cauchy region . As described earlier with respect to figure 6, an ingoing characteristic code can evolve a moving black hole with long term stability [119, 116]. This means that ccm might also be able to provide the inner boundary condition for cauchy evolution once stable matching has been accomplished . In this approach, the interior boundary of the cauchy evolution is located outside the apparent horizon and matched to a characteristic evolution based upon ingoing null cones . The inner boundary for the characteristic evolution is a trapped or marginally trapped surface, whose interior is excised from the evolution . In addition to restricting the cauchy evolution to the region outside the black holes, this strategy offers several other advantages . Although finding a marginally trapped surface on the ingoing null hypersurfaces remains an elliptic problem, there is a natural radial coordinate system (r,,) to facilitate its solution . Motion of the black hole through the grid reduces to a one - dimensional radial problem, leaving the angular grid intact and thus reducing the computational complexity of excising the inner singular region . (the angular coordinates can even rotate relative to the cauchy coordinates in order to accommodate spinning black holes .) The chief danger in this approach is that a caustic might be encountered on the ingoing null hypersurface before entering the trapped region . This is a gauge problem whose solution lies in choosing the right location and geometry of the surface across which the cauchy and characteristic evolutions are matched . There is a great deal of flexibility here because the characteristic initial data can be posed without constraints . This global strategy is tailor - made to treat two black holes in the co - orbiting gauge, as illustrated in figure 8 . Two disjoint characteristic evolutions based upon ingoing null cones are matched across worldtubes to a central cauchy region . The interior boundaries of each of these interior characteristic regions border a trapped surface . At the outer boundary of the cauchy region, a matched characteristic evolution based upon outgoing null hypersurfaces propagates the radiation to infinity . The cauchy evolution is matched across two inner worldtubes 1 and 2 to two ingoing null evolutions whose inner boundaries excise the individual black holes . The outer cauchy boundary is matched across the worldtube to an outgoing null evolution extending to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Ccm for binary black holes, portrayed in a co - rotating frame . The cauchy evolution is matched across two inner worldtubes 1 and 2 to two ingoing null evolutions whose inner boundaries excise the individual black holes . The outer cauchy boundary is matched across the worldtube to an outgoing null evolution extending to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Present characteristic and cauchy codes can handle the individual pieces of this problem . Their unification offers a new approach to simulating the inspiral and merger of two black holes . The individual pieces of the fully nonlinear ccm module, as outlined in section 5.2, have been implemented and tested for accuracy . The missing ingredient is long term stability in the nonlinear gravitational case, which would open the way to future applications . When an artificial finite outer boundary is introduced there are two broad sources of error: the outer boundary conditionwaveform extraction at an inner worldtube . The outer boundary condition waveform extraction at an inner worldtube . Cauchy - characteristic extraction (cce), which is one of the pieces of the ccm strategy, offers a means to avoid the second source of error introduced by extraction at a finite worldtube . In current codes used to simulate black holes, the waveform is extracted at an interior worldtube which must be sufficiently far inside the outer boundary in order to isolate it from errors introduced by the boundary condition . There the waveform is extracted by a perturbative scheme based upon the introduction of a background schwarzschild spacetime . This has been carried out using the regge - wheeler - zerilli [200, 254] treatment of the perturbed metric, as reviewed in, and also by calculating the newman - penrose weyl component 4, as first done for the binary black hole problem in [17, 195, 66, 18]. In this approach, errors arise from the finite size of the extraction worldtube, from nonlinearities and from gauge ambiguities involved in the arbitrary introduction of a background metric . The gauge ambiguities might seem less severe in the case of 4 (vs metric) extraction, but there are still delicate problems associated with the choices of a preferred null tetrad and preferred worldlines along which to measure the waveform (see for an analysis). Cce offers a means to avoid this error introduced by extraction at a finite worldtube . In cce, the inner worldtube data supplied by the cauchy evolution is used as boundary data for a characteristic evolution to future null infinity, where the waveform can be unambiguously computed in terms of the bondi news function . By itself, cce does not use the characteristic evolution to inject outer boundary data for the cauchy evolution, which can be a source of instability in full ccm . A wide number of highly nonlinear tests involving black holes [46, 255, 256] have shown that cce is a stable procedure which provides the gravitational waveform up to numerical error which is second order convergent . Nevertheless, in astrophysical applications which require high resolution, such as the inspiral of matter into a black hole, numerical error has been a troublesome factor in computing the news function . The cce modules were developed in a past period when stability was the dominant issue and second order accuracy was considered sufficient . Only recently have they begun to be updated to include the more accurate techniques now standard in cauchy codes . There are two distinct ways, geometric and numerical, that the accuracy of cce might be improved . In the geometrical category, one option is to compute 4 instead of the news function as the primary description of the waveform . In the numerical category, some standard methods for improving accuracy, such as higher order finite difference approximations, are straightforward to implement whereas others, such as adaptive mesh refinement, have only been tackled for 1d characteristic codes . A major source of numerical error in characteristic evolution arises from the intergrid interpolations arising from the multiple patches necessary to coordinatize the spherical cross - sections of the outgoing null hypersurfaces . More accurate methods have been developed to reduce this interpolation error, as discussed in section 4.1 . In a test problem involving a scalar wave, the accuracies of the circular - stereographic and cubed - sphere methods were compared . For equivalent computational expense, the cubed - sphere error in the scalar field \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal e}(\phi)$\end{document} was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\approx {1 \over 3}$\end{document} the circular - stereographic error but the advantage was smaller for the higher - derivatives (angular derivatives) required in gravitational waveform extraction . The cubed - sphere error \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal e}({\bar \eth\eth^2}\phi)$\end{document} was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\approx {4 \over 5}$\end{document} the stereographic error . In order to appreciate why waveforms are not easy to extract a simple approach to penrose compactification is by introducing an inverse surface area coordinate = 1/r, so that future null infinity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is given by = 0 . In the resulting x = (u,, x) bondi coordinates, where u is the retarded time defined on the outgoing null hypersurfaces and x are angular coordinates along the outgoing null rays, the physical space - time metric g has conformal compactification \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}_{\mu \nu}} = {\ell ^2}{g_{\mu \nu}}$\end{document} of the form 49\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hat g_{\mu \nu}}d{x^\mu}d{x^\nu} = - \alpha d{u^2} + 2{e^{2\beta}}dud\ell - 2{h_{ab}}{u^b}dud{x^a} + {h_{ab}}d{x^a}d{x^b},$$\end{document} where,, u and hab are smooth fields at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The news function and weyl component 4, which describe the radiation, are constructed from the leading coefficients in an expansion of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}_{\mu \nu}}$\end{document} in powers of . The requirement of asymptotic flatness imposes relations between these expansion coefficients . In terms of the einstein tensor \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}_{\mu \nu}}$\end{document} and covariant derivative \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \nabla}_\mu}$\end{document} associated with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}_{\mu \nu}}$\end{document}, the vacuum einstein equations become 50\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$- {\ell ^2}{\hat g_{\mu \nu}} = 2\ell ({\hat \nabla _ \mu}{\hat \nabla _ \nu}\ell - {\hat g_{\mu \nu}}{\hat \nabla ^\alpha}{\hat \nabla _ \alpha}\ell) + 3{\hat g_{\mu \nu}}({\hat \nabla ^\alpha}\ell){\hat \nabla _ \alpha}\ell.$$\end{document} asymptotic flatness immediately implies that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}^{\ell \ell}} = ({{\hat \nabla}^\alpha}\ell){{\hat \nabla}_\alpha}\ell = o(\ell)$\end{document} so that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is a null hypersurface with generators in the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \nabla}^\mu}\ell$\end{document} direction . From (50) there also follows the existence of a smooth trace - free field \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \sigma}_{\mu \nu}}$\end{document} defined on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} by 51\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hat \sigma _ {\mu \nu}}: = \underset{\ell \rightarrow 0}{\lim}{1 \over \ell}({\hat \nabla _ \mu}{\hat \nabla _ \nu}\ell - {1 \over 4}{\hat g_{\mu \nu}}\hat \theta),$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\hat \theta: = {{\hat \nabla}^\mu}{{\hat \nabla}_\mu}\ell$\end{document} is the expansion of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. The expansion \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \theta}$\end{document} depends upon the conformal factor used to compactify \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. In an inertial conformal bondi frame, tailored to a standard minkowski metric at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +}, \hat \theta = 0$\end{document}. But this is not the case for the computational frame used in characteristic evolution, which is determined by conditions on the inner extraction worldtube . The gravitational waveform depends on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \sigma}_{\mu \nu}}$\end{document}, which in turn depends on the leading terms in the expansion of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat g}_{\mu \nu}}$\end{document}: 52\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h_{ab}} = {h_{ab}} + \ell {c_{ab}} + o({\ell ^2}),\quad \beta = h + o(\ell),\quad {u^a} = {l^a} + o(\ell).$$\end{document} in an inertial conformal bondi frame, h = q (the unit sphere metric), h = l = 0 and the bondi news function reduces to the simple form 53\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n = {1 \over 4}{q^a}{q^b}{\partial _ u}{c_{ab}},$$\end{document} where q is a complex polarization dyad on the unit sphere, i.e. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${q^{ab}} = {q^{(a}}{{\bar q}^{b)}}$\end{document} the spin rotation freedom q eq is fixed by parallel propagation along the generators of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, so that the real and imaginary parts of n correctly describe the and polarization modes of inertial observers at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. However, in the computational frame the news function has the more complicated form 54\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n = {1 \over 2}{q^\alpha}{q^\beta}\left({{{\hat \sigma}_{\alpha \beta}} - \omega {{\hat \nabla}_\alpha}{{\hat \nabla}_\beta}{1 \over \omega} + {1 \over \omega}({\partial _ \ell}{{\hat g}_{\alpha \beta}})({{\hat \nabla}^\mu}\ell){{\hat \nabla}_\mu}\omega} \right),$$\end{document} where is the conformal factor relating hab to the unit sphere metric, i.e. Qab = hab . The conformal factor obeys the elliptic equation governing the conformal transformation relating the metric of the cross - sections of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} to the unit sphere metric, 55\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathcal r} = 2({\omega ^2} + {h^{ab}}{d_a}{d_b}\log \omega),$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\mathcal r}$\end{document} is the curvature scalar and da the covariant derivative associated with hab . By first solving (55) at the initial retarded time, can then be determined at later times by evolving it according to the asymptotic relation 56\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hat n^\alpha}{\partial _ \alpha}\log \omega = - {1 \over 2}{e^{- 2h}}{d_a}{l^a},\quad {\hat n^\alpha} = {\hat \nabla ^\alpha}\ell.$$\end{document} all of these procedures introduce numerical error which presents a a challenge for computational accuracy, especially because of the appearance of second angular derivatives of in the news function (54). Asymptotic flatness implies that the weyl tensor vanishes at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}, i.e. = o(). Let \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({{\hat n}^\mu},{{\hat \ell}^\mu},{{\hat m}^\mu})$\end{document} be an orthonormal null tetrad such that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat n}^\mu} = {{\hat \nabla}^\mu}\ell$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat \ell}^\mu}{\partial _ \mu} = {\partial _ \ell}$\end{document} at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Then the radiation is described by the limit 57\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \psi: = - {1 \over 2}\underset{\ell \rightarrow 0}{\lim} {1 \over \ell}{\hat n^\mu}{\hat m^\nu}{\hat n^\rho}{\hat m^\sigma}{\hat c_{\mu \nu \rho \sigma}},$$\end{document} which corresponds in newman - penrose notation to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$- (1/2)\bar \psi _ 4 ^ 0$\end{document}. The main calculational result in is that 58\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \psi = {1 \over 2}{\hat n^\mu}{\hat m^\nu}{\hat n^\rho}\left({{{\hat \nabla}_\mu}{{\hat \sigma}_{\nu \rho}} - {{\hat \nabla}_\nu}{{\hat \sigma}_{\mu \rho}}} \right),$$\end{document} which is independent of the freedom \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat m}^\mu} \rightarrow {{\hat m}^\mu} + \lambda {{\hat n}^\mu}$\end{document} in the choice of m. in inertial bondi coordinates, this reduces to 59\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \psi = {1 \over 4}{q^a}{q^b}\partial _ u^2{c_{ab}},$$\end{document} which is related to the bondi news function by 60\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \psi = {\partial _ u}n$$\end{document} so that 61\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${n_\psi} = n{\vert _ {u = 0}} + \int\nolimits_0^u {\hat \psi du,}$$\end{document} with n = n up to numerical error . As in the case of the news function, the general expression (58) for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \psi}$\end{document} must be used . This challenges numerical accuracy due to the large number of terms and the appearance of third angular derivatives . For instance, in the linearized approximation, the value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \psi}$\end{document} on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is given by the fairly complicated expression 62\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat \psi = {1 \over 2}\partial _ u^2{\partial _ \ell}j - {1 \over 2}{\partial _ u}j - {1 \over 2}\eth l - {1 \over 8}{\eth^2}(\eth \bar l + \bar \eth l) + {\partial _ u}{\eth^2}h,$$\end{document} where j = qqhab and l = qla . In the same approximation, the news function is given by 63\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n = {1 \over 2}{\partial _ u}{\partial _ \ell}j + {1 \over 2}{\eth^2}(\omega + 2h){. }$$\end{document} (the relationship (60) still holds in the linearized approximation but in the nonlinear case, the derivative along the generators of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\hat n}^\mu}{\partial _ \mu} = {e^{- 2h}}({\partial _ \mu} + {l^a}{\partial _ a})$\end{document} and (60) must be modified accordingly .) These linearized expressions provide a starting point to compare the advantages between computing the radiation via n or n. the troublesome gauge terms involving l, h and all vanish in inertial bondi coordinates (where = 1). One difference is that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \psi}$\end{document} contains third order angular derivatives, e.g. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\eth^3}\bar l$\end{document}, as opposed to second angular derivatives for n. this means that the smoothness of the numerical error is more crucial in the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\hat \psi}$\end{document} approach . Balancing this, n contains the term, which is a potential source of numerical error since must be evolved via (56). The accuracy of waveform extraction via the bondi news function n and its counterpart n constructed from the weyl curvature has been compared in a linearized gravitational wave test problem . The results show that both methods are competitive, although the 4 approach has an edge . However, even though both methods were tested to be second order convergent, there was still considerable error, of the order of 5% for grids of practical size . This error reflects the intrinsic difficulty in extracting waveforms because of the delicate cancellation of leading order terms in the underlying metric and connection when computing the o(1/r) radiation field . It is somewhat analogous to the experimental task of isolating a transverse radiation field from the longitudinal fields representing the total mass, while in a very non - inertial laboratory . In the linearized wave test carried out in, the news consisted of the sum of three terms, n = a + b + c, where because of cancellations n a/24 . The individual terms a, b and c had small fractional error but the cancellations magnified the fractional error in n. the tests in were carried out with a characteristic code using the circular - stereographic patches . The results are in qualitative agreement with tests of cce using a cubed - sphere code, which in addition confirmed the expectation that fourth - order finite difference approximations for the - operator gives improved accuracy . As demonstrated recently, once all the necessary infrastructure for interpatch communication is in place, an advantage of the cubed - sphere approach is that its shared boundaries admit a highly scalable algorithm for parallel architectures . Another alternative is to carry out a coordinate transformation in the neighborhood of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} to inertial bondi coordinates, in which the news calculation is then quite clean numerically . This approach was implemented in and shown to be second order convergent in robinson - trautman and schwarzschild testbeds . However, it is clear that this coordinate transformation also involves the same difficult numerical problem of extracting a small radiation field in the presence of the large gauge effects that are present in the primary output data . These underlying gauge effects which complicate cce are introduced at the inner extraction worldtube and then propagate out to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. Perturbative waveform extraction suffers the same problem . Lehner and moreschi have shown that the delicate issues involved at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} have counterparts in 4 extraction of radiation on a finite worldtube . They show that some of the techniques used at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} can also be used to reduce the effect of some of these ambiguities, in particular the ambiguity arising from the conformal factor . The analogue of on a finite worldtube can eliminate some of the non - inertial effects that might enter the radiation waveform . In addition, use of normalization conventions on the null tetrad defining 4 analogous to the conventions at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document} can avoid other spurious errors . This approach can also be used to correct gauge ambiguities in the calculation of momentum recoil in the merger of black holes . Although the bondi - sachs evolution algorithm can easily be extended to include matter, the advantage of a light cone approach for treating fluids is not as apparent as for a massless field whose physical characteristics lie on the light cone . However, results from recent studies of relativistic stars and of fluid sources moving in the vicinity of a black hole indicate that this approach can provide accurate simulations of astrophysical relevance such as supernova collapse to a black hole, mass accretion, and the production of gravitational waves . Miller and mota performed the first simulations of spherically symmetric gravitational collapse using a null foliation . Baumgarte, shapiro and teukolsky subsequently used a null slicing to study supernovae and the collapse of neutron stars to form black holes . The use of a null slicing allowed them to evolve the exterior spacetime while avoiding the region of singularity formation . Barreto s group in venezuela applied characteristic methods to study the self - similar collapse of spherical matter and charge distributions [20, 24, 21]. The assumption of self - similarity reduces the problem to a system of ode s, subject to boundary conditions determined by matching to an exterior reissner - nordstrm - vaidya solution . Heat flow in the internal fluid is balanced at the surface by the vaidya radiation . Their simulations illustrate how a nonzero total charge can halt gravitational collapse and produce a final stable equilibrium . It is interesting that the pressure vanishes in the final equilibrium state so that hydrostatic support is completely supplied by coulomb repulsion . Font and papadopoulos have given a state - of - the - art treatment of relativistic fluids which is applicable to either spacelike or null foliations . Their approach is based upon a high - resolution shock - capturing (hrsc) version of relativistic hydrodynamics in flux conservative form, which was developed by the valencia group (for a review see). In the hrsc scheme, the hydrodynamic equations are written in flux conservative, hyperbolic form . In each computational cell, the system of equations is diagonalized to determine the characteristic fields and velocities, and the local riemann problem is solved to obtain a solution consistent with physical discontinuities . This allows a finite differencing scheme along the characteristics of the fluid that preserves the conserved physical quantities and leads to a stable and accurate treatment of shocks . Because the general relativistic system of hydrodynamical equations is formulated in covariant form, it can equally well be applied to spacelike or null foliations of the spacetime . The null formulation gave remarkable performance in the standard riemann shock tube test carried out in a minkowski background . The code was successfully implemented first in the case of spherical symmetry, using a version of the bondi - sachs formalism adapted to describe gravity coupled to matter with a worldtube boundary . They verified second order convergence in curved space tests based upon tolman - oppenheimer - volkoff equilibrium solutions for spherical fluids . In the dynamic self - gravitating case, simulations of spherical accretion of a fluid onto a black hole were stable and free of numerical problems . Accretion was successfully carried out in the regime where the mass of the black hole doubled . Subsequently the code was used to study how accretion modulates both the decay rates and oscillation frequencies of the quasi - normal modes of the interior black hole . The characteristic hydrodynamic approach of font and papadopoulos was first applied to spherical symmetric problems of astrophysical interest . Linke, font, janka, mller, and papadopoulos simulated the spherical collapse of supermassive stars, using an equation of state that included the effects due to radiation, electron - positron pair formation, and neutrino emission . They were able to follow the collapse from the onset of instability to black hole formation . The simulations showed that collapse of a star with mass greater than 5 10 solar masses does not produce enough radiation to account for the gamma ray bursts observed at cosmological redshifts . Next, siebel, font, and papadopoulos studied the interaction of a massless scalar field with a neutron star by means of the coupled klein - gordon - einstein - hydrodynamic equations . They analyzed the nonlinear scattering of a compact ingoing scalar pulse incident on a spherical neutron star in an initial equilibrium state obeying the null version of the tolman - oppenheimer - volkoff equations . Depending upon the initial mass and radius of the star, the scalar field either excites radial pulsation modes or triggers collapse to a black hole . The transfer of scalar energy to the star was found to increase with the compactness of the star . The approach included a compactification of null infinity, where the scalar radiation was computed . The scalar waveform showed quasi - normal oscillations before settling down to a late time power law decay in good agreement with the t dependence predicted by linear theory . Global energy balance between the star s relativistic mass and the scalar energy radiated to infinity was confirmed . The approach initiated by font and papadopoulos has been applied in axisymmetry to pioneering studies of gravitational waves from relativistic stars . The gravitational field is treated by the original bondi formalism using the axisymmetric code developed by papadopoulos [186, 120]. Because of the twist - free property of the axisymmetry in the original bondi formalism, the fluid motion cannot have a rotational component about the axis of symmetry, i.e. The fluid velocity is constrained to the (r,) plane . In his thesis work, siebel extensively tested the combined hydrodynamic - gravity code in the nonlinear, relativistic regime and demonstrated that it accurately and stably maintained the equilibrium of a neutron star . As a first application of the code, siebel, font, mller, and papadopoulos studied axisymmetric pulsations of neutron stars, which were initiated by perturbing the density and -component of velocity of a spherically symmetric equilibrium configuration . The frequencies measured for the radial and non - radial oscillation modes of the star were found to be in good agreement with the results from linearized perturbation studies . The bondi news function was computed and its amplitude found to be in rough agreement with the value given by the einstein quadrupole formula . Both computations involve numerical subtleties: the computation of the news involves large terms which partially cancel to give a small result, and the quadrupole formula requires computing three time derivatives of the fluid variables . These sources of computational error, coupled with ambiguity in the radiation content in the initial data, prevented any definitive conclusions . A hybrid equation of state was used to mimic stiffening at collapse to nuclear densities and shock heating during the bounce . The initial equilibrium state of the core was modeled by a polytrope with index = 4/3 . Collapse was initiated by reducing the polytropic index to 1.3 . In order to break spherical symmetry, small perturbations were introduced into the -component of the fluid velocity . During the collapse phase, the central density increased by 5 orders of magnitude . At this stage the inner core bounced at supra - nuclear densities, producing an expanding shock wave which heated the outer layers . The collapse phase was well approximated by spherical symmetry but non - spherical oscillations were generated by the bounce . After the bounce, the bondi news function went through an oscillatory build up and then decayed in an = 2 quadrupole mode . However, a comparison with the results predicted by the einstein quadrupole formula no longer gave the decent agreement found in the case of neutron star pulsations . This discrepancy was speculated to be due to the relativistic velocities of 0.2c reached in the core collapse as opposed to 10c for the pulsations . However, gauge effects and numerical errors also make important contributions which cloud any definitive interpretation . This is the first study of gravitational wave production by the gravitational collapse of a relativistic star carried out with a characteristic code . It is clearly a remarkable piece of work which offers up a whole new approach to the study of gravitational waves from astrophysical sources . The pitt code has been coupled with a rudimentary matter source to carry out three - dimensional characteristic simulations of a relativistic star orbiting a black hole . A naive numerical treatment of the einstein - hydrodynamic system for a perfect fluid was incorporated into the code, but a more accurate hrsc hydrodynamic algorithm has not yet been implemented . The fully nonlinear matter - gravity null code was tested for stability and accuracy to verify that nothing breaks down as long as the fluid remains well behaved, e.g., hydrodynamic shocks do not form . The code was used to simulate a localized blob of matter falling into a black hole, verifying that the motion of the center of the blob approximates a geodesic and determining the waveform of the emitted gravitational radiation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${{\mathcal i}^ +} $\end{document}. This simulation was a prototype of a neutron star orbiting a black hole, although it would be unrealistic to expect that this naive treatment of the fluid could reliably evolve a compact star for several orbits . A 3d hrsc characteristic hydrodynamic code would open the way to explore this important astrophysical problem . The code was applied to the problem of determining realistic initial data for a star in circular orbit about a black hole . In either a cauchy or characteristic approach to this initial data problem, a serious source of physical ambiguity is the presence of spurious gravitational radiation in the gravitational data . Because the characteristic approach is based upon a retarded time foliation, the resulting spurious outgoing waves can be computed by carrying out a short time evolution . Two very different methods were used to prescribe initial gravitational null data: a newtonian correspondence method, which guarantees that the einstein quadrupole formula is satisfied in the newtonian limit, andsetting the shear of the initial null hypersurface to zero . Method 1 has only approximate validity in the relativistic regime of a star in close orbit about a black hole while method 2 completely ignores the gravitational lensing effect of the star . It was found that, independently of the choice of initial gravitational data, the spurious waves quickly radiate away, and that the system relaxes to a quasi - equilibrium state with an approximate helical symmetry corresponding to the circular orbit of the star . The results provide justification of recent approaches for initializing the cauchy problem which are based on imposing an initial helical symmetry, as well as providing a relaxation scheme for obtaining realistic characteristic data . A newtonian correspondence method, which guarantees that the einstein quadrupole formula is satisfied in the newtonian limit, and setting the shear of the initial null hypersurface to zero . One attractive way to avoid the computational expense of hydrodynamics in treating a star orbiting a massive black hole is to treat the star as a particle . This has been attempted using the pitt code to model a star of mass m orbiting a black hole of much larger mass, say 1000 m . The particle was described by the perfect fluid energy - momentum tensor of a rigid newtonian polytrope in spherical equilibrium of a fixed size in its local proper rest frame, with its center following a geodesic . The validity of the model requires that the radius of the polytrope be large enough so that the assumption of newtonian equilibrium is valid but small enough so that the assumption of rigidity is consistent with the tidal forces produced by the black hole . Characteristic initial gravitational data for a double null initial value problem were taken to be schwarzschild data for the black hole . The evolution equations for the particle were arranged to take computational advantage of the energy and angular momentum conservation laws which would hold in the test body approximation . The evolution was robust and could track the particle for two orbits as it spiraled into the black hole . Unfortunately, the computed rate of inspiral was much too large to be physically realistic: the energy loss was 10 greater than the value expected from perturbation theory . This discrepancy might have a physical origin, due to the choice of initial gravitational data that ignores the particle or due to a breakdown of the rigidity assumption, or a numerical origin due to improper resolution of the particle . These sources of error can be further aggravated by the introduction of matter fields, as encountered in trying to make definitive comparisons between the bondi news and the einstein quadrupole formula in the axisymmetric studies of supernova collapse described in section 7.2 . In the three - dimensional characteristic simulations of a star orbiting a black hole [48, 44], the lack of resolution introduced by a localized star makes an accurate calculation of the news highly problematical . There exists no good testbed for validating the news calculation in the presence of a fluid source . A perturbation analysis in bondi coordinates of the oscillations of an infinitesimal fluid shell in a schwarzschild background might prove useful for testing constraint propagation in the presence of a fluid . However, the underlying fourier mode decomposition requires the gravitational field to be periodic so that the solution cannot be used to test computation of mass loss or radiation reaction effects.
Consecutive outpatients were recruited at 2 primary healthcare centers and hospitalized patients at a governmental referral center (dr . All eligible auf patients (> 5 years of age) were included who met the following criteria: fever> 38c (central) for <14 days with no apparent other disease . The study was approved by the local medical ethical committee . A specific microimmunofluorescent antibody (ifa) assay for rickettsia spp . Was performed in marseille, france, by using whole - cell antigens of o. tsutsugamushi, r. japonica, r. heilongjiangensis, r. slovaca, r. honei, r. conorii subsp . Indica, rickettsia ati, r. helvetica, r. felis, r. typhi, and r. prowazekii . The assay results were considered positive when 1) antibody titers were> 256 for immunoglobulin (ig) g and> 64 for igm, or 2) seroconversion was observed, or 3) a> 4-fold increase in titers between the acute - phase and the convalescent - phase serum specimen was detected . Convalescent - phase samples were screened with the leptotek dri dot (biomrieux, marcy letoile, france). All positive samples were tested by the microscopic agglutination test (mat) and igg elisa (8). Additionally, a real - time pcr specifically targeting the secy gene of pathogenic leptospira spp ., a panel of 31 serovars was used containing 28 pathogenic and 3 nonpathogenic serovars . For patient samples tested by elisa or mat, a titer> 320 on a single sample was considered positive; also considered positive were those samples that showed seroconversion or a> 4-fold increase in titers between paired samples, as well as any patient sample with a positive pcr, irrespective of the serologic result . The main symptoms were headache (85%), myalgia (70%), nausea (64%), cough (44%), and abdominal pain (38%). Murine typhus and leptospirosis were found to cause auf in this clinical series (appendix table). In total, 9 patients (7%) had evidence of an acute infection with r. typhi; none showed a rash . Murine typhus could be diagnosed in 6 (9%) of 67 hospitalized patients; 3 (4%) of 70 outpatients had acute murine typhus . One patient showed evidence of a past infection with r. typhi (ifa igg / igm titer 128/0 in both serum specimens). Leptospirosis was diagnosed in 13 (10%) of 137 patients; results for 2 of these patients were positive only by pcr . Eleven leptospirosis patients were recruited in the hospital; 2 patients were recruited outside the hospital . Consequently, the percentage of auf caused by leptospirosis in hospitalized patients was 16% and in outpatients 3% . No dual infections were detected; however, 3 (23%) leptospirosis patients showed titers in the r. typhi ifa assay . We report that murine typhus and leptospirosis are important causes of auf in semarang, indonesia . A previous study from rural thailand identified both diseases in 2.8% and 36.9% of auf cases, respectively (6,7). In vientiane, the capital of laos, r. typhi was reported to cause fever in 9.6% of investigated persons, results that closely resembling our data (10). Unfortunately, leptospirosis was not investigated . In the present study, we expected leptospirosis to be a cause of auf because the dr . These cases were not included in the study because of the high clinical suspicion of leptospirosis on admission with jaundice, azotemia, and/or bleeding . A definite diagnosis of murine typhus and leptospirosis co - infections could not be made, but in 3 cases this scenario was plausible . We did not find evidence for scrub typhus, which we expected, because o. tsutsugamushi transmission occurs primarily in rural areas (11). Although sfgr have been reported in southeast asia and proof for their presence in indonesia is accumulating (2,12), these rickettsia were not identified as a cause of auf in the present study . From an epidemiologic point of view, semarang, indonesia, seems to encompass environmental circumstances that are prerequisites for r. typhi and leptospirosis transmission . Previous studies have shown that murine typhus is particularly prevalent in tropical port cities where rats are abundant (13,14). In the indonesian urban situation, r. rattus and r. norvegicus rats are likely to be the main hosts harboring r. typhi infected x. cheopsis fleas (12,15). These rats are also likely to be the maintenance hosts for pathogenic leptospira spp . In indonesia . Although serologic analysis might be hampered by cross - reactions and test sensitivity issues, we believe that our data are representative for the area . The chosen cut - off values are unlikely to cause false - positive results in a disease - endemic setting . In regard to leptospirosis serologic analysis this panel included serovars recommended by the world health organization and serovars that were previously isolated in indonesia . Moreover, most serogroups were represented in our panel, and cross - reactions are likely to detect missing serovars . Because of nonspecific clinical features, both diseases are difficult to diagnose on clinical grounds only . Therefore, rapid, cheap, and reliable diagnostic tests are needed to support clinical decision making . Clinical and laboratory data of patients with rickettsioses and leptospirosis, indonesia, february 2005-february 2006 *
The apoptotic cell death pathway, in its basic tenents, is conserved among metazoans . During development, resistance to apoptosis is a hallmark of cancer, whereas excessive programmed cell death is associated with degenerative diseases . Apoptosis is controlled and amplified through a variety of mechanisms, many of which converge on release of proapoptotic factors from mitochondria (danial and korsmeyer, 2004). (2011) now uncover how regulated trafficking of the proapoptotic protein bax off mitochondria and into the cytosol prevents mitochondrial permeabilization, reconciling discordant models for how antiapoptotic proteins on the mitochondrial surface inhibit proapoptotic proteins in the cytosol . The permeabilization of mitochondria is controlled by proteins belonging to the bcl-2 family, which is composed of anti- and proapoptotic members sharing homology in short stretches of amino acids called bcl-2 homology (bh) domains . The prosurvival members, including bcl-2, bcl - xl, and mcl-1, contain bh domains 14 and antagonize the prodeath members . The multidomain proteins, such as bax and bak, contain bh domains 13 and directly mediate mitochondrial permeabilization, whereas the bh3-only proteins (e.g., bid, bim, bad, etc .) Are thought to act as sensors of cellular stress (danial and korsmeyer, 2004). Despite clearly defined roles for the bcl-2 family members, two major questions have remained: how are proapoptotic members activated, and how do the antiapoptotic members inhibit them? The simplified rheostat model, first proposed in the early 1990s, suggests that pro- and antiapoptotic proteins directly counterbalance each other, with the more abundant protein dictating whether the cell dies or survives (danial and korsmeyer, 2004). Complicating this model is the existence of two types of bh3-only proteins: the activators that directly activate the proapoptotic multidomains and the sensitizers that neutralize the antiapoptotic inhibition of the multidomain proteins (letai et al ., 2002). A modified version of the rheostat model postulates that activator bh3-only proteins act as receptors for multidomain proapoptotic proteins on the mitochondrial outer membrane (om), whereas antiapoptotic proteins act like a sponge, soaking them off of the mitochondrial surface (lovell et al ., a mutually exclusive model postulates that the antiapoptotic proteins directly bind and inhibit bax and bak, with bh3-only molecules acting as inhibitors of the inhibitors (youle and strasser, 2008). This latter model suffered from the lack of evidence that could explain the spatial paradox of this direct inhibition: under normal circumstances, bax is in the cytosol, whereas bcl-2 is on the surface of mitochondria . Upon induction of apoptosis, bax accumulates on the mitochondrial om, undergoes conformational changes to expose a helical domain, and triggers the release of cytochrome c and other proapoptotic effectors . To gain insight into the regulation of bax, edlich et al ., (2011) examine the localization of a mutant version of bax that is constrained in its cytosolic conformation by engineered disulfide bridges . Surprisingly, this mutant protein, which does not interact with the mitochondrial antiapoptotic protein bcl - xl, localizes to mitochondria but does not induce apoptosis . Following up on this observation, the authors ask whether bax normally translocates to the mitochondrial surface . Using fluorescence loss in photobleaching, they monitor the localization of gfp - bax in cells and find that bax cycles on and off the mitochondria in healthy cells . The presence of antiapoptotic proteins in the mitochondrial om is required to constitutively retrotranslocate bax into the cytosol . Notably, the rate of retrotranslocation is almost doubled in cells overexpressing bcl - xl and requires the physical interaction between bcl - xl and bax . Once in the cytosol, bax quickly returns to its monomeric form, ready to cycle back to the mitochondrial surface . Edlich and coworkers observe an acceleration of the retrotranslocation rate upon the coexpression of bcl - xl and bax . Similarly, the bcl-2/bcl - xl inhibitor abt-737, as well as bh3-only proteins, disrupts the equilibrium between cytosolic and mitochondrial bax by reducing its retrotranslocation (figure 1). This new study provides a new paradigm for understanding bax regulation during apoptosis, but how the two categories of bh3-only proteins antagonize bax retrotranslocation by bcl - xl remains an open question . One possibility is that the activators (e.g., bid and bim) directly inhibit bax retrotranslocation by unmasking its 9 helix and anchoring bax in the mitochondrial om . Conversely, the sensitizer (e.g., bad) could obstruct the binding of bax to bcl-2 . The interplay between proapoptotic and antiapoptotic proteins is part of the normal regulation of the trafficking of bax in healthy cells . Under nonapoptotic conditions, the equilibrium between the cytosolic and mitochondrial pools of bax is maintained by its bcl - xl - dependent retrotranslocation . During apoptosis, however, this balance may be tipped by bh3-only proteins, which increase the insertion of bax into the mitochondrial om (figure 1). Thus, the retrotranslocation model is an important step forward, reconciling two opposing models of how bh3-only, multidomain, and antiapoptotic proteins cooperate to dictate whether a cell lives or dies (e.g., cheng et al ., 2001; lovell et al ., 2008 versus willis et al ., in addition to this new model, edlich and colleagues also find that the weak interaction between bcl - xl and bax is sufficient to extract bcl - xl from the mitochondrial om and transiently localize it to the cytosol . This highlights an unappreciated conformational change that facilitates the removal of an antiapoptotic protein from the mitochondrial om . Interestingly, the rate of bcl - xl retrotranslocation increases upon treatment with abt-737, raising the possibility that inactive bcl - xl might be predominantly cytosolic . It will be important to verify whether this is a feature common to extramitochondrial antiapoptotics, such as endoplasmic reticulum (er)-localized bcl-2/bcl - xl, and to understand the mechanism by which interaction with bax inactivates the transmembrane domain of bcl - xl . Interestingly, a fraction of inactive bax is associated with intracellular membranes, including mitochondria and er in transformed cells, but not primary hepatocytes (scorrano et al ., 2003). Understanding how oncogenic transformation influences the on and off rates of bax translocation could open new perspectives to apoptosis in cancer cells . From a teleological perspective, one might wonder why a cell would maintain a continuous flux of bax cycling on to the mitochondria and then back to the cytosol . One reason could be to prime cells to rapidly execute death, positioning bax in a ready to attack mode such that minor changes in the kinetics of bax retrotranslocation can swiftly induce apoptosis . Another possibility is that bax has additional functions that require its regulated presence on cellular membranes . Interestingly, inactive bax controls transient membrane events such as mitochondrial fusion (hoppins et al ., 2011) and, however, bax cannot remain on the organelle without becoming active and promoting apoptosis (nutt et al . The retrotranslocation model not only provides a paradigm for understanding how apoptosis is regulated, but also provides a platform for integrating the diverse functions of bax and other bcl-2 family members.
Oxygen - sensing mechanisms enable the cell to adapt to low oxygen environments and are also critical for normal development and apoptosis . Disruptions of oxygen - sensing pathways can lead to the development of certain forms of cancer [115]. Oxygen sensing is mediated partly via the egln hydroxylases (egln1/egln2/egln3) that are dependent upon molecular oxygen, iron (ii) and -ketoglutarate to perform proline hydroxylation on their target [1620]. Therefore these enzymes are considered oxygen and also metabolic sensors . The availability of molecular oxygen is absolutely required for the hydroxylation reaction, because it donates the oxygen atom to the hydroxyl group . But equally important for the hydroxylation reaction is the electron donor -ketoglutarate, a metabolite from the krebs cycle . Since egln is dependent upon metabolites that take part in the krebs cycle, they are directly tied into the cellular metabolic network (fig . Egln prolyl hydroxylases (also referred to as phd) belong to the 2-oxoglutarate - dependent oxygenase superfamily . The reaction proceeds via the incorporation of dioxygen into the prolyl residue forming hydroxyproline at the substrate . As electron donor for this reaction serves -ketoglutarate (= 2-oxoglutarate) which is oxidized to succinate and co2 . The dependency of the metabolite -ketoglutarate for catalytic activity makes these enzymes sensitive to -ketoglutarate levels and ties them directly into the metabolic network . The first identified substrate for the egln prolyl hydroxylases is the transcription factor hypoxia inducible factor (hif-) [16, 17]. The identification of hif- as a direct hydroxylation substrate provided the first and direct link between tumour suppressor function and oxygen sensing . The tumour suppressor von hippel lindau (vhl) acts as an ubiquitin ligase by targeting hydroxylated hif- for degradation when oxygen is available, as where under low oxygen environments, hif- escapes hydroxylation and subsequently escapes vhl recognition [35, 16, 2227]. The escape from vhl recognition allows hif- to accumulate and to transactivate its target genes that are important for adaptation to low oxygen environment including energy metabolism and angiogenesis [2830]. Both hif - dependent and hif - independent vhl functions contribute to vhl - defective tumorgenesis [3134]. The vhl disease is caused by inactivating germline mutations of the vhl gene and predisposes to a variety of tumours including haemangioblastoma of the retina and nervous system, clear cell renal carcinomas (rcc; the most common form of kidney cancer) and phaeochromocytomas / paragangliomas, tumours of the sympathoadrenal nervous system . Hif- deregulation appears to have a causal role in vhl - defective clear cell rcc and in vhl - defective blood vessel tumours haemangioblastomas (hb). Although hif- regulation has been a major focus of vhl research, the genotype phenotype correlation in the vhl disease gave insight into hif - independent vhl function (table 1). Specific vhl germline mutations corresponding to a specific subset of tumour phenotypes have a different relationship to hif- deregulation (categorized in type i / iia / iib / iic disease) [3537]. The type i disease associates with rcc and hb, and their tumours reflect deregulated high hif- expression, whereas a very different clinical outcome is observed in the type 2c - vhl disease . More importantly, hif- is not deregulated and maintains in low levels in the type 2c tumours [31, 32]. These findings suggest that hif- deregulation is not necessary for phaeochromocytma development in vhl disease . Phenotype correlation in the vhl disease interestingly, a second hydroxylation target of the prolyl hydroxylases egln has recently been identified . The 2-adrenergic receptor is a prototypic g protein - coupled receptor, which is hydroxylated by egln3 and also oxygen dependent degraded by pvhl . The discovery of another hydroxylation substrate by the egln enzymes not only broadens the functionality of prolyl hydroxylation, but also expands our understanding of cellular response to oxygen and its relationship to disease . Therefore, the identification of other egln substrates and the clues from the genotype phenotype correlation that emerged in the vhl disease clearly indicates hif - independent vhl functions . Of great insights are studies from egln3-mediated neuronal apoptosis during sympathetic neuronal development, which shed light onto the genesis of phaeochromocytoma by presumably hif - independent pathways . Phaeochromocytomas are rare, with only five to eight cases diagnosed per million people a year . Extra - adrenal phaeochromocytomas are sometimes referred to as paragangliomas . In short, these tumours are sympathetic nervous system tumours . The most frequent causes of phaeochromocytoma susceptibility are vhl missense mutations, activating mutations in c - ret, mutations in neurofibromatosis type 1 (nf1) and mutations in succinate dehydrogenase subunits of mitochondrial complex ii (sdh b / c / d). Loss of nf1 has been reported to promote survival of embryonic sympathetic neurons in the absence of the nerve growth factor ngf . Further, mutations in subunits of mitochondrial complex ii suggest impairment of mitochondria - mediated apoptosis and have led to the early hypothesis that failure of apoptosis in neuroendocrine precursor cells could result in the development of phaeochromocytoma and paraganglioma . Given the evidence that nf1 promotes sympathetic neuronal survival and further that these neoplasias originate from the sympathetic nervous system, a closer look into the sympathetic neuronal development reveals important clues to the genesis of these tumours . Since the discovery by rita levi - montalcini and viktor hamburger of the neurotrophic factor ngf, our understanding of developmental apoptosis in the sympathetic nervous system has greatly increased . During neuronal development competition for ngf is an important developmental process for matching the size of a neuronal population, especially in the peripheral nervous system . As much as 50% of neurons produced during embryogenesis die by apoptosis during neuronal development . Abnormal ngf signalling has been linked to paediatric nervous system tumours such as neuroblastoma [44, 45] and disease - associated mutations such as nf1 have been shown to enhance signalling by ngf receptors and promote neuronal survival in the absence of ngf . In the last decades it became evident that jnk / c - jun signalling is required for apoptosis when ngf is limiting in the developing nervous system [4650]. Egln3 is induced in sympathetic neurons deprived of ngf and further has pro - apoptotic activity when overexpressed . Given that egln3 is known to hydroxylate hif- and has been implicated in developmental apoptosis in sympathetic neurons, the following questions arise: (i) does egln3-mediated apoptosis depend upon its hydroxylation activity? (ii) does it depend upon hif hydroxylation or does it involve hydroxylation of unknown substrates? (iii) is failure of egln3-mediated apoptosis implicated in the genesis of phaeochromocytomas and other tumours arising from the neural crest origin? The ability of egln3 to induce neuronal apoptosis appears to be unique among the egln family members . Induction of apoptosis is dependent upon egln3 hydroxylation activity, because catalytic impaired egln3 fails to induce apoptosis [9, 52]. Importantly, egln3-induced apoptosis is not diminished in the presence of stable hif1 or hif2 variants that cannot be hydroxylated on their prolines . This suggests that hydroxylation targets of egln3 other than hif- are crucial for apoptosis function . Egln2 and egln3 are induced by hypoxia and dampen the hif- response under chronic hypoxia [5357]. However, egln1 appears to be the primary hif prolyl hydroxylase under normal conditions . Consistent with this, mice lacking egln2 or egln3 are viable and grossly normal whereas mice lacking egln1 are not viable . Conditional inactivation of egln1 in mice leads to polycythemia due to hif- stabilization and increased transcription of hif target genes including erythropoietin [60, 61]. Further, patients carrying egln1 mutations have been reported to develop polycythemia concluding that egln1 couples hif- stability in vivo[13, 62, 63]. Consistent with the unique role of egln3 in neuronal apoptosis are studies obtained from the egln3-deficient mice that clearly demonstrates the requirement for egln3-mediated apoptosis during the sympathetic neuronal development . Egln3 deficient sympathetic neurons are resistant to apoptosis after ngf withdrawal, as well as certain neurotoxins . Consistently, egln3 mice have an increased number of cells in the super cervical ganglia and in the adrenal medulla and show abnormalities in the sympathoadrenal system including systemic hypotension . In fact, it appears that some of the adrenal abnormalities are caused through deregulation of the 2-adrenic receptor (2-ar). Loss of egln3 results in 2-ar up - regulation and accordingly, hypoxia stabilizes the 2-ar . This is consistent with the observations from the type 2c vhl patients that develop phaeochromocytoma . Type 2c patients often show excessive secretion of catecholamines (endogenous 2-ar ligands) and increased sympathonervous system activity . In summary, this points towards distinct and unique functions within the family of egln prolyl hydroxylases . Identification of novel egln hydroxylation targets will open new oxygen sensing pathways independent of what we have learned from hif-. Given the evidence that (i) phaeochromocytomas are sympathetic nervous system tumours and that (ii) egln3 is critical for apoptosis during sympathetic neural development, an attractive hypothesis emerged in which failure of egln3-mediated apoptosis during sympathetic development predisposes to the genesis of phaeochromocytomas and perhaps other neoplasia arising from neural crest origin . Interestingly, apparently unlinked phaeochromocytoma lesions (vhl, nf1, c - ret and sdh) all appear to act on a single common pathway by decreasing egln3-mediated apoptosis at the time during development when levels of ngf become limiting (fig . Egln3 appears to act downstream of the c - jun in the ngf signalling pathway . Therefore, a single pathway was established in which the genetic phaeochromocytoma defects act either directly on egln3 or upstream of egln3 to impair apoptosis (fig . 2). For instance, the genetic defect in sdh acts directly on egln3-mediated apoptosis function . Sdh inhibition results in accumulation of intracellular succinate, which in turn can product - inhibit the egln prolyl hydroxylases [9, 71]. This succinate - mediated inhibition not only results in hif stabilization, but also importantly inhibits egln3-mediated apoptosis . The succinate - mediated product - inhibition of egln3 was overcome by re - addition of -ketoglutarate restoring ngf - mediated apoptosis . This suggests that sdh inhibition acts on the prolyl hydroxylases via succinate and not as alternatively suggested through the generation of reactive oxygen species ., ngf is required for neuronal survival but is also limiting . Neurons that successfully compete for ngf survive whereas unsuccessful neurons undergo c - jun - dependent apoptosis . C - jun transcriptionally activates prolyl hydroxylase egln3 . As ngf levels become limiting, familial phaeochromocytoma mutations (vhl, nf1, c - ret, sdh and kif1b) all decrease apoptosis mediated by egln3 . In addition to the predisposing sdh genetic lesion, other phaeochromocytoma lesions have been implicated upstream of egln3 to promote neuronal survival in the ngf signalling pathway . In the case of vhl predisposing lesion, pvhl suppresses junb and all vhl mutants tested (including type iic vhl disease mutant) abrogate its ability to do so . Junb acts as an antagonist of c - jun and increased junb levels attenuate the induction of c - jun - mediated apoptosis in sympathetic neurons deprived from ngf . Finally, previous evidence indicated that ret (the receptor for glial - derived neurotrophic factor) and nf1 could act through this pathway by modulating the action of the ngf receptor trka . Activation of c - ret, like loss of pvhl, leads to the induction of junb and attenuates apoptosis when ngf becomes limiting . Thus, when mutated, all the genetic phaeochromocytoma lesions (sdh, vhl, c - ret and nf1) impair ngf - mediated apoptosis in neuroendocrine precursors during development . These findings provide an explanation as to why the mutations in tumour suppressors that are found in familial phaeochromocytoma are rare in sporadic phaeochromocytoma . This is because the pathway is no longer critical once development is completed . In summary, all the genetic lesions associated with phaeochromocytoma act on a single common pathway that impinges upon egln3 apoptotic activity during sympathetic neuronal development . Mutations in sdh, vhl, c - ret and nf1 would allow sympathetic neuronal precursors to escape from developmental apoptosis and set the stage for their neoplastic transformation . It will be interesting to determine not only if egln3 is mutated in these neoplasias, but also, if failure of egln3 developmental apoptosis plays a broader role in paediatric cancers and other forms of hereditary cancer . An important challenge is to identify the link between this enzyme and apoptosis, which presumably involves hydroxylation of a protein other than hif-. Early studies indicated that egln3 mrna and protein expression (at that time referred as sm-20) increases shortly after removal of ngf in primary sympathetic neurons and peaks between 10 and 15 hrs at a time when cells undergo apoptosis . Overexpression of egln3 is sufficient to promote cell death in ngf - maintained sympathetic neurons [51, 73]. Cell death is caspase dependent and accompanied by an increase of cytochrome c in cytosolic and mitochrondria - enriched subcellular fractions . The mechanism underlying egln3-induced cytochrome c is not known although it appears to involve an increase in cytochrome c mrna . Other studies have identified egln3 as a growth factor inducible gene in vascular smooth muscle cells and later proposed, that it might function in growth arrest, differentiation and cell death during muscle differentiation [76, 77]. Expression of egln3 was also reported in fibroblasts upon activation of a temperature - sensitive form of p53 . However, egln3-induced apoptosis appears not to be impaired in cells lacking p53 or expressing a dominant negative p53 protein, indicating that p53 might not function downstream of egln3 to induce apoptosis . In addition, a recent study indicated that egln3 s ability to induce apoptosis correlates with the formation of aggresome - like structures . Recently, an unbiased genome - wide approach has been undertaken to understand how egln3 causes neuronal cell death . This led to the identification of the kinesin kif1b as a downstream effector of egln3 . Kif1b, a member the kinesin 3 family, consists of two major splice variants and . Kif1b and kif1b are motor proteins implicated in anterograde transport of mitochondria and synaptic vesicle precursors, respectively [79, 80]. The recent study, which identified kif1b as an egln3 downstream target showed how kif1b is both necessary and sufficient for neuronal apoptosis when ngf becomes limiting, but it remained unclear how egln3 regulates kif1b. Interestingly, kif1b maps on to the chromosomal region 1p36.2, a region of the genome that is frequently deleted in neural crest - derived tumours including neuroblastomas . The existence of one or more human tumour suppressor genes on chromosome 1p has been suspected for decades [8284]. Further suggestion that kif1b acts as a 1p36 tumour suppressor comes from the current model for phaeochromocytoma development . Phaeochromocytomas develop when sympathetic neuronal precursors escape from egln3-mediated developmental apoptosis, suggesting that mutations in kif1b may be relevant to phaeochromocytoma and other tumours of neuronal origin . Indeed, inherited loss - of - function kif1b missense mutations have been identified in phaeochromocytomas and neuroblastomas and an acquired loss - of function mutation in a medullablastoma, arguing that kif1b is a pathogenic target of these deletions . Nonetheless, it has been further reported that the remaining kif1b allele in 1p deleted tumours and cell lines is often wild - type, contrary to the knudson twohit scenario [64, 8588]. Also, the existence of multiple neuroblastoma and phaeochromocytoma suppressor genes on 1p has been suggested [89, 90]. Additional studies are needed to address how often it is deregulated, epigenetically or genetically, in cancer . A spotlight is now on understanding the mechanistic basis of how egln3 regulates kif1b and how this translates into cell death . Kif1b appears not be a direct egln3 hydroxylation target, but egln3 hydroxylation activity is required for kif1b regulation . Therefore egln3 presumably involves hydroxylation of a protein that in turn might regulate kif1b to induce apoptosis . In 1924, otto warburg observed that cancer cells are highly glycolytic in the presence of oxygen and have reduced rates of oxidative phosphorylation . Recent studies have argued that cancer cells might benefit from this persistence of high lactate production in the presence of oxygen, referred to as aerobic glycolysis or pseudo - hypoxia [92, 93]. From a bioenergetic perspective, it remains a conundrum how highly metabolic proliferative cancer cells undergo a pathway that results in 10 times less atp production compared to their normal counterparts that oxidize their glycolysis endproduct within the mitochondria via the krebs cycle (pyruvate conversion into acetyl - coa, fig . Interestingly, three major enzymes of the krebs cycle (sdh, fh, idh) have been recently identified as bonafide tumour suppressors, but more importantly, inactivation in either of them directly affects egln activity [9, 10, 14, 71]. This is because the egln activity is not only dependent upon molecular oxygen to perform their hydroxylation reaction, but also require the krebs cycle metabolite -ketoglutarate as electron donor, which ties them directly into this metabolic network . Therefore an attractive hypothesis arises: cancer cells favour aerobic glycolysis to inhibit egln activity in order to escape either certain oncogenic apoptotic signals and/or activate oncogenic hif-. About 70% or more of low - grade gliomas bear loss of function mutation in idh1 and idh2 [94, 95]. Subsequently egln prolyl hydroxylases are inhibited in their hydroxylase activity with the consequences of hif- stabilization . Presumably egln3-mediated apoptosis might be impaired in these settings as well, but this has not been investigated yet . Further, these studies follow the discoveries that germline mutation in sdh are linked to phaeochromocytoma and that fh mutation lead to leiomyosarcoma and renal cell carcinoma, both of which affect also the egln prolyl hydroxylase activity [9, 10, 71]. This is because loss of function mutation in sdh and fh increases the accumulation of succinate and fumarate respectively . The excess of these metabolites inhibits the proline hydroxylases with the consequences in either accumulation of oncogenic hif- and/or blunting egln3-mediated apoptosis [9, 10, 71, 96]. The krebs cycle enzymes (sdh, fh and idh) are tumour suppressors and linked to egln activity . The egln prolyl hydroxylases are dependent upon the krebs cycle metabolite -ketoglutarate as electron donor for hydroxylation activity . Loss of function mutation in idh decreases -ketoglutarate levels and subsequently impairs egln function . On the other hand, mutation in sdh or fh leads to accumulation of succinate or fumarate respectively, which in turn product - inhibits egln activity . Loss of function of sdh, fh or idh would lead to impaired krebs cycle activity and impaired oxidative phosphorylation with the consequence of enhanced glycolysis (embden meyerhof pathway) to generate atp . The generated pyruvate during glycolysis is redirected away from the krebs cycle by conversion into lactate . Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by otto warburg in 1924 . Despite the striking difference in atp production, cancer cells might favour aerobic glycolysis to escape from egln hydroxylation, resulting in the accumulation of oncogenic hif. And/or resistant to egln3-mediated apoptosis . The recently identified mutations in the metabolic / mitochondrial enzymes (fh, sdhb / c / d, idh1/2) provide convincing evidence that alteration in cellular metabolism contributes to the pathogenesis of cancer . Hence, 43 years later we are beginning to learn the depth of otto warburg s foresight that (in his words) cancer cells should be interpreted as a mitochondrial dysfunction and that the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar[97, 98]. Even if this is the prime cause for some cancers, the underlying mechanistic basis remains controversial . However, recently it has become more evident that the inactivation of the egln prolyl hydroxylases is a downstream effector of these mitochondrial alterations . The identification of sdh and fh mutations has already led to the development of cell permeable -ketoglutarate derivates with the potential to suppress the transforming effects of these mutations through reactivation of the eglns . The exciting part of identifying metabolic - enzyme mutations in specific cancers is that they are druggable. They might provide new opportunities as therapeutic targets that would be more susceptible and more effective than existing cancer therapies . Future work is needed to further elucidate the direct impact of cancer metabolism in prolyl hydroxylase functioning.
Descriptive epidemiologic statistics assist public health planning and provide valuable information about the burden of illness to policy makers, funding agencies, resource planners, healthcare insurers, and manufacturers . Information on malignancies that is compatible with their clinical classifications is of particular value to clinicians and public health professionals and increasing efforts are being made to collect data at this detailed level (e.g., the haemacare project). It is challenging, however, to assemble a database of descriptive epidemiologic statistics from the peer - reviewed literature alone . Cancer registries are used worldwide to collect and analyze demographic, diagnostic, and survival data . Some registries are fraught with poor quality and infrastructure; however, there is no standardized system for the collection and reporting of descriptive statistics worldwide [2, 3]. Comprehensive reviews of descriptive epidemiologic statistics, such as this one, are warranted to better understand the totality of the currently available data and to optimize the utility of such data in the future . This paper uses a particular cancer subtype b - cell malignancies to evaluate a novel approach to assembling a database of worldwide, national - level, descriptive epidemiologic cancer statistics . This approach incorporates information from various sources, including the peer - reviewed literature, online reports, and query systems from cancer registries and health agencies, and direct contact with cancer registries, to provide a current, comprehensive database for a representative group of countries worldwide . The major b - cell malignancies were chosen as the cancer subtype to test this approach largely because their epidemiology has not been well characterized . Further, some b - cell malignancy subtypes require detailed diagnostic evaluation, and this paper allowed us to broadly assess the extent to which detailed diagnoses are currently being reported to cancer registries . This is important because an understanding of these subtypes may inform the development of novel treatments . B - cell malignancies emerge in cells of the bone marrow, blood, or other tissues at various stages of b - lymphocyte differentiation and represent a rare (3% of all malignancies) and heterogeneous group of lymphohematopoietic malignancies (table 1). In 2001 and 2008, the world health organization (who) classified lymphoid hematologic neoplasms into 4 major categories based on the cell linage of the malignancy or the normal cell type that the tumor most resembles: b - cell malignancies, t - cell malignancies, natural killer (nk) cell malignancies, and hodgkin's disease . Further divisions were based on cell maturity (e.g., precursor versus mature b - cell neoplasms), as well as morphologic, genotypic, genetic, immunohistochemical, and clinical criteria . The major b - cell lymphoid malignancies include diffuse large b - cell lymphomas (dlbcls), follicular lymphomas (fls), and plasma cell or multiple myeloma (mm). It is estimated that 85% to 90% of non - hodgkin's lymphomas (nhl) are of b - cell origin, including dlbcl and fl . Certain leukemias also arise from the b cells and are categorized based on (1) whether the disease is acute or chronic and (2) what type of cell is infected . The leukemias associated with abnormalities of b - cell formation in the blood include b - cell acute lymphoblastic leukemia (all) and chronic lymphocytic leukemia (cll) or small lymphocytic lymphoma . All represents one of the most common forms of leukemia among children . In this paper, a novel, comprehensive approach to gathering descriptive, epidemiologic statistics was used to develop a current worldwide database of the major b - cell malignancies . The intent of this paper was to evaluate the feasibility of this novel approach for assembling a database of descriptive statistics and to broadly assess the level of detail collected by cancer registries worldwide . We also briefly describe our findings of the descriptive epidemiology of b - cell malignancies . We collected the most recent information available on the incidence, prevalence, and survival of the following b - cell malignancies: dlbcl, fl, cll, mm, adult b - cell all, and pediatric b - cell all . These data were collected for a representative selection of countries worldwide, categorized into the following groups: north and south america (brazil, canada, and the united states), the european union-5 (france, germany, italy, spain, and the united kingdom), asia (china, india, japan, and south korea), and australia . Descriptive statistics for the countries constituting the united kingdom (england, scotland, wales, and ireland [including northern ireland and the republic of ireland]) were also collected . We employed three general strategies: a structured literature review of the published, peer - reviewed literature in pubmed, a review of online documentation from cancer registries and relevant health agencies, and, finally, direct contact via email with personnel at cancer registries and key experts in the field . The first 2 strategies were employed in tandem; direct contact was used to address any remaining gaps and clarify the reasons behind missing data . Various search strings (see table s1 in supplementary material available online at doi:10.5402/2012/129713) were used in the pubmed search to identify general articles describing the epidemiology of these malignancies in the countries of interest . The search was restricted to human studies published from january 1, 2000 to july 13, 2011 . Approximately 5600 citations were retrieved, and the titles and abstracts of these publications were reviewed . Studies that appeared to report descriptive epidemiologic statistics (i.e., incidence, prevalence, or survival) for the b - cell malignancies of interest were included . Articles reporting national - level data on the statistics, malignancies, and countries of interest were identified and the relevant information was extracted . In addition, case reports, case series, and letters to the editor were excluded . Relevant full - text articles were retrieved and reviewed for inclusion . Worldwide, national, and regional cancer registries and online documentation for these registries (including reports and online query systems) the websites of other relevant health agencies and government departments were also searched for reports and references to the data of interest . Registries and relevant agencies were identified from previous experience, online research into the structure of public health in each country, and involvement in organizations such as the international association of cancer registries, globocan, and the european network of cancer registries . For some non - english language speaking countries (france, germany, spain, china, and japan), persons proficient in the language searched online data and translated some of the published literature; a translation of non - english language material (including online documentation and published studies) was not performed, however . The goal was to identify the most recent estimates of the incidence, prevalence, and survival associated with the relevant b - cell malignancies . If relevant data were reported by more than 1 source, the source with the most recent data and/or the most relevant data (e.g., data specific to b - cell all versus all) was used . Once gaps in the availability of the data of interest were identified, cancer registry personnel and key experts in the field were contacted via e - mail to discuss the availability of missing statistics (these contacts are referred to as personal communication). Table 2 describes the cancer registries and organizations that were contacted and the outcome of each respective communication . We did not collect data from globocan or the cancer incidence in five continents (ci5) series . These iarc programs provide a valuable resource for modeling estimates on the incidence and mortality of common cancers, including nhl and mm . However, since modeling estimates may not reflect the most recent national - level statistics, we did not rely on these databases for nhl and mm data . There are also standardized efforts in europe to report descriptive epidemiologic data: eurocare and the automated childhood cancer information system . Similar to ci5; however, these standardized systems are not based on complete ascertainment from registries in the underlying countries, for example, only select regional registries participate in spain, italy, and france . As such, we only used data from these european programs in the absence of national - level data . The following information was extracted from each data source: source of the data, country or countries, malignancy type, including any available information on international classification of diseases (icd) codes, and data relevant to incidence, prevalence, and/or survival . We sought to assess the availability of recent statistics; therefore, only post-2000 diagnoses were assessed for incidence and survival, and only prevalence estimates for a date after 2000 were collected . Incidence projections for future years were collected only in the absence of actual incidence estimates . Limited - duration prevalence rates and prevalence proportions estimated on a date after 2000 were collected for all available time frames (e.g., 5 y, 10 y). Relative survival rates were collected for all time periods (e.g., 5 y survival, 10 y survival) for diagnoses post-2000; it was noted whether survival rates were estimated using period methods . Relative survival is defined as the ratio of the proportion of observed survivors in a cohort of cancer patients to the proportion of expected survivors in a comparable set of cancer - free individuals . In the absence of data on relative survival, data were collected by gender and methods for age standardization and any age restrictions were noted . Data were reported from multiple citations for a particular malignancy and country if different information sources reported supplementary information . Large gaps in the availability of national - level statistics on b - cell malignancies were observed at the outset of our review process and few descriptive statistics were available for the b - cell nhls (i.e., dlbcl and fl) and b - cell all . We, therefore, extended the review to the larger diagnostic categories of nhl and all . Data on diffuse nhl, a diagnostic category of nhl that includes dlbcl, were available from numerous countries, and data were collected for this larger diagnostic category in the absence of data specific to dlbcl . As a result, we collected data on the larger diagnostic category lymphoid leukemia . Finally, national - level statistics were lacking in several countries (e.g., brazil, india, and china) because of the status of cancer registration in these countries . To compensate for this deficiency, table 3 provides an overview of the availability of statistics by malignancy type and country; notations are provided where proxy data (as defined above) were used . Supplementary tables 2 to 7 provide the most recent statistics by country and statistic type for nhl, dlbcl and fl, adult all, pediatric all, mm, and cll, respectively . Cancer registries are well recognized as a valuable information source for public health planning and epidemiologic and therapeutic research because they provide essential epidemiologic data on the current burden of disease . Nonetheless, we found that the implementation of cancer registries has been slow in some countries, and data were not made readily available or updated in a timely manner in others . In 2006, almost 80% of the world's population was not covered by a population - based cancer registry; most of this unrepresented population was from low- and middle - income countries . The uncoordinated nature of cancer registration worldwide has resulted in wide variation in the geographical areas covered, the methods used to estimate descriptive statistics, and the level of detail collected . This differential evolution of cancer registration worldwide yielded notable between - country variation in the availability of descriptive statistics on b - cell malignancies (table 3). The use of online resources and direct contact with cancer registries greatly expanded our database . The us surveillance, epidemiology, and end results (seer) registry provided the most complete data on b - cell malignancies . The seer registry provides incidence, prevalence, and survival data by icd - o-3 code using the seer*stat software . We were, therefore, able to calculate statistics for the b - cell malignancies dlbcl and b - cell all, which require icd - o-3 coding . Nearly all countries in our paper had national - level incidence statistics for nhl and mm, although prevalence and survival data for these malignancies were less complete . Data on dlbcl and fl were only available in the united states, the united kingdom, germany, and australia, but supplementary data were available on diffuse nhl from several countries (brazil, england, ireland, and germany). Since the course of leukemia varies by its tissue of origin and whether it is acute or chronic, the reporting of data by specific leukemia subtypes is preferable . Lymphoid leukemia incidence was often used as a proxy for all and cll, however, and prevalence and survival data were scarce for all categories of lymphoid leukemia . In many developed countries (e.g., canada, the united kingdom, and germany), only the prevalence of leukemia as a large diagnostic group was reported . France provided no national - level estimates of all incidence, except rates estimated using a statistical method based on unreported mortality data (belot a and mitchell m, personal communication with universit lyon, january 27, 2011). In germany, descriptive statistics were only available for leukemia as a large diagnostic group . Regional statistics for lymphoid leukemia were found in the bavaria and bremen population - based cancer registries (i.e., registries that collected data on every person with incident cancer within a defined geographic region). Italy, on the other hand, provided pooled data from its 28 regional cancer registries on adult and pediatric all and adult cll . Among the asian countries, only japan had national - level data on all, whereas china and south korea had national - level data on lymphoid leukemia . The united states and the republic of ireland were the only countries with incidence data specific to adult b - cell all (1.0 and 0.22 (s. deady and m. wagner, personal communication, with national cancer registry of ireland, september 24, 2010), respectively, per 100,000 persons). In the united states, these data suggest that b - cell all comprises approximately 60% of all adult all . The united states was the only country with statistics on pediatric b - cell all, with an incidence of 1.77 (95% ci, 1.701.84) per one million children (representing only 5% of pediatric alls). Of note, france, germany, and spain reported the incidence of mature b - cell lymphoid leukemias (international classification of childhood cancer [iccc] code ia2) among children 0 to 14 years of age (1.5, 1.0, and 0.6 per million children per year, resp . ); these data demonstrate that the vast majority of lymphoid leukemias in children are of the precursor cell type [1921]. Cancer registries in some countries indicated that the provision of descriptive data on specific nhl and leukemia subtypes was not possible because of small population sizes and the associated uncertainty with small numbers . The northern ireland cancer registry, for example, could not provide statistics by nhl or leukemia subtypes because of small numbers (d. donnelly and m. wagner, personal communication with northern ireland cancer registry, september 27, 2010). The national cancer registry of the republic of ireland, on the other hand, provided these data after e - mail contact was initiated with the registry (s. deady and m. wagner, personal communication with national cancer registry of ireland, other cancer registries could not provide the level of detail required for nhl and leukemia subtypes because the data were not coded appropriately to permit such analyses . For example, noted that it is not currently possible to calculate statistics for various hematologic subtypes in france because of issues with reliably classifying cases using a consistent coding scheme . Similarly, the office of national statistics (which releases descriptive data for england) stated that they were only able to provide data by icd-10 codes because of variability in the coding information provided by regional registries and issues with mapping icd - o-3 (introduced in 2008) to icd - o-2 (n. jakomis and m. wagner, personal communication with national cancer intelligence center on descriptive cancer statistics, october 11, 2010). Icd-10 codes do not allow for the calculation of statistics on b - cell all or dlbcl . We were able to capture statistics for fl with icd-10 code c82 and for diffuse lymphoma with icd-10 code c83, however . The icd - o-3 coding system provides the most relevant information for descriptive epidemiologic statistics because malignancies are coded by cell lineage and maturity, as well as morphologic, genotypic, genetic, immunohistochemical, and clinical behavior . Icd - o-3 has been adopted by the us and most european countries, but the report of descriptive statistics by icd - o-3 coding is limited by the recent conversion from icd - o-2 in 2000 and the small numbers associated with these rare hematologic malignancies . One author noted that, even considering the whole of europe, some entities were too rare to calculate statistics by icd - o-3 coding . To address this issue, haemacare, a project funded by the european commission to improve the standardization of population - based data on hematologic malignancies, collected incidence data 20002002 from 48 european registries in 20 countries using a grouping system based on who recommendations and the icd - o-3 morphology codes . Thus, categories represent cell lineage and prognosis, which are useful for epidemiologic and public health purposes . The haemacare coding system contains detailed information on mature b - cell malignancies, including dlbcl, fl, and cll . In their first publication of incidence data from the haemacare network, sant et al . Reported european age - standardized incidence rates (per 100,000 persons) for dlbcl, fl, and cll of 3.81 (95% ci, 3.733.89), 2.18 (95% ci, 2.122.24), and 4.92 (95% ci, 4.835.01), respectively . Compared to these haemacare european estimates, the incidence of dlbcl is considerably higher in the united states (6.83; see table s3) and the incidence of fl is higher in both the united states (3.7) and australia (4.1; see table s3). However, the haemacare european estimate of cll incidence is similar to rates observed in the united states (5.0), canada (5.0), and australia (4.9; see table s7). Sant et al . Noted geographic variability in the incidence of hematologic malignancies across europe, which they attributed to differences in diagnostic and registration criteria . This first publication from the haemacare network suggests that data on dlbcl, fl, and cll are increasingly available in certain registries across europe, but that cancer registries are reluctant to regularly publish and/or provide these statistics . Another limitation of the diagnostic classification system used for reporting cancer statistics is the use of the iccc for childhood cancers . In the third version of the iccc, all is categorized along with other lymphatic leukemias in the category iccc-1a, which includes morphology codes 9820, 9823, 9826, 9827, 98319837, 9940, and 9948 . Although the vast majority of lymphatic leukemias in childhood are acute in nature, estimates using iccc-1a only provide a proxy for actual all rates . There was wide variation between countries in the methods used for estimating national - level descriptive statistics . Some countries used direct estimates from national cancer registries, collecting information on all cancer diagnoses nationwide into 1 registry (i.e., wales, northern ireland, republic of ireland, scotland, and south korea) or a coordinated group of regional registries (i.e., england, canada, and australia). Other countries calculated estimates of the national - level experience using statistical methods to extrapolate data from regional registries to the national level (i.e., united states, france, japan, and china). Another group of european countries estimated the national - level cancer experience by pooling data from regional registries (i.e., italy, germany, and spain). This variability in the methods used to estimate national - level statistics limits the comparability of statistics between countries . One statistical method used to predict cancer incidence at the national - level is the use of mortality rates (which are often readily available nationwide) as a geographic correlate of incidence . Observed incidence and mortality data are modeled in this approach using age - cohort techniques to provide estimated incidence rates . This method was recently used to estimate cancer incidence in france and china [22, 25] and is frequently used by globocan . While this method is known to be generally reliable, there are some questions about the representativeness of the data used . However, the reliability of this method to estimate the incidence cancers with high survival rates (e.g., pediatric all) is less clear . The vast majority of registries were population - based, but some countries had registries that collected data only on cancer patients seen at a particular hospital (i.e., hospital - based registries). Hospital - based registries are limited compared with population - based cancer registries because the source population is not well - defined . Brazil has several hospital - based cancer registries, and south korea has a nationwide, hospital - based cancer registry (the korea central cancer registry) that reportedly captures 90% of all cancer cases nationwide . The national registry of childhood tumors is a population - based cancer registries of malignancies and benign brain tumors diagnosed in children living in england, wales, or scotland . The french national registry of childhood hematopoietic malignancies has recorded all cases of hematologic malignancies, including lymphoma, since 1990; the french national registry of childhood solid tumors has recorded all cases of solid tumors, except lymphomas, since 2000 . The german childhood cancer registry has collected data on cancer cases nationwide, with an estimated coverage of about 95% . Finally, the australian pediatric cancer registry has provided full coverage of all australian states and territories since 1983 . A substantial number of countries do not have a nationwide cancer registration program . Rather, cancer registration consists of a group of regional cancer registries, collecting complete data on cancer cases in their respective geographic regions and coordinated by an overarching network . The level of standardization and coordination between these regional registries varies substantially by country . In the united states, seer currently collects and publishes cancer data in a standardized fashion from 17 population - based cancer registries covering approximately 28% of the us population there are 21 regional cancer registries in france coordinated under the association of french cancer registries (francim) that cover approximately 16% of the population . In italy, there are 28 registries that cover approximately 26% of the population coordinated through the italian association of cancer registries (airtum). In germany, there are regional registries in 13 states and 1 administrative district, coordinated through the robert koch institute . Japan has approximately 30 population - based cancer registries that are coordinated through the japan cancer surveillance research group . Some developed countries still lack coordination of their regional registries, which limits what can be done to predict the national - level experience of cancer . Spain, for example, currently has 13 regional registries that cover approximately 27% of the population, but no network that coordinates and standardizes these registries; a network is currently under development, however . In less developed countries, where financial, resource, and logistical challenges dominate, coverage is typically limited to metropolitan areas, and differences between regional registries preclude the development of national estimates . Substantial efforts are being made, however, to organize and standardize these regional registries into a coordinated network . In brazil, for example, efforts are being made to collect data from an expanding network of regional registries and to improve data quality to support nationwide cancer prevention and planning . Epidemiology surveillance was formally organized and standardized in 1999 and now includes 22 population - based cancer registries, most of which include capital cities and surrounding areas . These population - based cancer registries vary in the source (hospital, laboratory, etc .) And quality of their data . Because of this variation, pooled data cannot be reported and statistics are provided only by individual population - based cancer registries for brazil . The age - adjusted incidence of nhl, for example, varied from 2.0 to14.1 per 100,000 among males in the ten population - based cancer registries with more than 2 years of data . The authors of the latest report cautioned that these results are not generalizable to brazil as a whole because the population - based cancer registries are still of heterogeneous quality, validity, and completeness . Underreporting also remains a serious concern in brazil and other less developed countries . Despite these limitations, some brazilian population - based cancer registries provided data on detailed diagnostic categories diffuse nhl, follicular lymphoma, and pediatric all . However, although incidence data were readily available from brazilian population - based cancer registries, more complex data (prevalence and survival) were not . Similarly, india has 26 population - based cancer registries and 6 hospital - based cancer registries coordinated under the national cancer registry programme network and covering 7% of the population; statistics are only reported by population - based cancer registries . China also has no national - level coordination of its 50 population - based cancer registries, which cover 5.7% of the country . This survey represents the status of data that were available as of mid - july 2011 using comprehensive methods . It is clear from our paper, however, that the breadth and specificity of data reported by cancer registries is continually evolving, and this evolution will ultimately entail the reporting of data at the national level for detailed cancer subtypes . In france, for example, national level data were reported for the first time in 2003 for cancer incidence, in 2006 for cancer survival, and in 2008 for cancer prevalence . Lacour et al . Described the incidence of childhood cancers nationwide in france for the first time in 2010 . Population - based cancer registration in germany improved considerably after the federal cancer registry data act of 2009, which called on all federal states to report complete data to the german centre for cancer registry data at the robert koch institute, thus expanding the breadth of cancer statistics in germany from data reported only by the saarland cancer registry to pooled analyses of data from cancer registries meeting the minimum requirements for data completeness . Current estimates of cancer incidence are based on data from 13 states and 1 administrative district, and the gekid cancer survival working group recently published their first comprehensive monitoring of cancer survival in germany based on a collaboration between 13 population - based cancer registries . Some basic patterns in the descriptive epidemiology of b - cell malignancies emerged from reviewing the available data . Specifically, the incidence rates of the b - cell neoplasms included in this paper were generally higher in the united states, australia, and the european union-5, compared with asia and brazil (possibly reflecting differences in the ascertainment of cases). Further, the incidence of nhl was generally higher in men, and nhl survival rates were slightly higher in women (see table s2). The higher incidence of hematologic malignancies in men versus women may be explained by the traditionally higher exposures of men to occupational and environmental carcinogens . Many cancer registries reported that the requested statistics were not readily available or were available at a cost (e.g., additional data were available at a cost from the office of national statistics, the welsh cancer intelligence service, and the australian institute of health and welfare). It is, therefore, possible that statistics beyond those reported in table 3 are available . Some attempts to communicate with key experts and cancer registries failed, thus limiting our understanding of the true availability of certain data (table 2). For example, our attempts to contact the northern and yorkshire cancer registry and information service (nycris) and the cote d'or hematological malignancies registry (the lead registries for hematologic malignancies in england and france, resp .) Yielded no responses . We were also unable to establish contact with cancer registries in italy, japan, and south korea . We limited the data we gathered to diagnoses post-2000 to allow for representation of the availability of recent descriptive statistics . Some data were only available, however, for diagnoses that occurred in the late 1990s (e.g., survival data in ireland and france and pediatric all incidence in scotland). This paper was also limited to select countries, which precludes a full evaluation of the worldwide availability of data and an analysis of geographic differences . A comparison of statistics across countries is also limited by differences in the structure of cancer registries, age standardization methods, methods for estimating the statistics (e.g., period estimates for survival), and the year(s) of diagnosis . Furthermore, the comparison of hematologic malignancies across time and place was significantly limited by regional differences in disease classification systems . This paper was also limited by reports of cancer data in non - english languages . Translations of non - english language material were restricted to languages in which the research team had proficiency and were not carried out in a systematic manner . Thus, it is possible that additional data is readily accessible in other languages . Updates are published continuously by cancer registries, and investigators are frequently publishing manuscripts with additional, relevant epidemiologic data . We attempted to capture the most recent data available as of approximately mid - july 2011 . At the time of the publication of this study, however, we were aware of numerous updates, namely the release of updated seer data . In addition, the nycris was planning to publish a report on hematologic malignancies for all english cancer networks in 2011 . This paper used a novel approach to gathering descriptive epidemiologic statistics on cancer by combining information from the biomedical literature, internet - based query systems and reports, and direct contact with cancer registry representatives and key experts to provide the most up - to - date, comprehensive picture of a particular malignancy type . This paper highlights the importance of international coordination between cancer registries (e.g., haemacare) to promote standardization in data collection and reporting . We also recommend continued coordination and standardization between regional registries in countries such as brazil, spain, and india to promote the development of nationwide estimates . This novel approach provided a more comprehensive database of the current patterns of b - cell malignancies compared with a simple literature search . In addition, gaps were identified that highlight the need for more detailed reporting to cancer registries and the importance of developing assumptions to estimate the burden of specific b - cell malignancies in the interim . Because research indicates that cellular lineage affects prognosis, treatment, and etiology, cancer registries should continue to work to improve reporting of detailed diagnostic information on malignancies.
A 37-year - old male underwent endothelial keratoplasty in the right eye for primary graft failure following large diameter (11.5 mm) therapeutic penetrating keratoplasty . The therapeutic penetrating keratoplasty was done for advanced fungal keratitis 6 months prior to dsaek . Following therapeutic keratoplasty the graft was edematous with 10 intact sutures covered by conjunctiva, the anterior chamber was deep with few peripheral anterior synechiae, pupil showed posterior synechiae, and lens showed early nuclear sclerosis . Considering the patient's age and the absence of significant cataract, simultaneous cataract surgery was deferred . The posterior lamellar graft was inserted by the push - in technique using the sheet iol glide (bd medical - ophthalmic systems, franklin lakes, nj usa) protecting the endothelium with sodium hyaluronate 1% . The graft clarity recovered in 1 month after the surgery . At 3 months postoperative visit, pre - operative evaluation by confocal microscopy (nidek, confoscan 4) revealed an endothelial count of 1937 cells / mm . Biometry was performed using the keratometry readings obtained from orbscan ii (bausch and lomb's, florida, usa) and iol power was calculated for a postoperative targeted refraction of 0.5 diopters of myopia . Irrigation was kept off while inserting the phaco handpiece into the anterior chamber to avoid dislodging the graft . During surgery, a dispersive ophthalmic viscosurgical device (hydroxypropyl methylcellulose 2%, viscomet, sun pharmaceuticals industries ltd ., on the first postoperative day, vision was 20/160, improving to 20/80, and the graft was compact [figs . 1 and 2]. He was treated with ofloxacin 0.3% eyedrops 6 times / day for a week and prednisolone acetate 1% eyedrops 8 times / day for the initial 1 week, followed by tapering of one drop a week and finally maintained on 3 times / day . One month after the cataract surgery, his vision was 20/50 . At the last visit, 3 months later, the graft was compact and the best corrected visual acuity was 20/30 with a 0.75 d sph/1.50 d cyl at 160. table 1 shows the visual acuity, donor thickness [assessed by anterior segment optical coherence tomography (oct), visante: carl zeiss, germany] and visual acuity pre- and post - cataract surgery . Slit - lamp photograph of the right eye after dsaek (day 1 post op) post - op day 1 anterior segment oct image showing well - attached, compact graft pre and post - cataract surgery parameters endothelial cell density changes cataract surgery following penetrating keratoplasty is a safe and effective procedure, with a low but definite risk of corneal graft failure . However, the safety and efficacy of intraocular intervention in eyes that have undergone dsaek is not well established . Post - dsaek surgical interventions may be fraught with the risk of endothelial cell damage or donor graft detachment or subsequent endothelial rejection . Therefore, patients with visually significant cataract often undergo cataract surgery at the time of endothelial keratoplasty . It may be deferred in eyes where the anterior chamber cannot be properly visualized . In eyes with crystalline lens or early nuclear sclerosis that have undergone only endothelial keratoplasty, cataract formation may be hastened due to surgical manipulation, air tamponade at the time of surgery and use of postoperative steroid medications . It is likely that all the mentioned factors might have led to accelerated cataract progression in this case . The specific surgery - related issues to be considered in these eyes are: the site of internal incision in relation to the edge of the lamellar graft, turbulence during insertion of the phaco handpiece with irrigation, which can potentially lead to graft dislodgement, reduced working space in the anterior chamber, and endothelial cell loss during surgery . An endothelial cell loss of 14.7% was noted at 3 months after dsaek and an additional loss of 19.3% was noted from the time of cataract surgery to 3 months later . The overall cell loss at 6 months post - dsaek was 31.2%, which is comparable to the endothelial cell density changes reported after dsaek in various series . This case shows that subsequent intraocular surgery with appropriate precautions can be performed safely in post - dsaek eyes . However, the long - term effect on the endothelium and graft survival needs to be better understood.
Sd, a 45-year - old caucasian male, was referred for evaluation for liver transplantation because of end - stage liver disease caused by hepatitis b. he had been well until six months prior to his initial visit, when he started losing weight and developed severe fatigue . During the six - month period, he had lost 25 kilograms . His past medical history was significant for non - hodgkin's lymphoma that was diagnosed eleven years ago . He was treated with 8 cycles of chop chemotherapy and had been in remission since then . On examination, the patient appeared mildly icteric . The abdomen was soft with some tenderness in the right upper quadrant and ascites . Complete blood count showed a leukocyte count of 2500 cells / mm, hemoglobin of 9.8 gm / dl, and platelet count of 29 000 cells / mm . His liver function tests were abnormal with a total bilirubin of 5 mg / dl (normal, 0.21.2 mg / dl), alt: 361 u / l (normal, 1763 u / l), ast: 750 iu / l (normal, 1540 u / l), total protein: 15.4 g / dl (normal, 6.17.9 g / dl), albumin: 2.9 g / dl (normal, 3.94.8 g / dl), and an inr of 1.9 . Serological tests for viral hepatitis were as follows: hepatitis b surface antigen positive (hbsag), hepatitis b surface antibody negative (antihbs), hbe antigen negative, hbe antibody positive, igm antibody to hepatitis b core antigen positive, igg antibody to hepatitis b core antigen positive, and hbv dna 4 150 000 iu / ml (by pcr). Ct scan and ultrasound examination of the abdomen showed a small liver with moderate ascites, splenomegaly, patent portal, and hepatic veins . The striking elevation in his globulin fraction (12.5 g / dl) was further worked up . Serum protein electrophoresis showed the presence of a large monoclonal protein spike (m protein) in the gamma region which on immunofixation electrophoresis (ife) was identified to be iga - kappa type (see figure 1). The iga levels were increased at 6730 mg / dl (reference range 70400 mg / dl) and the igg and igm levels were decreased at 466 mg / dl (reference range 7001600 mg / dl) and 25.6 mg / dl (reference range 40320 mg / dl), respectively . A bone marrow biopsy on two separate occasions showed normocellular bone marrow with less than 2 percent population of plasma cells . Full body positron emission tomography (pet) scan and ct scan were performed, and no focus suspicious for lymphoma or myeloma or infection was identified . Whole body mri was unremarkable . On retrospective review, the patient had an elevated total protein (12 g / dl) approximately six months prior to initial visit . Within the next few weeks, the patient continued to deteriorate, and he developed worsening ascites with hyponatremia . It was clear that the patient had progressive liver disease from chronic hepatitis b with a relatively high meld score of 23, and he would benefit from a liver transplantation . Our dilemma before proceeding with liver transplantation was whether he had an underlying hematological malignancy that was causing the monoclonal gammopathy . We were also concerned about potential vascular complications after liver transplant given his increased viscosity . However, due to his deterioration and absence of any clear - cut evidence of malignancy, it was decided to place the patient on the liver transplant wait list . Anti - hbv therapy was initiated with a combination of entecavir (0.5 mg / day) and tenofovir (300 mg / day) to decrease his hbv dna to the lowest possible level before transplantation . However, he was on this antiviral therapy for less than a month as he received a deceased donor liver transplant ten days after listing . In the period between listing and liver transplantation, his bilirubin continued to trend up from 3.3 mg / dl to 4.6 mg / dl, and his total protein level fluctuated between 12.5 g / dl and 13.5 g / dl ., he received two doses of rabbit antithymocyte globulin at 1.5 mg / kg during the time of transplant and on postoperative day 2 . Subsequently, the mycophenolate mofetil was withdrawn, and he is currently on tacrolimus monotherapy with trough levels between 35 mg / ml . He continues to receive hepatitis b immunoglobulin infusion every month along with entecavir (0.5 mg / day), and his hepatitis b antibody titre has been kept above 300 miu / ml . He is currently 9 months posttransplant, his total protein levels range 4 - 5 g / dl, and he has had no detectable m protein spike . The temporal relation of the liver transplant with disappearance of hepatitis b surface antigen and normalization of the monoclonal spike implicates hepatitis b as the possible cause of the m protein spike . There is very little data and uncertainty regarding the incidence and natural history of m proteins in a person with hepatitis b. it has been suggested that the immunological response elicited against hepatitis b virus in the host rather than the direct cytopathic effect of the virus may be the basis for the pathogenesis of hepatic and nonhepatic manifestations . Hepatitis b virus seems particularly well suited to initiate a chronic immune disease because of its tendency to persist in spite of a good immune response . This may cause clonal expansion of the immunoglobulin secreting cells and may explain the above phenomenon . Alternately, the m protein spike could represent increased serum immunoconglutinin titres . It has been previously suggested that elevated serum immunoconglutinin titers in patients with acute and chronic hepatitis b virus infection may represent a physiological autoimmune response to hbsag / anti - hbs immune complexes . Although our patient did not have the anti - hbs antibody, it has been suggested that in the light of massive antigenemia present in chronic hbsag carriers, it is possible that anti - hbs exists in the form of hbsag / anti - hbs immune complexes . This would make it difficult to detect anti - hbs in serum samples by conventional serological methods . The primary issue was whether he was eligible for liver transplantation in the presence of an increasing m protein . The fact that he had lymphoma 10 years ago complicated this evaluation . Additionally, an association between hepatitis b infection and non - hodgkin's lymphoma has been previously described . However, he had had regular follow - ups every six months after remission of lymphoma, and also his malignancy workup prior to transplant was negative . Moreover, the m protein spike was noticed only 6 months prior to transplant and this coincided with the elevation of his liver function tests . Surprisingly, his igm antibody to hepatitis b core antigen was positive, as was his total core antibody . He did have active hepatitis and this can be deduced by the fact that he had an alt level that was 10 times the upper limits of normal; his explanted liver showed significant inflammatory activity . It was felt that most likely he had a flare of his chronic hbv, which might have triggered the elevation in m protein . Because of his elevated m protein and an urgent need for liver transplantation, we decided to initiate dual therapy with a nucleoside (entecavir) and nucleotide (tenofovir) analogue to decrease his hbv dna . However, he was on therapy less than a month before a suitable organ became available . We decided to accept the organ because of high meld score rather than to wait for hbv dna disappearance . Since we were planning to continue therapy posttransplant along with hbig (target anti - hbs titers> 500 iu / ml first three months and levels of> 300 iu / ml after three months), we believed that the risk of recurrence of hbv was very small . Unfortunately in the time period before the transplant and after initiation of antiviral therapy, we failed to follow his iga levels and hbv dna levels . However, his protein levels remained unchanged suggesting that there may have been very little change in his gammopathy, and this is not surprising given the fact that nucleos(t)ide therapy seldom leads to hbsag clearance in that time frame . After liver transplantation and with clearance of the hepatitis b surface antigen, the m protein levels became normal . One and the most likely reason is the removal of antigen source with native hepatectomy . His hepatitis b surface antigen, m protein, and total protein levels remain within normal limits 1 year after transplantation . The second possibility is the impact of thymocyte, given at the time of transplant, on plasma cells . Antithymocyte globulin has been used in treatment of plasma cell dyscrasias due to its activity against several plasma cell antigens . This is less likely in our patient because thymocyte globulin was used only as induction therapy at the time of transplantation . The elimination half time of thymoglobulin is 2 - 3 days and is unlikely that its effect on plasma cell is sustained for a year . In conclusion, this case demonstrates that monoclonal gammopathy can be associated with chronic hepatitis b infection and can be eliminated after successful transplantation.
It originated in germany in the 1920s and was defined by rules of a specific technique evolved in the usa in the years 19301950, employing elements of african and native american folklore . Modern dance employs all the techniques of ballet; however, a modern dancer is not obliged to uncritically follow the classical rules of execution of these techniques . Cohan writes that the most important elements of a modern dancer s work are: ground work, work with the center of gravity, and motion in space . He mentions that the most important thing in a well performed movement is self - awareness, and that modern dance consists of centering, gravitation, balance, posture, gestures, rhythm, motion in space, and breathing . Although ballet is a form of art, it has much in common with professional sports . Modern style choreographers often propose very dangerous dancing movements from the point of view of biomechanics of the locomotor system . Movement designers are often the result of absence of basic knowledge about the capabilities and limitations of the human locomotor apparatus . Modern dancers in many shows perform falls, using their upper limbs as shock absorbers, they roll around the stage, women lift men, and men not infrequently lift persons heavier than themselves, they wear heavy costumes, they walk on stilts, hang from ropes, etc . In addition, the performances often take place outdoors or in halls that are not prepared for such events . Research shows that some of them, particularly during the landing phase (during the eccentric phase of muscle work related to shock absorption), generate high values of the vertical component of ground reaction force (grf), which may reach 7.4 bw [13]. Peak forces of impact phases occur after a few dozen milliseconds . According to luke, solomon, liederbach, and nicholas [48], the largest percentage of injuries affecting professional dancers are chronic injuries, such as soft - tissue inflammation, injuries resulting from overload, and muscle strains and tears . Nevertheless, ankle joint, foot, spine, hip joint, and knee joint are the most frequently listed regions of the body injured by dancers . Research by gorwa shows that modern dancers are primarily exposed to injuries of the spine, achilles tendon, knee joint, and hip joint . Studies performed by gorwa also show that up to 93% of modern dancers (female and male) suffer from chronic injuries directly related to their profession . Around 30% of persons who reported these conditions associated them with specific movements . Female modern dancers complained of spine conditions such as neck pain and lumbar or sacral spine region pain; they associated them with sudden twists and the very expressive movements so common in modern dancing style . Male modern dancers listed pain and injuries of the spine, feet, and knees as chronic conditions and indicated these problems were caused by lifting dancing partners, as well as by bizarre choreography . Since correct technique is a factor that significantly decreases the risk of injury, and the degree of motor habit control (e.g., the proper execution of a given sports technique) determines the values of forces recorded during the landing phase, we observed kinematic values and recorded the technique of execution of a modern dance movement referred to as the stag jump . The purpose of this study was to conduct a thorough analysis of kinematic structure of the landing phase in a selected expressive dance movement called the stag jump performed as a case study of 1 male and 1 female professional modern dancer . Researchers who compared the technique and morphological differences between modern and ballet dancers were bronner and ojofetimi, solomon and micheli, and solomon et al . . The methodology and measurements presented in this work are part of a project whose purpose will be to devise guidelines for new training methods to prevent injuries in dancers . The conducted kinematic analysis consisted in establishing such characteristic parameters as trajectories of joint centers and movement sequences of lower limbs, pelvis movement, angle changes in hip, knee, and ankle joints in the sagittal plane, as well as changes of foot position in relation to the ground . Synchronization of kinematic measurements with the measurement of vertical components of ground reaction forces in the landing phase during this stage of the study allowed us to identify impact loads on the locomotor apparatus of the studied dancers and to forecast methods for minimizing such loads . This work is a case study of 2 professional modern dancers (a female and a male) who work full time in dance theatre, 6 days per week on average, and do not perform any other job . It should be stressed that although artists in ballet dance groups in poland have identical education (national ballet schools: primary and secondary) around 7% of members of modern dance groups begin their dance education after age 18 . The tests used for this study were carried out in the biomechanical laboratory of the chair of biomechanics at the university school of physical education in poznan, poland . Kinematic values were computed with the use of apas motion analysis system and grfs were measured with the use of kistler force plate sampling at 1000 hz . The motion of test participants was recorded by 4 basler digital cameras with recording frequency of 200 hz . The images recorded by the cameras were transferred to a computer, where, with the use of apas software, the films were processed and the locations of markers positioned on dancers bodies were determined . At the same time, grfs acting during the landing phase were recorded . Due to the extensiveness of performed dancing movements, the cameras were arranged to allow us to precisely determine the kinematics of the pelvis and the right lower limb during the landing phase . The number and location of markers (figure 1) made it possible to determine the positions of right lower limb joint centers and, in consequence, the relative angular motion of particular lower limb segments and the pelvis . This work is a case study of 2 professional modern dancers (a female and a male) who work full time in dance theatre, 6 days per week on average, and do not perform any other job . It should be stressed that although artists in ballet dance groups in poland have identical education (national ballet schools: primary and secondary) around 7% of members of modern dance groups begin their dance education after age 18 . The tests used for this study were carried out in the biomechanical laboratory of the chair of biomechanics at the university school of physical education in poznan, poland . Kinematic values were computed with the use of apas motion analysis system and grfs were measured with the use of kistler force plate sampling at 1000 hz . The motion of test participants was recorded by 4 basler digital cameras with recording frequency of 200 hz . The images recorded by the cameras were transferred to a computer, where, with the use of apas software, the films were processed and the locations of markers positioned on dancers bodies were determined . At the same time, grfs acting during the landing phase were recorded . Due to the extensiveness of performed dancing movements, the cameras were arranged to allow us to precisely determine the kinematics of the pelvis and the right lower limb during the landing phase . The number and location of markers (figure 1) made it possible to determine the positions of right lower limb joint centers and, in consequence, the relative angular motion of particular lower limb segments and the pelvis . The measurements allowed the authors to determine values of lower limb joint angles and pelvis position within the coordinate system (figure 2). The following angle vs. time graphs flexion / extension, abduction / adduction, and rotation; for knee joint flexion / extension and rotation; for talocrural joint dorsal / plantar flexion and pronation / supination . The usage of force plates allowed us to establish ground reaction forces (grf) acting during the landing phase that followed the performed movement . Based on the results of the measurements, it was possible to assess movements performed by the dancers by analyzing the line graphs of grf and line graphs of angles in lower limb joints . This paper concentrates on the analysis of the stag jump movement performed by 2 modern dancers a woman and a man . The following diagrams (figures 310) present the results of measurements performed on modern dancers . The pelvis rotation in relation to the long axis of the body was 30 for the female dancer and 25 for the male dancer . Pelvis tilt in the sagittal plane is significant during the entire landing phase and fluctuates between 32 and 50 for the female dancer and 2131 for the male dancer . Both pelvis rotation in relation to the vertical axis and pelvis tilt in the sagittal plane result from specific positioning of the non - supporting limb (figure 6). The line graph of joint angles in the sagittal plane (figures 7 and 8) shows that they are similar in shape but differ in the range of motion (rom). The rom in the hip joint is 50 for the female and 36 for the male . In the knee joint (figure 8), rom of ankle joint (figure 9) in the sagittal plane was 60 for the female dancer and 59 for the male dancer . This study allowed us to describe the way a dancer positions (coordinates) the body during landing that follows a dance movement and the forces that act on the lower limb . The values of the vertical component of ground reaction reached the levels that equaled almost 4 times the body weight of man and a little over 3.2 times the weight of woman . Considering that the values of reaction in the lower limb joints exceed the values of ground reaction, one can observe the loads that a dancer s musculoskeletal system is exposed to . Such high loads are a frequent cause of injuries to dancers, as well as musculoskeletal problems that dancers already suffer from during their careers . Ground reactions measured during the landings of classical dancers in other studies were even higher than in modern dancers (2.65 bw for females and 3.7 bw for males). It is directly related to the requirements imposed on the technique of dance movements, which are much more rigorous in classical dance . Interestingly, the landing phase durations in this movement are very short . The impulsive character of the peak vertical component of ground reaction acting on feet during the landing phase that follows a ballet jump poses the greatest risk of joint injury, beginning with the lowest joints metatarsal and ankle joints . The short time allowed for the performance of a movement and the need to immediately transition to the following one leave dancers extremely little time to coordinate the entire body and prepare for receiving the requirement to perform each dance movement strictly according to the rules considerably limits the ability to properly absorb the impact during landing . This is why it is so important to define the best way (from the biomechanical point of view and taking into account the artistic design) to perform individual dance movements by dancers . It is obvious that the maximum value of the vertical component of grf depends on the way dancers will use their feet as a form of biological shock absorbers . The following observations can be formulated on the basis of the analysis of joint angles and grf peaks . At the moment the foot contacts the ground, it remains in plantar flexion in a manner that only the toes are in contact with the ground . When the vertical component of ground reaction reaches its maximum, the foot is resting flat on the ground (parallel to the ground). Therefore, the center of mass is transferred from toes and metatarsus to the vicinity of the ankle joint, which decreases the moment of vertical ground reaction forces (and thus the forces generated by muscles working around the ankle joint). In addition, the flat positioning of the foot lowers the center of the ankle joint, shortening the moment arms of the components acting in the horizontal plane . When the vertical component of grf reaches its peak, thighs and shins assume almost vertical positions . This leads to the conclusion that when the greatest loads act on the limb, these will be mostly compressive loads, for which the long bones of the lower limb are well prepared (they possess the best strength in the longitudinal direction). The analysis of foot angle in relation to the ground shows that after landing on his toes, the male dancer bends his foot dorsally and for a short period of time (about 0.2 s) puts weight on the entire foot and then stands on the toes . The female dancer lands in a different manner after touching the ground with the toes, she puts weight on the entire foot and keeps the foot in this position throughout most of the support phase . One of the most significant factors that lead to injuries is high dynamic loads (forces) of an impact nature that occur particularly during the landing phases of numerous expressive jumps . Peak forces are generated over a very short time period, which makes coordinating such movement structures very difficult . It is important to realize that these forces are transmitted via the relatively small surface of the dancer s feet that are only weakly protected by characteristic footwear the forefoot region and toes . Properly mastering movement technique is tremendously important in minimizing injuries . The knowledge of dynamic loads acting on the dancer s body and kinematics of movement may represent a significant contribution in the description of the proper technique of dance movements . The following values that best describe the landing phase of the stag jump were established: loads transmitted to the dancer s foot; line graphs of joint angles in the lower limb; changes in position of the foot in relation to the ground; values of joint angles, as well as the angle between the foot and the ground when the highest value of the vertical component of ground reaction occurs . This allowed us to analyze the influence of movement technique on the values of external loads (grf). Analysis of the conducted research reveals significant differences in the technique of the same dance movement ., as well as broner and ojofeitimi, suggests that these differences in technique probably do are not due to the dancer s sex, professional experience, or educational path of the dancer . The female dancer went through all levels of ballet education in poland (primary and secondary national ballet school), and the male dancer began his education as an adult at the danceweb workshop in vienna . However, such a conclusion needs to be confirmed by further research with a larger sample . Observed correctly that certain dance movements can be mastered only until puberty, when the body can still be molded . During the next phases of research, the results of these measurements will be used to determine loads transmitted by musculoskeletal system when performing dancing movements . For this purpose, a mathematical model of lower limb movement will be prepared, allowing us to determine the forces generated by muscles and the forces transmitted by joint surfaces . The values thus obtained will complement the results of the research . On the basis of these results, works are being conducted in cooperation with ballet teams, choreographers, and dance teachers to devise training methods using the methodology and the potential of a biomechanical laboratory . These training methods would be aimed preventing injury and physical overload, as well as increasing dancers knowledge of biomechanics and kinesiology of the locomotor apparatus in dancing.
Rabies is a major zoonotic viral disease which is regarded as a health problem around the world (1). In asia, eight countries have been reported as rabies free, including japan, malaysia, hong kong, singapore, taiwan, qatar, bahrain, and the united arab emirates (2). This disease is caused by a group of neurotropic viruses of the family rhabdoviridae, order mononegavirales, genus lyssavirus (1). According to cross protection tests and molecular biological analysis, the genus lyssavirus is classified into seven distinct genetic lineages: the classical rabies virus (rabv), lagos bat virus (lbv), mokola virus (mokv), duvenhage virus (duuv), european bat lyssavirus type 1 (eblv-1), european bat lyssavirus type 2 (eblv-2), and the australian bat lyssavirus (ablv) (3). Recently discovered lyssaviruses, aravan (arav), khujand (khuv), irkut (irkv) and west caucasian bat virus (wcbv) have also been introduced (4). The rabies virus includes five structural proteins, a nucleoprotein (n), a phosphoprotein (p), a rna polymerase (l), matrix protein (m) and glycoprotein (g). The n, p and l proteins together constitute the nucleocapsid (nc) (5). Prevention and control of rabies in human and animals (especially in the wild life), is based on the use of rapid and specific diagnostic techniques and is established according to clinical, epizootiological and laboratory assessments (6). Rabies should still be considered as an important zoonotic health problem in iran . However, currently the disease is being controlled if we compare the situation with 40 years ago (2, 7). Although official reports claimed that the principal maintenance host is domestic dogs, an investigation by the pasteur institute of iran (collaborating with the world health organization for reference and research on rabies) revealed that wolves are responsible for rabies transmission to humans (7, 8). The world health organization (who) and the international office of epizootics (oie) recommend the fluorescent antibody technique (fat), which is done on specimens from central nervous system, skin biopsy and salivary glands as the preferable laboratory method and gold standard for diagnosis of rabies (6, 9, 10) the nucleoprotein (np) with an approximate molecular weight of 50 kda, consists of 450 amino acids, and it is the most preserved antigenic protein for different rabies strains (1, 5). It is the main component of the rabies virion that contains group - specific antigenic determinants, which are important in diagnosis of rabies infection (1, 11). This study was conducted to extract np from baby hamster kidney cell clone (bsr) infected with the pasteur vaccine strain (pv) and to purify this np in order for it to be used in the future for producing a fluorescent anti - np conjugate with high sensitivity and specificity, which can be used in fat . The virus strain used in this study was the pv strain that is the standard rabies virus vaccine strain; the virus was propagated in bsr cells in the presence of dulbecco modified eagle s medium (dmem) (sigma - aldrich, usa) containing 10% fetal bovine serum (fbs) (sigma - aldrich, usa). Viral titration was carried out on bsr cells according to the who instructions (12, 13). Incubation was performed in an open system for 48 hours at 37c with 5% carbon dioxide (co2). The final titer of the virus was calculated according to the following formula: titer = [average number of fluorescent foci in the last dilution where fluorescence is still visible] [dilution 3] [20, 000] (12,16). Two milliliters of 10/ml bsr cells were added to each bottle and incubated at 37c until a monolayer was formed . After three days, the medium was poured out and the monolayer of the cells was washed with 20 ml phosphate buffered saline (pbs) (sigma - aldrich, usa) solution at ph 7.4 . Then, 9 ml of pv suspension was inoculated on to each monolayer and incubated for 1 hour at 37c with 5% co2 and the content of the bottles were gently stirred every 10 - 15 minutes to increase virus adsorption . After virus adsorption, the cell monolayers were washed with 20 ml pbs by adding dulbecco's modified eagle's medium(dmem) containing 10% fbs; the volume was adjusted to 50 ml . The battles were incubated for 24 hours at 37c with 5% co2 and then for 48 hours at 34c with 5% co2 and subsequently, the supernatant was discarded and the monolayer was washed with 20 ml of pbs . The cells were collected using a cell scraper with a blade length of 20 mm (midsci, tpp, usa) and were suspended in ice - cold 0.5 m sodium chloride / tris - hcl (nt) buffer at ph 7 and centrifuged twice at a speed of 900 g and 4c for 10 minutes (16, 17). Cell lysis was carried out by adding 5 ml of ice - cold, sterile, deionized water containing 25 l of aprotinin per milliliter (18). This mixture was incubated for 1 hour at 4c and clarified by centrifugation at a speed of 1,000 g and 4c for 20 minutes . The supernatants from each round were combined and clarified by centrifugation at a speed of 12,000 g and 4c for 10 minutes and finally, the supernatant was separated . According to the who instructions, rabies virus np was purified by ultracentrifugation (beckman type 90ti, usa) in cscl gradient (18). For each 3 ml of supernatant of np, 2 g of cscl was added to 5 ml polycarbonate tubes (beckman type 90ti, usa). The final volume was adjusted to 5 ml by adding nt buffer at ph 7.6 and centrifuged at speed of 50,000 rpm for 10 hours at 4c . The band (approximately 0.5 ml) was collected through the top of the tube using a 23-gauge needle (reno, nevada, usa). The fraction was dialyzed against ph 7.6 nt buffer for 24 hours at 4c . Then, the protein concentration was measured by a spectrophotometer at a wave length of 280 nm . In order to confirm np presence, 5 l of the sample of purified rnp was loaded on a 12.5% sds - page gel, with 5% stacking and stained with coomassie blue (merck, germany) (10, 15). We also performed a modified western blot analysis using only the bio - rad conjugate (bio - rad, france) to stain a fixed blot of the extracted np, as this conjugate is a specific antibody against the rabies np . The virus strain used in this study was the pv strain that is the standard rabies virus vaccine strain; the virus was propagated in bsr cells in the presence of dulbecco modified eagle s medium (dmem) (sigma - aldrich, usa) containing 10% fetal bovine serum (fbs) (sigma - aldrich, usa). Viral titration was carried out on bsr cells according to the who instructions (12, 13). Incubation was performed in an open system for 48 hours at 37c with 5% carbon dioxide (co2). The final titer of the virus was calculated according to the following formula: titer = [average number of fluorescent foci in the last dilution where fluorescence is still visible] [dilution 3] [20, 000] (12,16). Two milliliters of 10/ml bsr cells were added to each bottle and incubated at 37c until a monolayer was formed . After three days, the medium was poured out and the monolayer of the cells was washed with 20 ml phosphate buffered saline (pbs) (sigma - aldrich, usa) solution at ph 7.4 . Then, 9 ml of pv suspension was inoculated on to each monolayer and incubated for 1 hour at 37c with 5% co2 and the content of the bottles were gently stirred every 10 - 15 minutes to increase virus adsorption . After virus adsorption, the cell monolayers were washed with 20 ml pbs by adding dulbecco's modified eagle's medium(dmem) containing 10% fbs; the volume was adjusted to 50 ml . The battles were incubated for 24 hours at 37c with 5% co2 and then for 48 hours at 34c with 5% co2 and subsequently, the supernatant was discarded and the monolayer was washed with 20 ml of pbs . The cells were collected using a cell scraper with a blade length of 20 mm (midsci, tpp, usa) and were suspended in ice - cold 0.5 m sodium chloride / tris - hcl (nt) buffer at ph 7 and centrifuged twice at a speed of 900 g and 4c for 10 minutes (16, 17). Cell lysis was carried out by adding 5 ml of ice - cold, sterile, deionized water containing 25 l of aprotinin per milliliter (18). This mixture was incubated for 1 hour at 4c and clarified by centrifugation at a speed of 1,000 g and 4c for 20 minutes . The supernatants from each round were combined and clarified by centrifugation at a speed of 12,000 g and 4c for 10 minutes and finally, the supernatant was separated . According to the who instructions, rabies virus np was purified by ultracentrifugation (beckman type 90ti, usa) in cscl gradient (18). For each 3 ml of supernatant of np, 2 g of cscl was added to 5 ml polycarbonate tubes (beckman type 90ti, usa). The final volume was adjusted to 5 ml by adding nt buffer at ph 7.6 and centrifuged at speed of 50,000 rpm for 10 hours at 4c . The band (approximately 0.5 ml) was collected through the top of the tube using a 23-gauge needle (reno, nevada, usa). Then, the protein concentration was measured by a spectrophotometer at a wave length of 280 nm . In order to confirm np presence, 5 l of the sample of purified rnp was loaded on a 12.5% sds - page gel, with 5% stacking and stained with coomassie blue (merck, germany) (10, 15). We also performed a modified western blot analysis using only the bio - rad conjugate (bio - rad, france) to stain a fixed blot of the extracted np, as this conjugate is a specific antibody against the rabies np . Several serial passages were done to obtain the 10 focus - forming dose (ffd) titer of the virus . The volume of lysate was 15 ml and after purification; it became 2.5 ml, with a concentration of 3.25 mg / ml . The extracted rabies virus np from infected bsr cells was at the same molecular weight (50 kda) as rabies virus np by sds / page . The obtained clear band with an approximate molecular weight of 50 kda is depicted in figure 1 . There were also some non - specific bands on sds - page, which usually form after purification of intra cellular proteins such as viral proteins with no interference with the specific np band . Rabies virus causes the formation of specific aggregates of viral material, which are called negri bodies (5, 6, 16). These structures are found in the cytoplasm of infected neurons or in cell cultures and are considered as the most important factor for confirmation of rabies virus infection . Negri bodies are usually between 0.24 - 27.0 m in size and 2 - 10 m in diameter (5). Electron microscopy observations showed that negri bodies are made - up of a matrix of filamentous material comprised of viral np (19). Conserved antigenic sites on the np result in identification of all rabies virus strains by using anti - rabies antibody conjugates (3). At present, the method of choice for detection of these cytoplasmic structures is fat (6). Fluorescent antibody technique is one of the most rapid techniques for rabies antigen identification in the brain and salivary glands of human and animals suspected of infection by the rabies virus (12, 15). Using a good quality conjugated antiserum should be considered as one of the main requirements for precise diagnosis of rabies infection (17). The fat has approximately 100% specificity and sensitivity if a proper conjugate is used (6). In 1973 dean and abelseth, used inactivated rabies infected mice brain suspensions as a source of viral antigens for immunization of animals for obtaining concentrated specific antibodies (12). In 1974 atanasiu and his colleagues reported the first production of high - titer conjugates using rabies virus nc (13, 14). Since fat by using the anti - np conjugate is much more sensitive than using conjugates specific to the total virion, we attempted to obtain this protein in its purified form, in order to produce the conjugate in the future (6, 12). In this study the virus was obtained in a similar titer as previously described by a brazilian study (10). The extracted rabies virus np was confirmed by sds - page and western blotting as the viral strain used in this project was the standard rabies virus strain (pv). There was also some partial purification of np, which resulted in a few minor bands on sds - page after purification with no probability of interference with the specific conjugate (10, 12). Commercial conjugates are against the total virion or specific to the rabies np (such as the conjugate from bio - rad, france), or they are prepared from monoclonal antibodies (3). Rabies virus is amongst health problems in iran (2, 20). In 2006, more than 130,000 people received post - exposure prophylaxis thus, a sensitive surveillance system is necessary to detect rabies infections using the most accurate laboratory methods recommended by who (12, 15). At the pasteur institute of iran (who collaborating center for reference and research on rabies), a rabies conjugate is provided by bio - rad with a high price . In addition to cost, there are other problems such as delays in delivery, changes in quality and affinity of conjugate and sometimes lack of access . Here, we tried to overcome these problems and we successfully achieved the first step for producing the anti - np conjugate by extraction and purification of rabies virus np from cell culture.
Superparamagnetic iron oxide nanoparticles (nps), with core diameters of 315 nm, are used in a rapidly expanding number of applications in the biomedical field; the most common include cell labeling, hyperthermia, drug delivery, and as contrast agents for magnetic resonance imaging . For these applications, the iron oxide cores are coated with polymer, lipid, or other dispersants to enable dispersion of nps in aqueous solutions containing biomolecules . The most common method to stabilize iron oxide nps for biomedical applications has been to enwrap them in a weakly adsorbed shell of high molecular weight polymer (often dextran) or amphiphiles such as block copolymers or lipids . However, weakly adsorbed shells lead to low polymer densities on the particle surface that are further reduced with time under dilute conditions, resulting in low colloidal stability . Chemically grafted polymer shells are physically and chemically stable; they can also be made denser than physisorbed shells, and therefore they have received increasing attention . With recent improvements in the synthesis of nps there has been a push toward more well - defined, core spherical iron oxide nps can now be synthesized with monodisperse size (sd <5%), but they are as - synthesized capped with a strongly adsorbed, hydrophobic shell of oleate that is difficult to replace; these nps require stabilization with a shell of linear, end - grafted polymer dispersants of sufficient thickness and grafting density to ensure that the particles remain monodisperse during application . Defined core shell architectures enable the prediction of all colloidal properties and can be used to define biological interactions through the attachment of organic ligands . The main advantage of grafting dispersants to the core is that the hydrodynamic size of the nps, the stability of the shell, and the presentation of ligands can be precisely controlled, in contrast to for nps with shells consisting of physisorbed high molecular weight dispersants; this critically determines np performance in a biological fluid . Precise control over monodisperse size, purity, and colloidal interactions is crucial for nps in biomedical applications to control biodistribution and self - assembly of drug delivery structures . Achieving high dispersant grafting density on monodisperse iron oxide nanoparticles is challenging since the dispersant has to densely replace the (oleic acid) organic shell carried by the as - synthesized particles . In addition, a major problem for the synthesis and characterization of core shell nanoparticles is the efficient separation of excess dispersants . The peg - dispersant density on nanoparticles is difficult to determine since continuous and complete removal of excess dispersants from nanoparticle dispersions can lead to particle aggregation and precipitation; alternatively, not all free ligands are removed, which leads to overestimation of the dispersant density on the nanoparticles . The actual stability and correct core shell structure cannot be determined if excess dispersants are not removed . Efficient separation methods can also contribute to nanoparticle degradation and should be identified and avoided . An irreversibly grafted shell of> 2 chains / nm of peg(5 kda) on polydisperse and irregularly shaped single nps was shown to be required for colloidal stability under application conditions . Np curvature facilitates polymer grafting by grafting - to of presynthesized polymer coils and chain grafting densities up to 2.5 chains / nm have been claimed on iron oxide nps . Until recently, only 0.5 chains / nm of peg(5 kda) was reported grafted on spherical fe3o4 nps after ligand replacement by nitro - dihydrohyphenylalanine (nitro - dopa)-peg(5 kda) on monodisperse nanoparticles synthesized using oleylamine as capping agent . Lak and colleagues recently synthesized large, 25 nm in diameter, fe3o4 ferrimagnetic nanoparticles that were stabilized through ligand replacement with nitrodopamine (nda)-peg . Due to their size, these multidomain particles are optimized for applications where brownian relaxation mechanisms are desired, while particles with weaker magnetic interactions and nel relaxation as the dominant relaxation mechanism require iron oxide nps smaller than 15 nm . These large particles form small aggregates in cell culture and pbs, which might be due to the stronger long - range core quantitatively addressed the problem of replacing oleic acid by ligand exchange on superparamagnetic iron oxide nanoparticles . Full ligand replacement was not shown, although the superiority of dopa and nitro - dopa anchor groups to displace oleic acid from the surface was demonstrated . These and other previous studies did not address the effect of purification from excess ligands on the results . Shell nanoparticles under relevant conditions such as biological buffers and elevated temperature was also not investigated . We present protocols to replace oleic acid for a dense spherical brush peg - shell on monodisperse, monocrystalline, and spherical iron oxide nanoparticles in the superparamagnetic size range of 310 nm diameter at physiological temperatures . The colloidal stability in both phosphate buffered saline (pbs) and biofluids of these densely pegylated core shell particles significantly exceeds what has previously been described for particles synthesized by ligand exchange . Importantly, we also present an evaluation of different purification methods to separate pegylated particles from free peg, and we suggest a protocol for iterated solvent precipitation and magnetic extraction as most suitable to completely purify these nanoparticles at high yield . Only through this advancement is the high grafting density and colloidal stability conclusively demonstrated, with important implications for correct comparison of previous and future results on np ligand density . All chemicals (see supporting information for details) were purchased from sigma - aldrich, all solvents were from roth, and peg was from jenkem and used as received without further purification . All nps and nitrocatechol ligands originated from the same respective batch to ensure maximum reproducibility . Tem studies were performed on a fei tecnai g2 20 transmission electron microscope operating at 120 or 200 kv for high resolution imaging . Samples were prepared by dropping toluene dispersions (as - synthesized nps) or aqueous dispersions (pegylated nps) onto a 300-mesh carbon - coated copper grid and subsequently evaporating the solvent in air . Size distributions were evaluated using the pebbles software package with a local intensity - model fitting algorithm . Thermograms were recorded on a mettler - toledo tga / dsc 1 star system in the temperature range 25650 c with a ramp of 10 k / min in synthetic air stream of 80 ml / s in order to ensure complete combustion of ligands as nda was found to polymerize by pyrolization under n2 . 70 l aluminum oxide crucibles were filled with 0.51.5 mg of sample, and the total organic content (toc) was evaluated as the mass loss fraction at 500 c by horizontal setting . H and c solution spectra were collected on a bruker dpx operating at 300 mhz in cdcl3 and d6-dmso as solvents using tms as an internal standard . Mid - ir powder spectra of the lyophilized samples were collected on a single reflection bruker tensor 37 ftir spectrometer with bruker platinum diamond single - reflection atr equipment at a resolution of 4 cm by averaging 32 scans . Samples were measured in type 0500 glass capillaries (hilgenberg, germany) with nominal diameter of 1 mm and wall thickness of 10 m . Measurements were carried out using a rigaku s - max 3000 saxs system equipped with a copper - target microfocus x - ray tube micromax-002 + (45 kv, 0.88 ma) with an energy of 8.05 kev, collimated through three pinholes (400, 200, and 700 m) to achieve a beam diameter at the sample position of 210 m (fwhm) and a triton 200 2d multi wire gas - filled x - ray detector (200 mm diameter of active area, spatial resolution 200 m). Data were acquired in the q - range from 0.01 to 0.26 with a measurement time of 28.800 s for each scattering pattern at vacuum conditions better than 10 mbar . Subsequent data treatment included background correction based on the measured transmission and radial integration with the saxsgui 2.8.03 software package . Superparamagnetic oleic acid (oa)-stabilized magnetite nanoparticles were synthesized by thermal decomposition of an iron precursor according to a slightly modified heat - up procedure described by hyeon et al . Briefly, for 9.6 nm nps a mixture of 50 ml of dioctyl ether (oct2o) and 7.04 ml of oa was heated to 100 c under n2 . 1 ml of iron pentacarbonyl (fe(co)5) was injected rapidly, and the reaction mixture was heated to 290 c with a temperature ramp of 3 c / min . After aging for 1 h the np dispersion was allowed to cool to room temperature and precipitated thrice with ethanol (etoh) from toluene in order to remove excess oa . The size was controlled by the fe(co)5:oa ratio; details can be found in the supporting information (table s1). 6-nitrodopamine hemisulfate (nda - hso4) was synthesized according to literature with slight modifications . Nda - peg(5 kda) was synthesized by (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (comu)-mediated peptide - coupling reactions (see supporting information for experimental details). Typically, 1.3 g of wet (from etoh washing) nps (containing 0.11 g cores) were dispersed in 30 ml of dmf; the desired amount of nda - peg(5 kda), usually a 10-fold excess (with respect to the grafting density of 1 nda - peg / nm expected from preliminary measurements of the maximum achievable grafting density by ligand replacement, e.g., 1.9 g nda - peg(5 kda) for 9.6 nm nps), was dissolved in dmf and mixed with the nps . Note that only a low excess is needed for the ligand exchange, but to allow for fast ligand exchange and easier determination of the efficiency of the purification methods, a large excess was used . The dispersion was sonicated for 26 h at slightly elevated temperature (35 c). Preliminary tests showed that 26 h was sufficient to replace oa, and longer sonication did not improve the exchange . Subsequently, the mixture was extracted thrice with n - hexane (30 ml each) in order to remove released oleic acid . Afterward, the solvent was evaporated; the pegylated nps were lyophilized for 24 h and stored as dry powder for further analysis and are referred to as nps were obtained as a light brown powder in 97% yield with respect to the amount of cores . All purification methods were applied after the extraction of the nps with n - hexane . All nps were characterized and analyzed before and after purification by tem, tga, ir, and dls . Additionally, nps purified by precipitation and magnetic decantation were analyzed by saxs . 0.3 g of the nps after ligand replacement (nps raw) was dissolved in 2 ml of h2o and sonicated for 15 s. sephadex was swollen overnight in milli - q water at 40 c and manually packed into a 25 2 cm column . The length of the column was sufficient to split the np sample in six to seven identifiable, differently colored fractions (figure s1). The eluates were collected and freeze - dried for further analysis . For all np sizes fraction ii could be identified as the main fraction . For two np sizes (9.6 and 7.1 nm) this main fraction was reapplied onto a fresh column, and the eluate was again characterized . The 1000 kda molecular weight cutoff (mwco) membranes were equilibrated for 10 min in 10% v / v etoh and 30 min in h2o . 0.3 g of the raw nps were dissolved in 5 ml of milli - q and dialyzed against 5 l of milli - q . To monitor the dialysis progress, equivalents of 0.5 ml were taken after 1, 2, 4, 6.5, and 24 h and freeze - dried . The 9.6 and 7.1 nm nps were further dialyzed for 48 h and the small nps (4.9 and 3.5 nm) for 70 h. the dispersions were lyophilized for further analysis by tem, tga, ir, and dls . Typically, 0.15 g of raw nps were dispersed in 2 ml of milli - q and loaded into a 50 kda amicon - ultra-15 membrane centrifugation filter unit and centrifuged at 4500 g for 15 min . The centrifuge effluent was collected, and the nps were redispersed in 1 ml of milli - q . This procedure was repeated up to 15 times; equivalents were taken after 5, 7, and 11 centrifugation steps to track the removal of excess nda - peg . All dispersions and the collected effluents were freeze - dried for further analysis by tem, tga, and dls . Typically 0.3 g of the raw nps (containing 0.019 g cores for 9.6 nm nps) were dissolved in 10 ml of etoh by sonication and slight heating with a hot gun to 60 c . The clear dispersion was poured into a small beaker and mixed with 10 ml of ice - cold petrolether whereupon it got slightly turbid . The beaker was placed on a 5 5 cm 1 t magnet in the fridge at 4 c, and the nps were soaked from the cloudy mixture to the bottom of the beaker within a few minutes, dependent on the np size . The supernatant was decanted by holding back the nps with the magnet, collected, combined, and freeze - dried for tga measurements . This procedure was repeated 79 times; after each step a small sample of nps was kept and freeze - dried for tga analysis to monitor the removal of excess ligands and impurities . The final products after the seventh step were freeze - dried and further analyzed . The nps were obtained as dark brown powder in 96% yield with respect to the cores . Freeze - dried samples of all purification methods were stored at room temperature without protection from light . After 6 months dry storage, suspensions of nps in milli - q water and pbs of the nps purified by dialysis (4.9 nm cores) and magnetic decantation (7.1 nm cores) were prepared (4 mg / ml) and characterized by tem and dls . The np dispersions were stored for 4 months at 4 c to prevent microbe growth and analyzed . Additionally a sample of 7.1 nm cores purified by magnetic decantation was dissolved in pbs and water at 3 mg / ml; 10% bovine calf serum was added to investigate if the nps aggregate upon nonspecific protein adsorption . Dls was measured over a temperature cycle from 207020 c in 5k steps with an equilibration time of 5 min at each temperature . Spherical, monodisperse, and single - crystalline superparamagnetic iron oxide nanoparticles were synthesized using a modified heat - up method introduced by hyeon and co - workers . The np size could be precisely tuned in the range of 310 nm without any further size selection process by varying the fe(co)5 to oleic acid molar ratio (see table s1 in the supporting information for details). The obtained nps were characterized by low- and high - resolution tem, tga, and atr - ftir . Figure 1 shows tem micrographs of monodisperse 3.5 0.3, 4.9 0.4, 7.1 0.4, and 9.6 0.4 nm magnetite cores, calculated with pebbles by evaluation of 900 nps from multiple hr and lr - tem micrographs of the respective batches . The hr - tem (inset in figure 1c) shows the single - crystallinity of the nps with lattice spacing in the (311) direction of 0.26 nm . The ring diffraction pattern (inset figure 1d) reveals the highly crystalline structure of the nps . The ratio of d - spacings in the obtained pattern show good agreement with the jcpds database numbers for maghemite or magnetite . Transmission electron micrographs of as - synthesized monodisperse oa - capped fe3o4 nps: (a) 3.5 nm . (c) 7.1 nm; inset: high resolution tem image showing that the particles are single - crystalline . (d) 9.6 nm; inset: electron diffraction pattern of 9.6 nm nps reveal the highly crystalline nature of the nps . The grafting of nda - peg to the nanoparticle cores was performed in large excess of peg in order to ensure fast and full ligand replacement and to provide an easily distinguished baseline for evaluation of different methods to remove unreacted dispersant . The separation of all excess free peg - dispersant after grafting is challenging, as indicated by previous work; we performed a thorough comparison and analysis of different methods that have been applied for core shell nanoparticle preparation: cross - linked dextran size - exclusion column separation, dialysis, membrane centrifugation and precipitation with magnetic decantation . The different methods were compared on their ability to remove excess peg to a stable value determined by tga and their effect on nanoparticle stability . The tga results with corresponding nda - peg(5 kda) grafting density are summarized for all purification methods and core sizes in table 1 . The total organic content (toc) was determined as the mass loss fraction up to 650 c and converted to nda - peg(5 kda) dispersant grafting density using the known molecular weight of nda - peg, the average iron oxide core area determined by tem, and a core density of 5.17 g / cm . As described in the materials and methods section, the nps were after column purification collected in 67 dark brown, red, orange, and yellow fractions . The same colors and amounts were observed for all np core sizes (see figure 2b). All samples passed the column without visible sticking to the dextran matrix, which demonstrates a high and homogeneous peg shell density on the particles . Fraction 2 could be identified as the main fraction for all sizes; it had the largest amount of nps with the smallest amount of nda - peg(5 kda). Figure 2 shows the results of the column purification in terms of grafting density based on measurement of organic content by tga . For two np sizes (9.6 and 7.1 nm) the main fraction was dispersed in mq and reapplied onto a fresh sephadex column . For the 3.5 and 4.9 nm nps the amounts contained in fraction 2 were too small for a second pass through the column . The reapplied samples passed the column as a single band with a very small fraction of particles that passed the column faster . The organic content of the reapplied fractions could be decreased from 66% to 49% (9.6 nm nps) and 91% to 73% (7.1 nm nps) by the second pass . For the 9.6 nm nps this translates into a grafting density of 1.0 nda - peg(5 kda)/nm, while for 7.1 nm nps a grafting density of 2 nda - peg(5 kda)/nm is calculated . (a) total organic content of the column purification, measured by tga and converted into nda - peg(5 kda)/nm using the known molecular weight of nda - peg(5 kda) and average core surface area . Toc includes large excesses of free peg and sephadex and was converted into dispersants / nm for comparison with the other methods but does therefore not reflect the actual grafting density of nda - peg . (b) fractions of nps after column purification dispersed in water (9.6 nm, upper) and freeze - dried (7.1 nm, lower). Dialysis has to be performed with membranes having a pore size sufficiently large to allow diffusion of the dispersant through the pores; the mwco of membranes is however typically given for equivalent protein or dextran sizes and cannot be used directly for comparison to highly solvated polymer molecular weights for which the radius of gyration is much bigger than for e.g. Protein of the same molecular weight . A rule of thumb of choosing a pore size> 5 that of the dialyzed molecule is often invoked . This was confirmed by attempting dialysis of dissolved peg(5 kda) (0.2 g / ml against 5 l of milli - q) in a membrane of 1214 kda mwco, i.e., nominally 23 times the molecular weight of the nda - peg, which was used in previous publications studying grafting - to and ligand replacement of pegylated nps . After 24 h only 50% of the free peg was removed, and only 60% had been removed after 5 days despite exchanging milli - q in the reservoir five times . Thus, choosing a too low mwco results in remaining free dispersant and subsequent overestimation of the dispersant grafting density by tga . We therefore changed to cellulose - based membranes with a mwco of 1000 kda, which nominally corresponds to a cutoff hydrodynamic size of 37 nm (assuming a scaling related to equivalent sizes of dextran). Nps with a hydrodynamic radius rh between 15 and 23 nm (for 7.1 nm cores) were still retained while free nda - peg(5 kda) with an rh of 4 nm was presumably removed from the dispersion . Figure 3a shows the removal of excess ligands by dialysis for all sizes of nps . Large - core nps (7.1 and 9.6 nm) dialyzed for 24 h yielded an average grafting density of 1 nda - peg(5 kda)/nm determined by tga . However, nps also started to stick to the dialysis membrane after 24 h. tem of large nps (7.1 and 9.6 nm) extracted from the dialyzed dispersion after 24 h showed aggregated morphology (figure 3b). The 24 h dialyzed samples could be redispersed in water but showed poor long - term stability . Visible aggregates were obtained after 1 week . Continued dialysis of the large cores led to an even lower grafting density (0.5 nda - peg(5 kda)/nm) and increased aggregation . (a) removal of excess ligands by dialysis with a 1000 kda mwco membrane . Total organic content was measured by tga and converted into nda - peg(5 kda)/nm using the known molecular weight of nda - peg(5 kda) and average core surface area determined by tem . After 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size, but core size - dependent aggregation and precipitation were observed . Dialysis for 70 h lowered the grafting density to 0.3 nda - peg(5 kda)/nm, resulting in colloidally unstable dispersions . (b) typical tem of nanoparticles with 9.6 nm core size that had visible aggregates (inset) grafted with nda - peg(5 kda) after 24 h dialysis against water using a 1000 kda mwco cellulose membrane . Nps with small core sizes (4.9 and 3.5 nm) had grafting densities of 0.8 and 1.7 nda - peg(5 kda)/nm, respectively, after 24 h dialysis and showed good colloidal stability . The small nps were dialyzed also for 70 h, which resulted in lowering of the average grafting density to 0.3 nda - peg(5 kda)/nm . During dialysis the np suspension underwent visible color change from light brown to almost black (figure s2 in the supporting information), indicating that as dialysis proceeds, surface - bound nda - peg is ripped off in addition to the initial removal of free nda - peg . In contrast to diffusion - driven dialysis, a mwco of 50 kda was sufficient to remove excess nda - peg . This can be ascribed to the convective flow and high forces induced by the centrifugation that deform the free polymer to flow through narrow pores . Figure 4 shows the results of the membrane centrifugation for the different np sizes . (a) removal of excess ligands by repeated membrane centrifugation with amicon 50 kda centrifugation filter units . After 11 repetitions all sizes show an average grafting density around 0.50.7 nda - peg(5 kda)/nm . (b) more centrifugation steps lead to a decrease in grafting density and colloidal instability . Left to right: 4.6 nm nps after 5, 4.6 nm nps after 15, and 7.1 nm nps after 11 centrifugation steps . The average grafting density at this point was measured to be 1 nda - peg(5 kda)/nm by tga, but the resuspended nps were colloidally unstable, as can be seen by the visible aggregates sticking to the vial in figure 4b . In order to study if the grafting density stays constant, the centrifugation steps were repeated up to 15 times . As shown in figure 4a, the grafting density did not stay constant but could be reduced to 0.4 nda - peg(5 kda)/nm . These nps could be redispersed in milli - q but showed visible aggregation and precipitation after 2 days (figure 4b). Analysis of the combined centrifugates showed a 99.8% organic composition, meaning that only ligands were removed from the dispersion and all cores were retained by the membrane . All nps purified by this method showed strong aggregation and were therefore not analyzed further . Both column separation and filtration (dialysis and membrane centrifugation) are established methods, but they failed to produce a high yield of colloidally stable nanoparticles for all sizes when the peg - shell was grafted through ligand replacement . We therefore attempted a variation on precipitation that exploits the possibility to magnetize the nanoparticle cores . While hydrophilic, polymer - stabilized superparamagnetic nanoparticles cannot be pulled out of aqueous solution using a fixed magnet, such extraction is possible in solvent / nonsolvent mixtures in which the poor solubility leads to formation of small aggregates . By dispersing the particles in a solvent mixture of etoh with an addition of petrolether, a cloudy suspension of poorly dispersed core the poorly suspended nps could be pulled to the container wall by application of a fixed magnet . The decantation step has to be repeated several times due to the poor solubility also of the free peg dispersant in the etoh / petrolether . Tga was therefore performed after each decantation step to evaluate the stepwise reduction in dispersant concentration (figure 5a). After the third precipitation and magnetic decantation step the grafting density stayed constant, interpreted as that no further dispersant removal is possible and that all remaining dispersants are strongly bound to the iron oxide core surface . The grafting density is within the measurement error independent of core size at 1 nda - peg(5 kda)/nm . Standard deviations were calculated based on results measured on 67 different samples for each core size . The combined supernatants were analyzed with tga, and 1.5% inorganic material was found, which means that this purification method removes free nda - peg and no cores . After reaching the constant grafting density by precipitation and magnetic decantation the retained product included 98% of the cores initially added for ligand replacement independent of np size . (a) step - by - step removal of excess ligands by repeated precipitation / magnetic decantation total organic content was measured by tga and converted into nda - peg(5 kda)/nm . After three precipitations with magnetic extraction the free dispersant is removed for all core sizes . A stable grafting density of 1 nda - peg(5 kda)/nm independent of core size remains with a yield of 98% purified nps . (b) representative tem micrograph of well - dispersed, nda - peg - grafted nps with 9.6 nm core diameter . In comparison to oa - capped nps it is possible to dry the nps well dispersed and without order on the grid due to the steric repulsion of the added peg shell (cf . Tem micrographs of smaller cores purified by this method can be found in the supporting information (figure s7). Compared to the oa - capped nps (figure 1d), the interparticle distance is increased due to the bulky nda - peg shell, and importantly an inspection of thousands of particles did not reveal any aggregated cores . This strongly suggests that the peg shell was formed around individual cores and remained homogeneous . The morphology of the np cores did not change after oa was replaced by nda - peg . A color change from light brown to dark brown the difficulty of achieving complete exchange of oa for new dispersants previously reported by us and others demonstrates the need to establish that the total organic content determined by tga is traced exclusively to nda - peg . Significant amounts of other dispersants on the core surface might alter the physicochemical properties of the core shell particles . Atr - ftir analysis was performed to verify the composition of the dispersant shell after purification (figure 6a; insets can be found in figure s4). Ir spectra were also recorded on the complete supernatant collected from the precipitation and magnetic decantation method . The presence of nda - peg on the np surface could be confirmed by several marker bands below 1000 cm as well as bands in the region from 1345 to 1240 cm and from 1144 to 840 cm that can be mainly attributed to ethylene glycol ch2 and c o / c c stretching vibrations, respectively . (a) atr - ftir spectra of 7.1 nm iron oxide nanoparticles: (a) as - synthesized cores with oa ligands, (b) nda - peg - nps after ligand exchange before purification, (c) peg - nda - nps purified by precipitation and magnetic decantation, (d) peg - nda - nps dialyzed 24 h, (e) peg - nda - nps purified through sephadex g75 column (fr ii), (f) sephadex g75, (g) peg - nda, and (h) combined supernatants of all magnetic decantation steps . Labeling: () 1960 cm oh - bending from peg - cooh, (+) 1740 cm coor or cooh, () 1702 cm (c = o) free oa, () 1640 cm amide conh or nh2, () 1074974 cm sugar band, () 965 cm ((c = o)oh) free oa; 680400 cm np bands . (b) representative tga (solid lines) and dsc (dashed) graphs of 4.9 nm nps measured in synthetic air: oa - capped nps (orange), nda - peg - nps raw after ligand exchange before purification (black), precipitation and magnetic decantation (green), dialyzed (light blue), membrane filtration with amicon (dark cyan), column purified (dark yellow), and pure ligand nda - peg (navy). The amide stretching vibrations at 1640 (co) and 1550 cm (nh) (figure 6a ()) that can be found in all spectra except for the oa - np are a good indication of successful coupling of nda to peg and its presence at the np surface; this peak is slightly larger for nps purified by magnetic decantation compared to dialyzed nps . A weak peak (figure s4b ()) at 1491 cm fits monodeprotonated nitrocatechol and is attributed to surface - bound nda - peg . The band at 1728 cm (figure s4a ()) arises from residual carboxylic carbonyl groups of free peg(5 kda) that has not been coupled to nda and is found only in the nonpurified sample and the combined supernatants . A similar trend for the band at 1960 cm (figure 6a ()) suggests a common origin; we therefore attribute this vibration to an overtone or combination vibration of the out - of - plane oh bending mode of peg carboxylic acid . The bands at 1730 and 1740 cm (figure 6 (+)) that were found in all samples except in the precipitation purified nps and oa - nps might originate from peg - cooh or coor groups from unreacted peg or cross - reacted nda - peg (o - ph). However, these byproducts were removed by precipitation / magnetic decantation . An important observation is that the nps purified by column show deviations from the ir absorption spectrum for iron oxide nanoparticles purified by other means . The column material sephadex dextran shows strong similarities (bands at 1074974 and 790740 cm, figure 5a (), oh vibrations at 36003000 cm) with these additional peaks in the spectrum, indicating that nps purified by column are additionally covered with small amounts of dextran . A final important question is the presence of significant amounts of weakly or strongly bound oleic acid after ligand replacement and purification . In particular, free or weakly bound oleic acid can greatly influence the colloidal stability . The presence of free oleic acid can be determined from absorptions at 1702 cm in the ftir spectra (figure 6 ()) assigned to the c = o stretch of h - bonded (dimeric) alkylcarboxylic acids and from other bands characteristic for free acid groups such as the oh in - plane and out - of - plane bending at 1410 and 965 cm which is overlaid (figure 6 ()). The absence of the bands at 1702 cm in all samples (also the raw sample) demonstrates that free oa was successfully removed by the dmf hexane extraction . The presence of oleate can be evaluated at 1605, 1520, and 1410 cm corresponding to two asymmetric and one symmetric stretching vibrations of the carboxylate headgroup . Whereas the peaks at 1605 and 1520 cm are overlaid by bands from the peg, a slight shoulder of the band at 1410 cm can be found in all samples . This band, however, is more prominent in the raw and the dialyzed samples than in the precipitated samples . For the column purified nps this peak multistep tga profiles observed below 400 c have been attributed alternatively to vaporization of physisorbed oleic acid, to chemisorbed oleate species with different binding strengths, or to partial cleavage of the capping agent . Our data (figure 6b) show that a significant second step in tga only is observed when free or weakly bound impurities such as physisorbed oleate or dextran are present . A one - step profile is only observed for nps purified by magnetic decantation . We conclude from the ir measurements that the ligand replacement is not complete in any sample . This is also suggested by that the measured high surface coverage of 1 nda - peg(5 kda)/nm is still lower than the theoretical maximum surface coverage of> 2 nda - dispersants / nm . However, for samples purified by precipitation and magnetic extraction only a minor amount of strongly complexed oleate remains on the particle surface shielded by a dense peg brush . If oleate with a negligible desorption rate is distributed on the particle surface, its short extension combined with the high peg - brush density still results in excellent colloidal stability as shown in the next section . The long - term stability of samples that were purified by dialysis and magnetic decantation was investigated with tem, dls, and saxs . Long - term stability of dialyzed samples was only investigated for 4.9 nm core size as the dispersions of nps with larger core diameters showed visible aggregation after a few days . Also, samples purified by membrane filtration were not subjected to long - term stability studies as the dispersions showed visible aggregates after 2 days . For long - term studies of core shell nps purified by precipitation and magnetic decantation, 7.1 nm nps were dispersed at a high concentration of 4 mg / ml that speeds up aggregation in both h2o and pbs . Pbs tests the stability at physiological ionic strengths and is additionally known to cause aggregation and precipitation of poorly stabilized iron oxide nps due to the strong interaction of phosphates with iron ions . Tem, dls, and saxs were measured after 1 week and 4 months . For nps in water and pbs a rh of 16 and 23 nm (intensity weighted), respectively, was measured by dls (table 2), which is in good agreement with previous findings for individually stabilized core shell np with nda - peg monolayer, considering the length of hydrated nda - peg(5 kda) to be 4 nm and the core 7.1 nm, resulting in a rh(theoretical) of 15 nm . These values are significantly smaller than in other recent publications investigating direct ligand replacement on oleic acid - capped nanoparticles with peg dispersants . The narrow size distributions strongly support the interpretation of individually stabilized and colloidally stable core no precipitation and negligible change in the hydrodynamic radius were found for nps in milli - q as well as pbs after storage of the dispersions for 4 months at 4 c (table 2). The rh of dialyzed 4.9 nm cores was measured to be 16 nm and increased slightly after 4 months to 18 nm . Tem imaging of the nanoparticles stored for 4 months in milli - q and pbs showed no trace of core aggregation or aging of the particles (figure s5). The stability of the dialyzed sample during storage, but not to further dialysis, indicates a degradation mechanism related to the dialysis membranes . Scattering curves and fits for samples in milli - q and pbs fresh and after 4 months are shown in figure 7b . The high monodispersity of the cores is evident from the scattering curves . A core size of 6.8 0.4 nm could be fitted to the data . This is in excellent agreement with the core size determined by image analysis of the tem data of the same particles (7.1 0.4 nm). After storage for 4 months at 4 c there is no significant deviation in the scattering attributable to aggregates, such as a structure factor peak and/or a change in the low q range . (a) dls measurements of 7.1 nm nps purified by magnetic decantation; 3 mg / ml in pbs with 10% v / v fetal calf serum added . Temperature cycle from 20 to 70 c (circles) and back (triangles) with 5 min equilibration time at each temperature . (b) saxs curves of 7.1 nm nps in pbs and h2o, freshly prepared and after storage for 4 months at 4 c . Inset: clear colloidal dispersion of nps from measurement (a) stored in pbs / fcs 10% v / v for 2 months at 4 c after temperature cycling . The colloidal stability of the nps was further challenged by heating the nps (3 mg / ml) up to 70 c in pbs with 10% v / v fetal calf serum (fcs). This test is applied to distinguish between particles that show stability in serum but interact with denatured protein aggregates and those for which interactions between core and denatured proteins also do not occur.figure 7a shows the evolution of the hydrodynamic radius with temperature . No increase in rh or precipitation of nanoparticles during the temperature cycle was observed . The inset in figure 7b shows a picture of the same np dispersion subjected to temperature cycling after being stored for 2 months at 4 c . Aggregation or precipitation also cannot be observed after storage; the dispersion remains clear and colloidally stable . Tem micrographs (figure s6) show that the nps in pbs / fcs remain well - dispersed after storage for 2 months in serum . Dialysis has to be performed with membranes having a pore size sufficiently large to allow diffusion of the dispersant through the pores; the mwco of membranes is however typically given for equivalent protein or dextran sizes and cannot be used directly for comparison to highly solvated polymer molecular weights for which the radius of gyration is much bigger than for e.g. Protein of the same molecular weight . A rule of thumb of choosing a pore size> 5 that of the dialyzed molecule is often invoked . This was confirmed by attempting dialysis of dissolved peg(5 kda) (0.2 g / ml against 5 l of milli - q) in a membrane of 1214 kda mwco, i.e., nominally 23 times the molecular weight of the nda - peg, which was used in previous publications studying grafting - to and ligand replacement of pegylated nps . After 24 h only 50% of the free peg was removed, and only 60% had been removed after 5 days despite exchanging milli - q in the reservoir five times . Thus, choosing a too low mwco results in remaining free dispersant and subsequent overestimation of the dispersant grafting density by tga . We therefore changed to cellulose - based membranes with a mwco of 1000 kda, which nominally corresponds to a cutoff hydrodynamic size of 37 nm (assuming a scaling related to equivalent sizes of dextran). Nps with a hydrodynamic radius rh between 15 and 23 nm (for 7.1 nm cores) were still retained while free nda - peg(5 kda) with an rh of 4 nm was presumably removed from the dispersion . Figure 3a shows the removal of excess ligands by dialysis for all sizes of nps . Large - core nps (7.1 and 9.6 nm) dialyzed for 24 h yielded an average grafting density of 1 nda - peg(5 kda)/nm determined by tga . However, nps also started to stick to the dialysis membrane after 24 h. tem of large nps (7.1 and 9.6 nm) extracted from the dialyzed dispersion after 24 h showed aggregated morphology (figure 3b). The 24 h dialyzed samples could be redispersed in water but showed poor long - term stability . Visible aggregates were obtained after 1 week . Continued dialysis of the large cores led to an even lower grafting density (0.5 nda - peg(5 kda)/nm) and increased aggregation . (a) removal of excess ligands by dialysis with a 1000 kda mwco membrane . Total organic content was measured by tga and converted into nda - peg(5 kda)/nm using the known molecular weight of nda - peg(5 kda) and average core surface area determined by tem . After 24 h the surface coverage was 1 nda - peg(5 kda)/nm independent of size, but core size - dependent aggregation and precipitation were observed . Dialysis for 70 h lowered the grafting density to 0.3 nda - peg(5 kda)/nm, resulting in colloidally unstable dispersions . (b) typical tem of nanoparticles with 9.6 nm core size that had visible aggregates (inset) grafted with nda - peg(5 kda) after 24 h dialysis against water using a 1000 kda mwco cellulose membrane . Nps with small core sizes (4.9 and 3.5 nm) had grafting densities of 0.8 and 1.7 nda - peg(5 kda)/nm, respectively, after 24 h dialysis and showed good colloidal stability . The small nps were dialyzed also for 70 h, which resulted in lowering of the average grafting density to 0.3 nda - peg(5 kda)/nm . During dialysis the np suspension underwent visible color change from light brown to almost black (figure s2 in the supporting information), indicating that as dialysis proceeds, surface - bound nda - peg is ripped off in addition to the initial removal of free nda - peg . First - generation, hydrophilic iron oxide nps were mainly stabilized with dextran or carbohydrate derivatives physically adsorbed without using a separately defined anchor such as nitrodopamine . A second generation of polymer - coated iron oxide nps could be said to have polydisperse cores but defined polymer shells end - grafted with defined anchor groups . Shell architecture allows for exact characterization and functionalization, which is crucial to medical applications . A third generation with monodisperse iron oxide cores to perfectly tailor uniform properties several mutually contradicting conditions during the ligand replacement reaction have to be fulfilled: (1) to dissolve the capping agent (oleic acid), (2) to solubilize the dispersant, (3) to keep the dispersant at low coil size, quantitatively described by e.g. Rg, which determines the grafting footprint, and (4) to provide the right conditions (protonation) of the anchor group to irreversibly bind to the core . We could optimize the ligand exchange procedure within the boundaries of the contradicting requirements and thus produce nps with grafting densities consistently around 1 nda - peg(5 kda)/nm . The grafting densities of 1 nda - peg(5 kda)/nm for monodisperse nps were shown to be sufficient to render the nps long - term stable in biologically relevant media . This is in contrast to what was reported earlier for polydisperse systems . For polydisperse cores a grafting density of 2 nda - peg(5 kda)/nm was found as the threshold for colloidal stability at temperatures above body temperature and in biological fluids . We hypothesize that the accurate exchange of ligands into a homogeneously coated shell is what enables the improved stability at a lower average grafting density for monodisperse, spherical nps . On the other hand, we were unable to achieve grafting densities higher than 1 nda - peg(5 kda)/nm by direct ligand replacement . That the grafting density was constant with respect to core size indicates that surface curvature is not the determinant of the ultimate grafting density by ligand replacement in the superparamagnetic size range . This is similar to previous observations for ligand replacement of hydrophobic dispersants and nda - peg grafting in the melt, for which within the experimental error a difference in grafting density with respect to curvature could not be demonstrated . In the present case the constant grafting density is lower and suggests that the size of the peg - coil is the main determinant of the grafting density . Recent work by e.g. Mefford and co - workers reported very high ligand densities from ligand replacement in their study of oleic acid displacement . In their study, one precipitation step followed by dialysis with low mwco membranes was used to remove excess dispersant . Our results show that this is insufficient to remove excess dispersants so their unusually high dispersant density in comparison with literature can likely be traced to this fact . A higher polydispersity of the cores can also have contributed to uncertainty in the grafting density ., large monodisperse cores were grafted with nda - peg and purified by repeated ultracentrifugation from aqueous solution, which is only feasible with very large cores . They report a grafting density of 1 nda - peg(5 kda)/nm, which is inconsistent with the presented tga results that yield a grafting density within a large interval of 528 nda - peg(5 kda)/nm . This organic content, which corresponds to an order of magnitude higher grafting density than the theoretical maximum, demonstrates that also a single centrifugation step and dialysis are insufficient to purify also large core nps . These samples even showed low colloidal stability, which is typically associated with a low grafting density . However, one should keep in mind that stabilizing ferrimagnetic nanoparticles with large cores using thin polymer brushes is more challenging than in the superparamagnetic range . A different ligand replacement protocol in this study might also have left a higher fraction of destabilizing or heterogeneously distributed oleate bound to the np surface . Finally, it was recently shown that grafting of polymer shells under solvent - free, polymer - melt conditions can lead to a near complete coverage of end - grafted polymer dispersants, with unique shell structure and colloidal properties as the result . While this might seem superior we show here that optimized protocols for grafting - to can yield the required colloidal properties for standard medical applications . Our grafting - to by one - step ligand replacement has the benefits of (a) providing near - total yields (98%) under optimal conditions, while melt - grafting requires considerable excess dispersant and was reported with a maximum yield of 30% calculated on nanoparticle cores; (b) multifunctional particles can be constructed by molar mixing of differently functionalized dispersants without risking degradation of the functional group under the melt - grafting conditions; and (c) direct ligand replacement is easier to scale up for synthesis of large batches . Purification of as - synthesized core shell nps is crucial for proper determination of np properties, and we emphasize the thorough analysis that we have done of the efficiency and effect of different purification methods as a central result of the presented work . To our knowledge, there has been no previous study on the colloidal stability of core we report a strong time - dependent degradation of the particles when dialyzed through high mwco regenerated cellulose membranes . Cellulose ester should be inert and not the cause of the observed particle degradation, which from tem clearly included the particle core . A similar trend was found for nps purified by membrane filtration: np degradation in terms of removal of surface - bound nda - peg was observed, and a colloidally stable product could not be obtained . Degradation of the core can occur by that a dispersant with a strongly binding anchor group leaves the core surface and removes the surface iron ion with it . Since the other purification methods do not lead to this degradation, i.e., the nda anchor is stably bound to the core, we hypothesize that the degradation during dialysis and membrane filtration involves an osmotic / mechanical strain related to dispersant interaction with the pores of the membranes where the particles are also found to precipitate . Stretching of the peg chain leads to a spring - like increase in energy; it could effectively weaken the bond to the surface and lead to polymer detachment and the observed surface dissolution . Size - exclusion column chromatography with cross - linked dextran as column material is often used for purification of biotechnological samples in the size range of nanoparticles; it was previously advocated by us and others as a suitable way to purify core shell nanoparticles after grafting - to and simultaneously testing their grafting density and colloidal stability . Dextran has high affinity to iron oxide and has been used in commercial contrast agent coatings of iron oxide nanoparticles . Our new results show that for highly but not extremely densely grafted nps dextran can transfer from the column to the nps . The cross - linked dextran employed in a size - exclusion column should not transfer to the nps, but the ir results unambiguously show its presence in the eluted np fractions even after passing the same fraction through fresh columns twice . Although dextran was not found by ir for the same type of np cores with grafting densities> 2 nda - peg(5 kda)/nm, a lower grafting density seems to result in transfer of column material to the free core area . The high peg grafting density of 1 nda - peg(5 kda)/nm obviously prevents further np aggregation or sticking to the column . We interpret this result as that transferred dextran also can stabilize nanoparticles that are not extremely densely grafted . This additional physisorbed dextran coating can explain the high organic content in other particle fractions that passed the column . High - molecular - weight polymer physisorbed to nps induce bridging interactions that cluster multiple cores; this leads to effective differences in size and column retention time, which spreads the nps over several fractions and pushes down the yield of the main fraction . Thus, despite the stability of the nps purified by column chromatography, the application of cross - linked dextran as column material cannot be generally recommended for purification that should yield particles with controlled shell properties . The choice of another column material with less strong interaction with iron oxide could remedy this drawback . However, the yield and throughput would based on our study remain low compared to magnetic decantation . Among the many investigated purification methods, our study suggests a combination of precipitation and magnetic decantation repeated in multiple cycles as the preferred choice . This method reproducibly yields nps with exceptional long - term colloidal stability, at high yield and without free dispersant . This very high yield is far superior to the inherent losses of nps when column purification or dialysis is used and can be attributed to the efficient magnetic extraction . Similar approaches have previously been applied in combination with centrifugation, which is sensitive to the choice of solvent ratio and polymer shell molecular weight for their success and do not make use of the superparamagnetic core . The crucial step for successful separation by magnetic decantation was to discard the supernatant prior to sedimentation of precipitated excess peg, since peg and densely peg - grafted nps have similar solubility . Aggregation of the superparamagnetic cores enables rapid collection of the nps within 25 min depending on their size; the spontaneous precipitation and sedimentation of the free peg takes 10 min . The petrolether used in the magnetic decantation also aided removal of remaining oa from the nanoparticle sample that cannot be removed by dialysis or centrifugation, since oa was found in the removed supernatant . A final major advantage of the magnetic decantation method is that in addition to its high yield and purity it can be scaled up and automated . Large volumes and samples can be handled in a short time (<1 h for> 3 cycles necessary for free dispersant removal), while dialysis is inherently time - consuming (relying on diffusion), membrane centrifugation is limited by the volume of the filtration units, and columns are difficult to scale . We show magnetic decantation for synthesis of gram quantities and further scaling up is possible . We conclude that direct ligand replacement of oleic acid - coated monodisperse iron oxide nps can be optimized to consistently yield grafting densities of 1 nda - peg(5 kda)/nm regardless of core size in the investigated superparamagnetic range (diameter 310 nm). Shell nps colloidally stable over many months in biologically relevant media as well as withstanding demanding tests such as temperature cycling in serum . The choice of purification method is as important as the synthesis protocol for reproducible results and greatly affects yield, purity, speed, and amount that can be produced . The best purification method that we found was magnetic decantation, which compared to dialysis, centrifugation filtration, and sephadex size - exclusion chromatography provides the highest quality product and a means to verify purity; it also provides the highest yield and sample volumes that can be extracted per time unit . The methods described in this work form the fundament for development of a whole set of diversely functionalized nps that are suitable for biomedical applications such as targeted mri, multifunctional superparamagnetic nps, and drug delivery.
Igg4-related diseases are systemic syndromes characterized by elevated serum levels of igg4 and igg4-positive lymphoplasmacytic infiltrative lesions in the body . It was first observed that mikulicz's disease correlated with igg4-related disease and later determined that igg4-related disease can occur in any ocular adnexal tissues [25]. Here, we review the clinicopathological features, differential diagnosis, and treatments of orbital igg4-related disease on the basis of a meta - analysis of 42 patients including 3 case series studies . The median age of patients with orbital igg4-related disease is 59 years (range: 30 to 86 years) with a 1: 1 male - to - female ratio [35]. Notably, there is a 1: 3 for bilateral lacrimal lesions similar to finding in mikulicz's disease . Although orbital igg4-related disease can occur in men and women of any age, many patients have a history of allergic diseases such as asthma and allergic rhinitis . The signs and symptoms of orbital igg4-related disease are chronic lid swelling (figure 1) and proptosis (figure 2), but otherwise there are only mild signs, or no signs of inflammation or periocular pain . Ocular motility is restricted mildly if at all, despite the presence of one or more enlargements of the large extraocular muscles (figure 1). There are generally no visual disturbances, although they may occur due to apical orbital lesions (figure 2). Imaging studies show infiltrative lesions in ocular adnexal tissues such as the lacrimal glands (figure 1) [25], extraocular muscles (figure 1) [3, 4], infraorbital nerves (figure 2), optic nerve sheath, lacrimal sac, and even cavernous sinus (figure 2) or the intracranial extension . In cases of orbital igg4-related disease, 62% have bilateral lesions, 69% have lacrimal gland involvement, and 48% have bilateral lacrimal gland involvement [35]. Patients with orbital igg4-related disease sometimes have systemic igg4-related lesions in their submandibular glands (29%), lymph nodes (14%), pancreas (5%), or bile ducts (5%) [35]. Igg4-related lesions in the thyroid and pituitary may also be present [7, 8]. Chronic rhinosinusitis with orbital igg4-related lesions has similar histology (figure 3), although nonspecific chronic rhinosinusitis can be also associated with igg4-positive plasma cells in the lesions . Laboratory data of patients with orbital igg4-related disease show elevated serum levels of igg4 and ige, as well as hypergammaglobulinemia [25]. High igg4 levels are associated with elevated levels of the soluble interleukin (il-2) receptor . Sclerosing pancreatitis and cholangitis, considered as igg4-related diseases, have lymphocytes that signal for il-4 and il-10 in situ . Although an etiology for the igg4-related group of disease has been not determined yet, one plausible hypothesis for these serological and immunological abnormalities is that they result from in vivo activation of the immune system by activated th2 cells [3, 10]. The histology of orbital igg4-related disease includes different degrees of lymphoplasmacytic infiltration with dominant sclerosing lesions or reactive lymphoid follicle (reactive lymphoid hyperplasia, figure 3) [25]. Rarely, orbital igg4-related disease may have an infiltration by lymphoplasmacytic cells and macrophages containing eosinophilic material . Igg4-related diseases in the body are characterized histologically by obliterative phlebitis; however, it is rare in orbital igg4-related disease . Immunohistochemical analysis shows igg4-positive plasma cells (figure 3), which differentiate igg4-related disease from other inflammatory conditions arising from the ocular adnexa [3, 4]. For optimal treatment and resolution, orbital igg4-related disease must be differentiated from the following: idiopathic orbital inflammation, idiopathic orbital myositis, marginal zone b - cell lymphoma, antineutrophil cytoplasmic antibody- (anca-) mediated systemic vasculitis (such as churg - strauss syndrome and wegener granulomatosis), and reactive lymphoid hyperplasia without igg4-positive plasma cells (figure 4). They are characterized by sudden onset of orbital inflammation, periocular pain, swelling and redness of the eyelids, proptosis, ptosis, and ocular motility restrictions . However, some cases of idiopathic orbital inflammation have atypical signs and symptoms, that is, they have lacked acute onset and inflammatory signs . In such cases, ocular adnexal marginal zone b - cell lymphomas make up the majority of lymphomas arising from the ocular adnexa . They are characterized histologically by the presence of reactive follicles in up to 64% of cases, sclerosis in up to 20% of cases, and plasma cells in up to 35% of cases . In addition, 9% of patients with ocular adnexal marginal zone b - cell lymphomas have infiltration of igg4-positive plasma cells and elevated serum level of igg4 . Evidence of light chain restrictions by in situ hybridization and immunoglobulin heavy chain gene rearrangements by southern blot analysis can differentiate between marginal zone b - cell lymphomas with igg4-positive plasma cells and igg4-related inflammatory disorder . Ocular adnexal lymphomas are reported to arise in igg4-related sclerosing dacryoadenitis, indicating a possible link between the two conditions . The symptoms of patients with orbital lesions include periocular pain, which can differentiate these patients from those with orbital igg4-related disease thus, anca - related vasculitis may not only include nonspecific inflammatory lesions, but also have abundant igg4-positive plasma cells . Finally, mikulicz's disease includes symmetrical bilateral lacrimal gland enlargements and frequently correlates with igg4-related disease . However, symmetrical bilateral lacrimal gland enlargements do not always indicate igg4-related lesions (figure 4). Treatments for patients with orbital igg4-related diseases may include systemic steroids, radiotherapy, or rituximab [35]. Studies of each of these treatments involved relatively few patients, making it difficult to evaluate the treatment outcomes via meta - analysis . Orbital igg4-related diseases resolve after systemic steroid therapy, but relapse is often observed following therapy discontinuation . In mikulicz's disease, relapses of lesions are observed when steroids were discontinued . Therefore, it may be best to continue prednisolone at 5 to 10 mg / day or to combine prednisolone with an immunosuppressant such as azathioprine . Rituximab treatment leads to prompt clinical and serologic improvement in patients with refractory igg4-related diseases, although recurrence is also observed after rituximab treatment ends in some patients with orbital igg4-related disease . Orbital igg4-related disease has several unique characteristics that distinguish it from other orbital inflammatory conditions . Orbital igg4-related disease differs from other igg4-related diseases in the body in that it arises from nonglandular lesions and is not associated histologically with obliterative phlebitis.
In summary, we have developed a novel dna binding motif consisting of two dna binding fragments of natural tfs connected via an at - hook linker, which allows the selective recognition of designed, extended dna sequences (up to 13 bp). The success of this design relies on the ability of the at - hook moiety to act as a bidentate minor groove - anchoring device that delivers the dna binding tf fragments to appropriate positions for insertion in their respective major grooves . The peptidic nature of the at - hook allowed an easy installation of each of the dna binding peptides at the c- and n - terminus of the anchor . The construct represents the first demonstration of an engineered synthetic dna binder that reaches two consecutive major grooves across the minor groove . The tripartite (major minor major groove) recognition introduces a novel dna binding motif that lacks a natural counterpart . This approach promises to be applicable to other dna binding tf fragments addressing different sites, and introduces a novel way of targeting specific and long dna sequences . Electronic supplementary information (esi) available: peptide synthesis, full experimental procedures and analytical data of the peptides and products obtained.
Before the third edition of dsm, substance and alcohol dependency were considered as symptoms of personality disorders (pds). However, in the third edition, drug consumption was identified as substance use disorders (sud) (1). From then on, psychologic comorbidities have been viewed as an important subject in drug consumption (2). The pds are rigid, inflexible, and maladaptive behavior patterns, which are severe enough to cause significant impairments in functioning or internal distress . They are enduring and persistent style of behavior and thought, not atypical episodes (3). The high prevalence of dsm - iv pds among those with suds was evidently indicated in the research literature (4, 5). A recent literature review (6) summarized prevalence rates and concluded that estimates of the overall axis ii prevalence range from 34% to 73% in treated substance users, with and borderline pds as the most prevalent ones . A short review on previous studies revealed individuals with certain pds, especially the antisocial pd, were more prone to sud (7 - 9), and persons dependent to different types of drugs showed more pds comorbidity (10, 11). One meta - analyze has shown that patients under medical treatment for sud display more comorbidity with pds than normal individuals do (12). (13) showed the chronic drug consumers had higher scores in schizoid pd and antisocial pd than the functional consumers did . (14) found the most frequently diagnosed pds in heroin substance users undergoing methadone maintenance treatment were antisocial and borderline pd . In addition, grana et al . (13) found that antisocial and schizoid traits are the most outstanding traits of heroin substance users and multidrug consumers . However, cocaine use (15) is often associated with psychiatric comorbidity such as antisocial pd (24%), major depressive disorder (18%), and posttraumatic stress disorder (12%). Applying millon clinical multi - axial inventory - iii (mcmi - iii), craig (16) focused on range of pds in 443 individuals with drug dependency . In both groups, antisocial (60%), passive - aggressive (34%), and depressive pds (32%) were the most prevalent pds . In a study on heroin users in taiwan, 58.5% of men and 62.5% of women had at least one axis - i psychiatric disorder or antisocial pd (17). It is generally accepted that substance users prefer drugs that have best effects on healing their personality problems and use other kind of drugs only when they are deprived of the preferred ones (6, 18). Evident reveals that drugs may be choose by a substance user due to their self - medicating effects (19). Robbins (20) points out that substance users use drug to reduce their social anxieties . However, these suggestions about the specific association between psychological problems and drug choosing have not been scrutinized . Yet drugs are the most powerful means to change one s mental state because of their effects on minimizing psychological problems . Some patients abuse drugs to reduce anxieties, irritation, and depression . Therefore, in cases of self - medication, it would be better to consider a proper plan to solve fundamental problems (19). This study aimed to determine the association between pds and types of drugs in iranian substance users . The pds of cluster b, especially borderline and antisocial pds, and sud and its intensity were studied largely in previous studies . No research has concentrated on comparing narcotics (including opium, shireh, heroin, and crack, a kind of compacted heroin made in iran) with stimulants (as amphetamine, methamphetamine and cocaine) in iran . Undoubtedly, any knowledge about the personal characteristic of these groups of drug users sheds new lights on the more effective medical and psychological treatment for better outcomes . Thus, if routine personality assessment improves outcomes of sud treatment, the clinical implication will be increasing the use of pd assessment in sud treatment settings (21). Current study was a correlation study . To determine the sample size, the cochran s sample size formula for correlation studies (22) was used (equation 1, in which t = 1.96, p = 0.5, q = 0.5, and d = 0.11). The minimum sample size needed to obtain statistically valid results was 79, and we added 51 more samples to obtain higher validity . The samples were male substance users aging 18 to 45 years, who met the dsm - v (23) criteria for suds and were in diverse treatment programs such as methadone maintenance therapy, buprenorphine therapy, and detoxification in private clinics for substance use treatment . The sample group was 260 substance users, 130 narcotics dependents and 130 stimulant dependents . Private clinics of substance dependency treatment were classified into four groups of north, east, west, and south clinics in tehran, iran . Then in each region, two clinics were selected conveniently regarding the clinic manager s tendency to participation in the study . Thus, patients were recruited from centers for substance dependency treatment in four regions of tehran . Patients were recruited by general practitioners (gps) working in these centers until the required sample was reached, without a quota of patients assigned for each center . The inclusion criteria were as follows: being male, 18 age of to 65 years, being able to understand and read persian language, and willing to participate in the study . Exclusion criteria included schizophrenia and psychosis, incomplete questionnaire, and relapse in the preceding two days . Receiving medical treatment was not an exclusion criterion; however, treatment was not modified or increased during the study period . Diagnosis of drug dependency was made with structured interview according to addiction severity index (asi) and confirmation of the technical manager of the clinic, who were mostly gps or psychiatrists . All of participants filed out mcmi - iii to assess type of drug and pds . Then, a psychiatrist or a clinical psychologist confirmed the result from mcmi - iii by interviewing the patient . 1) asi: the asi is a semi - structured interview for substance abuse assessment and treatment planning . The asi is designed to gather valuable information about areas of a client s life that may contribute to their substance - abuse problems . The asi was developed in 1980 by a. thomas mclellan, along with collaborators from the university of pennsylvania s center for the studies of addiction . The asi was the first standardized assessment tool of its kind to measure the multiple dimensions of substance abuse . It has three categories: 1) socio - demographic: sex, age, years of education, and main source of income; 2) consumption: main substance, type of treatments, and years of consumption; and 3) severity of dependency . Overall, studies typically conclude that the asi is a consistent and accurate tool for assessing clients and their substance abuse issues . The asi is able to successfully identify the client s problem area in which they are experiencing the greatest difficulties, such as alcohol or drug addiction, or legal or familial problems . Once a client s psychosocial issues are identified, an appropriate course of treatment should be administered . Severity ratings are based on the following ten - point scale (range, 0 - 9): 0 - 1, no real problem; treatment is not indicated . 2 - 3, slight problem; probably treatment is not necessary . 4 - 5, moderate problem; the severity ratings scale allows the interviewer to determine the severity of a client s problem . The higher the score is, the greater the need for treatment in each area or immediate intervention will be . The asi scores can be used to profile a client s problem areas and then plan an effective course of treatment (24). 2) mcmi - iii: the mcmi - iii is a psychologic assessment tool intended to provide information on psychopathology, including specific disorders outlined in the dsm - iv . It is intended for adults (18 years old) with at least an eighth - grade reading levels who are currently seeking mental health services . The mcmi was developed and standardized specifically on clinical populations, ie, patients in psychiatric hospitals or people with existing mental health problems, and the authors are very specific that it should not be used with the general population or adolescents (25). However, there is a strong evidence that shows it still retains validity on non - clinical populations . The mcmi differs from other personality tests in that it is based on theory and is organized according to a multi - axial format . Updates to each version of the mcmi coincide with revisions to the dsm (26). It is composed of 175 true / false questions that reportedly take 25 to 30 minutes to complete . It was developed by theodore millon, carrie millon, roger davis, and seth grossman (27). The test is modeled on four scales: 14 personality disorder scales; 10 clinical syndrome scales; five correction scales including three modifying indices (which determine the patient's response style and can detect random responding) and two random response indicators; and 42 grossman s personality facet scales, based on seth grossman's theories of personality and psychopathology . The mcmi - iii was updated in 2008, with a new norming sample of 752 individuals (52.8% female; 76% caucasian) with a wide range of clinical disorders . The scale development stage consisted of 600 of these individuals (48.8% male; 84% caucasian), and the cross - validation stage consisted of the remaining 398 individuals (49.5% male, 81.7% caucasian) (28). They are broken down further into 11 basic clinical personality patterns (scales 1 through 8b) and three severe personality pathology scales (scales s through p) (table 1). The final validation stage includes convergent and discriminative validity of the test, which is assessed by correlating the test with similar / dissimilar instruments . Positive predictive value is the likelihood of being right given a test positive, which ranged from 0.30 (masochistic) to 0.81 (dependent). Sensitivity or the proportion of individuals that have a condition and are correctly identified ranged from 0.44 (negativistic) to 0.92 (paranoid). Patients' raw scores are converted to base rate (br) scores to allow comparison between the personality indices . The br scores are essentially where each score fits on a scale of 1 through 115, with the median score of 60 . Conversion to a br score is relatively complex, and there are certain corrections that are administered based on each patient's response style . The modifying indices are scored using a complex system in which the patient's responses in the other scales are compared and the raw and br scores are taken from this . However, the invalidity index is an exception and is not converted to a br score (25 - 27). In iran, before they give their consent, the patients were provided with a general overview of the aims and characteristics of the study . They were also informed that they were participating voluntarily, and that they could choose to withdraw at any time with the guarantee that they will continue to receive the treatment considered most appropriate by their clinic . The study was approved by the ethical review board of the behavioral sciences research center of shahid beheshti university of medical sciences, tehran, iran (11.12.2013; ref: pi10/039). The results were used anonymously and all of the data were kept confidential in this study . The statistics were analyzed both descriptively and inferentially . Applying pearson correlation coefficient and linear regression in the inferential part, hypothesis of the research were examined . 1) asi: the asi is a semi - structured interview for substance abuse assessment and treatment planning . The asi is designed to gather valuable information about areas of a client s life that may contribute to their substance - abuse problems . The asi was developed in 1980 by a. thomas mclellan, along with collaborators from the university of pennsylvania s center for the studies of addiction . The asi was the first standardized assessment tool of its kind to measure the multiple dimensions of substance abuse . It has three categories: 1) socio - demographic: sex, age, years of education, and main source of income; 2) consumption: main substance, type of treatments, and years of consumption; and 3) severity of dependency . Overall, studies typically conclude that the asi is a consistent and accurate tool for assessing clients and their substance abuse issues . The asi is able to successfully identify the client s problem area in which they are experiencing the greatest difficulties, such as alcohol or drug addiction, or legal or familial problems . Once a client s psychosocial issues are identified, an appropriate course of treatment should be administered . Severity ratings are based on the following ten - point scale (range, 0 - 9): 0 - 1, no real problem; treatment is not indicated . 2 - 3, slight problem; probably treatment is not necessary . 4 - 5, moderate problem; the severity ratings scale allows the interviewer to determine the severity of a client s problem . The higher the score is, the greater the need for treatment in each area or immediate intervention will be . The asi scores can be used to profile a client s problem areas and then plan an effective course of treatment (24). 2) mcmi - iii: the mcmi - iii is a psychologic assessment tool intended to provide information on psychopathology, including specific disorders outlined in the dsm - iv . It is intended for adults (18 years old) with at least an eighth - grade reading levels who are currently seeking mental health services . The mcmi was developed and standardized specifically on clinical populations, ie, patients in psychiatric hospitals or people with existing mental health problems, and the authors are very specific that it should not be used with the general population or adolescents (25). However, there is a strong evidence that shows it still retains validity on non - clinical populations . The mcmi differs from other personality tests in that it is based on theory and is organized according to a multi - axial format . Updates to each version of the mcmi coincide with revisions to the dsm (26). It is composed of 175 true / false questions that reportedly take 25 to 30 minutes to complete . It was developed by theodore millon, carrie millon, roger davis, and seth grossman (27). The test is modeled on four scales: 14 personality disorder scales; 10 clinical syndrome scales; five correction scales including three modifying indices (which determine the patient's response style and can detect random responding) and two random response indicators; and 42 grossman s personality facet scales, based on seth grossman's theories of personality and psychopathology . The mcmi - iii was updated in 2008, with a new norming sample of 752 individuals (52.8% female; 76% caucasian) with a wide range of clinical disorders . The scale development stage consisted of 600 of these individuals (48.8% male; 84% caucasian), and the cross - validation stage consisted of the remaining 398 individuals (49.5% male, 81.7% caucasian) (28). They are broken down further into 11 basic clinical personality patterns (scales 1 through 8b) and three severe personality pathology scales (scales s through p) (table 1). The final validation stage includes convergent and discriminative validity of the test, which is assessed by correlating the test with similar / dissimilar instruments . Positive predictive value is the likelihood of being right given a test positive, which ranged from 0.30 (masochistic) to 0.81 (dependent). Sensitivity or the proportion of individuals that have a condition and are correctly identified ranged from 0.44 (negativistic) to 0.92 (paranoid). Patients' raw scores are converted to base rate (br) scores to allow comparison between the personality indices . The br scores are essentially where each score fits on a scale of 1 through 115, with the median score of 60 . Conversion to a br score is relatively complex, and there are certain corrections that are administered based on each patient's response style . The modifying indices are scored using a complex system in which the patient's responses in the other scales are compared and the raw and br scores are taken from this . However, the invalidity index is an exception and is not converted to a br score (25 - 27). In iran, the questionnaire has been normalized by sharifi et al . Before they give their consent, the patients were provided with a general overview of the aims and characteristics of the study . They were also informed that they were participating voluntarily, and that they could choose to withdraw at any time with the guarantee that they will continue to receive the treatment considered most appropriate by their clinic . The study was approved by the ethical review board of the behavioral sciences research center of shahid beheshti university of medical sciences, tehran, iran (11.12.2013; ref: pi10/039). The results were used anonymously and all of the data were kept confidential in this study . The statistics were analyzed both descriptively and inferentially . Applying pearson correlation coefficient and linear regression in the inferential part, hypothesis of the research were examined . The average age of the stimulant and narcotics dependents was respectively 28.8 and 33.1 years . Half of the narcotic dependents were married (n = 67), one was divorced, and the rest were single (n = 63). In stimulant dependents, most of stimulant dependents were single while the married and single ones were nearly equal in narcotic dependents . Most of narcotic and stimulant dependents had diploma (certificate for secondary education graduates in iran) or were secondary education undergraduates . In addition, frequency of narcotic dependents with diploma, associate degree, and bachelor degree was lower than that of stimulant dependents . In order to explore the association between pds and type of used drugs, pearson correlation coefficient was applied with the results presented in two ways: for each pd and for each cluster . As is shown in table 2, there was a significant correlation between stimulant consumption and histrionic pd (p <0.001), and antisocial pd and narcissistic pd (p <0.05). In addition, there were significant correlations between narcotic consumption and histrionic, narcissistic, and avoidant (p <0.05 for all), antisocial, borderline, and depressive pds (p <0.001 for all). According to table 3, examining correlation coefficients between each cluster and type of drug revealed a significant correlation between cluster b and narcotics and stimulant consumption (p <0.001). On the other hand, the table shows a significant correlation between cluster c and narcotic consumption (p <0.001). The results of linear regression in table 4 shows that avoidant, histrionic, narcissistic, depressive, antisocial, and borderline pds play significant role in narcotic consumption . The aim of this study was to determine the association between pds and types of drugs in iranian male drug users . The research findings revealed that there was a significant association between pds in cluster b and both narcotic and stimulant consumption, while pds in cluster a had no significant association with narcotic and stimulant consumption . On the other hand, pds in cluster c also showed significant association only with narcotics (e.g. Opium, heroin, and crack in iran) consumption . As it was suggested, analyzing the association between certain types of drugs with each pd proved that narcotic consumption was associated with histrionic, borderline, depressive, avoidant, antisocial, and narcissistic pds, and stimulant usage was associated with narcissistic, antisocial, and histrionic pds . Study of pds and drug of choice in a meta - analysis indicated that borderline and antisocial pds in stimulant users, dependent, avoidant, schizoid, and borderline pds in narcotics users, and in general, histrionic and narcissistic pds were prevalent among patients with sud (6, 30). For example, antisocial and borderline pds, as the most common pds in patients with sud (6), have comorbidity of 18% and 17%, respectively (31), and half of patients consuming buprenorphine have borderline pd (32). Cluster b and c are the most common comorbidities in heroin users (33 - 38). On the other hand, cocaine users show more characteristics of schizoid and avoidant traits (39), or antisocial pd (19). It can be said that individuals in cluster c have different reasons for their tendency to narcotics in comparison with individuals in cluster b. in fact, persons in cluster b because of their illegal behavior (40), and persons in cluster c due to their low self - esteem, self - dependency, self - sufficiency, and inferiority in social association are drown to narcotics consumption (41). Some studies have proved that heroin users are more impulsive and aggressive than cocaine users (42, 43), while other have expressed no significant difference in personality traits between cocaine and heroin (44) or cannabis abusers (45). Researches consistently have provided the evidence of higher impulsivity and emotional instability in multi - drug abusers, and they had higher levels of antisocial, borderline, and passive aggressive pds (41). Others have signified that narcotic users share characteristics including irresponsibility, snobbishness, ignoring others, and have pds, especially antisocial and histrionic pds (46). Other enquiries have shown that personality characteristics including reclusiveness, being antisocial, sensitive, and anxious, irresponsibility, and poor social adjustment and empathy are associated with risky behaviors such as drug use as a way to overcome negative moods and feelings (47). Most surveys have concluded that behaviors such as risky sexual behaviors and shared injections are associated with the above - mentioned characteristics (48). (49) argued that risky behaviors are in fact, an attempts to reduce anxiety levels and obtain transitory tranquility and pain relief, which are resulted from negative emotions and feelings . These results suggest that most people who drink alcohol hazardously, or use drugs such as cocaine, marijuana, heroin and other narcotics have anxiety, isolation, and loneliness (50). Armstrong and costello (51) found that 60% of drug users, drug abusers, and drug dependents have comorbid pds . As long as having a psychiatric disorder (52, 53), especially pds (6, 54), can significantly increase the risk of relapse, drop out, or poorer treatment outcomes, these patients need more specific treatment plans (55 - 57). In conclusion, results of the present research were in accordance with previous ones in terms of pds and types of consumed drugs . Antisocial and borderline pds are prevalent in stimulant users, avoidant, dependent, schizoid, and borderline pds in alcohol and narcotic users, and in general, histrionic and narcissistic pds are prevalent in those with suds . In general, 34.8% to 73% of the treated substance users have comorbid pds (6). According to craig (16), compton et al . (58), and grant et al . (59), in each group, the prevalence of antisocial pd was significant . Armstrong and costello (51) acknowledged the findings in teenagers . In this research, in comparison with stimulant consumption, narcotic consumption had higher rate of pds . Finally, this conclusion could be drawn that self - medication effects of narcotics, due to the pain relieving and anxiety - reducing characteristics, can cover more pds, while stimulants have more limited self - medicating effects . The current study was the first study conducted on both narcotics and stimulant users about pds on iranian population; however, it study has some limitations . For instance, it has been conducted on men in tehran and cannot be generalized to women or substance users of other cities . It included population between 18 to 45 years old and cannot generalize the results to teenagers or elderly . The same study can be conducted on women, teenagers, and population in other cities.
The experimental models of myocardial infarction have largely contributed to a better understanding of the pathophysiology of myocardial infarction . Studies on large animal models revealed many mechanisms involved in myocardial injury and repair [1, 2]. In the last few years, transgenic mice and gene targeting technologies allowed profound studies of the underlying pathomechanisms . For this purpose, we developed murine models of myocardial ischemia and reperfusion and used them to study the mechanisms involved in ischemic myocardial injury [3, 4]. We described pathological features of reperfused myocardial infarction in mice, that is, rapid formation of granulation tissue and subsequent development of a stable scar . Our studies also revealed an important role for macrophages and their associated inflammatory and remodeling - related mediators [5, 6]. A profound understanding of these mechanisms is crucial for projects utilizing novel cellular therapies, since the local microenvironment seems to exerts a strong influence on the cells applied in our model of myocardial cryoinfarction [79]. This study compares the pathology and the sequence of cellular and molecular events in the two mechanistically different lesion models of myocardial cryoinfarction and reperfused infarction and reveals substantial differences in myocardial remodeling between them . Both models show a transient inflammatory response associated with induction of chemokines, cytokines, and remodeling - related mediators . The cryolesioned heart showed a prolonged remodeling with postponed development of granulation tissue and scar formation, which was associated with persistent macrophage infiltration in the injured tissue when compared to reperfused infarction . Wild - type c57/bl6-mice (charles river, sulzfeld, germany) of 18 to 25 g weight and 8 to 10 weeks old were used in our infarction models . Group size in reperfused infarction model was n = 8 mice and in cryoinfarction n = 6 mice . All experiments were performed in accordance with an animal protocol approved by the local governmental authorities . In an initial surgery (2.5 g / g; merial, halbermoos, germany) as previously described . Then, mice were intubated with a pe-90 tube (becton dickinson, sparks, md, usa) connected to a small animal respirator (rodent ventilator mod . Millis, ma) and ventilated at a frequency of 110/min with tidal volume of 0.25 ml . Pericardium was dissected and partially removed, and a prolene 8 - 0 suture (ethicon, norderstedt, germany) was placed around the left descending coronary artery (lad). Suture ends were threaded through a sterile pe-10 tube (becton dickinson) of 3 mm of length, exteriorized through the thoracic wall, and then stored subcutaneously . The thorax was closed with interrupted prolene 6 - 0 stiches, and the skin was closed with a running prolene 6 - 0 suture . At the end, metamizol (100 mg / kg; novalgin) was given for analgesia in a mixture with cefuroxim as antibiotic prophylaxis i.p . (100 mg / kg, zinacef; bristol - myers squibb, munich, germany). The skin was reopened, and the ends of the lad - ligature were connected to heavy metal picks . Pulling on the ligature ends induced lad - occlusion, and this ischemia was maintained for 60 minutes . After removal of the lad - ligature, the blood flow was restored in the reperfusion . Ecg monitoring of einthoven lead ii during and after lad - occlusion confirmed successful ischemia and reperfusion . One - hour ischemia was followed by reperfusion for 6 hrs, 1, 3, 7, and 14 days . Mice were euthanized using an overdose of pentobarbital i.p . ; hearts were excised and fixated in zinc - paraformaldehyde (z - fix, 4%; anatech, battle creek, mi, usa) for histology or stored in rna - later solution (qiagen, hilden, germany) for mrna - studies . Animals were sedated by brief exposure to narcotic gas containing 50% o2, 50% n2o, and 3 to 4 vol-% of isoflurane as previously described . Thereafter, mice were positioned on a temperature - controlled plate (37c) in supine position, intubated, and ventilated using a small animal respirator (harvard apparatus) as described in the previous section . An anterolateral skin incision was performed 5 mm above the costal margin, the anterolateral thoracic muscles were transected, and the thorax was opened in the fifth intercostal space . The pericardium was opened, and the apex of the heart was exposed cryocoagulation was performed by placement of a copper probe (3 mm diameter, cooled in liquid nitrogen for 2 min) to the free left ventricular wall (3 times for 20 seconds) in order to achieve reproducible, large transmural myocardial lesion . After induction of the cryoinfarction, a 22-g chest drain (optiva, johnson & johnson, new brunswick, nj, usa) was inserted into the left pleural cavity to prevent pneumothorax . The thorax was closed with prolene 6 - 0 sutures, and air was drained using negative pressure on chest drain before extubation ., the hearts were excised after the above - mentioned time periods and further processed for histological and molecular analysis . After excision, hearts were washed in cardioplegic solution containing 4 g nacl, 3.73 g kcl, 1 g nahco3, 2 g glucose (all from berlin chemie, berlin, germany), 3 g 2,3-butandion monoxime (sigma - aldrich, munich, germany), 3.8 g ethylenglycol tetra acetic acid (sigma), 0.2 mg nifedipine (sigma), and 10 ml heparin (1000 iu / ml; ratiopharm, ulm, germany), all of which were dissolved in 1 l of isotonic nacl (berlin chemie). Hearts were fixated in zinc - paraformaldehyde for 24 hrs and further processed using standard paraffin embedding . Hearts where cut from basis to apex, and at every 250 m, a set of ten 5 m sections were mounted on glass slides . Myocardial sections below insertion of the papillary muscles were further used for histology and immunohistochemistry . Photographic images were recorded on a computer system equipped with a digital camera (dp70, olympus, hamburg, germany), and planimetric evaluation was performed using analysis software (olympus). Picrosirius red staining of total collagen was used to evaluate development of fibrosis in the myocardial scar . Cell density was measured by cell count of immunohistochemically stained cells in infarcted myocardium and was expressed as number per mm . We used the following primary antibodies for immunohistochemistry: alpha smooth muscle actin mouse monoclonal antibody (clone 1a4; sigma, st . Louis, mo, usa) for myofibroblasts, f4/80 rat monocyte / macrophage antibody (serotec, duesseldorf, germany) for macrophages and mca 771 g rat monoclonal antibody for neutrophils (serotec). Furthermore, samples were stained for macrophage maturation marker osteopontin-1 using a goat polyclonal antibody (p-18, santa cruz, heidelberg, germany) and for macrophage elastase using a rabbit monoclonal anti - mmp12 antibody (abcam, cambridge, uk). Immunohistochemical staining was performed using appropriate vectastain elite abc kits and diaminobenzidine (axxora, loerrach, germany). Excised hearts were cleared of large vessels and atria and placed in rna - later solution (applied biosystems, foster city, ca, usa). Hearts were minced using a tissue tearor (tissue tearor modell 398; biospec, bartlesville, ok, usa), and mrna was isolated using standard phenol / chloroform extraction method (trizol, applied biosystems). Mrna was transcribed into cdna using high - capacity cdna transcription kit (applied biosystems) with random hexameric primers as described in the manufacturer's protocol . The mrna - expression was determined by taqman real - time quantitative pcr (rt - qpcr, applied biosystems). Cdna was diluted 1/10 and then used for measurement of gene induction according to the manufacturer's instructions on an abi prism 7900ht sequence detection system using sds2.2 software (applied biosystems). All murine primers were commercially available and measured with fam tamra chemistry using the relative standard curve method . At the end of rt - qpcr cycle, dissociation curve analysis was performed to ascertain the amplification of a single pcr product . Two - tailed, unpaired student's t - test was used to determine a significant difference between two groups . Histopathological features of cryoinfarction and reperfused myocardial infarction in our model of cryoinfarction showed a transmural lesion with sharp infarction borders early after injury . The cryoinjury presented after 3 days with extensive cellular infiltration and partial granulation tissue formation starting at the periphery of infarction (figure 1(a)). The central part of the cryoinjury at the copper probe application site contained numerous dead cardiomyocytes and cellular debris . The formation of loose granulation tissue was almost finished after 7 days (figure 1(b)). The subsequent scar formation was largely completed after 14 days, but a substantial cellularity was still present in some parts of the cryoinjury whereas revascularization of large vessels was not observed (figure 1(c); arrow). In contrast, the reperfusion of myocardial infarction in our closed - chest model led to a nontransmural lesion . Complete formation of granulation tissue was found after 3 days (figure 1(d)) and followed by a compacted scar formation with low cellular content after 7 days reperfusion (figure 1(e)), as we published before . The cryoinfarction showed large areas with loose collagen deposition after 7 days (figure 1(f)) in contrast to compacted collagen - rich scar in reperfused infarction at the same time point (figure 1(g)). This difference in scar formation was further underlined by a vast number of myofibroblasts in cryoinfarction after 7 days (figure 1(h)) whereas only few myofibroblasts were found in the reperfused infarction (figure 1(i)). The collagen was largely compacted in cryoinfarction after 14 days, but this was still associated with low myofibroblast persistence in the scar . The cryoinjury led to prolonged scar formation probably due to a longer time period needed for debris clearance, which could only progress from the periphery of the injury due to completely cryodamaged vasculature in the scar . In contrast, the reperfusion of myocardial infarction was associated with rapid scar formation due to an intact vascular network, which provided synchronous cell migration into the entire infarcted area . Immunohistochemical staining of neutrophils revealed a strong myocardial infiltration early after the injury, suggesting a comparable myocardial debris clearance in both models . We found a significant difference in neutrophils density in infarcted area between the two models after 3 days (figures 2(a) and 2(b)) and slightly longer neutrophils persistence in cryoinfarctions after 7 days (figure 2(c)). We found no difference in cellular density of neutrophils in noninfarcted remote myocardium in both models (figure 2(d)). Macrophage staining showed a persistent, strong infiltration of cryoinjured myocardium after 7 days whereas only few f4/80-positive cells were found after 7 days reperfusion of infarction, as previously published (figures 3(a) and 3(b)). We investigated macrophage activity using elastase staining after 7 days and found very strong signals in cryoinfarction, but no signals in reperfused infarction (figures 3(c) and 3(d)). Interestingly, after 14 days, cryoinfarction showed much lower elastase activity, even though the total macrophage cell density was still high . The macrophage cell density data strongly support a short, rapid course of tissue remodeling after reperfusion of murine infarction, in contrast to a persistent macrophage infiltration even 14 days after cryoinfarction (figure 3(e)). To our surprise, macrophage density was also significantly higher in the remote, noncryoinjured myocardium, thus suggesting a dysfunctional development of the infarction border zone in this model (figure 3(f)). We further investigated the maturation of macrophages and found after 7 days a stronger infiltration of osteopontin-1-positive cells in cryoinfarcted hearts than in reperfused myocardial infarction (figures 4(a) and 4(b)). Evaluation of osteopontin-1-positive cell density revealed a prolonged, but functional maturation of macrophages (figure 4(c)). The cell density of osteopontin-1-positive cells in the noninfarcted area was also comparable between the models (figure 4(d)). Taken together, the course of cellular events involves a prolonged infiltration of cryoinfarction with inflammatory cells, thereby explaining the later development of a stable scar in this model . In contrast, a transient, short infiltration of reperfused myocardial infarction with inflammatory cells is associated with a rapid resolution of myocardial remodeling . The course of molecular events in cryoinfarction and reperfused infarction was assessed using mrna - expression measurements of specific inflammatory and remodeling - related mediators . The myocardial damage leads to a rapid production of reactive oxygen species and induction of several scavenger enzymes, for example, heme oxygenase 1, glutathione peroxidase, and so forth . The expression of heme oxygenase 1 in reperfused myocardial infarction accompanied the transient short inflammatory reaction with a peak induction after 24 h of reperfusion and a rapid downregulation thereafter (figure 5(a)). The cryoinfarction led to a significantly different expression pattern with prolonged induction of heme oxygenase 1 . The cytokine tnf-, the next downstream mediator in the postischemic inflammatory cascade, presented with a significantly stronger mrna - induction early after reperfusion of infarction, which is probably triggering the rapid onset of inflammatory reaction (figure 5(b)). Tnf--expression decreased thereafter in reperfused infarction, in contrast to its maximal upregulation 3 days after cryoinfarction . We measured the induction of the anti - inflammatory cytokine il-10 to investigate negative feedback regulation of proinflammatory response (figure 5(c)) and found a comparable time course in both models with a significantly higher induction of it in cryoinfarction after 3 days . Based on our previous results regarding the role of chemokines in remodeling, we measured the expression of macrophage - related ccl2 and ccl4, as well as neutrophils - related ccl3 . To our surprise, we found a significantly higher induction of ccl2 in reperfused infarction whereas cryoinfarction did not lead to its significant upregulation at a later time point (figure 5(d)). In contrast, the mrna - expression of ccl3 showed a significantly stronger early induction in cryoinfarction and a second maximum after 3 days when compared to reperfused infarction (figure 5(e)). The chemokine ccl4 showed a comparable expression pattern between the models, but also significant differences at an early and later time point (figure 5(f)). Taken together, the expression of free radical scavenger enzymes, cytokines and chemokines, is directly influencing the course of cellular events in both models of infarction . Still, the expression of chemokines shows a different time course, probably due to their additional role in collagen deposition during advanced stages of remodeling . Since osteopontin-1 is not only related to macrophage maturation but also is involved in extracellular matrix formation and remodeling, we measured its mrna - expression and found an earlier peak after 24 hrs in reperfused infarction whereas in cryoinfarction its maximum was reached after 3 days (figure 6(a)). Another marker of early remodeling, tenascin c, showed a significantly higher induction early after reperfusion of infarction, thus supporting the rapid granulation tissue formation (figure 6(b)). Tenascin c - expression was significantly higher after 3 days in cryoinjured myocardium, thus mediating the prolonged granulation tissue formation . We also investigated the mrna - expression of tgf- and found a later induction of predominantly profibrotic acting tgf-1 and -2 isoforms (figures 6(c) and 6(d)). Interestingly, we found a significantly higher induction of rather antifibrotic acting tgf-3 isoform 7 days after cryoinfarction, thus probably representing a strong signal for the resolution of myocardial remodeling (figure 6(e)). These data reveal specific mediators responsible for the prolonged granulation tissue formation and delayed remodeling process after cryoinfarction . Our data also confirm our previously published findings on induction of tgf- isoforms in reperfused infarction and give us additional novel insights in osteopontin-1- and tenascin c - expression in the reperfused infarction model . Studies using large experimental models of myocardial infarction brought a detailed analysis of pathology and some insights into mechanisms involved in myocardial ischemia . Recent developments in transgenic mice and gene - targeting technologies provided tools for profound studies of the pathomechanisms and specific genes involved in cardiac repair . We developed a chronic, closed - chest murine model of myocardial ischemia followed by reperfusion, which leads to infarction with rapid scar formation . This closed - chest model has minimized the influence of the initial surgery trauma and is, therefore, particularly useful for studies of inflammatory response and early remodeling . We used this model in cellular therapy experiments and found a very poor engraftment of the i.v . Injected whole bone marrow cells (unpublished observation) in contrast to a very good engraftment found in another model, that is, cryoinfarction . Therefore, we assumed differences in myocardial remodeling and specifically in local microenvironment of injured myocardium . In order to investigate these differences, we compared the pathology and the course of cellular and molecular events during myocardial remodeling between the two models . In this study, we confirmed our previous findings on rapid development of granulation tissue and the time course of mediators involved in transient inflammatory reaction, as well as in subsequent remodeling [3, 4]. We found a transient, strong increase in proinflammatory cytokines and chemokines leading to a rapid tissue infiltration with neutrophils and macrophages, which was also described in large animal models . In addition, we observed a transient induction of heme oxygenase 1, which is caused by a massive production of reactive oxygen species during early reperfusion and precedes the cytokine release in the ischemic heart . Development of granulation tissue was associated with anti - inflammatory action of il-10, with transient upregulation of early remodeling mediator tenascin c, and was followed by induction of macrophage maturation marker osteopontin-1 and, as previously published, upregulation of tgf- isoforms . These mediators lead to differentiation of myofibroblasts and subsequent collagen deposition, thus resulting in the stable scar formation 7 days after reperfusion of murine infarction . The timely resolution of myocardial remodeling is also confirmed by a lack of macrophage elastase and osteopontin-1 staining after 7 days reperfusion . The pathology of cryoinfarction showed a similar course of events, but a longer duration of this process until formation of a stable myocardial scar is completed . The basic histology revealed that cryoinfarction leads to an early, strong cellular infiltration of the damaged myocardium, which slowly decreased to a lower level until 14 days postinjury . We observed a permanent damage and occlusion of the large vessels in cryoinjured myocardium, which mechanically prevented an evenly distributed cellular infiltration of the injured myocardium and thus rapid myocardial remodeling, as it is observed in reperfused infarction . In cryoinfarction, macrophages and neutrophils persist up to 14 days in the last area near the center of the epicardial copper probe application site . This seems to be one of the main factors influencing the pace of remodeling in the cryoinfarction model . Interestingly, we found a significantly prolonged macrophage infiltration of the noninjured area, which may be associated with a dysfunctional border zone formation and infarction limitation in this model . The concomitant appearance of elastase - producing macrophages and myofibroblasts 7 to 14 days postinjury represents an active myocardial remodeling process, which is further supported by not entirely compacted collagen in the scar . In consequence, this active myocardial remodeling is probably responsible for a good engraftment of the implanted cells in the cryoinfarction model . The mrna data provide novel insights into the time course of the expression of several mediators involved in remodeling of cryoinfarcted myocardium . The later induction of cytokines and remodeling - related mediators is leading to the prolonged remodeling in this model . In particular, the relatively high expression of heme oxygenase 1 after 3 days represents persistent reactive oxygen species production from cell debris, while the high ccl2 and ccl4 expression characterize the inflammatory response associated with strong macrophage activity . The concomitant upregulation of anti - inflammatory il-10 is acting towards resolution of the inflammatory reaction and formation of granulation tissue . The significantly higher expression of neutrophils - related chemokine ccl3 seems to reveal a strong migration stimulus into the damaged area, which is limited by the lack of intact vasculature as discussed above . Chemokines, particularly the ccl2, have also been associated with myofibroblasts activity and collagen production, which may provide additional explanation for its upregulation after 3 days in cryoinfarction . Using transgenic mice, we described a crucial role for the chemokine ccl2 and macrophages in timely resolution of myocardial remodeling and preservation of myocardial function . The later induction of tenascin c and osteopontin-1 as well as the tgf- isoforms represent the prolonged extracellular matrix production and collagen deposition in myocardial remodeling . This study shows substantial differences in local microenvironment and cellular and molecular events between the models of cryoinfarction and reperfused infarction . In a comparison between murine models of cryoinfarction and coronary ligation without reperfusion, a modest adverse remodeling was postulated for the cryoinfarction, and the cryoinfarction was suggested as a representative model for myocardial infarction encountered in clinical practice . We do not completely share this opinion, since the main goal in treatment of acute infarction is early revascularization with reperfusion . But for the experimental study purpose, the cryoinfarction indeed offers a very reproducible area at risk and infarct size whereas this is a major weakness of the reperfused infarction model due to the anatomical variability of coronary arteries . The prolonged myocardial remodeling seems to be beneficial in cell transplantation studies, since the cellular engraftment after direct application into injury is very good . Our data provide novel insights into expression of cytokines and chemokines in cryoinfarction, where persistent proinflammatory milieu may be favorable for the cell engraftment . On the other side, the rapid resolution of inflammatory response in reperfused infarction may minimize the time span of suitable local environment for cell engraftment, but this concept still has to be further investigated . In respect to the ongoing clinical trials, the reperfused infarction model could be seen as a more relevant model, but it has not been widely used yet . First, the findings are mainly of experimental interest since the model of reperfused myocardial infarction is comparable to a clinical situation in patients presenting with acute infarction, but it seems to be less favorable for the cell engraftment studies . On the other hand, the cryoinfarction model provides good conditions for cell engraftment and it is very reproducible, but of a limited value to the clinical practice . Second, we did not measure infarct size and one could argue that a difference in infarct size may explain the observed differences in pathological and molecular events between the two models . Based on previously published work, the model of reperfused infarction [4, 5] affects mostly a larger portion of left ventricle than the cryoinfarction, but the reperfusion still leads to a faster resolution of inflammation and scar formation than the cryoinjury . Therefore, the infarct size does not reflect the quality and pace of myocardial remodeling, and we did not found it to be necessary for explanation of the findings in this study . In conclusion, the cryoinfarction is associated with prolonged inflammatory response leading to a postponed granulation tissue formation and scar development, when compared to the reperfused myocardial infarction . Several inflammatory mediators and remodeling factors are involved in this process, and they all contribute to a specific, dynamic local environment in ischemic myocardium . These substantial differences in remodeling may affect and even be favorable for cellular engraftment and should therefore be considered in cell therapy studies.
The study was conducted between june 2007 and september 2007, and between december 2007 and february 2008 . The persons included in the present study were those who participated in a weight loss program at sendai health promotion center (weight loss program participants) and the staff of sendai health promotion center (weight loss program non - participants) in japan . The inclusion criteria for the intervention program were (1) both sexes and (2) age between 20 and 70 years . The exclusion criteria were history of diabetes mellitus, cancer, ischemic heart disease, stroke, or renal disease . The weight loss program was based on exercise program (exercise guidance, stretching exercise, and strength training) and nutritional program (nutritional guidance and cooking practice). With a meansd value of 57 g / ml in adiponectin, a minimum sample size of 50 subjects would be required to detect a difference (power=70%, two - sided =0.05). We asked 60 subjects to participate in this study and obtained informed consent from 51 subjects . The study protocol was reviewed and approved by the ethics committee of tohoku university graduate school of medicine . We used commercially available catechin - containing beverages (500 ml). According to the data provided by the manufacturer, the high - concentration beverage contained 400 mg catechin and the low - concentration beverage, 100 mg (table 1). Adherence to the study protocol was confirmed by asking the subjects to return the bottle caps and by reviewing their consumption records . Eligible participants were stratified by sex (men or women) and the weight loss program (participation or non - participation), and randomization was conducted by permuted block method using a four - person block . A total of 51 participants were randomly assigned by an epidemiologist (nn) to either the high catechin group (n=25) or the low catechin group (n=26) (fig . 1). The study participants were asked to stop gt consumption for 2 weeks (washout period), following which they were to start drinking the provided gt beverages everyday for 9 weeks . During the intervention period, the participants were asked not to drink any other catechin - containing beverage; other beverages were allowed . Both the catechin - enriched beverages had similar taste and appearance . At the end of the study components of the test beverages the outcome measures were changes in the adiponectin levels and cvd risk factors: body weight, body mass index (bmi), waist circumference, blood pressure (bp), and levels of total cholesterol (tc), ldl cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), triglyceride (tg), fasting plasma glucose, as well as aspartate aminotransferase (ast), alanine aminotransferase (alt), gamma - glutamyl transpeptidase (-gtp), uric acid (ua), and high - sensitive c - reactive protein (crp). Blood samples were collected into a tube containing ethylenediaminetetraacetic acid (edta)-2na and a tube containing heparin . Serum and plasma samples were obtained by a 10-min centrifugation at 3,000 rpm within 30 min of obtaining the sample . The samples were then transported frozen to the srl laboratory in hachioji, tokyo, japan, and stored at below 20c until analysis . Serum adiponectin level was determined by enzyme - linked immunosorbent assay (elisa; otsuka pharmaceutical, tokyo, japan), serum tc level, ceh - cdh - uv method (sysmex corporation, hyogo, japan), serum ldl - c level, liquid selective detergent method (sekisui medical, tokyo, japan), serum hdl - c level, accelerator selective detergent method (sekisui medical), tg level, enzymatic method without endogenous free glycerol (sekisui medical), fasting plasma glucose level, hk - g-6-pdh method (shino - test, tokyo, japan), ast, alt, and -gtp levels, jscc transferable method (kanto chemical, tokyo, japan), ua level, enzymatic method (sekisui medical), and the high - sensitive crp level was assayed by nephelometric immunoassay (siemens healthcare diagnostics, tokyo, japan). Bmi was calculated as body weight (kg) divided by squared height (m2). Bp was measured using a cuff placed on the upper arm of each participant in the sitting position . Nutrition surveys were carried out using a food frequency questionnaire (excel eiyokun) (21). Comparisons between the two groups were performed by student's t - test to assess the differences in the biochemical and anthropometric parameters at baseline . Effects of the intervention on serum adiponectin level and other outcome measures were tested using a paired t - test in each group before and after the intervention . Analysis of covariance was used to investigate the significance of the differences in the initial values as well as the net changes after the intervention between the two groups . We considered the following variables as potential confounders a priori: age at baseline in years (continuous variable), sex, and baseline level of each variable . In addition, stratified analyses according to weight loss program (participation or non - participation) were conducted . The study was conducted between june 2007 and september 2007, and between december 2007 and february 2008 . The persons included in the present study were those who participated in a weight loss program at sendai health promotion center (weight loss program participants) and the staff of sendai health promotion center (weight loss program non - participants) in japan . The inclusion criteria for the intervention program were (1) both sexes and (2) age between 20 and 70 years . The exclusion criteria were history of diabetes mellitus, cancer, ischemic heart disease, stroke, or renal disease . The weight loss program was based on exercise program (exercise guidance, stretching exercise, and strength training) and nutritional program (nutritional guidance and cooking practice). With a meansd value of 57 g / ml in adiponectin, a minimum sample size of 50 subjects would be required to detect a difference (power=70%, two - sided =0.05). We asked 60 subjects to participate in this study and obtained informed consent from 51 subjects . The study protocol was reviewed and approved by the ethics committee of tohoku university graduate school of medicine . We used commercially available catechin - containing beverages (500 ml). According to the data provided by the manufacturer, the high - concentration beverage contained 400 mg catechin and the low - concentration beverage, 100 mg (table 1). Adherence to the study protocol was confirmed by asking the subjects to return the bottle caps and by reviewing their consumption records . Eligible participants were stratified by sex (men or women) and the weight loss program (participation or non - participation), and randomization was conducted by permuted block method using a four - person block . A total of 51 participants were randomly assigned by an epidemiologist (nn) to either the high catechin group (n=25) or the low catechin group (n=26) (fig . 1). The study participants were asked to stop gt consumption for 2 weeks (washout period), following which they were to start drinking the provided gt beverages everyday for 9 weeks . During the intervention period, the participants were asked not to drink any other catechin - containing beverage; other beverages were allowed . Both the catechin - enriched beverages had similar taste and appearance . At the end of the study the outcome measures were changes in the adiponectin levels and cvd risk factors: body weight, body mass index (bmi), waist circumference, blood pressure (bp), and levels of total cholesterol (tc), ldl cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), triglyceride (tg), fasting plasma glucose, as well as aspartate aminotransferase (ast), alanine aminotransferase (alt), gamma - glutamyl transpeptidase (-gtp), uric acid (ua), and high - sensitive c - reactive protein (crp). Blood samples were collected into a tube containing ethylenediaminetetraacetic acid (edta)-2na and a tube containing heparin . Serum and plasma samples were obtained by a 10-min centrifugation at 3,000 rpm within 30 min of obtaining the sample . The samples were then transported frozen to the srl laboratory in hachioji, tokyo, japan, and stored at below 20c until analysis . Serum adiponectin level was determined by enzyme - linked immunosorbent assay (elisa; otsuka pharmaceutical, tokyo, japan), serum tc level, ceh - cdh - uv method (sysmex corporation, hyogo, japan), serum ldl - c level, liquid selective detergent method (sekisui medical, tokyo, japan), serum hdl - c level, accelerator selective detergent method (sekisui medical), tg level, enzymatic method without endogenous free glycerol (sekisui medical), fasting plasma glucose level, hk - g-6-pdh method (shino - test, tokyo, japan), ast, alt, and -gtp levels, jscc transferable method (kanto chemical, tokyo, japan), ua level, enzymatic method (sekisui medical), and the high - sensitive crp level was assayed by nephelometric immunoassay (siemens healthcare diagnostics, tokyo, japan). Bmi was calculated as body weight (kg) divided by squared height (m2). Bp was measured using a cuff placed on the upper arm of each participant in the sitting position . Nutrition surveys were carried out using a food frequency questionnaire (excel eiyokun) (21). Comparisons between the two groups were performed by student's t - test to assess the differences in the biochemical and anthropometric parameters at baseline . Effects of the intervention on serum adiponectin level and other outcome measures were tested using a paired t - test in each group before and after the intervention . Analysis of covariance was used to investigate the significance of the differences in the initial values as well as the net changes after the intervention between the two groups . We considered the following variables as potential confounders a priori: age at baseline in years (continuous variable), sex, and baseline level of each variable . Differences were accepted as statistically significant at p<0.05 . In addition, stratified analyses according to weight loss program (participation or non - participation) were conducted . All the study participants completed the study; 95.9% of the tea bottles were consumed in the high catechin group and 97.6%, in the low catechin group . Comparisons of baseline variables between the high- and low catechin groups are shown in table 2 . The proportion of women was approximately 65% in both the groups . With the exception of the baseline mean -gtp level, no other variable showed a significant difference between the two groups . Baseline characteristics of participants according to high - catechin group and low - catechin groupa values were expressed as meansd . P - values with chi - squared test for female ratio and for biochemical parameters, anthropometric parameters, nutrient intake, and energy expenditure, with student's t - test . There were significant reductions in total energy intake and carbohydrate intake in any of the groups . The intake of total fat, total dietary fiber, sodium chloride, and tocopherol was significantly decreased in the low catechin group . However, the net change between the groups was not significant . Change in nutrient intake and energy expenditure of participants according to high - catechin group and low - catechin group the change in high - catechin group minus the change in low - catechin group . The net differences were calculated by analysis of covariance . Adjusted for age (in years), sex, and individual baseline variables . After 9 weeks of catechin consumption, the meansd changes from baseline in the adiponectin level were 1.292.77 g / ml in the high catechin group and 1.001.87 g / ml in the low catechin group . We found no significant difference between the high- and low catechin group with respect to changes in the serum adiponectin level: 0.35 g / ml (95% ci: 1.03, 1.74). There were significant decreases in the body weight, bmi, and waist circumference in both the groups, but the net change was not significant for any of these variables . Furthermore, there were no significant differences in the net change in other variables as well . Change in serum adiponectin and cardiovascular risk factors of participants according to high - catechin group and low - catechin group the change in high - catechin group minus the change in low - catechin group . The net differences were calculated by analysis of covariance . Adjusted for age (in years), sex, and individual baseline variables . As for the net change in serum adiponectin, stratified analyses according to weight loss program (participation or non - participation) were conducted (table 5). Among weight loss program participants and weight loss program non - participants, there were no significant differences in the net change: 0.15 g / ml (95% ci: 1.54, 1.85) among weight loss program participants, and 1.49 g / ml (95% ci: 0.46, 3.43) among weight loss program non - participants . Change in serum adiponectin of participants according to high - catechin group and low - catechin group stratified by weight - loss program the change in high - catechin group minus the change in low - catechin group . The net differences were calculated by analysis of covariance . Adjusted for age (in years), sex, and baseline serum adiponectin . In this rct, we tested a hypothesis that consumption of catechin - enriched gt would affect the serum adiponectin level and cvd risk factors in apparently healthy subjects . After 9 weeks of catechin consumption, the meansd changes from baseline in the adiponectin level were significantly increased in both groups . However, we found no significant difference between the high- and low catechin group with respect to changes in the serum adiponectin level: 0.35 g / ml (95% ci: 1.03, 1.74). The cvd risk factors, namely, body weight, bmi, and waist circumference, were significantly decreased in both the groups, but the net change was not significant for any of these variables . There are at least three reasons that the changes from baseline in the adiponectin level were significantly increased in the both groups . First, more than half of the study participants participated in a weight loss program . Third, catechin was contained not only in high - concentration beverage (high catechin group; 400 mg) but also in low - concentration beverage (low catechin group; 100 mg). Therefore, if the low concentration of the catechins might be enough to increase the adiponectin levels, an increase would be observed in the both groups . In addition, we conducted stratified analyses according to weight loss program (participation or non - participation) because the change in adiponectin levels in both the groups may be related to the significant weight loss program . We also found that there were no significant differences in the net change among weight loss program participants and non - participants . Because the net change among weight loss program non - participants was greater than that among weight loss program participants, the change in adiponectin levels would be less affected by the weight loss program . To date, three rcts have examined the association between tea catechin consumption and adiponectin levels in humans (1820). These rcts recruited patients with diabetes mellitus and obesity . Because these patients might have had atherosclerosis before the study, the effect of tea catechin consumption on serum adiponectin level might not be well detected . Observed a change in the adiponectin level after the consumption of 900 ml of water containing 9 g of gt daily for 4 weeks in patients with type 2 diabetes mellitus (18). After 4 weeks, the meansd change in the adiponectin level from the baseline value was 6.033.71 g / ml in intervention group and 6.013.16 g / ml in control group, although the net change between the groups was not significant . Hsu et al . Observed a significant increase in the adiponectin level in obese women who consumed one capsule containing 491 mg of total catechin daily (19). After 12 weeks, the meansd change in the adiponectin level from the baseline value was 2.54.2 g / ml in intervention group and 2.05.4 g / ml in control group, although the net change was not significantly different . Observed a significant increase in the adiponectin level after the consumption of 582.8 mg of catechin daily in patients with type 2 diabetes mellitus (20). After 12 weeks, the meansd change in the adiponectin level from the baseline value was 1.320.61 mg / ml in intervention group and 0.340.48 g / ml in control group, although the net change was not significantly different . Thus, all the three rcts showed that the increase in the serum adiponectin level in the intervention group was greater than that in the control group, although the net change between the groups was not significant . Although we recruited healthy participants who did not have a history of diabetes mellitus, cancer, ischemic heart disease, stroke, or renal disease, our findings were consistent with those of the above reports . Although we found no significant difference in the net change in adiponectin level, several reasons should be considered in the interpretation of our results . First, it may be necessary to consider the difference in the catechin dose between the high- and low catechin groups and the intervention period . In the present study, the difference in the catechin dose between the high- and low catechin groups was only 300 mg / day and the intervention period was 9 weeks . Hsu et al . Adopted the difference in the catechin dose of 491 mg / day and intervention period of 12 weeks, but the net change in adiponectin level was not significant (19). Also, nagao et al . Adopted the difference in the catechin dose of 486.5 mg / day and intervention period of 12 weeks, but the net change in adiponectin level was not significant (20). Therefore, the difference in the catechin dose and the intervention period could not explain our observation . Second, because gt is consumed primarily in japan and china (22), habitual gt consumption may have the potential to affect study results . Although we adopted the washout period for 2 weeks, we did not find any apparent association between catechin - enriched gt consumption and adiponectin . Similarly, previous studies adopted the washout period for 2 weeks (19) and 4 weeks (20), but the net change in adiponectin level was not significant . Also excluded subjectswho had consumed gt regularly for over a month, but the net change in adiponectin level was not significant (18). Third, the compounds such as caffeine found in gt may have been responsible for the association between gt consumption and cvd risk factors . A previous observational study indicated the association between consumption of caffeine - containing coffee and adiponectin . No association between consumption of caffeine - containing coffee and adiponectin was indicated in either group (quartile 1: 0100 mg, quartile 2: 101237 mg, quartile 3: 237378 mg, quartile 4: 379967 mg) among non - diabetic subjects (23). Because the difference in the caffeine dose between the high- and low catechin groups was small (25 mg / day) in our study fourth, chocolate, red wine, apples, and berries are known as good source of catechin (24, 25). Although we asked participants not to drink any other catechin - containing beverage, we had no information on the intake of these food items during the 9 weeks . In addition, we had no data on the levels of the major dietary catechins (gallocatechin, epicatechin, epigallocatechin, etc .) And the total blood antioxidant levels . However, these factors may be divided equally between the high- and low catechin groups by successful randomization . Also, a previous study indicated that the half - lives of epigallocatechin-3-gallate, epigallocatechin, and epicatechin once ingested were 3.4, 1.7, and 2.0 h, respectively (26). Therefore, it is difficult to interpret the results of all - night fasting plasma levels of catechins, if measured . Finally, our rct design might yield a relatively small number of participants, although we made a power calculation regarding sample size . We found no significant difference between the high- and low catechin groups with respect to changes in the serum adiponectin level . Our study had a similar sample size to previous rcts, and our results were consistent with results of previous rcts (19, 20). Therefore, a larger sample size may be necessary to detect any effect of tea catechin consumption on serum adiponectin level . We found no significant differences in cvd risk factors between the high- and low catechin groups . Many studies have assessed the relation between gt consumption and cvd risk factors . In previous studies, the gt consumption showed statistically significant reductions in body weight, bmi, and waist circumference (2729). Our study showed that decrease in the anthropometric parameters in the high catechin group was greater than that in the low catechin group, although the net change between groups was not significant . The previous studies that suggested statistically significant changes had a larger sample size (28, 29). Therefore, a larger sample size may be necessary to detect the effect of tea catechin consumption on the anthropometric parameters . The effect of gt consumption on bp has been investigated in meta - analysis of previous studies . These data suggested that gt consumption did not show significant effects on systolic and diastolic bp (30). Our results on bp were consistent with the previous studies . Among previous studies that have examined the association between gt and blood cholesterol (tc, ldl - c, and hdl - c), gt consumption significantly lowered the tc and ldl - c level, but no effect on hdl - c was observed (20, 28, 30, 31). Our results on hdl - c were consistent with previous studies, but the inconsistent findings on the effect of gt consumption on tc and ldl - c were observed . There are several possible reasons for the discrepancy between our study and previous studies on tc and ldl - c . First, the subjects of the previous studies were not a healthy population (20). Therefore, one of the reasons for discrepancy might be explained by the difference in the study subjects . This rct showed that increase in serum adiponectin level in the high catechin group was greater than that in the low catechin group, although the net change between groups was not significant . Also, no significant difference was observed between the high- and low catechin groups with respect to changes in any cvd risk factors . This study was supported by a grant - in - aid for exploratory research (term of project: 20072008, project number: 19659157) from the japan society for the promotion of science (jsps), japan.
Hemorrhage after percutaneous nephrolithotomy (pcnl) is a significant complication that occurs in a small percentage of cases . However, arterial injuries sometimes require transarterial embolization to control the refractory bleeding . In patient presenting with refractory bleeding, pseudoaneurysm formation is the most common cause with a reported incidence of 0.6 - 1% . We report a case of early erosion of an embolization coil from the renal vasculature into the urinary collecting system causing urinary tract obstruction and urosepsis . A 23-year - old male underwent left urs and pcnl for a left upper ureteric calculus and a 2.5-cm lower pole renal calculus in a community hospital in december, 2010 . One week after nephrostomy tube removal, the patient experienced gross hematuria with clot retention . The patient underwent endoscopic bladder clot evacuation and dj stent removal in the same hospital . Two weeks later, again he developed gross hematuria with fever and a significant decrease in hematocrit . After resuscitation the patient was referred to our centre . On angiography, there was a pseudoaneurysm in the lower segmental renal artery . After 3 months of embolization, the patient presented with high - grade fever, left flank pain and burning micturiton . On x - ray kub 2 coils were seen in left renal area, whereas 1 coil was seen in l3 transverse process area . Axial noncontrast and contrast - enhanced ct images showed left hydronephrosis with dilated upper ureter and an embolization coil in the upper ureter [figure 1]. X ray kub shows 2 coils in left renal area, 1 coil in l3 transverse process area . Ct urogram shows left hydronephrosis with dilated upper ureter and 2 coils in left kidney & 1 coil in upper ureter in 1975, angioembolization for renal pseudoaneurysm was reported in a human patient when an autologous blood clot was used to embolize a bleeding pseudoaneurysm as a temporary measure before nephrectomy . We found only three case reports in which embolization coil migrated to the collecting system. [810] in all these case reports the coil eroded after 1 year following embolization . All the three patients presented with hematuria and renal colic . In our case, patient presented early (3 months) with complaints of renal colic and persistent fever after coil embolization . Hypothetically three mechanisms of coil erosion have been proposed . First, the diameter of arteriocaliceal communication might be larger than the embolization coil, and the coil gets extruded through the communication soon after placement . Second, the arteriocaliceal communication could get dilated over time because of inflammation due to infection or the constant irritation from the indwelling coil itself, leading to eventual erosion into the collecting system . Finally, rupture of the weakened wall of a pseudoaneurysm exposed to high arterial pressure may lead to erosion . According to the first hypothesis, if the arteriocaliceal communication was larger than the embolization coil than all three coils would have extruded through the communication and gross hematuria would have reappeared . The third mechanism would have lead to massive hematuria in addition to the coil migration . In this case the patient presented with high - grade fever and left flank pain but not hematuria . Therefore in this case the second mechanism seems to be the most likely cause of erosion of the embolization coil . One of the important but rare causes of urinary tract obstruction is the migration of embolization coil into urinary collecting system.
Drug reaction with eosinophilia and systemic symptoms (dress) is a life - threatening reaction that necessitates determination and discontinuation of the offending drug.the aromatic structure of teicoplanin is shared by most other medications involved in dress.the use of additional treatment including intravenous immunoglobulins, corticosteroids and antivirals is generally based on experience rather than proven benefits drawn from well - designed clinical trials . Drug reaction with eosinophilia and systemic symptoms (dress) syndrome is defined as an idiosyncratic, rare, and life - threatening reaction . The clinical features of the syndrome, including fever, rash, facial edema, lymphadenopathy, hematological abnormality, and internal organ involvement, arise 1030 days following drug exposure . This late onset of symptoms discriminates dress from some other drug - induced skin reactions such as erythema morbilliform [1, 2]. The most common suspected medicines causing dress include aromatic anticonvulsants (carbamazepine, phenytoin, phenobarbital, and lamotrigine), allopurinol, and antibiotics (sulfasalazine, vancomycin, and minocycline). To the best of our knowledge, there are limited reports of teicoplanin - induced dress in the literature [26]. Here a 37-year - old woman was admitted to hospital with redness and edema of inguinal area . The involved area was tender and warm on examination . With a presumptive diagnosis of cellulitis, after 24 h, due to the acceptable clinical state of the patient, treatment was planned to be completed in the ambulatory setting . Vancomycin was replaced with teicoplanin, considering its ease of administration as an intramuscular injection (400 mg every 12 h for three doses, then 400 mg daily). On the 14th day of treatment, the patient developed generalized maculopapular rash (fig . 1), accompanied by fever (39 c), wheezing, shortening of breath, and cervical and axillary lymphadenopathy . Lab tests revealed abnormal liver enzymes [alanine aminotransferase (alt) 134 iu / l, aspartate transaminase (ast) 141 iu / l], leukocytosis (white blood cell count 17,000/l) with eosinophilia to more than 8% (1360/l), a blood urea nitrogen (bun) value of 24 mg / dl, and a serum creatinine (scr) value of 0.8 mg / dl . The follow - up lab test performed 1 month later indicated resolution of liver dysfunction (alt 22 u / l, ast 18 u / l).fig . 1generalized maculopapular rash on the neck and trunk generalized maculopapular rash on the neck and trunk with respect to diversity in scoring systems and differential diagnoses, the exact incidence of dress, as a life - threatening skin reaction, remains unknown . This could be partially because there is no gold - standard test for diagnosis of dress, and as a result, the diagnosis remains a challenge and is mainly based on conventional proposed scoring systems . The most common scoring systems to stratify dress are regiscar, the japanese group s criteria for diagnosis of dress / drug - induced hypersensitivity syndrome (dihs), and a system proposed by kardaun et al . (table 1).table 1kardaun et al.s scoring system reproduced from kardaun et al ., with permissionitempresentabsentpatient s scorefever 38.5 c (101.3 f)010enlarged lymph nodes (> 1 cm size, at least two sites)101eosinophilia: 700 or 10% (leucopenia); 1500 or 20% (leucopenia)1; 201atypical lymphocytes100rash 50% of body surface area101rash suggestive (2 of facial edema, purpura, infiltration, desquamation)100skin biopsy suggesting alternative diagnosis100organ involvement: 1; 21; 202disease duration> 15 days020investigation for alternative cause (blood cultures, ana , serology for hepatitis viruses, mycoplasma, chlamydia), 3 done and negative100total score5total score <2: excluded; 23: possible; 45: probable; 6: definiteana antinuclear antibody kardaun et al.s scoring system reproduced from kardaun et al ., with permission total score <2: excluded; 23: possible; 45: probable; 6: definite * ana antinuclear antibody dress is classified as a type iv drug - induced hypersensitivity reaction that is characterized by delayed onset of symptoms . The rising of eosinophil count and non - necrotizing lesions differentiate dress from other type iv drug - induced hypersensitivity reactions such as stevens - johnson syndrome / toxic epidermal necrolysis (sjs / ten). In regard to delayed onset of signs and symptoms including skin rash (more than 50% of body surface area), fever (more than 38.5 c), and enlarged lymph node (more than 1 cm in two sites), dress was highly suspected . These findings are in concordance with previous reports of teicoplanin - induced dress [3, 4, 6]. Additional work - up was performed to evaluate hematological abnormalities and organ involvement, which revealed leukocytosis with eosinophilia and liver involvement . Chest x - ray or computerized tomography (ct) scan and skin biopsy were not performed due to patient non - compliance . Testing for human herpesvirus-6, human herpesvirus-7, and epstein - barr virus antibodies was not requested because of limited resources . In general, our presumptive diagnosis was mainly based on clinical signs and symptoms and accessible lab tests . On the basis of the scoring systems mentioned above, the reaction was rated as probable (score = 4) according to regiscar and possible (score = 5) according to kardaun et al.s scoring system . Since presence of atypical lymphocytes and reactivation of human herpesvirus were not investigated, dress was not confirmed by the japanese group s criteria for diagnosis of dress / dihs . The aromatic structure of vancomycin and teicoplanin may explain the occurrence of dress with these agents . In this case, teicoplanin was used instead of vancomycin according to the summary of product characteristics (available at http://www.sanofi.com.au). Given the similar structure of vancomycin and teicoplanin, . The implementation of additional treatment including intravenous immunoglobulins, corticosteroids and antivirals is generally based on experience rather than proven benefits drawn from well - designed clinical trials [1113]. Administration of corticosteroids in severe pulmonary involvement seems to be reasonable according to results from some studies [13, 14]. However, we did not implement these interventions, considering the lack of proven benefit and the patient s overall state of health . Sholeh ebrahimpour, mehdi mohammadi, and kheirollah gholami declare that they have no conflicts of interest relating to the content of this article.
Potential candidates include l - lactate, -hydroxybutyrate, d - lactate, salicylate, formate and oxalate in toxicological situations, pyroglutamate, semisynthetic penicillins, sulphate and hippurate in renal failure, and occasionally urate and amino acids with catabolic states and total parenteral nutrition . Reports of increased tricyclic acid (tca) cycle anions in shock are now emerging . Their presence is often inferred from the anion gap (ag), calculated as [na] + [k] - ([cl] + [hco3]). When its reference range is exceeded, a search for unmeasured anions should commence, irrespective of the overall metabolic acid - base status, because a competing metabolic alkalosis can mask their presence . Likely culprits vary with the clinical scenario, but the search usually starts with l - lactate and -hydroxybutyrate . During this process, stoichiometry is tracked between ag (measured ag normal ag) and the summed concentrations of suspect anions (always in meq / l, because we are dealing in electrical neutrality). If ag both sensitivity and specificity are reduced by perturbations of albumin (remembering that albumin negative charge forms the bulk of the normal ag), ph, [ca], [mg] and [phosphate]. The most promising alternative is the strong ion gap (sig) like the ag, the sig quantifies unmeasured anions minus unmeasured cations, but unlike its predecessor it is insulated from variations in [albumin], [phosphate], ph, [l - lactate], [ca] and [mg]. In the previous issue of critical care, bruegger and colleagues combine sig calculations with capillary electrophoresis, and report that anions associated with the tca cycle, specifically citrate and acetate, contribute to the metabolic acidosis of canine haemorrhagic shock . Their data originate from an earlier experiment designed to investigate the benefits of a perflurocarbon - based oxygen carrier during resuscitation from a predefined oxygen debt . Capillary electrophoresis on specimens before shock, during shock and on resuscitation revealed maximal citrate elevations of 1.9 meq / l, whereas the peak acetate increase was 3.4 although these findings fuel ongoing speculation concerning tca anions in shock, several potential confounders are worthy of comment . During preparation, the animals acquired major metabolic perturbations, with severe baseline hypoalbuminaemia (1.5 g / dl) and impressive hyperchloraemia (130 mmol / l), but (from the parent study) only mild anaemia (11 g / dl). Most surprising in this context was a massive baseline plasma acetate (2.4 meq / l), which is 40 times the level reported from a previous study in dogs (0.06 mmol / l). The postshock acetate peaked at 5.8 meq / l, over 30 times that in the previous report (0.19 mmol / l). To our knowledge such prodigious acetate levels are unprecedented outside the setting of exogenous administration . In the parent study, ringer's solution 15 ml / kg per hour was documented as infused during all but the shock phase . If this was ringer's acetate, and if the animals had received both saline (as stated by bruegger and colleagues) and ringer's acetate, then this would explain much . Of relevance is a report that exogenous acetate can elevate hepatic citrate . Although the authors acknowledge that they re - infused blood containing citrate phosphate dextrose solution during the shock phase, thus introducing exogenous citrate a final caveat is that charge and dissociation indices for human albumin used in this study differ from those for canine albumin, although the effect on sig calculations is probably small . Until now, talk of unmeasured ions in critical illness has largely been speculative, based on discrepancies in ag or sig . Nonetheless, since the late 1960s reports have emerged of accumulating tca cycle intermediates in shock and dysoxic states . The pattern reported by forni and colleagues in human metabolic acidosis differed substantially from the findings reported by bruegger and coworkers, with relatively small increases in isocitrate, -ketoglutarate, malate and d - lactate, and in some cases citrate and succinate . Only on aggregate were these sufficient to inflate the ag . It is insufficient to invoke' tissue stress' to explain such increases in tca anions . Elevations must be considered within the context of anaplerosis and cataplerosis, which combine to maintain adequate concentrations of tca intermediates . Ag = anion gap; sig = strong ion gap; tca = tricyclic acid.
Laryngeal cancers represent about 30% of head and neck cancers with a high incidence for the glottic location (from 25 to 85%) [1, 2]. There is currently no recommendation from the uicc (international union against cancer) for pre - therapeutic assessment (endoscopy, computed tomography, and magnetic resonance imaging) specifying in particular the need to involve them . Now the evaluation of the cartilaginous extension, especially thyroid, is an important element in the pre - therapeutic assessment of the endolaryngeal cancer . In case of infiltration the tumor is classified ct4a, and the patient is amenable to an aggressive therapy given the high risk of local recurrence and low radiosensitivity . The purpose of this study is to evaluate, by comparing, the existing correlation between preoperative radiological classification of endolaryngeal tumors involving the anterior commissure (endoscopy, ct scan) and postoperative classification (pt pathology). This is a single - center retrospective study (19982005) conducted at the croix - rousse hospital on 127 patients with endolarynx cancer involving the anterior commissure . All these patients were surgically treated and 32 of them with a total laryngectomy, the 95 other patients with a partial laryngectomy . Squamous - cell carcinoma represented the main pathology (124 patients) with a case of adenosquamous carcinoma, a case of pseudosarcomatous carcinoma, and a last case of verrucous carcinoma . All patients underwent a nasofiberscopy (including a verification of chordal mobility) followed by panendoscopy using optical at 0 and 30. radiological examinations were performed in seven different centers in 2 mm maximum sections, in all cases after injections of contrast . The tumor extension should be clarified to the following areas: sub glottis, paraglottic area, preepiglottic area, and cartilages (thyroid, cricoid). The patients were 58 years old in average, 93% of them were smokers, and more than half had a regular alcohol consumption . Table 1 summarizes the comparison of ct and pt stages for all tumors in the study . Only 76% of tumors have been classified correctly with the endoscopy and ct scan combination . It is more likely to correctly classify tumors with a limited size since only 64% of t3 and t4 tumors were correctly classified preoperatively . 24.6% of ct2 and 33.3% of ct3 tumors are actually reclassified pt4 after histopathological examination . This shows that the cartilage invasion is frequently under - estimated by the radiological assessment with endoscopy and cervical ct scan . This underestimation of the cartilage infiltration is more common than on the assessment of the subglottis, paraglottic areasc and preepiglottic extension . Only 2% of ct2 tumors were reclassified pt3, and 5% of the ct3 tumors were reclassified pt2 after pathological examination of the specimen . A decrease in the survival rates of laryngeal cancer in the united states, the terms of support, especially for the advanced stages, are currently controversial . If no conclusion of this study can be drawn in a formal and dogmatic manner, the proliferation of treatment options (radical surgery, partial surgery, transoral surgery, and laryngeal preservation protocols) requires more accurate and right pretreatment staging . One of the major means of all these studies, randomized or not, is the selection mean with the inclusion of patients with faulty pretreatment radiological staging . The underestimation of the thyroid cartilage invasion by cervical ct is found in many studies with variable rates . In a prospective study related to the cartilage invasion, realized on 40 patients, zbren et al . Found a 67% sensitivity and a 87% specificity of ct . When studies involve all endolaryngeal tumors, it appears that the anterior commissure of the underestimation is greatest . Thus, on nakayama and brandenburg's cases, 50% of t3 tumors were subclassified by breach of a micro - cartilage invasion . 90% of this situation involved tumors of the anterior commissure . The special case of the thyroid cartilage invasion via the anterior commissure was also clearly demonstrated . Barbosa et al . Then found a 25% overall underestimation of the ct stage of the anterior commissure tumors by the combination ct / endoscopy . However, reports show a stronger underestimation for smaller tumors classified as t1 and t2 (respectively 50% and 62% of correct estimation). Showed that the redefinition of radiological criteria allowed the increase in accuracy of radiological conclusions in two successive audit cycles by increasing the accuracy rate of 45% to 71% . This also shows that our balance sheet allows us to achieve correct figures and that theses are not related to poor quality of the examinations . Table 2 summarizes the various studies on the reclassification of endolaryngeal tumors after postoperative pathologic examination (initial radiological staging ct / endoscopy). Some scan signs to look for can increase the relevance of this paper . For the thyroid cartilage, specificity (ability to correctly identify individuals who are not affected by the disease) would be about 93% for erosion or lysis the combination of these signs causes a high sensitivity (ability to detect cases of a disease) and reinforces the negative predictive value (probability of being healthy if negative). Zbren et al . Also studied retrospectively the reclassification of the endolaryngeal recurrent lesions . In these cases, it appears that the combination ct / endoscopy is even less accurate to correct a tnm stage assessment . They report indeed a 48% sensitivity on the study of the thyroid cartilage invasion and 47% for the cricoid invasion . Our study did not use any mri as an examination to detect the cartilage infiltration . With a 8994% sensitivity and a 7488% specificity, its negative predictive value (npv) is around 9496%, but its positive predictive value (ppv) reaches only about 71% (probability of getting sick if positive). They are then many false positives with a significant risk of inadequate treatment by overestimation [1215]. This difficult pretreatment evaluation of the cartilaginous extension of endolaryngeal tumors leads us to suggest a surgical approach with resection of exposed cartilaginous portions as soon as the anterior commissure and the anterior portion of the subglottis are getting invaded . Our study shows that radiological pretreatment classification (ct) of laryngeal cancer involving the anterior commissure is often inaccurate when compared with postoperative pathology (pt). The mri appears to offer a more effective accuracy but still below the pt . This finding should lead to a transdisciplinary consideration from the moment the treatment choice is not directed towards a first surgical resection.
Malaria is a serious and sometimes fatal disease caused by a parasite, which is commonly seen in developing countries . Parenteral artesunate is the first line treatment for severe malaria worldwide, however it is not yet licensed in the united states . We report a case of a returning traveler with fever and delayed hemolytic anemia from artesunate given while in nigeria . A 68 year old african american woman presented with a three day history of fever to 102 f, periods of intermittent confusion, fatigue, anorexia, and myalgia . The patient was treated with artesunate for malaria in nigeria 3 weeks prior to admission . The temperature was 101.6 f, with a pulse of 106 beats / min, blood pressure of 125/80 and oxygen saturation of 99% on room air . Laboratory examination revealed a leukocyte count of 21,000/l with 70% neutrophils, 23% lymphocytes, 4% monocytes and 3% eosinophils, a hemoglobin of 8.4 g / dl which dropped to 7.0 on the fifth day of hospitalization, and a platelet count of 306,000 . The serum in the chemistry tube was grossly hemolyzed, with a potassium of 15 meq / l and normal renal function . However, the peripheral smear was remarkable for rouleaux formation and erythrocytes with atypical diamond - shaped central blanched pitting [fig . 1]. Further laboratory testing on hospital day 2 revealed an indirect bilirubin of 3.2 mg / dl, total bilirubin 4.4 mg / dl, ldh 965 iu / dl and haptoglobin 326 mg / dl (normal 43212). The anemia and hemolytic picture worsened over the next few days, before improving [table 1]. On hospital day 3, the patient was treated with hydroxychloroquine, despite having several peripheral smears negative for parasites . It was later discontinued because the patient developed a diffuse puritic rash thought to be a hypersensitivity reaction . On hospital the patient was discharged to home on hospital day 11 with oral anticoagulation for the pulmonary embolus . Severe malaria remains a serious clinical condition with considerable mortality . According to the world health organization, there were an estimated 198 million cases of malaria worldwide (range 124283 million) in 2013, and an estimated 584,000 deaths (range 367,000755,000) an estimated 207 million clinical cases of malaria each year and more than 600,000 malaria related deaths, the burden of disease remains unacceptably high . Artesunate is a derivative of the quing hao, or sweet wormwood plant, which has been used worldwide for more than 20 years for the treatment of malaria . It is used as first line therapy for severe malaria in sub saharan africa and southeast asia, but is not yet licensed in the united states . Although other agents are fda - approved for the treatment of uncomplicated malaria, intravenous quinidine gluconate is currently the only non - oral drug that is fda approved and available to treat severe malaria in the united states . In 2007, intravenous artesunate was only approved as an investigational new drug (ind) protocol by the fda in the united states . There have been 19 reported cases of delayed hemolytic anemia after treatment of severe malaria with artesunate during 20102012 . Worldwide, there have been 37 reported cases of hemolysis associated with the use of artemisinin derivatives in the treatment of severe malaria . To our knowledge, there have been only two similar cases of delayed hemolysis due to artesunate therapy described in the united states . Two distinct patterns of hemolysis after artesunate therapy have been described: a delayed onset or a persistent pattern of hemolysis . Persistent hemolysis is defined as continuing hemolysis starting around day 7 of artemisinin treatment and persisting beyond day 14 . Delayed hemolysis typically occurs 23 weeks after completion of artesunate therapy, as in our patient . A delayed hemolytic pattern is defined by a decrease in hemoglobin associated with a low haptoglobin or increase in ldh occurring at least 7 days following artesunate treatment . These latter findings were demonstrated in our patient, and after reviewing the clinical presentation and peripheral smear, it was determined that fever and delayed hemolysis were more likely to have been secondary to recent artesunate therapy in nigeria . One proposed mechanism is a splenic process called pitting, a process where artesunate exposed parasites are expelled from infected erythrocytes in the spleen . The erythrocytes then reseal (instead of lysing) and reenter circulation as pitted erythrocytes where they have a shortened lifespan . The delayed clearance of these pitted erythrocytes by the spleen may explain the features of post artesunate delayed hemolysis . A recent french study analyzed the hematologic parameters of 123 travelers treated with arteusnate for severe malaria . Among 60 nontransfused patients observed for more than 8 days, 13 (22%) the onset of hemolysis in the second or third week after treatment means that it is most likely to occur after a patient has been discharged from the hospital . . Physicians should be aware of this delayed side effect in order to prevent unnecessary further treatment of presumed malaria . Cdc has amended the artesunate ind protocol and now recommends that persons treated for severe malaria with artesunate be followed for 4 weeks after treatment and evaluated for hemolytic anemia . Still, in many parts of the world where malaria is endemic, artemisinin - based drugs are considered miracle drugs for resistant malaria . A multidisciplinary team approach is important for early diagnosis and appropriate management of this clinical presentation . In returning travelers from countries in which malaria is endemic, physicians in the u.s.a . Parenteral artesunate is only released by the cdc when parenteral quinidine is contraindicated, not tolerated or not available . Hemolysis probably results from delayed clearance of once infected erythrocytes which continue to circulate after artesunate has killed the parasites.
Association of osteomalacia or rickets with neurofibromatosis has been documented only rarely . As a rule, osteomalacia in neurofibromatosis is characterized by later onset in adulthood, renal phosphate loss with hypophosphatemia, and multiple pseudofractures in the typical cases . The hypophosphatemic conditions that interfere in bone mineralization comprise of many hereditary or acquired diseases, all of them sharing the same pathophysiological mechanism - reduction in the phosphate reabsorption by the renal tubuli . This process leads to chronic hyperphosphaturia and hypophosphatemia, associated with inappropriately normal or low levels of calcitriol, causing rickets in children and osteomalacia in adults . A 43-year - old woman presented to the orthopedic department of our institute with progressive bone pain and difficulty in walking since two years . The patient was a known case of neurofibromatosis-1, with multiple cutaneous nodules, from her early childhood . She was otherwise healthy until the age of 40 years, when she started experiencing progressive bone pain affecting her thighs, pelvis, and left forearm . In recent times, she could walk only with the help of crutches . On examination, tenderness and deformity were present in the regions of her left forearm, bilateral thigh, left knee, and leg . Multiple skin nodules were present all over her face, back, and abdomen [figure 1]. 43 yrs old, female known case of von recklinghausen neurofibromatosis with multiple skin nodules all over her face and legs her skeletal survey revealed generalized osteopenia, coarse trabeculations, and nodular shadows in the soft tissue . In addition, a radiograph of her left forearm with elbow revealed fractures of the upper shafts of the radius and ulna [figure 2]. A radiograph of her left knee revealed pseudofractures (looser's zone) in the distal femur, upper shafts of the tibia / fibula and mid shaft of the fibula [figure 3]. A radiograph of the pelvis with bilateral hips showed a deformed, triradiate pelvis, with bilateral symmetrical fractures of the upper shaft of the femur and pseudofractures of the inferior pubic rami [figure 4]. And lateral revealed diffuse osteopenia with fractures of upper shafts of radius and ulna (arrows) radiograph of left knee with leg a.p and lateral revealed generalized decrease in bony density with pseudo fractures (looser's zone) in the distal femur, upper shafts of tibia / fibula and mid shaft of fibula (white arrows). Note nodular shadows in soft tissue (black arrows) radiograph of pelvis - a.p . View . Triradiate pelvis with osteopenia and bilateral, symmetrical fracture of upper shafts of femur (white arrows) with bilateral, symmetrical pseudo fracture of inferior pubic ramii (black arrows) the laboratory data in our institute were as follows: serum calcium was 8.7 mg / dl (normal 8.5 - 10.5 mg / dl). Serum phosphorus was 1.5 mg / dl (normal: 2.5 - 4.5 mg / dl). Alkaline phosphatase was 650 lu / l (normal 44 - 147iu / l). The 24-hour urinary excretions of calcium and phosphorus were 98 mg/24 hours (normal: 0 - 300) and 440 nmol/24 hours (normal: 13 - 42), respectively . Her serum parathyroid hormone (pth) and 25-(oh) vitamin d were within normal range . On the basis of the radiological and laboratory findings, a final diagnosis of hypophosphatemic osteomalacia in a patient of von recklinghausen disease was made . She has been prescribed a high dose of calcitriol and oral phosphate and in view of innumerable neurofibromas, surgical resection was not advised . Her constitutional symptoms have improved, but the fractures have shown no radiological signs of healing in the last three months of follow - up . Oncogenic hypophosphatemic osteomalacia (oho) is a rare endocrinological paraneoplastic syndrome, characterized by defective bone mineralization from renal phosphate loss . It is an unusual condition, but probably still is the most common cause of acquired hypophosphatemic osteomalacia in adult males . The affected age group range is between seven and seventy - seven years with a male: female ratio of 1.2:1 . Patients characteristically present with joint deformities, waddling gait, bone pain, muscle weakness, anorexia, fatigue, and occasionally long - bone fractures . The initial clinical presentation may be mistaken for rheumatoid arthritis, muscular dystrophy or primary neurological disorder in some cases . If the biochemical profile of the patient is that seen in hypophosphatemia, namely low phosphate in the serum and high phosphate excretion in the urine, and the patient is not responding adequately to oral calcium and vitamin d therapy, then the possibility of an underlying cause of either renal or oncogenic or hereditary x - linked hypophosphatasia must be entertained . The most common tumor described is hemangiopericytoma, but other tumor types described include fibrous dysplasia, osteosarcoma, chondroblastoma, chondromyxoid fibroma, malignant fibrous histiocytoma, giant - cell tumor, hemangioma, paraganglioma, prostate cancer, and oat - cell carcinoma of the lung . Hypothesize that putative melatonin deficiency in cases of neurofibromatosis-1 may play a role in the pathogenesis of hyperphosphaturia, by decreasing the sodium - phosphate cotransport, increasing the level of cyclic adenosine monophosphate (camp) and the un - antagonized effect of dopamine on phosphate reabsorption, and increasing the glucocorticoid levels . Parathyroid overactivity that may occur secondary to osteomalacia may have synergistic effects with dopamine and further exaggerate the phosphate loss in urine . On the other hand moreover, in the presence of hypophosphatemia, hypercortisolism may further inhibit melatonin secretion that may lead to progression of bony deformities in these cases . In recent times, it has been suggested that in the epidermal nevus syndrome and type-1 neurofibromatosis, hypophosphatemic rickets / osteomalacia is probably due to increased secretion of fibroblast growth factor 23 (fgf-23) by cells from the nevus or neurofibromas . X - linked hypophosphatemic rickets, autosomal dominant hypophosphatemic rickets, tumor - induced osteomalacia (tio), fibrous dysplasia, and the mccune albright syndrome share a common underlying pathophysiological condition: increased phosphorus renal loss secondary to augmented fgf-23 plasma levels and activity . (a gene on the x chromosome that codes for a zn - metaloendopeptidase proteolytic enzyme, which regulates the phosphate). Fgf-23, the largest member of the fibroblast growth factor family (fgf), contains 251 amino acids and is encoded by a gene in 12p13 . The metabolic abnormalities in oncogenic osteomalacia are hypophosphatemia, hyperphosphaturia, low or normal serum calcium, raised alkaline phosphatase, low concentrations of 1,25 dihydroxy vitamin d, decreased tubular resorption of phosphates, normal paratharmone levels, and normal urinary calcium . These are believed to be the result of mesodermal dysplasia, intrinsic to the disease and they appear early in life . They cause bone deformities, and are not associated with disturbances in calcium and phosphate metabolism . In contrast, osteomalacia of neurofibromatosis is very rare, presents in middle age, and is associated with marked disturbance of phosphate metabolism . The presence of multiple symmetrical pseudofractures (looser's zone), high alkaline phosphatase, low serum phosphate, and generalized demineralization of the bones pointed to the diagnosis of hypophosphatemic osteomalacia . If complete excision of the tumor is achieved, it can lead to improvement in the clinical course of the disease as well as the biochemical markers, and may be curative . In patients with neurofibromatosis and osteomalacia, although innumerable neurofibromas are present, it is probably the largest ones or those with recent growth that cause oho and their surgical removal should be tried, to achieve permanent cure . High doses of calcitriol and oral phosphate salts are indicated, similar to those used in the treatment of x - linked hypophosphatemia . Osteomalacia in neurofibromatosis is a very rare entity and distinct from the more common dysplastic skeletal affections of this disease, as a rule, being characterized by later onset in adulthood, with renal phosphate loss, and with hypophosphatemia and multiple pseudofractures in typical cases . In patients with neurofibromatosis, although innumerable neurofibromas are present, it is probably the largest ones or those with recent growth that cause oho, and their surgical removal should be tried, to achieve permanent cure, along with high doses of calcitriol and oral phosphate.
Chronic pain affects up to 20% of the population in developed nations.14 this represents a profound impact on individuals and their families alongside the sizeable burden on employers, health care systems, and society in general.3 when chronic pain occurs, it has the potential to become disease itself, and subsequently, chronic pain has emerged as a distinct phenomenon.5 management of chronic pain varies greatly between nations and even within nations . Literature supports a multidisciplinary approach as the standard of care, although various health care systems may not always support this concept consistently.2 the current standard of care for chronic, noncancer pain typically includes many disciplines with the clinician developing an individualized treatment plan with the options of utilizing surgical interventions, pharmacology, and psychological and physical therapies . Opioid analgesics are often prescribed, despite the lack of clinical evidence supporting their long - term use in the management of chronic pain.6 however, for many patients, this multidisciplinary approach is inadequate or ineffectual or is accompanied by the burden of side effects that are unacceptable and debilitating . Only at this late stage, the field of neuromodulation for the treatment of pain has developed rapidly since the seminal paper on the electrical inhibition of pain by the stimulation of the dorsal column almost 50 years ago.7 the original term of dorsal column stimulation has evolved to become known as spinal cord stimulation (scs).8 scs has been particularly effective as an adjunct in treating mixed neuropathic / nociceptive and neuropathic / radicular pain conditions such as failed back surgery syndrome (fbss) and complex regional pain syndrome (crps). Neuromodulation therapies offer a treatment option that has minimal side effects and that is relatively safe and potentially reversible.9 scs has been used to treat various pain conditions for many decades.8,1013 in traditional scs therapies, the objective has been to replace the pain sensation with paresthesia that requires mapping of stimulation to the region of pain.14 the anticipation is that the electrical current alters pain processing by masking the sensation of pain with a comfortable tingling or paresthesia . Although patients mostly cope with paresthesia, a significant proportion report that the sensation is unpleasant, particularly with positional changes . The stimulation is provided either through electrodes that are placed percutaneously into the epidural space or through a surgical paddle lead that is delivered via a laminotomy.8 these devices are capable of delivering pulse frequencies in the range of 21,200 hz but are regularly utilized at 4060 hz . Patients typically undergo a trial of neuromodulation with an externalized power source and if this trial proves to be positive and compelling, they subsequently have a subcutaneously implantable pulse generator for the long - term therapy . In recent years, the next phase in the evolution of neuromodulation has become available with the development of dorsal root ganglion (drg) scs and the emerging use of two novel advances in stimulation frequencies, being high - frequency scs (at 10,000 hz) and burst scs.1419 these recent advances have improved the efficacy and expanded the applicability of scs . Drg scs is a highly targeted form of neuromodulation therapy.20 studies indicate that the drg plays a key role in both nociceptive and neuropathic pain.14,21 drg scs is particularly useful in treating focal areas of pain, in particular those that have been difficult to target with traditional scs systems such as groin and foot pain, by applying an innovative lead configuration and delivery system around the drg.8,9,15 high - frequency 10 scs (hf10) presents a significant development in the evolution of scs technologies.19 this involves application of a unique waveform at 10,000 hz at a subthreshold level and therefore provides pain relief without any paresthesia.18,22 the majority of patients have a clear preference for paresthesia - free stimulation, and hf10 has been approved for clinical use in australia and europe since 2011 and has received food and drug administration approval for the united states in 2015 for patients with chronic refractory pain of the trunk and/or limbs.22,23 burst scs offers another novel mode of stimulation whereby conventional frequency parameters are provided in bursts of five pulses . The burst frequency is 40 hz, and the pulse frequency is 500 hz . Amplitude is reduced to try and achieve subthreshold stimulation, thereby providing pain relief with either reduced or no paresthesia.16,17,24 the most recent systematic and comprehensive review of the effectiveness of scs in treating chronic spinal pain demonstrated that there is a significant (level i ii) evidence for scs as a treatment for lumbar fbss, where conventional medical management has failed.23 furthermore, there is now level i evidence for high - frequency stimulation but only limited evidence for burst stimulation.23 in another recent and extensive review and meta - analysis of conventional scs, more than half of all patients experienced significant pain relief.25 the authors observed that this was maintained for a mean follow - up period of 24 months.25 these reviews demonstrate that traditional scs is an effective treatment option for a cohort that is notoriously difficult to treat . The existing scs literature has a large number of case series reports and only a limited number of high - quality, large prospective, consecutively recruited, randomized, or controlled comparative trials (table 1).8,23,25 furthermore, the literature, when viewed historically, must be tempered by the developments in skills, application, and technological advances.26 hence, the traditional scs papers have often reported successful pain relief as an undifferentiated generic pain that is not specific to the site of the primary or greatest pain (eg, back or leg).25 this observation is important because conventional scs therapy has historically been prescribed for limb pain and has had only limited success in managing back pain.10,2729 indeed, predominant back pain has been an exclusionary factor in many studies.25 recent studies that have included back pain as the primary source have involved hf10 therapy at 10,000 hz; this therapy has evolved to better capture significant back, leg, and radicular pain.10,22 tolerance to scs has been observed in patients where pulse amplitude needs to be increased to achieve the same analgesic benefit over time and/or efficacy has been lost.30,31 tolerance cannot be predicted, and although the rate has not been widely reported in the literature, one study that researched over 10 years found it to be in the order of 29%.32 possible causes for stimulation tolerance include neuroplasticity of pain transmission pathways, cellular or fibrotic changes in the tissues around the electrodes, patients reframing their pain over time, and psychological or psychiatric affective disorders.30 however, data pertaining to hf10 scs have demonstrated no tolerance at this point.22 despite strict criteria for patient selection, a substantial number of patients fail to achieve optimal pain relief with scs.30,33 a number of factors have been identified as possible indicators for treatment failure including tobacco and drug use, age, and lengthy delay between times of original pain onset to scs implant.30,33 food and drug administration requirements for labeling the recent advanced iterations of scs systems have led, for the first time, to level i noninferiority comparative studies being undertaken to achieve labeling . Drg scs has been demonstrated as effective in multiple etiologies, including fbss, crps, and chronic postsurgical pain.15 a recent study reported 1 year outcomes for drg with overall pain scores reducing from 77.6 to 33.6 (p<0.005). Back pain reduced from 74.5 to 39.7 (p<0.05), and leg pain reduced from 74.6 to 28.7 (p<0.0005). The most compelling pain reduction happened for foot pain with scores reducing from 81.4 to 22.0 (p<0.05).15 approximately 60% of the drg scs patients reported> 50% improvement in their pain, and the pain localized to the back, legs, and feet was reduced by 42%, 62%, and 80%, respectively.15 other outcome parameters including quality of life, mood, and satisfaction were improved and maintained throughout the 12 months.15 the accurate study is a us pivotal, noninferiority, randomized controlled trial (rct) between drg scs and traditional scs medtronic system (medtronic, inc ., fridley, mn, usa). It is the largest rct in the history of crps and causalgia, running from 2013 with primary completion estimated for 2018 . The sample size for the study is 152; with 76 randomized to drg scs and 76 to the control arm using medtronic traditional scs . The inclusion criterion was leg pain for more than 6 months duration with a visual analog scale (vas) score> 6/10.34 in the intention - to - treat analysis, superiority was demonstrated in the drg scs group with 81% of patients achieving> 50% pain reduction and meeting the primary endpoint at the 3-month mark, and 74% maintaining that primary endpoint at 12-month follow - up . The traditional scs arm demonstrated 56% of patients having> 50% pain reduction at 3 months and 53% maintaining this through 12 months.34 in the subset analysis of the implant - only group, the data were even more impressive with 93% of the drg scs group meeting the primary endpoint at 3 months and 86% at 12 months . The traditional scs arm had 72% meeting the primary endpoint and 70% at 12 months.34 the statistical analysis demonstrated noninferiority and, beyond that, superiority in the intention - to - treat, modified intention - to - treat, and implant - only groups . Furthermore, it was noted that 70% of patients achieved> 80% pain reduction in the drg group versus 52% in the medtronic group . Target specificity for stimulation and pain relief was achieved in 94.5% of the drg group and 61% of the medtronic group.34 the sunburst study is set to run from 2013, with primary completion in 2016 . It is a prospective randomized, non - inferiority controlled trial with the st jude medical company (st jude medical, inc . Patients with intractable pain were randomized for the order they would receive either traditional or burst scs . The study was performed with a one - to - one crossover at 12 weeks to the alternate therapy . The study was applied to an enriched cohort with patients who required to have pre - existing pain scores> 6/10 and a> 50% pain reduction in a traditional scs trial using tonic stimulation . The mean age of patients was 59 years, with a median duration of pain being 13 years.35 the trial demonstrated noninferiority, and further statistical analysis demonstrated superiority for burst stimulation over tonic stimulation (p=0.035).35 the mean difference in burst pain reduction compared with tonic stimulation was 6 mm vas points . This difference, while being statistically significant, does not meet the well - defined criteria for minimal clinical important difference.36 approximately 65% of the burst cohort experienced paresthesia - free stimulation and 69% of the cohort chose a preference for burst, with the majority of these having their preference related to no paresthesia, more so than better pain reduction.34 the senza rct is a level i study design run from 2012 with an estimated primary completion in 2015.22,37 this is the first - ever rct of two scs therapies with patients randomized to hf10 scs (senza system; nevro corp ., redwood city, ca, usa) or traditional scs commercially available, precision plus, scs system (boston scientific corporation, marlborough, ma, usa). It is a noninferiority study with the statistical capability of demonstrating superiority supervised by the food and drug administration, and patients were monitored and programmed by the technicians associated with the respective devices . One hundred and ninety - eight patients were randomized with 101 to the hf10 scs group and 97 to traditional . Of these, 90 hf10 scs patients and 81 traditional scs patients were subsequently implanted . The primary endpoint of> 50% back pain reduction at 3 months was achieved in 80.9% of the hf10 scs group versus 42.5% of the traditional scs group.37 this met the criteria for noninferiority and statistical superiority (p<0.001). Furthermore, at 12 months, this primary endpoint was met in 78.7% versus 51.3% of the patients . Similarly, the primary endpoint for leg pain reduction was met in 80.0% of the hf10 scs group versus 49.4% of the traditional scs group.37 the responder rates for> 50% leg pain reduction at 3 months was 83.1% in the hf10 scs group and 55.0% in the traditional scs group . The 12-month outcome data for the same groups were 78.7% versus 51.3% (superiority p value, p<0.001).37 this study demonstrated superiority of hf10 scs to traditional scs in all primary and secondary endpoints that has led to the labeling of hf10 therapy as superior to traditional low - frequency scs by the food and drug administration . In further analysis of pain etiology, it was demonstrated that for the conditions of fbss, radiculopathy, degenerative disc disease, and spondylosis, the relative ratio of patients meeting the primary endpoint with hf10 scs was approximately 2.0 times the traditional scs (eg, fbss 85.7% versus 41.4%).37 further subset analysis of patients who achieved vas pain scores of 2.5 showed that for those with back pain, relief was maintained for 12 months 68.5% of the time for hf10 scs, but only 35.8% of the time using traditional scs . Whereas, for those with leg pain it was achieved 67.4% of the time for hf10 scs versus 42.5% of the time with traditional scs . Superiority p - values for hf10 were significant (p<0.001).37 these data demonstrate compelling evidence for treating complex back pain that was previously unheralded in the literature . Furthermore, paresthesia - free options allow the patient to keep stimulation on potentially 24 hours a day and hence sleep with the stimulator on and also perform activities such as driving . Health care policy and funding decisions require evidence of clinical efficacy and information around cost - effectiveness of treatments.38,39 consequently, there have been a number of studies considering the economic factors associated with scs . Most recently, a 2015 study investigating the cost - effectiveness of conventional medical management with or without scs in patients with fbss compared a summary of the total direct and indirect costs incurred in the 12 months prior and 24 months following scs.40 the costs were scaled to values of 2,009 . The year of implant incurred a significant increase in costs of 20,902/patient - year, mainly attributed to the high cost of the scs devices . In the following 1214 months, scs implant had dropped to 5,430/patient - year.40 applying the current united kingdom national health service threshold (intervention considered not cost - effective if the incremental cost - effectiveness ratio is higher than 45,000/quality - adjusted life year [qaly]), scs with conventional medical management would be considered cost - effective around 40% of the time.40 however, if the willingness - to - pay threshold was shifted to 60,000/qaly, scs would be considered cost - effective by the national health service with an average of 80% of the time . In 2013, a study developed models to evaluate the cost - effectiveness of scs and conventional medical management together compared with conventional medical management alone for patients with fbss and crps.41 health effects were expressed as qalys and costs were expressed as canadian dollars (can$) scaled to 2012 . The models were extrapolated over a 20 year - time period with 3.5% discounts annually (as per national institute of clinical excellence suggestion). The modeling data showed that scs with conventional medical management is cost - effective compared with conventional medical management alone for all presentations, with a cost - effectiveness ratio for scs of can$9293 for fbss and can$11,216 for crps per qaly gained.41 in a study in 2010, the cost - effectiveness of scs with conventional medical management was compared with conventional medical management alone in crps patients.42 this study models economic costs using a simulated model population, employing parameters and assumptions set from previously published randomized trials . Here, scs was shown to be cost - effective in select crps patients, with a probability exceeding 80% that scs is cost - effective where the willingness to pay is set for a maximum of 30,000 per qaly.42 another cost - effectiveness study of scs was performed in 2010 using a fbss cohort.42 here, the authors compared scs versus conventional medical management versus reoperation . This study showed that in selected patients, scs is cost - effective both as an adjunct to conventional medical management and as an alternative to reoperation; that the likelihood scs would be cost - effective versus conventional medical management and versus reoperation exceeds 80%, where the willingness to pay is set for a maximum of 20,000 per qaly.42 in 2008, a systematic review for cost - effectiveness of three studies where fbss patients had been treated with scs demonstrated that scs is both more effective and less costly than conventional medical management alone in the long - term but there are high upfront implant costs associated with scs implantation and maintenance.43 in 2008, another study reported the generic health - related quality of life and costs of scs at 6 months follow - up (using data from the process trial) compared to the quality of life, resource consumption, and costs of conventional medical management alone in patients with fbss.39 the study found that the mean total health care costs for the scs group were significantly higher (12,653) than the conventional medical management group (2,594), when scaled for uk 20052006 national data.39 this result reflects the high upfront costs of scs over the limited 6-month follow - up period . The authors showed that 15% of the additional mean cost of scs was offset within 6 months by a reduced use of drug and nondrug therapies . They also demonstrated a gain in quality of life over the same period which was significantly greater for the scs group . The study concludes that, over the short term, scs treatment results in greater healthcare costs but also generates important health improvements for the patients over the same period.39 cost - efficacy studies show that despite significant initial costs, scs compared with other conventional treatments available to chronic pain patients results in long - term reductions in health care costs, which offset the high initial treatment costs over time.44 in the literature, scs is reported as a safe procedure due to its reversible and minimally invasive characteristics.30 although catastrophic complications are possible, they are very rare . However, the incidence of minor complications of scs has been reported at around 30%40%.13,30,31,4547 these minor complications tend to occur within 12 months of implantation and are readily reversible and generally resolved.13 the complications are divided into three main categories: mechanical, biological, and technique - related.32 complications of a mechanical origin are more common than those of biological origin.31,45 historically, hardware - related complications occurred at a rate of between 24%50%, whereas adverse biological events occurred in 7.5% of cases.30 mechanical complications include lead fracture or disconnection, which has a reported incidence of between 5% and 9%; lead migration has a reported incidence between 0% and 27%; implantable pulse generator failure occurred at a reported frequency of 1.7%.11,30 these complications can be minimized by using appropriate leads, anchoring, and suturing techniques . Furthermore, minimizing patient movements in the first 3 months after surgery allows for postoperative scarring of leads into place.30 kapural et al22 demonstrated that lead migration of significance and requiring intervention in both the hf10 and traditional scs arms occurred <5% . This most likely reflects improvements in both lead design and the anchoring systems used (figures 13).14,22,26,48 biological complications include infection, allergic reaction, pain at implant site, implantable pulse generator seroma, epidural fibrosis, epidural hematoma, dural puncture, and, rarely, neurological injury.30,31,4952,5760 the most common biological complication is infection with a rate between 3% and 8%, and the majority of these are superficial.30 as is seen in the more recent literature,30 incidence of infection can be minimized via pre and postoperative antibiotic use and appropriate skin preparation . The occurrence of dural puncture is reported as between 0.3% and 2%.53 other adverse biological events such as epidural fibrosis, compressive phenomenon, or spinal cord injury, while serious, are rare . The neuromodulation appropriateness consensus committee has recommended a number of criteria and adaptations to practice to help reduce complications: physician training and mentoring, the appropriate and careful selection of patients, a continued focus on equipment development and innovation, as well as the dissemination and application of practice and research - based advances.30 as this field strives to provide high - quality, transparent, and independent empirical evidence for scs therapies, it is possible to overlook the primary rationale for scs treatment in the first place: patient benefit . Improvements to training in pain management are needed, beginning at the undergraduate medical level . At present, the time allocated to pain management is generally inadequate; for instance, in the united kingdom, the median time spent on pain management by a medical student is 13 hours, and sometimes it is as little as 6 hours.2 indeed, when the undergraduate training of all healthcare professionals is analyzed, education about the identification, assessment, and treatment of pain represents less than 1% of university - based teaching yet pain is the most common reason for patients to consult their general practitioner.2 the undergraduate training of all healthcare professionals is analyzed, education about the identification, assessment, and treatment of pain represents less than 1% of university - based teaching yet pain is the most common reason for patients to consult their general practitioner.2 the ensuing repercussions may include inadequate diagnosis, inappropriate treatments, and extended periods of mismanagement . Further complicating the treatment of this cohort has been the inclusion of neuromodulation therapies as a treatment of last resort.54 yet research has shown that shifting scs forward in the treatment algorithm of refractory chronic pain is associated with better patient outcomes.44,55 in recent years, many researchers and practitioners have included patient satisfaction (alongside empirical measures) as a clinically useful metric for assessing scs treatment success.23 when reported, overall patient satisfaction is high for the vast majority of scs patients, perhaps reflecting the efficacy of scs treatment, but may also reflect factors such as limitations of alternative treatments, safety, and tolerability of scs and the rapidity of onset and durability of scs treatment.56 moreover, under current arrangements, most patients access scs treatment through private health insurance schemes or through compensatory bodies such as workers compensation schemes.57 for patients who do not qualify for these funding systems, the immediate cost outlay would likely post a significant financial barrier to treatment.57 the scs specialty should aim to support the provision of equitable and accessible treatment for all . Significant evidence exists for traditional scs as a safe, clinical, and cost - effective treatment for many chronic pain conditions . Indeed, the field is rapidly evolving, and there is now level i evidence for newer techniques including hf10 scs and drg scs, which demonstrate dramatic improvements in overall efficacy in reducing pain in specific conditions, including failed back surgery, back pain, neuropathic leg pain, crps, and causalgia . Furthermore, the field has increasingly met the challenge of not only having newer devices to achieve these outcomes but concurrently reducing the risks of complications and adverse events . The data supporting scs in its multiple forms are compelling and have reached a level that now demands that this therapy be considered earlier in the treatment continuum and to be no longer regarded as simply an end - stage salvage therapy.
Testicular germ cell tumors comprise the most common group of solid malignancies in male population of 15 to 35 years old . These tumors account for approximately 95% of all testes cancers . The incidence of testicular tumors varies in different countries and ethnic groups with the highest reported incidence in scandinavia region (about 9 in 100,000 males). Few reports are present about the incidence and epidemiologic features of testis cancer in the male population of asian and latin american countries, which indicate that testicular germ cell tumors are less common in these regions than most western countries . A recent study has shown that the incidence of testis cancer is 1.3 in 100,000 male population in iran . These include positive family or personal history of testis tumor, cryptorchidism and intratubular germ cell neoplasia (itgcn). Earlier studies have claimed that concomitant itgcn is seen in 80%90% of testes affected by germ cell tumors using immunohistochemistry (ihc). Thus, itgcn is also considered as a premalignant lesion and prompt treatment is essential if it is incidentally discovered in testis biopsy . In addition, in cases of testis sparing surgery, itgcn may be observed in the remnant tissue . In these situations,, we aimed to assess the prevalence of itgcn in the patients who have underwent radical orchiectomy due to testicular tumor, as well as its correlations with pathologic features of concomitant testis cancer and patients' epidemiologic characteristics . To our knowledge, this is the first report on itgcn in association with testicular germ cell tumor in our region, also the first study which is looking for its prognostic role . This is a descriptive analytical and cross sectional study which was performed in our center . The study population was the patients who underwent radical orchiectomy due to testis tumor from 2003 to 2015 . Serum tumor markers had been measured before the surgery and consisted of alpha - fetoprotein (-fp), and beta - human chorionic gonadotropin (-hcg). Cd-117 (c - kit) and placental - like alkaline phosphatase were two immuno - reactive agents that were used to identify itgcn via ihc . Data about patients' age at the time of surgery, predisposing factors (such as family history, cryptorchidism), testis atrophy, and serum tumor marker before surgery were recorded and gathered for further analysis . Pathologic reports were categorized as pure seminoma, mixed germ cell tumors (mgcts; defined as containing at least two pathological subtypes of germ cell tumors), pure mature teratoma, embryonal carcinoma, yolk sac tumor, lymphoma and benign lesions (leydig or sertoli cell tumor). Data was gathered and analyzed using ibm spss ver . 18.0 (ibm co., armonk, ny, usa). Independent sample t - test was obtained to assess any relationship between itgcn and quantitative factors . For qualitative factors we used chi - square test and fisher exact test as needed . A p - value less than 0.05 was considered as significant . The institutional review board of iranian urology and nephrology research center approved the study design . Mean age of the patients was 34.5 years old (range, 182 years). Eighty - four point four percent (84.4%) of patients (n=151) were in the range of 20 to 50 years and 2.2% (n=4) were under 12 months old . History of testis atrophy (confirmed by ultrasound study) was observed in 8 cases (4.5%). Right and left testicles were involved by tumor in 96 and 83 cases, respectively . Calcification (diagnosed by ultrasound study) was observed in 87 patients (48.6%). Pathologic reports showed that pure classic seminoma and mgcts were the most common histologic types . Considering the pathologic components of all cases (including those with mgct), we found that seminoma was also the most prevalent component . Pathologic features of the patients are demonstrated in table 1 . While primary study of the specimens by light microscopy leaded to diagnosis of itgcn in only 21 patients (11.7%), further analysis by ihc staining increased this figure to 85 patients (47.5%). So, it seems that light microscopy missed about three - fourths (75%) of patients with itgcn . With exclusion of lymphoma and benign lesions, the prevalence of concomitant itgcn was determined as 49.4% . There was not a statistically significant difference in mean age, histologic type, histologic components, cryptorchidism, and lymphovascular invasion between the 2 groups (p=0.151, p=0.11, p=0.233, p=0.413, and p=0.14, respectively). However, calcif ication of testis parenchyma, as identif ied by ultrasonography, was lower in patients who had concurrent itgcn (35% vs. 59%, p=0.001). The prevalence of itgcn was significantly lower in the patients with stage t1 than those with higher stages (t2). Among the 150 patients with stage t1, 62 (41%) had itgcn, while 18 out of 29 patients (62%) with stage t2 and t3 had itgcn (p=0.03). Since patients with pure seminoma do not present with elevated serum tumor markers, any relationship between itgcn and tumor markers was assessed in 98 patients with nonseminoma histology . Fifty - two patients with nonseminoma pathology had concomitant itgcn, of which 44 patients (84.6%) had elevated serum -fp before the surgery, while in the other group, only 24 patients (52%) had elevated serum -fp level . Thus, elevated serum -fp level is much common in patients with itgcn (p<0.001). Prevalence of elevated serum -hcg was 58% and 64% in patients with and without itgcn, respectively; which did not indicate a significant difference (p=0.6). There are several risk factors that contribute to testis cancer, including family or personal history, cryptorchidism, and itgcn . Skakkebaek have noticed that almost all types of testicular germ cell tumors (except germ cell tumors in children and spermatocytic seminomas) originate from itgcn . The microscopic features of itgcn resemble that of seminoma and ihc is needed for exact diagnosis . The results of our study shows that light microscopy might underestimate itgcn in patients with testicular germ cell tumors . It is higher in western countries and scandinavia region, and lower in african americans and asian countries . The incidence of testicular cancer is about 5.3 cases per 100,000 in the united states, but the highest rate of testis cancer has been reported from scandinavia region as about 9 in 100,000 . Several studies have shown that the incidence of testicular germ cell tumors is increasing . A recent study by basiri et al . Has shown that the incidence of testis cancer is lower in iran than western countries . In 2009, since itgcn is a major risk factor and precursor to most testicular cancers, we decided to evaluate the prevalence of itgcn and its correlations with other histologic factors in an iranian population who had testis tumor . Earlier studies by skakkebaek and montironi indicated that 80%90% of patients who undergo radical orchiectomy have simultaneous itgcn in the adjacent tissue . Another study by klein et al . Have shown that 82% of seminomas and 75% of nonseminomas are associated with itgcn . Although mean age of patients was lower in itgcn group (32.6 years old), it was not significantly different from the overall group . . The reason may be that both itgcn and seminomas both originate from primordial germ cells (gonocytes). Current data in the literature supports the idea that microlithiasis is not an independent predisposing factor for testicular cancer, although it may accompany other possible premalignant features . A study by meissner et al . Concluded that microlithiasis may be accompanied by itgcn, and thus, healthy patients with several risk factors who present with microlithiasis need to undergo routine testicular biopsy to rule out itgcn or testis tumor . This study and other previous surveys, evaluated the significance of microlithiasis and its correlation with itgcn in healthy patients who did not have testis cancer at the time . However, the results of our study indicate that in patients who suffer from testis cancer, microlithiasis does not accompany itgcn . In support of this idea, a recent literature review revealed that there are 5 conditions in relation to microlithiasis and testicular cancer: down syndrome, mccune - albright syndrome, cryptorchidism, infertility and familial disposition of testicular cancer . Itgcn was much common in patients with higher tumor stage (i.e., t2, t3). This may indicate that itgcn is related with more aggressive tumors, which is a new finding . In addition, in patients with nonseminoma germ cell tumors, elevated serum -fp level was closely related to itgcn . More than 84% of patients with itgcn had high serum levels of -fp before orchiectomy . This study was the first to evaluate the associations of itgcn with epidemiologic and pathologic factors of testis cancer . High volume, multi center studies with larger sample size are needed to get more constant and reliable information about itgcn . The prevalence of concomitant itgcn in the iranian population who undergo radical orchiectomy is lower than most western countries . Itgcn is more common in higher tumor stages and is accompanied with elevated serum -fp levels before the surgery . Since the prevalence of itgcn is higher in the regions with high incidence of testis cancer and in high stages of germ cell tumors, presence of itgcn in adjacent tissue may suggest a negative cancer behavior.
A cross - sectional study was conducted to assess the level of radiation safety practices and awareness among 300 general dental practitioners in trivandrum district, kerala, india . A questionnaire comprising 18 questions in clinical, radiographic practice was formulated . Information regarding demographic data such as age, sex, qualification, and years of experience was also obtained . After obtaining clearance from the institutional research and ethical committee, the questionnaire was distributed among general practicing dentists and collected back with their response . An awareness brochure pertaining to radiation safety independent samples t - test, at p = 0.05 significance level, to compare mean scores between qualifications, and years of experience, was used for analysis . The results were assessed by chi - square statistical test and other software (microsoft excel + spss 20.0 trail version, ibm corp . Released 2011 . Independent samples t - test, at p = 0.05 significance level, to compare mean scores between qualifications, and years of experience, was used for analysis . The results were assessed by chi - square statistical test and other software (microsoft excel + spss 20.0 trail version, ibm corp . Released 2011 . Total sample size of the study was 300 general practitioners (247 female and 53 male), of which 83% of the dentists had been practicing for more than 5 years and 259 out of 300 dentists were general dental practitioners with no specialized qualification . Intraoral radiographic machines with conventional x - ray films were widely used by them in their practice (63.3%), followed by radiovisiography (rvg) (17.7%) and osteoprotegerin (2%) and 17% were using a combination of radiographic machines and techniques in their practice . Most of the dentists (71%) instruct their patients to hold the intraoral periapical (iopa) film with their fingers while carrying out the radiographic exposure and only 16.7% were using holders in their practice while 12.3% of the clinicians used other methods for placing iopa films . On statistical analysis, p = 0.579 for practical score was obtained based on qualification [table 1], and based on years of experience, p = 0.834 for practice score was obtained [table 2]. Independent samples t - test to compare mean scores between qualifications independent samples t - test to compare mean scores between years of experience many of the practitioners (28.3%) followed exclusively the position distance rule while 22% were found to be using lead barrier . Sixteen percent used lead aprons in their practice, and 33.3% have used a combination of safety techniques such as lead apron and lead barrier . However based on the qualification, an attitude score with p = 0.081 is obtained [table 1], and by year of experience, p = 0.307 is obtained [table 2]. Only 22% of the practitioners were aware of special situation such as pregnant women and children who are more susceptible to the hazardous effect of radiation . Most of the dentists (84.3%) reported that they were aware of as low as resonably achievable (alara) principle, but 66.7% were not aware of the atomic energy regulatory board (aerb) recommendations . Ninety - eight percent of the practitioners were aware of the function of thermoluminescent dosimeter (tld) badges, but only 2% of them were using tld badges in their practice . A mere 14.7% of the dentists were aware of annual whole body radiation limit for radiation worker and only 21% of the practitioners knew about radiation dose limit for the patient . Majority of the dentists were interested in updating their knowledge about radiation hazard and safety using various modes, 52.3% by continuing medical education programs, 7.3% by articles and journals, and 13% by internet update and the remaining 27% through a combination of continuing medical education programs, journals, workshops, and internet updates . Based on their qualification, a knowledge score of p = 0.924 [table 1] was obtained, and based on the years of experience, a knowledge score of p = 0.216 [table 2] was obtained . A study done on radiation protection by svenson and petersson among swedish dental practitioners revealed their knowledge, attitude, and practices in the field as dentist having higher level of knowledge with 525 years of experience than those with lesser or greater years of experience . The study also revealed that specialists have a better knowledge than other general practitioners . In our study too, we found that practitioners with more than 5 years of clinical experience had better awareness regarding radiation hazards and safety but scored poorly when it came to practices pertaining to the same [table 2]. All the practitioners included in the study were found to have facilities for radiographic investigation in their clinic . Among them, in that 63.3% were using intraoral radiographic machine with conventional x - ray films . Hayakawa et al . Have done a study on radiation dosage reduction in general dental practice using digital intraoral radiographic systems, which concluded that radiation exposure is reduced to 4060% in rvg than using e - speed intraoral films . International recommendation for radiological protection also recommends using rvg by which radiation exposure can be reduced by 60% as compared to e - speed intraoral films . Comparison among the machines commonly used for radiographic investigation revealed that intraoral periapical machine was used by 63.3% of the dental practitioner according to the aerb, safety code for the installations of medical diagnostic x - ray equipment mentions that all x - ray using institutions should have a separate x - ray room and specific instructions for the particulars in the room . Among 300 general practitioners, 80.3% had a separate section for radiographic examination in their clinic . Since 1977, the international commission on radiological protection started to implement risk / benefit concept . All radiation exposure done in medicine must be based on the alara principle (as low as reasonably achievable). In the present study, ilgy et al . In their study to check dentist's knowledge about radiographic equipment, dose reduction techniques, and quality of dental radiographic service in general dental practice in turkey indicate that for reducing any unnecessary radiation, attempts should be made to improve dentist's knowledge about radiation dose reduction techniques . Analysis of the awareness about alara principle revealed that 84.3% of the dentists are aware of it a study by sheikh et al . Reported poor radiation protection practices among indian dental practitioners . Our study revealed that majority of the practitioners (28.3%) still followed the position and distance rule, 22% were found to be using lead barriers while 16% mere used lead aprons and 33.3% were using a combination of various safety techniques [graph 3]. Moreover, shielding includes both protective barriers such as lead shield and personnel protective measures such as lead apron . Ninety - eight percent reduction in scattered radiation and attenuate dose to 0.04 r can be achieved using lead aprons . Patient should wear thyroid collar during radiation exposure as it reduces attenuation of scattered radiation to 92% . Proper shielding from radiation and by increasing the distance from source protect radiographer as well as patient for unnecessary exposure to radiation . According to position distance rule, radiographer position should be at least 6 feet from the source at an angle of 90 to 135 to the central ray of x - ray beam . A study by noohi among radiographers in kerman (iran) concluded that percentage of protective shields used for patient and radiographers is 0.01% and 15.7%, respectively . However, another study by mojiri and moghimbeigi among radiographers in hamandan city shows significantly higher results of 78.9% and 83.1%, respectively . Analysis of the type of protective measures being used for radiation safety revealed that a maximum of 28.3% of practitioners were following position and distance rule the present study concludes that 90.3% of general practitioners were not providing any safety measure for their patients [graph 4]. Shahab et al . In their study among iranian dentists concluded that most of the dentists are not using proper method, material, and equipment for reducing unnecessary radiation exposure to patients . Film holders not only help radiographers to position the film but also help patients to avoid unnecessary exposure to their fingers . Our study shows that 71% of the dentists are instructing their patients to hold the iopa films with their fingers and only 16.7% are using holders in their practice and 12.3% of clinicians use other methods for placing the iopa films [graph 5]. Analysis of the attitude of the general practitioners revealed that 90.3% of them are aware about radiation safety measures for patient comparison of the techniques being used for taking intraoral periapical radiograph revealed that 70.9% of the practitioners are asking the patient to hold the intraoral periapical film during the x - ray exposure radiation exposure to pregnant women causes several biological effects on fetus such as intrauterine death, developmental abnormalities, and mutagenic carcinogenic effects . In pregnancy, it is better to avoid radiation exposure during the first trimester, i.e., during 815 week of pregnancy . If radiological investigation is inevitable, it should be carried out during the second and third trimester with proper protection by means of lead apron, thyroid collar, etc . Our study reveals that only 22% of dentists were aware of their patients who are more susceptible to radiation, such as pregnant ladies and children . Arnout and jafar done a questionnaire - based study on 57 undergraduate dental students reveal that about 3060% of future dentists do not undertake any radiographic procedure for pregnant patients, without considering patient's trimester or level of emergency . Another study by razi et al . On 250 general dentists in tabriz concluded that dentists do not have sufficient awareness regarding radiation risk associated with pregnancy . They monitor and provide data regarding exposure dose limits and proper undertaking of protective measures . In india, lithium borate, calcium fluoride, and calcium sulfate are materials used in tld badges which emit light by the stimulation of heat . The two regions where these badges to be worn are on the trunk, at the level of the radiographer's waist on the anterior aspect or on the collar area of upper chest region on the anterior aspect . Our study revealed that over 90% of the dentists were aware of tld badges but a paltry 2% were using it in their practice . Recommended occupational dose limit by the aerb is 20 msv / year the present study revealed that only 14.7% of the dentists were aware of annual radiation dose limit for radiation workers . Another study by amirzadeh and tabatabaee among radiation employees reveals that 51.2% of the workers were aware of radiation dose limit . A study done by mojiri and moghimbeigi among radiographers in hamandan city revealed 58% of radiographers to be aware of radiation dose limit . Biological effects of ionizing radiation are grouped into stochastic and deterministic effects . Above a particular determined dose of radiation biological damage stochastic effect is that in which there is no particular dose level above which biological change occurs in the body . Ionizing radiation causes both the effects depending on radiation doses and body's response to these radiations . Dentists as well as patients are more prone to the risk of stochastic effects because of its lack of a dose threshold limit . The positive aspects of disease diagnosis and disease detection should be considered while evaluating the risks of the biological effects of radiation . Over 88% of the dentists in our study concluded that 70% of the general practicing dentists in their group were unaware of the biological hazards of radiation . It is very important for dentist to update their knowledge about new trends in diagnostic techniques, protective measures, etc ., this can be achieved by means of continuing education, journals, workshops, and other media . The distribution of the preferred method of updating their knowledge and awareness is illustrated in [graph 6]. Studies by aps to assess general dental practitioner's knowledge about dental radiography and radiation protection in belgium show the results that clearly indicated the need for continued education on the subject . A study by amin tavakli et al . In shahid beheshti university of medical science also revealed that the dentist believes they need continuing education programs in radiology to update their awareness . Analysis of methods by which dentists wish to update their awareness revealed more than 50% of the dentists wish to update through continuing education programs the study suggests that practicing dentists need to be enticed and encouraged to conform to the rules of protection from x - rays; implementing and enforcing the matter via enforcement agencies will take us a long way in this context . Protection of one's self and patient from all kinds of health hazards is the hallmark of concerned doctors . The aerb recommendations should reach out through the dentists platform (e.g., ida) to the dental practitioner . Better safe than sorry remains no more a virtue but a fundamental necessity.
Reliable information on morbidity and mortality is the key for evidence - based public health practice . More emphasis on estimating burden of diseases was given at global level, especially since the launch of global burden of disease study . While the decision - making priorities of global health authorities are dependent on these projected data, developing countries often lack the local data for proper public health planning . Health professionals still fights to establish vital registration, while the morbidity surveillance is still in its initial stage . In countries where information is available, data from ambulatory care services, primary healthcare centers, and outpatient departments (opd) are often lacking . Economic impacts of the mild to moderate ill health conditions are considerable given the large number of patients utilizing these services . As an example, in sri lanka, more than 43 million visits are made to the opds in government hospitals annually . Given that the only 50% of outpatient care is delivered through public sector, the total outpatient visits are estimated to be more than 80 million, four times the sri lanka population of 20 million . Despite this heavy burden, information on opd morbidities are not routinely collected and used in healthcare planning . As a preliminary study to assess the burden of disease and morbidity pattern in opd, we conducted a rapid survey in a tertiary care hospital in sri lanka . Study sample consisted of opd patients visiting teaching hospital peradeniya, which is the teaching hospital for second largest medical school in sri lanka . Three of the six authors physical presented at the opd during the study period, explained the procedure to patients, and consented patients filled a self - administered short questionnaire . A half - page questionnaire was used and the main problem which patients sort medical advice was inquired . A total of 3817 patients attended opd during the study period and 1908 were recruited as the study sample using the systematic sampling strategy . Of them, 1439 completed the questionnaire with a response rate of 75.4% . A significantly higher number of females (924,64.2%) visited the opd compared to males (515,35.8%) (chi square for goodness of fit 116, p<0.001). The age of the patients attending the opd ranged from 1 year to 89 years . The mean age of the study sample was 40 years (sd = 19 years). Majority of the females attending the opd were of middle age while males were distributed evenly among all age categories . The mean age of females was significantly higher than that of males (t = 4.03, p<0.001). Housewives constituted the major occupational group among females, accounting for 52% of the total sample, followed by adolescents (15.9%). Ten leading cause of opd visits were nonspecific body aches and pains 225 (15.6%); respiratory symptoms (cough and cold) 151 (10.5%); abdominal pain 122 (8.5%); fever - 117 (8.1); backache and joint pains 112 (7.8); skin conditions - 106 (7.4); constipation - 84 (5.8); chest pain 66 (4.6); diarrhea - 59 (4.1); and dog bite / s 51 (3.5). Table 1 shows the leading morbidities according to sociodemographic profile of the patients . Among children (10 years)/school children, cough and cold was the commonest complaint followed by dog bites . Constipation (14.5%) was the commonest presenting complaint among tamils (6.3%) but was not in the leading five commonest complaints among the other ethnic groups . Main presenting symptom among adolescents was various skin conditions, which was third commonest condition among young adults . Commonest presenting complaint by demographic characteristics among patients attending outpatient department of teaching hospital peradeniya, sri lanka (n = 1 439) this could be explained partly from the so - called male - female health survival paradox (i.e., males report better health than females, but encounter higher mortality at all ages). There is growing evidence to conclude that men are healthier, but have substantially higher mortality rates . However, this could be also due to a gender difference in health - seeking behavior as shown in previous studies. [46] children less than 10 years accounted for only 5% of the opd patients, which shows a marked difference from the private sector utilization in sri lanka where 32% of encounters were reported as children, which was completely different from the findings of the present study . Previously, respiratory tract problems were reported as the commonest encounter in government phc setting as well as in gp practice in sri lanka, with higher percentages ranging from 20% to 55%. [79] in our study, respiratory problems was the commonest among younger age groups . However, nonspecific body aches and pains was the leading encounter . However, this problem needs to be studied to provide meaningful services . Despite having low annual numbers of rabies, dog bite showed to be a leading problem among children . Reported constipation encounters as leading reason for opd care seeking among tamil ethnic group these observations from a single hospital could not be generalized to sri lanka or other developing countries . However, our study provides early evidence for probability of changing morbidity patterns with demographic transitions that should be taken into account in public health program planning.
A combination of fracture shaft of humerus with ipsilateral shoulder fracture dislocation is rare injury . Only few cases are reported in the english literature till date having anterior dislocation of shoulder and ipsilateral closed humeral shaft fracture [110]. There are cases of two - part fracture dislocation associated with humeral shaft fracture [2, 58, 10] and cases of three - part fracture dislocation associated with humeral shaft fracture . We report an even rarer combination of injury where there was a two - part fracture dislocation of shoulder and ipsilateral compound fracture of humerus shaft . We have discussed the probable mechanism of injury, and share our difficulties while managing this case and have reviewed the literature . A 30-year - old female came to the emergency department at midnight following a road traffic accident with severe pain and swelling over the left shoulder and arm . Her arm was trapped when the vehicle in which she was travelling skidded off the road . On examination she had degloving injury over posteromedial aspect of the arm extending to axilla with acute bend over arm and loss of shoulder contour (fig . 1b) showed anterior dislocation with displaced greater tuberosity fracture and transverse fracture of humerus in its middle third . An external fixator was applied on humerus and then shoulder was reduced without difficulty . After reduction, greater tuberosity fracture was found displaced so it was fixed with a cannulated cancellous screw through a deltoid splitting approach (fig . Skin grafting over the raw area was done on 10th day after the wound bed was ready . There was poor take over axillary region so another grafting was done after 1 week . After 3 weeks, wound healed completely and then, passive movement at shoulder joint was started . After two and half months, the external fixator was removed and functional brace was applied . After 4 months, the fracture had united completely (fig . She had a useful range of motion at the time of brace removal, with abduction and flexion of around 90. external rotation was 20 while internal rotation was normal . At the last follow - up of 11 months, she had a good range of motion with abduction and flexion of 160flexion and full rotation . (b) preoperative x - ray of left shoulder with arm anteroposterior view showing dislocation of shoulder, greater tuberosity fracture and humerus mid - shaft fracture . (c) postoperative x - ray showing shoulder reduction, greater tuberosity fracture fixation with a cannnulated screw and humerus fracture fixed with external fixation . (d) four months postoperative x - ray showing fracture union . (a) clinical picture at the time of presentation to emergency . (b) preoperative x - ray of left shoulder with arm anteroposterior view showing dislocation of shoulder, greater tuberosity fracture and humerus mid - shaft fracture . (c) postoperative x - ray showing shoulder reduction, greater tuberosity fracture fixation with a cannnulated screw and humerus fracture fixed with external fixation . Fracture shaft of humerus associated with ipsilateral dislocation is rare injury, whereas isolated shoulder dislocation or humerus fracture is very common . Anterior dislocation is relatively more common than posterior, in association with humerus shaft fracture . Of the reported cases, almost all so, this case, where we have the fracture dislocation with ipsilateral compound fracture of humerus shaft, is probably one of the few reported cases and is important due to three aspects . Sankaran - kutty and sadat - ali suggested that force exerted along the axis of the humerus simultaneously fractured the humerus and dislocated the shoulder, whereas kontakis et al . Proposed that the shoulder dislocated first and subsequent, bending or torsion forces fractured the shaft of humerus . We believe that the mechanism of injury in our patient was as described in the second case by kontakis et al ., and this is supported by the fracture pattern that is transverse, acute bending of arm during emergency presentation and degloving of skin flap over arm with compounding of fracture . Closed reduction is sometimes assisted by steinman pin as reported by barquet et al . And winderman . Sankaran - kutty and sadat - ali used limb lengthening apparatus to assist in closed reduction of shoulder . Open reduction was required in cases reported by kontakis et al . And baker . Only in few cases, the authors have reduced the shoulder prior to fracture fixation, otherwise all have fixed fracture before reduction of the shoulder . Splint, casting [1, 3, 4, 9, 10], plates and screw [4, 5, 8], external fixator and even intra - medullary nail [2, 3] all have been used with consistent result in fracture union . In our case too, we fixed the fracture prior to reduction . We were bound to use external fixator in our case due to compound nature of injury . We fixed the greater tuberosity fragment through a deltoid splitting approach after thorough debridement of wound and did skin grafting on the raw wound later . We believe that external fixator is not ideal for humerus fracture with shoulder dislocation as it is cumbersome, it affects mobilization and hence the outcome of shoulder movement but it has consistent results on fracture union . The presence of wound required skin grafting later and this also affected early mobilization of the shoulder . Almost all reported cases have reported satisfactory outcome (some degree of restriction of abduction and flexion) despite different modalities of management . We also had a good outcome in our patient despite having more severe injury, open fracture and associated fracture of greater tuberosity and the fact that she was managed with external fixator initially . We believe that following the basic principles of trauma care was the key for good outcome in this case.
Metastases in the head neck region from renal cell carcinoma (rcc) are seen in less than 15% of patients with metastatic disease . Majority of the times, it is a manifestation of widespread metastases in a known case of rcc, but such lesions can be the initial presentation of the disease . Primary localizing sites in the head neck region include nodal and soft tissue metastases, sinonasal region, nasopharynx, parotid, larynx, thyroid gland, orbit, and tongue . Lingual metastases on the dorsum, ventrum, lateral border, and tip are more frequently reported than metastases to base tongue . A 48-year - old gentleman was diagnosed as a case of left - sided rcc in june 2004, which was treated with radical nephrectomy and adjuvant radiotherapy at an outside hospital . In october 2006, he presented to our hospital for second opinion in view of progressive metastatic deposits in both the adrenals, lungs, and mediastinum . He was started on interferon (inf-, 5 miu daily subcutaneous doses), which he continued till december 2007 . He was advised external beam radiotherapy for the base tongue lesion but refused treatment due to personal reasons . In december 2009 he was barely able to swallow liquids orally and had lost 2 - 3 kg of weight . On clinical examination, he had a large (5 5 cm) exophytic swelling in the oropharynx with epicenter in the right base of tongue [figure 1]. It involved the tonsillar fossa and adjacent soft palate on right side, crossing midline . Computerized tomography (ct) scan of the face, neck, thorax, abdomen, and pelvis was suggestive of widespread visceral and osseous deposits apart from the base tongue lesion [figure 2]. No adenopathy was noted (which corroborated with secondary from rcc as it is uncommon for large oropharyngeal primary of squamous carcinoma to present without neck nodes). In view of predominant symptom of dysphagia and exophytic growth, the patient received radiotherapy to a dose of 50 gy in 25 fractions over 5 weeks with limited portals . Protracted regimen (prolonged fractionated course of radiotherapy over 5 weeks as compared to the most commonly used short course of palliative radiotherapy) was chosen expecting sustained and relatively quicker relief of symptoms . By the end of treatment subsequently, he was referred to medical oncologist for targeted therapy . Till the time of last follow - up (3 months postradiotherapy), durable response at the base tongue lesion was documented but unfortunately he was yet to start targeted therapy due to financial issues . (b) regression of base tongue metastases after palliative radiotherapy contrast - enhanced ct scan of face and neck illustrating the right base tongue metastasis the kidney is third common primary infraclavicular location of malignant neoplasms metastasizing to head and neck . Tongue base is a peculiar favorable site for metastases due to its rich vascular supply . Early lesions may be asymptomatic or present with some discomfort in the throat, while moderate to large size lesions may present with either dysphagia and change in voice (as in the present case) or develop bleeding and pain at the local site . Review of base tongue metastases from renal cell carcinoma the rarity of this bizarre site of metastases can create a diagnostic dilemma . Primary malignant clear cell tumors of the head and neck arising from the salivary, thyroid, or parathyroid glands and secondaries from lung and female genital tract form the diagnostic differentials . Cross - sectional imaging would be helpful for lingual metastases which show high signal due to their vascular nature or occurrence of intralesional hemorrhage . Renal cell carcinoma even with metastatic disease has a protracted clinical course and patients survive relatively longer as compared to metastatic disease from other histologies . Hence renal metastatic disease is comparatively treated more aggressively with an intent to contain the disease for relatively longer time and achieve sustained palliation . Based on the reports in literature, surgical excision is recommended as the primary treatment for base tongue metastases, provided good structural and functional outcome can be achieved . However, prognosis for patients with lingual metastasis of rcc with disseminated disease has been reported to be poor despite aggressive local treatment . The aim is primarily to provide relief from dysphagia and pain while preventing bleeding and infection . Radiation therapy and immunotherapy or immune - chemotherapy for palliation of lingual metastases has produced encouraging results . Contrary to the traditional belief of relative radio- and chemo - resistance of rcc metastases, secondary lingual lesions from rcc have responded remarkably to 5 weeks of protracted radiation therapy as well as to 1 month of inf- therapy . Infact, it was concluded by the authors that tongue is a possible sensitive site to ifn- therapy as better response as compared to pulmonary, bone, and lymph node metastases was noted . With regards to the efficacy of radiation therapy for base tongue metastases per se, authors have not come across any report in literature . Considering the possibility of avoiding a major surgical intervention and expecting a better functional outcome, radiation therapy is an acceptable palliative strategy . On the other hand, as compared to inf- palliative radiotherapy produces prompt relief of pain, bleeding, and dysphagia . The kidney is third common primary infraclavicular location of malignant neoplasms metastasizing to head and neck . Tongue base is a peculiar favorable site for metastases due to its rich vascular supply . Early lesions may be asymptomatic or present with some discomfort in the throat, while moderate to large size lesions may present with either dysphagia and change in voice (as in the present case) or develop bleeding and pain at the local site . Review of base tongue metastases from renal cell carcinoma the rarity of this bizarre site of metastases can create a diagnostic dilemma . Primary malignant clear cell tumors of the head and neck arising from the salivary, thyroid, or parathyroid glands and secondaries from lung and female genital tract form the diagnostic differentials . Cross - sectional imaging would be helpful for lingual metastases which show high signal due to their vascular nature or occurrence of intralesional hemorrhage . Renal cell carcinoma even with metastatic disease has a protracted clinical course and patients survive relatively longer as compared to metastatic disease from other histologies . Hence renal metastatic disease is comparatively treated more aggressively with an intent to contain the disease for relatively longer time and achieve sustained palliation . Based on the reports in literature, surgical excision is recommended as the primary treatment for base tongue metastases, provided good structural and functional outcome can be achieved . However, prognosis for patients with lingual metastasis of rcc with disseminated disease has been reported to be poor despite aggressive local treatment . The aim is primarily to provide relief from dysphagia and pain while preventing bleeding and infection . Radiation therapy and immunotherapy or immune - chemotherapy for palliation of lingual metastases has produced encouraging results . Contrary to the traditional belief of relative radio- and chemo - resistance of rcc metastases, secondary lingual lesions from rcc have responded remarkably to 5 weeks of protracted radiation therapy as well as to 1 month of inf- therapy . Infact, it was concluded by the authors that tongue is a possible sensitive site to ifn- therapy as better response as compared to pulmonary, bone, and lymph node metastases was noted . With regards to the efficacy of radiation therapy for base tongue metastases per se, authors have not come across any report in literature . Considering the possibility of avoiding a major surgical intervention and expecting a better functional outcome, radiation therapy is an acceptable palliative strategy . On the other hand, as compared to inf- palliative radiotherapy produces prompt relief of pain, bleeding, and dysphagia . The kidney is third common primary infraclavicular location of malignant neoplasms metastasizing to head and neck . Tongue base is a peculiar favorable site for metastases due to its rich vascular supply . Early lesions may be asymptomatic or present with some discomfort in the throat, while moderate to large size lesions may present with either dysphagia and change in voice (as in the present case) or develop bleeding and pain at the local site . Primary malignant clear cell tumors of the head and neck arising from the salivary, thyroid, or parathyroid glands and secondaries from lung and female genital tract form the diagnostic differentials . Cross - sectional imaging would be helpful for lingual metastases which show high signal due to their vascular nature or occurrence of intralesional hemorrhage . Renal cell carcinoma even with metastatic disease has a protracted clinical course and patients survive relatively longer as compared to metastatic disease from other histologies . Hence renal metastatic disease is comparatively treated more aggressively with an intent to contain the disease for relatively longer time and achieve sustained palliation . Based on the reports in literature, surgical excision is recommended as the primary treatment for base tongue metastases, provided good structural and functional outcome can be achieved . However, prognosis for patients with lingual metastasis of rcc with disseminated disease has been reported to be poor despite aggressive local treatment . The aim is primarily to provide relief from dysphagia and pain while preventing bleeding and infection . Radiation therapy and immunotherapy or immune - chemotherapy for palliation of lingual metastases has produced encouraging results . Contrary to the traditional belief of relative radio- and chemo - resistance of rcc metastases, secondary lingual lesions from rcc have responded remarkably to 5 weeks of protracted radiation therapy as well as to 1 month of inf- therapy . Infact, it was concluded by the authors that tongue is a possible sensitive site to ifn- therapy as better response as compared to pulmonary, bone, and lymph node metastases was noted . With regards to the efficacy of radiation therapy for base tongue metastases per se, authors have not come across any report in literature . Considering the possibility of avoiding a major surgical intervention and expecting a better functional outcome, radiation therapy is an acceptable palliative strategy . On the other hand, as compared to inf- palliative radiotherapy produces prompt relief of pain, bleeding, and dysphagia . It should be considered in the differential diagnosis of malignant tumors with clear cell histologic pattern . Base tongue metastases from rcc are exquisitely responsive to both radiation therapy as well as immunotherapy and form acceptable palliative strategies.
Glucose homeostasis is maintained through the efficient modulation of insulin production and release by the pancreatic beta - cells coupled to a correct response of insulin - sensitive cells to the hormone . Failure of the beta - cells to produce adequate amounts of insulin triggers progressive glucose intolerance and eventually overt type 2 diabetes mellitus (t2 dm) (1). T2 dm is a global public health problem, affecting nearly 285 million individuals; prevalence of diabetes is projected to rise to 435 million by 2030 (international diabetes federation, diabetes atlas . The pathophysiological mechanisms underlying beta cell failure remain poorly understood, limiting the availability of novel approaches to treat or prevent t2 dm . Monitoring the transcriptome of functional and disturbed beta - cells might reveal genes and pathways involved in the maintenance of normal beta - cell functional capacity . In march 2006, a consortium of recognized european experts in the field of t2 dm initiated eurodia, an integrated project devoted to understanding the biology of the pancreatic beta - cell . Several transcriptomics experiments were planned using two different technologies, custom spotted arrays and affymetrix chips, on three organisms: human, mouse and rat . The eurodia database has been developed as a tool to integrate heterogeneous gene expression datasets, to enable sharing of data and to provide efficient analysis methods to mine the information content . Several public datasets from arrayexpress (2), ncbi gene expression omnibus (3) and the betacell gene bank (4,5) were first integrated into the system and, as the project evolved, new unpublished experiments were added and combined with public data for analysis . To stimulate collaboration, once published in the database, these experiments were shared freely between members of the consortium . At the time of publication, the eurodia database contains 38 curated experiments (441 hybridizations), 13 of which were produced by members of the eurodia project . To ensure continuous access to this valuable data collection after the formal end of the project, the eurodia database has now been opened to the whole t2 dm research community for both consultation and contribution . The eurodia database has been built using gene expression data analysis interface (gedai), a flexible framework for storing, analyzing and sharing gene expression data and results . Gedai was originally developed for eurodia, and is currently being used as a gene expression data storage and analysis pipeline for several other research projects . The eurodia database is a web - accessible resource for storing and analyzing gene expression data from pancreatic beta - cells . Raw and processed data files quantified from individual hybridization scans are grouped into experiments which are briefly described with a name, description, type (one or more per experiment) and ownership . Experiments are grouped into projects and are related to an organism (human, mouse or rat) whose genome is annotated with ncbi entrez gene data (6). These genome annotations enable comparisons between experiments studying either the same organism but using different array designs, or experiments studying different organisms . Additional annotations for the affymetrix mouse 430_2 array based on probe exon mapping, signal intensity and uniqueness on the mouse genome have also been included (7). At the time of publication eurodia and public. The eurodia project contains experiments performed by members of this european consortium and the public project contains experiments imported from public repositories . All eurodia experiments have both raw and normalized data available (table 1), whereas some of the public experiments have only normalized data . Table 1.list of experiments contained in the eurodia databasenamegrouptechnologyreferencespancreatic islet implanted transgenic (expressing survivini) in micepublicaffymetrixbeta cells (min6) treated with amylin at different times and doses and growth at different concentrations of glucosepublicaffymetrixbeta cell specific ablation of foxa2 (hnf-3b) in micepublicspottedmin6 cells treated with exendin-4 over timepublicspottedpancreas versus isletspublicspottedtranscription profiling of mouse islet growth after partial pancreatectomy and exendin-4 treatmentpublicspottedmouse islets treated with pancreatic derived factor for 48 or 72 hpublicspottednon - diabetic and diabetic obese mice isletspublicaffymetrixhsl ko and wtpublicspottedtungstate - induced beta cell mass increase in stz ratspublicaffymetrixidentification of tissue restricted and interferon alpha regulated transcripts in human islet tissuepublicaffymetrixlcm human islet beta cellspublicaffymetrixnestin positive cells versus human isletspublicaffymetrixpancreas versus isletspublicaffymetrixhuman pancreatic islets from normal and type 2 diabetic subjectspublicaffymetrixtranscription profiling of hnf4 alpha null mice to investigate the phenotype of dis - regulated insulin secretion and abnormal beta cell growthpublicspottedcyclophosphamide - induced beta cell destruction in nod micepublicaffymetrixppar gamma overexpression and activation in rat pancreatic isletpublicaffymetrixpancreatic beta cell lines overexpressing wild - type mody genes or mutant hnf1b allelespublicaffymetrixislet amyloid polypeptide effect on pancreatic cell line: time course and dose responsedpublicaffymetrixtranscription profiling of rat pancreatic islets after culture in low, intermediate and high (glucose)publicaffymetrixdetailed transcriptome atlas of the pancreatic beta cellpublicaffymetrixgsis study of rat ins1 cell linespublicspottedlipotoxicity study of rat ins1 cell linespublicspottedfoxa1 and beta cell functionpublicspottedidentification of novel cytokine induced genes in rat pancreatic beta - cellseizirikaffymetrix(26)global profiling of double stranded rna- and ifn - gamma - induced genes in rat pancreatic beta cellseizirikaffymetrix(27)cytokine - induced and nuclear factor - kappa b - dependent genes in primary rat beta - cellseizirikaffymetrix(28)global gene expression profiling in cytokine - treated rat pancreatic beta - cellseizirikaffymetrix(29)global alternative splicing profiling in cytokine - treated rat pancreatic beta - cellseizirikaffymetrix(29)gene expression differences between control and tcf1/hnf1alpha knock - out pancreatic isletsferreraffymetrix(30)gene expression differences between control, pancreas - specific hnf4alpha knock - out(p - hnf4ako, mediated by expression of pdx-1cre transgen),tcf1/hnf1alpha heterozygous (hnf1ahet) and double mutant (p - hnf4a;hnf1ahet) pancreatic isletsferreraffymetrix(31,32)gene expression data from murine islet samples comparing transgenic mice with beta cell specific hormone sensitive lipase (hsl) overexpression with wild - type miceholmaffymetrixcalcineurin inhibitors on human isletsmarchettiaffymetrix(33)type 2 diabetic versus non - diabetic isletsmarchettiaffymetrixbeta cells of type 2 diabetic subjects versus beta cells of non - diabetic controlsmarchettiaffymetrix(34)islet s / a lf versus hf dietschuitaffymetrix(35)islet wt versus islets s / a control dietschuitaffymetrix(35)hf - diet versus normal diet in islets of wt c57bl6 miceschuitaffymetrixtime - course hfd study in wt miceschuitaffymetrixdouble gip - r + glp1-r ko mice pancreatic isletsthorensspotted(36,37)comparison glp-1 or glp-1+diazoxide treatment of betatctet cells for 7h . Pancreatic beta cell linethorensspottedtreatment of betatctet cells with glp-1 (glucagon - like peptide 1) for 0, 45 min, 3 h and 7 hthorensspotted(38)direct comparison of glp-1 and gip treatment for 7 h on betatctet pancreatic beta cell linethorensspottedhsl: hormone sensitive lipase, ko: knock - out, wt: wild - type, lcm: laser capture microdissection, gsis: glucose stimulated insulin secretion, s / a: eif2-ser51 is mutated to encode an alanine, lf: low fat, hf: high fat, hfd: high fat diet . List of experiments contained in the eurodia database hsl: hormone sensitive lipase, ko: knock - out, wt: wild - type, lcm: laser capture microdissection, gsis: glucose stimulated insulin secretion, s / a: eif2-ser51 is mutated to encode an alanine, lf: low fat, hf: high fat, hfd: high fat diet . To upload an expression dataset, the user provides experiment annotation in a predefined microsoft excel template and uploads this file together with the raw data files (cel files for affymetrix; genpix, imagene for spotted arrays) zipped into an archive . Once uploaded, the raw data are then normalized using rma (8) for one color arrays or loess (9) for two channel arrays and several quality control plots are generated to assist the user in identifying poor quality hybridizations . Raw data can be downloaded either as binary (affymetrix) or text (spotted) files . Normalized data annotated at the probe level with the manufacturer provided annotations can be downloaded as text files . Additionally, both raw and processed data can be downloaded as a bioconductor (10) expressionset object, which can be easily loaded into an r session to perform analyses that are not included in the eurodia database . The browse page of the eurodia database (figure 1) provides a convenient way to access an experiment . From this page the user can visualize quality control graphs, download data or perform statistical analysis for a dataset . Experiments are grouped by experiment type (for example: time series, dose response, compound treatment or genetic variation), project, laboratory affiliation, organism or array design, thus providing multiple entry points for a user to access a particular experiment . (a) experiments can be grouped by type, laboratory affiliation, project, organism or array design . Clicking on one experiment name reveals quality control plots (b) and the list of related hybridizations . (d) experiments can be downloaded in three forms: normalized data with probe annotation, raw data and bioconductor expressionset object . Web interface . (a) experiments can be grouped by type, laboratory affiliation, project, organism or array design . For each category, clicking on one experiment name reveals quality control plots (b) and the list of related hybridizations . (d) experiments can be downloaded in three forms: normalized data with probe annotation, raw data and bioconductor expressionset object . In addition to providing a data repository for a variety of pancreatic beta - cell experiments, the eurodia database contains several analysis tools for mining the data . Through the web interface, a user can identify differentially expressed genes by fitting a linear model for each gene and evaluating the fold change and moderated t - statistics p - values (11). It is possible to use one of several web forms designed to describe common experimental set - ups; the different web forms available are for (i) group designs allowing to compare two or more conditions, (ii) factorial designs of type two by two for comparing the combined effect of two conditions or treatments (e.g. The combined effect of a treatment and a mutant background) or (iii) paired samples where hybridizations are compared by pairs . The latter may be used, for example, to identify differentially expressed genes between alpha and beta cells of six pancreas samples, each alpha cell sample being paired with the beta cell sample from the same organ . A few additional filters can also be set to correct the obtained p - values for multiple testing using either holm, benjamini - hochberg or the storey - tibshirani false discovery rate (fdr) methods and to exclude probes showing low expression and/or variance . Results are presented as tables that can be sorted, filtered and downloaded together with probe annotations provided by the array manufacturer (figure 2). The order of the boxes reflects the flow of analysis steps . From top to bottom and left to right, the experiment name and description, the selection of the type of analysis, the form to describe analysis design (for this example, paired samples design), the list of differentially expressed probes with their relevant gene symbol, expression ratios and p - values of differential expression and the list of altered go categories is provided . The order of the boxes reflects the flow of analysis steps . From top to bottom and left to right, the experiment name and description, the selection of the type of analysis, the form to describe analysis design (for this example, paired samples design), the list of differentially expressed probes with their relevant gene symbol, expression ratios and p - values of differential expression and the list of altered go categories is provided . Once a set of differentially expressed genes has been identified, the next step is often to explore the biology around these genes . The eurodia database provides several tools to help extract valuable information and knowledge from gene expression data . From the web interface a user can evaluate whether the differentially expressed genes are enriched for particular gene ontology (go) (12) categories, kegg pathways (13) or reactome metabolic maps (14). The results are presented as a table of significant categories or pathways with the relevant p - values for enrichment . In addition to these enrichment analyses, a user can also perform gene set enrichment analysis (gsea) (14) using ordered gene lists to identify enriched pathways or functionally related groups of genes . The gene lists for gsea can either be selected from msigdb (14) or imported by the user . As an alternative, for some experiments a user might be interested in identifying subgroups of genes and conditions that share similar expression profiles (expression modules). The eurodia database interface offers the possibility to identify expression modules using the iterative signature algorithm (1517). For each expression module, a go category, kegg pathway and chromosomal location enrichment is computed (18). It is possible to combine multiple experiments of the same platform type (affymetrix or spotted) by merging probes using either their unique probe identifier (to combine data from two versions of the same platform), ncbi gene index (to combine data from the same organism on different platforms) or ncbi homologene i d (to combine data from different organisms). To address the problem of variability between measurements originating from different laboratories, expression ratios between conditions are not calculated for merged experiments, the rank products algorithm (20) is used to compare the co - occurrence of one gene amongst the most up or down regulated genes of all the compared hybridizations . Finally, to provide a more global view of the data, tools have been incorporated to display the expression profile of a particular gene across the whole database and to measure the correlation of the global expression profiles of two genes . The web interface of the eurodia database is generated using a combination of javascript and php scripts that form the gedai framework . The majority of statistical analyses are performed using packages from r / bioconductor (10). Quality controls and normalizations of two channel arrays are performed with the marray (19) and limma (9) packages, and the affy (20) and affyplm (8) packages are used to process affymetrix arrays . Affymetrix gene and exon arrays are normalized using the affymetrix power tools, a set of command line programs from affymetrix . Normalized experiments are stored as bioconductor expressionset objects that can be efficiently processed to identify differentially expressed genes . These functions are integrated in the limma (11), qvalue (21), rankprod (22) and eisa (18) packages . Go categories and kegg pathways enrichments are computed by functions of the gostats (23) package . The gsea analysis is performed by a speed - improved version of the original gsea algorithm . Most of the r scripts used to run analyses can be downloaded from the web interface . The eurodia database is a unique collection of beta - cell gene expression datasets generated by a consortium of european experts . Because of the need to provide users in the different laboratories with a way of uploading and sharing data that is both time - efficient and user - friendly, we opted not to include all experimental annotations that would be required to make the database miame (24) compliant . However, by offering a user - friendly interface to several well - accepted r / bioconductor packages, the eurodia database enables to rapidly evaluate the quality of a dataset, to identify differentially expressed genes and to reveal potentially altered biological pathways or molecular functions . Having the data repository integrated with the analysis tools also avoids the cumbersome steps of data extraction, reformatting and loading of data into an external tool . A unique feature of our database is its ability to combine studies performed using different array platforms, or even different organisms, in a single analysis . Other similar diabetes resources such as t1db (4,5) or epcondb (25), whilst being valuable data repositories, often lack the flexibility of eurodia, such as the integration of quality controls and the ability to perform re - analysis . The eurodia database was built using the gene expression data analysis interface (gedai) framework, which supports gene expression data measured with affymetrix gene chips, agilent arrays, illumina gene chips and custom spotted arrays . Currently gedai is used for several independent research projects and handles gene expression data from arabidopsis, ant, mouse, rat and human . The eurodia database will be maintained in the coming years and will evolve to integrate new gene expression datatypes like rnaseq . Supported by the european union (integrated project eurodia lshm - ct-2006 - 518153 in the framework programme 6[fp6] of the european - community). Funding for open access charge: european union (integrated project eurodia lshm - ct-2006 - 518153 in the framework programme 6 [fp6] of the european - community).
The nerves and their structures change and are subject to the law of neuroplasticity: they adapt to reflect the stimulus which is present (mechanical, biochemical, electrical, or metabolic).1 the peripheral nervous system is subjected to a daily mechanical tensile load, as when a joint moves, undergoing compression and stretching . Compression happens when the surrounding tissues create a longitudinal force to a nerve, such as when the muscles are stretched, while stretching occurs when a force is parallel to the nerve, for example, when the elbow is flexed, the ulnar nerve is stretched.2 this mechanical stress can leave the physiological confines when there is direct trauma to the nerve or surrounding tissue.2 the physiological tensile load allows the nerve to regenerate, through autocrine and paracrine substances, generated by the same nervous structure . When the nerve is subjected to stress in elongation, the schwann cells can proliferate and synthesize many neurotrophic factors, which play roles in nerve repair and remodeling.3 the synthesized molecules are varied, with different functions depending on the site of production and the quantity and based on the combinations that occur; their direction and propulsion force also change.4,5 the synthesized molecules from nerve structure can travel along the nerve and stimulate the various receptors, activating or switching them off, so as to or not to interact with the molecule produced; the same molecules can behave in different modes based on the area where they were synthe - sized.4,5 in order for the nervous tissue to reshape correctly, it must have the ability to retain its elasticity . The correct sliding of the fascial structures that make up nerves and the sliding of nerves between the various tissues that cross and innervate it become fundamental, so that the mechanical stress can be communicated properly and has the ability to adapt and regenerate the nerves.6 an impediment to this slide will lead to dysfunction and pathology.7 the biochemical information carried by the nerve does not merely have the function of operating on the nervous system, but it is also able to assist trophism and tissue function that crosses the nerve and innervates it . Nerves can carry multipotent cells for repair and remodeling of tissues and organs, which are innervated by the same nerves.8 the fascial structure of a nerve is divided into three layers: the endoneurium, perineurium, and epineurium . All the layers are innervated and have a subtle but potentially important plexus of nociceptors.9 the epineurium is composed mainly of type i and iii collagen, fibroblasts, mast cells, and fat cells . The inter - fascial epineurium is loosely attached to the perineurium, facilitating sliding of the various fascia . There is abundant epineurium in larger nerves, in order to disperse most of the compressive forces . The epineurium is attached to paraneural fascial components of the connective tissue that surrounds the nerve and is an extension of the dura mater . The vessels enter the epineurium in rolled - up form (to adapt better to the strain in elongation of the nerve) and periodically along its length, forming the vasa nervorum . Therefore, the epineurium supports the blood vessels while keeping the microvascularity of the nerve constant and also supports the nervi nervorum (sensory nerve fibers) of the nerve itself . It contributes to the tensile strength of the nerve and to the sliding, but it does not provide a barrier function.10,11 the perineurium surrounds the axon filaments with a dense connective tissue and the same perinerium is formed by up to 15 layers of flat perineural cells . We can encounter collagen type i and ii, and other cells with different carriers modulating the tension recorded from the nerve, thereby regulating intraneural pressure . Perineural cells synthesize several substances and are in close contact with laminin and collagen type iv, further acting as a shock absorber . Another key task of the perineurium is to act as a blood barrier, ie, not allowing all the filtered substances to reach the endoneurium . Arterioles that reach and penetrate the perineurium form an oblique angle; these vessels have developed little smooth muscle and therefore do not possess a great intrinsic ability to regulate blood flow . Nevertheless, the perineurium performs important functions in the repair of nerve tissue by managing the traffic of molecules inside and out of the neural environment by active transcytotic transport.10,11 a study has shown the presence of many pinocytotic vesicles for this transcytosis, but more research is needed to draw conclusions.11 molecules can also be transported via specific membrane receptors.11 the endoneurium contains individual axons wrapped several times by schwann cells; this sheet is in turn wrapped by type iv collagen cells, fibronectin, laminin, and proteoglycans . Inserted between the various filaments, collagen type i and ii with a longitudinal orientation, mast cells and macrophages, and endoneurial fluid (70% water) can be found . The capillaries that penetrate the endoneurium increase in size, allowing the blood flow to head in different directions . The arterioles, however, are tightly wrapped by the endoneurium cells to create an additional barrier to the blood . It is most likely that the blood flow comes into contact with the endoneurium by diffusion . Venules carry the blood back to the venous system . The lymphatic system, however, is present only in the epineurium; there are no lymphatic drainage vessels into the nerves (figure 1).10,11 the nervi nervorum, or a nerve s nerves, can evoke local neuroinflammation when there is nonphysiological damage or mechanical stress to the nerve tissue, to assist in its repair.9 the fascial nerve system innervated by the nervi nervorum may become a source of local pain.9 another cause of pain by the fascial nerve system, connected to axonal and physiological dysfunctions and capable of generating dysesthetic or distant pain (for example, pain resulting from traction of the sciatic nerve, when the leg is stretched during tests), follows directly from the fascial structures of the axon . These fascial structures become more sensitive to mechanical stimulation and, after a few days of local inflammation, they are able to generate potential action similar to the mechanical stimulus that causes dysfunction . This mechanism is known as ectopic electrogen - esis.9 such nonphysiological situations may require from 1 week to 2 months to disappear and only involve the epineurium and perineurium.9 there is a strong hypothesis that impeded sliding of a nerve between its various layers and tissues that cross its fascial system will generate local and dysesthetic pain . When there is an impediment to the sliding of a nerve, the rigidity of its fascial structures during joint movement is increased, and this is even more true when the joint is moved swiftly, which often occurs with everyday gestures.10 an elongated nerve causes a reduction in its diameter, defined as transverse contraction, with an increase in the pressure of the endoneurial compartment.10 its return to size and length at rest happens due to the elasticity of the perineural tissue, while the endoneurial tissue has less elastic compliance.10 a decrease in the nerve s ability to stretch may damage the endoneurial integrity before a lesion to the epineurium is visible.10 repetitive elongation of a nerve with reduced fascial elasticity properties brings about further inability of the nerve to slide, decreasing blood flow and likely leading to ischemic problems.10 for example, use of a computer mouse for prolonged periods leads to a reduction of the flow capacity of the median nerve at the level of the carpal tunnel, with possible pathological consequences of nerve compression, including the formation of edema, inflammation, and the production of adhesions, along with pain and reduced axonal flow (figure 2).10 edema is found at the intraneural level and is a common response to trauma, compression, tension, or excessive vibration . A slight trauma may give rise to edema, more superficially, at the epineural level; this can be transformed into intraneural edema if compression continues, creating a mini - compartmental syndrome of the nerve, due also to the absence of lymphatic vessels in the endoneurium . All this will lead to fibrous adhesions, decreasing the sliding of the intrafascicular tissue . The fibrosis increases the compression both inside and outside the tissues with a thickening of the nerve . This is even more noticeable when the nerve passes through small areas.12 the situation described will still generate pain symptoms . Many peripheral nerves can be palpated and directly moved, but at the same time, they are more susceptible to trauma than the central nervous system, which is protected by the cranium.9,13 many peripheral nerves can be palpated and directly moved for anatomical reasons.9 there are very few texts in the scientific literature on manual treatment of the peripheral nervous system . Such therapy is used by manual operators with the intent of allowing adequate sliding of the peripheral nerve and surrounding tissue, using two methods in particular: the sliding technique or a technique that alters the tension of the nerve path.13 studies on the human model are scarce, and there are no precise data on the method of movement that should be performed on a patient s nerve path in order to optimize the results . Specific work protocols are lacking, and a positive clinic outcome is not always observed.1419 within the field of osteopathy, there are no scientific articles that deal with palpation or manual techniques applied directly to the peripheral nerve, although there is a book that describes and shows how the osteopathic operator should palpate and treat emergencies superficial to the nerve path.20 the purpose of these therapies and treatments is to alter the mechanical properties of fascia, such as density, stiffness, and viscosity, so that the fascia can more readily adapt to physical stresses . In fact, some osteopathic physicians and manual therapists report local tissue release after the application of a slow manual force to tight fascial areas; these reports have been explained as a breaking of fascial cross - links, a transition from gel to sol state in the extracellular matrix, and other passive viscoelastic changes of fasciae.21 the fascial osteopathic technique is the application of a low - load, long - duration stretch into the myofascial complex that is intended to restore the optimal length of this complex.21 the operator places his or her hand on the fascial restriction identified previously by palpation, using different approaches, until the perceived resistance disappears or greatly decreases, inducing or stopping the preferential direction of the tissue.21,22 the time required for the technique varies according to the response of the patient.22 there are reports in the literature that improvement in the sliding of the different fascia layers, via manual manipulation, may decrease pain symptoms and reduce inflammation at the local level.21,23 palpation of the peripheral nerves is feasible and is also used to evaluate nerve function, because these nerves are very often superficial.24,25 our clinical experience encourages us to support osteopathic fascial treatment of the peripheral nerve, providing a strong incentive to launch new research aimed at understanding what happens to the nerve using fascial osteopathic techniques, in addition to quantifying the benefit derived by the patient . It is important to remember that evidence - based medicine involves not only scientific research highlighted in an article, but also the patient s experience of treatment and the clinical experience of the operator.26 to provide some examples of treatment of peripheral nerve fascia, a finger or fingers are placed on the nerve emergence, either at the elbow on the cubital tunnel, where it passes the ulnar nerve (figure 3), or in the middle third lateral of the humerus, where the radial nerve passes (figure 4).24 ulnar compression difficulties may be encountered at the cubital tunnel level, while the humeral area is subject to direct trauma that might damage the radial nerve.2729 the method of treating any emergence of the peripheral nerve is relatively simple and noninvasive . Once the fingers are in contact with the emergence and the most superficial area of the nerve branches, a fascial osteopathic technique is used, during which the operator waits for the tissues of the fabric to release under the fingers.21 it is common for the area being treated to be characterized by tissue hardness, with layers that slide with difficulty under the fingers: the technique ends when the tissue in the area has been restored, as close as possible, to a softer, more flexible tissue . We do not know the exact scientific reasons for this fascial release, despite many studies showing that an osteopathic fascial treatment is useful in many clinical conditions.21 an in vitro study demonstrates how osteopathic techniques can influence the metabolic behavior of fibroblasts, such as proliferation and inflammatory response.21 another possible explanation is that improved sliding of the various fascial layers would allow resetting of the afferents of the free nerve endings, resulting in a physiologic response of the efferents.21 further studies are expected in order to facilitate choosing the best osteopathic approach to ensure the well - being of the patient . The peripheral nerves are composed of several layers of fascial tissue, which can become a source of pain if they fail in their ability to slide . It is only recently that fascial osteopathy research has been aimed at understanding what happens to the fascia following treatment and, as a result of the first studies, it has been possible to highlight some of the benefits, including a reduction in local pain and inflammation . The osteopathic approach to the fascial system of the peripheral nerve does not have a grounding in scientific research, being based only on the clinical experience of individual operators, despite the use of peripheral nerve palpation as a method for the evaluation and testing of the nerve s function . The authors wish to encourage the initiation of new research in the fields of academic and clinical osteopathy that is aimed at quantifying the possible benefits a patient may derive from osteopathic treatment of the peripheral nerve.
All patients were part of the lung cancer group cologne (http://www.cio-koeln-bonn.de) cohort and analyzed as part of our routine molecular diagnostics program according to the local ethical guidelines and reviewed by the institutional ethics committee . For squamous cell carcinomas, all consecutive patients with sufficient tumor material from january 2010 until june 2011 were analyzed, and cases with other histological subtypes were selected at random from the same period in time . Three - hundred sixty - seven primary pulmonary carcinomas were included: 307 squamous cell carcinomas, 47 adenocarcinomas, and 13 carcinomas of other differentiation types (i.e., 6 undifferentiated large cell carcinomas, 4 adenosquamous carcinomas, 2 large cell neuroendocrine carcinomas, 1 combined small cell and squamous carcinoma). Adenosquamous carcinomas and the combined small cell and squamous carcinoma were included to answer the question whether tumors with only partial squamous differentiation may also be amplified for fgfr1 . Since emerging data from expression profiles provide evidence that some pulmonary large cell carcinomas might represent a dedifferentiation end point of squamous carcinomas, we further investigated a small number of these tumors . An additional cohort of 53 primary adenocarcinomas of the lung, which were sent to the lung cancer group cologne for molecular testing from october 2011 until february 2012, was examined in a confirmatory study . Thus, a total of 420 cases were included (307 squamous cell carcinomas, 100 adenocarcinomas, and 13 carcinomas of other differentiation types). All diagnoses were reviewed by two experienced pathologists and confirmed by immunostainings, if appropriate . For squamous cell carcinomas, these include positive cytokeratin 5/6 (clone d5&16b4, cellmarque; dilution 1:50) and p63 (a4a, zeta corp . ; positive stainings for cytokeratin 7 (ov - tl 12/30, dako; 1:800), ttf1 (8g7g3/1, cellmarque; 1:200), and napsin a (rabbit polyclonal antibody, cellmarque; 1:200) were performed . Stainings for cytokeratin 20 (ks20.8, cellmarque; 1:400) and cdx2 (cdx2 - 88, biogenex; 1:200) were used to exclude pulmonary metastases of intestinal adenocarcinomas . Diagnoses and grading of tumors were made in accordance with the current who classification system . For fish, tumor tissue from biopsies or surgical resection specimens three to four m tissue sections were mounted on sialinized slides and hybridized overnight with the zytolight spec fgfr1/cen 8 dual color probe (zytovision, bremerhaven, germany). Briefly, deparaffinization, protease treatment, and washes were performed on the half - automated vp2000 processor system (abbott molecular, wiesbaden, germany). After pretreatment, the slides were denatured in the presence of 10 l probe for 5 min at 75c and hybridized at 37c overnight . Post - hybridization ssc washes were performed at 72c and the slides stained with dapi before analysis . Normal tissue including vessels, fibroblasts, or non - tumor lung tissue served as internal positive control . Cases were only further evaluated if control tissue nuclei displayed one or two clearly distinct signals of each color . Tumor tissue was scanned for amplification hot spots by using 40 or 63 objectives (dm5500 fluorescent microscope; leica). If the fgfr1 signals were homogeneously distributed, then random areas were used for counting the signals . Twenty contiguous tumor cell nuclei from three hot spots or random areas, resulting in a total of 60 nuclei, were individually evaluated with the 100 or 63 objectives by counting green fgfr1 and orange centromer 8 (cen8) signals . The fgfr1/cen8 ratio, the number of cells with 5 and 15 fgfr1 signals and the average fgfr1 copy number per cell were calculated . For statistical analysis having evaluated the first 50 tumors of our series, we became aware of different signal distribution patterns (figure 1). Fgfr1 signals were heterogeneously distributed in most tumors, and small clusters of amplified cells often occurred . We observed high - level cluster amplifications with 15 fgfr1 signals in a small subset of tumors whereas small clusters (microclusters') with 5 fgfr1 signals on average were found more frequently . The majority of tumors displayed polysomy with> 2 cen8 signals, sometimes leading to an fgfr1/cen8 ratio below 1.0 despite an increase in absolute numbers of fgfr1 signals compared with normal tissue . Fgfr1 signal doublets and triplets were counted as one signal, but closely spaced groupings of signals were considered as small clusters of fgfr1 signals according to the reading criteria for most other fish probes (e.g., her2 and egfr). Therefore, we developed the following reading and evaluation strategy that was derived from our observations in lung cancer and fgfr1 data published previously from studies with other tumor entities: assessment of hybridization quality: evaluate only samples and areas with sharp borders of nuclei, no signs of overdigestion, non - overlapping nuclei, bright and specific green and orange signals in internal control tissue and in the tumor area.scanning the slide: scan the entire tumor area for hot spots of increased fgfr1 copy numbers.reading the slide: count 20 tumor cell nuclei in three areas, either in three hot spots or in three random areas in case of homogeneous signal distribution . Count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas.count only clearly distinct signals as two separate signals . Count fgfr1 signal doublets and triplets as one signal . In cases of signal clusters give cluster estimation in steps of five signals, for example, 15, 20, or 25 fgfr1 signals . Assessment of hybridization quality: evaluate only samples and areas with sharp borders of nuclei, no signs of overdigestion, non - overlapping nuclei, bright and specific green and orange signals in internal control tissue and in the tumor area . Scanning the slide: reading the slide: count 20 tumor cell nuclei in three areas, either in three hot spots or in three random areas in case of homogeneous signal distribution . Count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas . Count fgfr1 signal doublets and triplets as one signal . In cases of signal clusters give cluster estimation in steps of five signals, for example, 15, 20, or 25 fgfr1 signals . Cases were considered as fgfr1 positive (amplified') under one of the following conditions: the fgfr1/cen8 ratio is 2.0;the average number of fgfr1 signals per tumor cell nucleus is 6;the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10%the percentage of tumor cells containing 5 fgfr1 signals is 50%,with (13) representing a high - level and (4) a low - level amplification . The fgfr1/cen8 ratio is 2.0; the average number of fgfr1 signals per tumor cell nucleus is 6; the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10% the percentage of tumor cells containing 5 fgfr1 signals is 50%, in all, 347 out of 367 tumors (95%) were evaluable, that is, 290 squamous cell carcinomas, 44 adenocarcinomas, and 13 carcinomas of other differentiation types . In all, 58 of 290 squamous cell carcinomas (20%) were fgfr1 amplified according to our fish scoring definition, whereas 0/44 adenocarcinomas and 2/13 tumors of other differentiation were amplified . The latter two positive cases were one tumor with adenosquamous differentiation and one undifferentiated large cell carcinoma, in which a high - level amplification was found . In a confirmatory study, 53 adenocarcinomas were additionally examined; all of them were fish negative . Thus, a total of 97 pulmonary adenocarcinomas did not show an amplification of fgfr1 . Among the 60 positive tumors, 13 cases revealed low - level amplification as defined by category (4) of our scoring system (table 1). Thus, low - level amplifications represent 22% of all fish - positive cases . Among the squamous cell carcinomas, the average fgfr1 copy number per nucleus (figure 2) ranged from 1.1 to 16.5 (mean: 3.6). The mean fgfr1/cen8 ratio was 1.4 (range: 0.48.8), the mean percentages of tumor cells with 5 and 15 fgfr1 copies were 22 and 3%, respectively (figure 2). Fgfr1/cen8 ratio (t - test, p=0.01), average fgfr1 copy number per nucleus (t - test, p<0.001), and percentage of tumor cells with 5 fgfr1 copies (t - test, p<0.001) were significantly higher in squamous cell carcinomas compared with all other tumor types . The percentage of tumor cells with 15 fgfr1 copies was significantly higher in squamous cell carcinomas compared with adenocarcinomas (p<0.001). Among evaluable squamous cell carcinomas, a high - level amplification was detected at a frequency of 16% (n=45) and low - level amplification at a frequency of 4% (n=13). Importantly, the fgfr1 amplification status is not exclusively based on an fgfr1/cen8 ratio of 2, as some tumors fulfill only criteria (2) and/or (3) for high - level amplification despite an overall ratio below 2.0 (table 1). However, in the majority of cases in which a high - level amplification was determined at least two criteria were fulfilled . Ratio 2.0 and average number of fgfr1 signals per tumor cell nucleus 6 turned out to be the most consistent criteria for fgfr1 amplification . All fish - positive cases, low- and high - grade amplifications, had at least 50% of tumor cell nuclei containing five or more fgfr1 signals, thus, fulfilling criterion (4). Preliminary survival data indicate that there is no significant difference between outcome of fgfr1 amplified and non - amplified lung cancer patients . Having evaluated the first 50 tumors of our series, we became aware of different signal distribution patterns (figure 1). Fgfr1 signals were heterogeneously distributed in most tumors, and small clusters of amplified cells often occurred . We observed high - level cluster amplifications with 15 fgfr1 signals in a small subset of tumors whereas small clusters (microclusters') with 5 fgfr1 signals on average were found more frequently . The majority of tumors displayed polysomy with> 2 cen8 signals, sometimes leading to an fgfr1/cen8 ratio below 1.0 despite an increase in absolute numbers of fgfr1 signals compared with normal tissue . Fgfr1 signal doublets and triplets were counted as one signal, but closely spaced groupings of signals were considered as small clusters of fgfr1 signals according to the reading criteria for most other fish probes (e.g., her2 and egfr). Therefore, we developed the following reading and evaluation strategy that was derived from our observations in lung cancer and fgfr1 data published previously from studies with other tumor entities: assessment of hybridization quality: evaluate only samples and areas with sharp borders of nuclei, no signs of overdigestion, non - overlapping nuclei, bright and specific green and orange signals in internal control tissue and in the tumor area.scanning the slide: scan the entire tumor area for hot spots of increased fgfr1 copy numbers.reading the slide: count 20 tumor cell nuclei in three areas, either in three hot spots or in three random areas in case of homogeneous signal distribution . Count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas.count only clearly distinct signals as two separate signals . Count fgfr1 signal doublets and triplets as one signal . In cases of signal clusters give cluster estimation in steps of five signals, for example, 15, 20, or 25 fgfr1 signals . Assessment of hybridization quality: evaluate only samples and areas with sharp borders of nuclei, no signs of overdigestion, non - overlapping nuclei, bright and specific green and orange signals in internal control tissue and in the tumor area . Scanning the slide: reading the slide: count 20 tumor cell nuclei in three areas, either in three hot spots or in three random areas in case of homogeneous signal distribution . Count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas . Count fgfr1 signal doublets and triplets as one signal . In cases of signal clusters give cluster estimation in steps of five signals, for example, 15, 20, or 25 fgfr1 signals . Cases were considered as fgfr1 positive (amplified') under one of the following conditions: the fgfr1/cen8 ratio is 2.0;the average number of fgfr1 signals per tumor cell nucleus is 6;the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10%the percentage of tumor cells containing 5 fgfr1 signals is 50%,with (13) representing a high - level and (4) a low - level amplification . The fgfr1/cen8 ratio is 2.0; the average number of fgfr1 signals per tumor cell nucleus is 6; the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10% the percentage of tumor cells containing 5 fgfr1 signals is 50%, in all, 347 out of 367 tumors (95%) were evaluable, that is, 290 squamous cell carcinomas, 44 adenocarcinomas, and 13 carcinomas of other differentiation types . In all, 58 of 290 squamous cell carcinomas (20%) were fgfr1 amplified according to our fish scoring definition, whereas 0/44 adenocarcinomas and 2/13 tumors of other differentiation were amplified . The latter two positive cases were one tumor with adenosquamous differentiation and one undifferentiated large cell carcinoma, in which a high - level amplification was found . In a confirmatory study thus, a total of 97 pulmonary adenocarcinomas did not show an amplification of fgfr1 . Among the 60 positive tumors, 13 cases revealed low - level amplification as defined by category (4) of our scoring system (table 1). Thus, low - level amplifications represent 22% of all fish - positive cases . Among the squamous cell carcinomas, the average fgfr1 copy number per nucleus (figure 2) ranged from 1.1 to 16.5 (mean: 3.6). The mean fgfr1/cen8 ratio was 1.4 (range: 0.48.8), the mean percentages of tumor cells with 5 and 15 fgfr1 copies were 22 and 3%, respectively (figure 2). Fgfr1/cen8 ratio (t - test, p=0.01), average fgfr1 copy number per nucleus (t - test, p<0.001), and percentage of tumor cells with 5 fgfr1 copies (t - test, p<0.001) were significantly higher in squamous cell carcinomas compared with all other tumor types . The percentage of tumor cells with 15 fgfr1 copies was significantly higher in squamous cell carcinomas compared with adenocarcinomas (p<0.001). Among evaluable squamous cell carcinomas, a high - level amplification was detected at a frequency of 16% (n=45) and low - level amplification at a frequency of 4% (n=13). Importantly, the fgfr1 amplification status is not exclusively based on an fgfr1/cen8 ratio of 2, as some tumors fulfill only criteria (2) and/or (3) for high - level amplification despite an overall ratio below 2.0 (table 1). However, in the majority of cases in which a high - level amplification was determined at least two criteria were fulfilled . Ratio 2.0 and average number of fgfr1 signals per tumor cell nucleus 6 turned out to be the most consistent criteria for fgfr1 amplification . All fish - positive cases, low- and high - grade amplifications, had at least 50% of tumor cell nuclei containing five or more fgfr1 signals, thus, fulfilling criterion (4). Preliminary survival data indicate that there is no significant difference between outcome of fgfr1 amplified and non - amplified lung cancer patients . We and others recently described therapeutically targetable fgfr1 amplifications in smoking - associated human squamous lung cancers . This is a clinically important finding since worldwide this cohort of patients is steadily increasing in size and current therapies are very limited squamous cell lung carcinomas account for 25% of new lung carcinoma cases and 40 000 deaths per year in the united states . To translate this finding into the clinic patients eligible for treatment with fgfr inhibitors in clinical trials furthermore, their putative response data need to be carefully related to the specific pattern of fgfr1 amplification . In addition, molecular testing needs to be fast and reliable on frequently very small biopsies . Therefore, we evaluated 290 squamous cell carcinomas by a dual color fish assay and found that 20% of cases showed high- or low - level amplifications . The percentage is very close to data observed in two cohorts analyzed by snp 6.0 by us (n=155, frequency 10%) and fish (n=153, frequency 22%) and others (n=57, frequency 21%). Importantly, the distribution of fgfr1 copies is heterogeneous in most tumors, and small clusters (microclusters') as well as high - level amplification in only few isolated tumor cells and co - amplifications of fgfr1 and cen8 signals are common features . Our reading and evaluation strategy is able to address the issue of intratumoral heterogeneity and other special features of fgfr1 in pulmonary carcinomas . Thus, our scoring system might serve as a standardized screening tool to identify patients for ongoing and upcoming clinical trials with fgfr1 inhibitors . Several compounds have already entered early clinical trials giving precise fgfr1 diagnostics high clinical impact . In the setting of these clinical trials, we believe that our fish score might be helpful for identifying patients with significant increase in fgfr1 gene copy numbers . However, it still needs to be clarified by future response data derived from these studies whether our criteria are equally good or even better predictor of response than traditional amplification criteria . Thus, our work represents a proposal for standardized screening for clinical trials, which potentially needs to be modified against the background of future clinical response data . However, it appears likely that fgfr1 fish assay is going to represent a companion diagnostic and there is a need for definition of interpretation criteria that could be implemented in the clinical practice . Having carefully assessed a large cohort of pulmonary carcinoma cases by applying our reading and evaluation strategy, we were able to divide positive cases into two types of amplification patterns . Low amplification levels (as defined by 5 fgfr1 signals in 50% of tumor cells) occurred at a frequency of 4% in squamous cell carcinomas and account for 22% of all fish - positive cases . In all, 16% of squamous cell carcinomas (78% of fish - positive tumors) show high - level amplification (as defined by an fgfr1/cen8 2.0, or average number of fgfr1 signals per tumor cell nucleus 6, or the percentage of tumor cells containing 15 fgfr1 signals or large clusters 10%). Our previous data suggest that cancer cell lines with high focal amplifications respond better to treatment with fgfr inhibitors, which was also confirmed in an independent study . Therefore, it may be expected that tumors with high - level fgfr1 amplification will respond better to fgfr inhibitors . Very early and preliminary data from a phase i study with a selective pan fgfr inhibitor indicate a partial response in one patient whose pulmonary squamous cell carcinoma has been tested positive by applying the fgfr1 fish score . However, further results from ongoing early clinical trials need to be awaited . In any case, it will be important to record amplification levels obtained by fish in a reproducible manner as proposed by our score to correlate these clinical response data with amplification patterns . We found very rare fgfr1 amplifications in non - squamous lung cancer: only one single case each of undifferentiated large cell carcinoma and adenosquamous carcinoma, but not in adenocarcinomas of the lung . These data indicate that squamous cell carcinomas, adenosquamous, and undifferentiated large cell carcinomas should be included in fgfr1 amplification screening of lung cancer, but cases of pure adenocarcinoma may be omitted.
The combination of methods from the behavioral decision - making literature such as risky decision - making tasks derived from the classic work of kahneman and tversky (1979), and methods of neuroscience such as functional magnetic resonance imaging (fmri) and lesion studies has led to breakthroughs in both fields . Examples include how impairment in specific brain functions translate into disadvantageous decision - making inside and outside of the laboratory (bechara et al ., 1994, 1996, 1997, 1999) and how common decision - making biases and heuristics can be understood at the neural level (sanfey et al ., 2003; hsu et al ., 2005; kuhnen and knutson, 2005; de martino et al ., 2006; huettel et al ., 2006; a major contribution of this work has been a better understanding of how emotion, in combination with cognition, guides our decisions, particularly in the realm of risky decision - making where conflicts often arise in balancing the lure of reward and the fear of loss . Evidence is accumulating that emotional reactivity differs in response to risky gains and risky losses . Logical questions are whether risk - taking for gains and risk - taking for losses can best be understood as separate psychological processes, and ultimately, whether they rely on different brain structures . In this paper, we integrate findings from our own work and that of others to come to conclusions that have some generality but also allow for differences between studies based on methodology . In order to frame this investigation, we start with a model put forth to support the findings from two studies we conducted with patients with lesions to areas of the brain known to be critical to risky decision - making, namely the ventromedial prefrontal cortex (vmpfc), the amygdala, and the insula (bechara et al ., 1999; 2008). As summarized in figure 1 (from weller et al ., 2007), we propose that risky decision - making is influenced by the opposing forces of lure of gain and fear of risk . We operationalize the lure of rewards as either the potential for a relatively large gain in the gain domain (in comparison to the small sure gain from a riskless choice) or the potential for avoiding a loss altogether in the loss domain, and the fear of risk as arising from risking a relatively large loss in the loss domain (in comparison to the small sure loss from a riskless choice) or not winning anything in the gain domain . We suggest that the vmpfc subregions, the amygdala, and the insula each contribute in different ways to the processing and utilization of these two critical pieces of emotional information . The mere presence of uncertainty induces a primary fear response elicited by the amygdala, which has been associated specifically with fear processing and avoidance behavior (ledoux, 2000; trepel et al . This fear response activates the vmpfc whose function it is to mediate decision - making and allows for more careful deliberative processes by linking together working memory and emotional systems (damasio, 1994). Processing of primary inducers, mediated by the amygdala, triggers the ventromedial prefrontal cortex (vmpfc) system, which, in turn, conducts a more deliberative analysis of uncertainty . However, decisions involving potential losses may trigger redundant neural responding from structures such as the insula (anterior, posterior, or both) and the adjacent primary and secondary somatosensory cortices (si and sii), which are independent of the amygdala; these backup processes are represented here by dotted lines . While the amygdala has been studied extensively and shown to be a key substrate for triggering emotional responses, especially in connection with fear (ledoux, 2000), the fact remains that the triggering of emotional responses involves multiple neural regions, and not just the amygdala . Thus, structures such as the insula, which are independent of the amygdala, are also likely to impact decision - making under uncertainty (kuhnen and knutson, 2005; clark et al ., 2008; weller et al . In particular, we propose that the insula and the amygdala provide complementary systems for dealing with potential losses, which we attribute to the evolutionary significance of dealing with potential losses . Our ancestors learned to avoid situations that risked the loss of things essential for survival and it is reasonable to assume that our brains have been primed for avoiding losses . This account parallels the proposed dual systems approach of system 1 (experiential) and system 2 (deliberative) for decision - making (kahneman, 2003). According to the somatic marker hypothesis (bechara and damasio, 2005; reimann and bechara, 2010), after the amygdala triggers an automatic emotional response (or primary induction), the vmpfc subsequently prompts a more careful deliberative analysis that triggers secondary emotional responses (secondary induction) that help guide advantageous decision - making . Findings in support of the somatic marker hypothesis were key to new behavioral theories in which emotions play a pivotal role in decision - making (mellers et al . 2002). In the following sections of this paper, we review the evidence for our model based on studies involving the vmpfc, amygdala, and insula, but we also include studies involving other areas that have implications for addressing the basic question of whether there is evidence at the neural level of a distinction between risky decision - making in the gain and loss domains . We will provide evidence that separate psychological processes are involved in risk - taking for gains and losses in terms of both behavioral and neurological reactions that discriminate between risk - taking to achieve a gain and risk - taking to avoid a loss . We then address the more complex issue of whether distinct neural structures support these different reactions . In the case of fmri studies, we will see that results depend on when during the decision - making process the recordings are made . We start, however, with some more straightforward and well - known behavioral phenomena that motivate the search for neurological dissociations between risk - taking for gains and losses . It is typical to consider risk - taking as a unified behavioral concept when we talk about a person in terms such as she is a risk - taker or he likes to play it safe . However, it has been shown that risk - taking within the same individual varies across content domains such as monetary, health, and social risks (weber et al ., 2002). Within each of these domains, we may talk about an action as being risky because of the uncertainty of its outcome without differentiating between the potential for achieving benefits versus the potential for avoiding aversive consequences . Kahneman and tversky (1979) demonstrated a fundamental principle that sparked decades of later research: individuals were more likely to take a risk to avoid a loss than to achieve a gain of the same magnitude . Later work by the same authors revealed a fourfold pattern of risk - aversion for gains and risk - seeking for losses of high probability but risk - seeking for gains and risk - aversion for losses of low probability (tversky and kahneman, 1992). This was explained in terms of underweighting the likelihood of high probability but overweighting the likelihood of low probability events . This paper describes a relatively new component of this research: neuroscientific studies that provide additional sources of data that separate risk - taking to achieve a gain and risk - taking to avoid a loss . In presenting the most recent research in our laboratory, we focus on the cups task (levin et al ., 2007), which we developed specifically to separate risky decision - making for actual gains and losses, both in terms of overall riskiness and sensitivity to expected value (ev) differences between choice options . Gain trials involve some probability of an addition to the decision - maker s account while loss trials involve a possible reduction . Decision makers choose between one array of cups in which the outcome is constant (the riskless choice) and one array of cups in which the outcomes vary (the risky choice). Outcomes are displayed immediately after choices are made . By varying the number of cups and the amount to be won or lost, we create gain and loss trials with contingencies that either do or do not favor a risky choice (see figure 2). For example, a one - out - of - three chance of winning five coins is better in the long run than a sure gain of one coin but a one - out - of - three chance of losing five coins is worse in the long run than a sure loss of one coin . A key component of data analysis for the cups task is the extent to which an individual makes choices based on the consideration of relative ev between choice options, for both gain- and loss - related decisions . Ev sensitivity represents an index of advantageous decision - making because consistently choosing the option with a more favorable ev will yield more positive outcomes in the long run . As will be described later, a somewhat simpler version of the task was adapted for use in scanner research . Across many data sets, we demonstrated that kahneman and tversky s (1979) original finding of more risk - taking to avoid a loss than to achieve a gain of the same magnitude is reproduced in the cups task . Beyond the initial demonstration of greater risk - taking for losses than for gains, our recent research with the cups task showed age - related differences in risk - taking as a function of decision domain (risk - taking to achieve a gain versus to avoid a loss). Risk - taking in the domain of gains decreased monotonically from early childhood to older adulthood whereas overall risk - taking to avoid losses was remarkably constant across age groups (weller et al ., 2011). Within both domains, ev sensitivity increased from early childhood through adulthood with a slight decline for older adults note: in each case the riskless side is depicted on the left and the risky side is depicted on the right . In the experiments these were counterbalanced over trials . We turn to neuroscience for an exploration of brain functions that may help explain these gain / loss behavioral differences . Our approach in this paper is to provide a body of evidence that is consistent with the proposition that risky decision - making is separable in the gain and loss domains rather than providing a single critical test . Historically, the most fundamental functional division of the brain was thought to be the one that distinguished between approach and avoidance behaviors . However, many years of animal research failed to identify anatomically separate neural substrates neural systems underlying pain and pleasure seem to overlap considerably (e.g., craig, 2009). Later human behavioral studies found equivocal support for a separation of neural systems whereby the left hemisphere is predominantly concerned with approach behaviors and the lure of reward, whereas the right hemisphere is critical for avoidance behaviors and the fear of uncertainty (davidson et al ., more recently, neuropsychological research on the approach avoidance conflict evolved into studies of risky decision - making where the shift was to a more microscopic analysis of neural systems . Neuroimaging data have been used to gain new insights concerning risky decision - making . In particular, fmri studies use changes in blood flow that accompany neural activity in different parts of the brain to associate these areas to particular behaviors . For instance, in a recent meta - analysis of fmri studies of risky decision - making using young, healthy adults, mohr et al . (2010) found evidence common to all studies that risk processing is associated with activation of specific emotional systems in the brain such as the anterior insula, especially when potential losses are involved . The dorsolateral prefrontal cortex and parietal cortex are also activated when making decisions involving risk . Using fmri in conjunction with a paradigm in which individuals decided whether to accept or reject gambles offering a 50/50 chance of gaining or losing varying amounts of money, tom et al . (2007) found that activity in the ventral striatum and the vmpfc increased as potential gains increased but decreased as potential losses increased . Also, in the anterior insula, activity was found more strongly associated with the anticipation of losses than with anticipation of gains (knutson et al ., 2007). Earlier research showed increased arousal following losses than following gains (bechara et al ., 1999). Such results motivated us to classify study results based on whether activation was measured before, during, or after a decision was made . In order to get a more complete picture using the keywords fmri, gains, losses, risk, and uncertainty, table 1 summarizes the results of a number of fmri studies in terms of which areas of the brain were studied and at what point in time, and whether the study provided support for distinct mechanisms involved in risky decision - making for gains and losses . While the results are mixed, a pattern emerges when the studies are separated based on whether brain activation was measured before, during, or after a risky choice was made . Most noteworthy, while different regions were the focus of different studies, in 14 studies in which activation was assessed prior to a choice (i.e., anticipation), support for separate mechanisms was found in eight studies, four studies did not support separate structures, and two studies did not make claims about separate structures because they focused on a specific region only . For example, studies by kuhnen and knutson (2005) and knutson et al . (2008b) each found that the nucleus accumbens was activated in anticipation of a risky gain, whereas the insula was activated in anticipation of a risky loss . We think these results are particularly compelling because they suggest that different parts of the brain drive risky decision - making in anticipation of uncertain gains versus uncertain losses . Whereas activation during or after a risky choice can influence subsequent risky choices, activation prior to a choice is unique in its potential to influence the current choice . This table is sorted by time of measurement (before, during, or after decision - making) and by result (supportive of separate structures or not). In each category, the table is sorted first in chronological order, then in alphabetical order . Beside the dissociation at the pre - decision stage, recent evidence suggests that experienced gains and losses might also activate different regions, which then affect subsequent decisions making . In a recent study using the cups task, we found that at the feedback stage, experienced reward was associated with strong activation in the vmpfc and the ventral striatum, and the stronger reward - related responses in the vmpfc were positively associated with risk - taking (xue et al ., 2009). In a follow up study, we explicitly examined how neural and behavioral responses to gains and losses were associated with subsequent decisions . We developed a modified version of the cups task in which a single array of cups was presented on a given trial where one coin would be lost for all but one randomly selected cup, but multiple coins would be won if the other cup was drawn (xue et al ., 2011). The decision - maker indicated whether to take or not take the gamble . In one analysis, we focused on how an experienced gain versus an experienced loss could modulate subsequent risky decision - making, both behaviorally and neurally . We found that subjects took more risk after losing a gamble than after winning a gamble . At the neural level, we again found that at the feedback stage, win was associated with stronger activation than loss in the anterior cingulate cortex, the posterior cingulate cortex, the ventral striatum, and the insula . More importantly, decisions after loss were associated with stronger activation in the frontoparietal network, which was positively correlated with individuals increased tendency to take more risk . These results thus suggest that experienced gains and losses not only involve different brain regions, but also trigger differential neural responses and behaviors in subsequent decisions . Despite this suggested anatomical separation, the fact remains that the same structure, for example, the insula, has sometimes been implicated in the processing of both painful and pleasurable stimuli (e.g., craig, 2009). Indeed, when compared to a baseline of activation following trials on which the decision - maker decided not to take the gamble, both experienced gains and losses elicited strong insular activation, which then modulated subsequent decision - making (xue et al ., 2010). This calls for caution when making absolute determination about the anatomical separation of these pleasure (gain)loss (pain) systems . In particular, a proper baseline should be included in this analysis since the same regions might show opposite modulation by gains and losses (tom et al ., 2007). Thus, the stronger activation for gains or losses in some regions might not necessarily reflect distinct neural structures for gains and losses . Another reason for these difficulties in establishing absolute anatomical separations is that cellular physiological evidence of neurons responding to positive versus negative valence stimuli, at least within the amygdala, indicates separation, while anatomical evidence is highly inter - mixed (e.g., paton et al ., 2006). This explains why the neural systems for risky gains versus losses can be functionally separate, but finding clear - cut separation viewed at the global anatomical level is more difficult, given the proximity and overlap of these two systems . Next, we turn to lesion studies which are smaller in number in terms of addressing this issue but which should align with the anticipatory fmri studies because, of course, pre - existing brain damage would likewise serve to influence revealed choices . While neuroimaging studies argue whether a particular brain region is involved in a particular function, lesion studies test whether that brain region is necessary for that function, and thus form more direct tests of the model in figure 1 and our earlier reference to anatomically separate neural substrates . The logic here is that if a particular function is impaired in individuals with a localized lesion, then the affected neural region must play a crucial role in executing that function . Lesion studies seem to reveal little dissociation between the domains of gains and losses within the prefrontal cortex region, but such dissociations are more likely to be revealed when one considers two other neural systems, the insula and amygdala, which feed information into the prefrontal cortex . Indeed, within the prefrontal cortex, patients with damage to the vmpfc show deficits for both risky gains and risky losses (weller et al ., 2007) compared to healthy controls, vmpfc patients showed increased levels of risk - taking and decreased sensitivity to ev differences in both gain and loss domains . In contrast, amygdala patients showed impaired decision - making and exaggerated levels of risk - taking to achieve gains . However, in the loss domain amygdala damage was not associated with significantly increased risk - taking or decreased ev sensitivity . Given the abundance of literature suggesting that the amygdala is involved with avoidance of punishment, this finding suggests that other structures may act in concert with the amygdala to produce a signal that engages the vmpfc . When patients with insula damage were compared to controls, a different pattern emerged (weller et al ., 2009). Consistent with research suggesting that the insula is important for risk processing (preuschoff et al ., 2008), insula lesion patients like vmpfc and amygdala patients showed decreased sensitivity to ev differences between choice options for both risky gains and risky losses . However, these individuals showed lower levels of risk - taking compared to healthy controls, especially on gain trials . Thus the insula, with connections to the amygdala, ventral striatum, and the vmpfc, may serve the purpose of providing a gate to determine the effectiveness of excitatory and inhibitory motivational circuits, signaling approach or danger . Subsequently, insula damage may result in a blunted response toward risk, and would lead to insensitivity to changes in environmental contingencies signaling the approach or avoidance of a risk, regardless of domain . Because the amygdala and insula have long been implicated in the processing of negative emotions, evoked from stimuli that are particularly aversive and perhaps even a threat to survival (e.g., ledoux, 2000; paulus and stein, 2006; phelps, 2006), we argue that these emotional reactions may be processed by multiple neural structures and are thus more difficult to disrupt as a result of a focal lesion to the amygdala or the insula alone . Specifically, a person with a damaged amygdala but an intact insula can still make reasoned decisions in the domain of losses even when they cannot in the domain of gains . While a separation in processing gains and losses is achieved at the level of the amygdala versus insular cortex, the two neural systems may come closely together (and become more difficult to dissociate) by the time information reaches the prefrontal cortex, which responds similarly to risky gains and risky losses . Nevertheless, when considering the evidence from both insula and amygdala lesions, support for separate processes for risky decision - making in the gain and loss domains seems to emerge . Consistent with our model, the insula, in addition to its general role in processing risk, serves to especially aid in recruitment of the vmpfc to guide risky decisions in the more emotion - laden loss domain . Taken individually, each of the neuroimaging and lesion studies reviewed here has its limitations . Lesion studies are limited to the small sample of available participants who meet the criteria of damage to a targeted area . Furthermore, the complexity and length of tasks that can be conducted in a scanner are limited . Also, because different studies focus on different areas (see table 1), comparisons, and integration of findings can be difficult . Finally, for present purposes, the tasks used in the different studies differed in their ability to separate the gain and loss domains nevertheless, we believe that we can provide a meaningful summary of the findings reviewed here . A study comparing different age groups suggests different developmental trajectories for risk - taking in the gain and loss domains . Neuroimaging studies are sometimes inconclusive in mapping brain systems to differential reactions to risky gains and losses . For example, while there is evidence that a system such as the vmpfc or the striatum is involved in both risky gains and losses, different parts of the system may be differentially sensitive to gains and losses (xue et al ., 2009). In such cases, the more general hypothesis of separate processes underlying risk - taking for gains and losses is still supported . With regard to the stricter hypothesis of separate structures, a breakdown of fmri studies in table 1 shows the strongest evidence for this hypothesis when recordings capture pre - decisional or anticipatory processes . Although a more detailed meta - analysis is clearly warranted, table 1 shows that a wide variety of structures are involved in risky decision - making beyond those depicted in figure 1 . Nevertheless, we feel that the relatively simple depiction of the model represents a good start in capturing the different neurological underpinnings of risk - taking for gains and losses . The complementary roles of the vmpfc, amygdala, and insula depicted in the model are consistent with both the general hypothesis that separate processes underlie risk - taking for gains and losses, and the stricter hypothesis of separate neural structures coming together in different ways to guide risky decision - making in the gain and loss domains . In conclusion, we find that evidence of different neural responses underlying risk - taking for gains and losses favors the hypothesis that decision makers react differently to risky gains and losses, both in terms of overt risk - taking and neural activation . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Interstitial deletion of chromosome 3 is rare . To our knowledge, there are only 11 cases of 3p deletion reported in english literature . We report a girl with interstitial deletion of the short arm of chromosome 3 (46, xx, del 3 (p13 p21)) with features of joubert's syndrome (js). Js is a rare brain malformation characterized by the absence or underdevelopment of the cerebellar vermis- an area of the brain that controls balance and coordination . This saudi female baby born to g4 p3 mother at 38 weeks gestation by emergency caesarian section due to fetal bradycardia . Parents were non - consanguineous saudi couple and the mother is 34-year - old and father 40-year - old . Family history was unremarkable . During antenatal period, there was no history of medication intake or exposure to radiation . Birth weight was 1980 g (10 centile); length: 43 cm (10 centile); head circumference: 31.5 cm (10 centile). After birth, severe respiratory distress was encountered, which required intubation and mechanical ventilation . Bilateral choanal atresia diagnosed and surgical repair of choanal atresia was performed at 1 week . Poor and uncoordinated sucking was noticed, which necessitated tube feeding initially, and later on gastrostomy tube feeding . The subject had a broad fore head, low frontal hair line, hypertelorism, a short broad based nose with anteverted nares, bilateral microopththalmia, a short philtram, high and vaulted palate, small low set ears with hypoplasia of the upper part of helix . Finger positioning showed camptodactyly of the second finger with the third and fourth finger overlapping the index finger . Hands were positioned in an ulnar deviation and transverse crease was found in the palms . The magnetic resonance imaging showed molar tooth sign, absence of vermis with partial agenesis of corpus collosum, figure 3a and b. analysis of g - banded chromosomes showed an interstitial deletion of p13 to p21 in the proximal short arm of chromosome 3, figures 4 and 5 . Patient died at the age of 5 and 1/2 months due to escherichia coli sepsis . Facial features of the patient with 3p deletionsupine position features of the patientlateral view molar tooth sign in magnetic resonance imaging, absence of vermis with partial agenesis of corpus collosum magnetic resonance imaging showing the molar tooth sign an interstitial deletion of p13 to p21 in the proximal short arm of chromosome 3 to our knowledge, total of 11 cases of the proximal interstitial deletion of the short arm of chromosome 3 have been published, deletion of the short arm of chromosome 3 in association with js was not reported . Js is an autosomal - recessive disorder, characterized by hypotonia, ataxia, global developmental delay and molar tooth sign on magnetic resonance imaging . A variety of other abnormalities described in children with js, including abnormal breathing, abnormal eye movements, a characteristic facial appearance, delayed language, hypersensitivity to noise, autism, ocular and oculomotor abnormalities, meningoencephaloceles, microcephaly, low - set ears, polydactyly, retinal dysplasia, kidney abnormalities (renal cysts), soft - tissue tumor of the tongue, liver disease and duodenal atresia . Even within siblings the phenotype may vary, making it difficult to establish clinical diagnostic boundaries of js . Dagmar wieczorek et al . Reported a case of interstitial deletion of the short arm of chromosome 3 and charge like phenotype association . Our patient has overlapping chromosomal breaking points and strikingly similar facial appearance; in addition, our patient had bilateral hydronephrosis, but no coloboma of iris . Neri et al . Reported a proximal 3p deletion phenotype including four major manifestations; the first being a characteristic facial phenotype was characterized by a low fore head, epicanthic folds hypertelorism, broad nasal bridge, short stubby nose with anteverted nares, short philtrum, small mouth, micrognathia, low set and dysplastic ears . Thus, in comparison with proximal 3p deletion, our patient has recognizable facial phenotype . In 3 of the 5 reports with the proximal break points in 3p 13, the patient of kogame and kudo and wyandt et al . Illustrated, but show a different facial phenotype, neri et al . Reported 3 major manifestations of the proximal 3p deletion phenotype are limitations of joint movements, deformities, including ulnar deviation of the hands, comptodactyly and calcaneous feet and delayed psychomotor development . Other reported anomalies such as heart defects and intestinal malformations including agenesis of gall bladder, posteriorly placed anus and meckel's diverticulum seems to be non - specific . Being also present in other chromosomal rearrangements, they do not defining the proximal interstitial 3p phenotype . In addition to the clinical manifestations mentioned above, which fit into the spectrum of an interstitial deletion of 3p, our patient had bilateral choanal atresia, congenital heart disease (patent ductus arteriosus, atreal and ventricular septal defects). She had also growth retardation, bilateral hydronephrosis, low set, dysplastic protruding ears, absence of cerebellar vermis and partial agenesis of corpus collosum, which is consistent with js . Lin et al . Reported a case of direct interstitial duplication of chromosome 4 from 4q28.1 to 4q35 associated with bilateral choanal atresia . The child also had dysmorphic features including a broad nasal bridge, telecanthus, downward slanting palpebral fissures, prominent ears, and mild bilateral clinodactyly of the fifth fingers and bilateral hypoplasia of the second to fifth toenails . There was also a slightly dilated renal collecting system . At the age of 2.5 years, he had moderate global developmental delay, short, wide, tapering fingers and short toes with hypo plastic toenails . Reported a case of a 22-month - old boy with developmental and psychomotor retardation as well as craniofacial dysmorphism, including a cleft lip . Analysis of g - banded chromosomes of the propositus showed a de novo interstitial deletion of the short arm of chromosome 3, del (3) (p13p11). Hertz reported de novo interstitial deletion of the short arm of chromosome 3 prenatally diagnosed in a male fetus, karyotype 46, xy, del (3) (pter p14.2::p11qter). The fetus had craniofacial dysmorphisms, a single transverse palmar crease, ulnar deviation in the wrists, cardiovascular anomalies, a slight ureteric dilatation and a mobile caecum . Short et al . Reported deletions of 3p usually involve the terminal portion (3p25). An interstitial deletion of a proximal 3p segment (3p14) was detected at amniocentesis . The clinical and cytogenetic characteristics of this case and of three previously published cases are reviewed . Reported a girl with delayed growth in body height and weight, retarded psychomotor development, facial dysmorphism, high - arched palate, extension defects of elbows, and a probable hearing impairment is presented . A chromosome investigation by both conventional and high - resolution banding techniques revealed an apparently pure interstitial deletion of the proximal segment of the short arm of chromosome 3 (46, xx, del (3) (p11 p14.2) de novo). The paternal karyotype is 47, xyy . The clinical features of the patient are compared with those of two previously reported cases in the literature with an interstitial 3p deletion . He was found to have an interstitial deletion of band p14 from the proximal short arm of chromosome 3 . Examination of the father's chromosomes indicates an inserted para centric inversion in chromosome 3 as the probable origin of the deletion in the child . From the above discussion we believe that our patient with the typical phenotype features of 3p (p13-p21) has a unique findings in association the js, which is characterized by the dysgenesis of the cerebellar vermis with the brain stem malformation comprising the molar tooth sign in magnetic resonance imaging.
Allergic disease is an increasingly prevalent problem affecting up to one - third of the general population in industrialized countries . Immunotherapy is a treatment modality that can modify the immunological response of the allergy sufferer so that the affected individual will stop reacting to involved allergens . Immunotherapy is indicated for the treatment of allergic rhinitis (ar) and asthma, and it may prevent development of asthma in patients with ar [1, 2]. Immunotherapy can be administered by different routes amongst which we find injectable and oral vaccines . Injectable vaccines refers to the classical subcutaneous injection immunotherapy (scit) usually known as allergy shots . Oral vaccines refer to sublingual immunotherapy (slit) where the allergens are administered as drops to the sublingual area even though the term oral vaccines may also include allergy tablets . The purpose of this study is to compare the efficacy of treatment results in patients with nasal allergies, with or without asthma, that were treated with either one or the other of these two treatment modalities: scit or slit . There is a voluminous body of scientific evidence that proves that these two treatment modalities are efficacious for the management of allergic conditions but the issue of these two modalities having similar efficacy has not yet been fully addressed . A review of the literature reveals only a few articles that directly address this issue [410]. In five of these reports [59] scit and slit is found to have better results, and one report finds both equally effective for ar patients but scit more effective for asthmatic patients . In our own experience, slit and scit appear to be of similar efficacy in this report the efficacy of one will be compared against the other . Scit is a well - established treatment modality that has been successfully used for many decades and is relatively well tolerated . Slit is also a very old treatment modality (earliest description is from 1900) and yet, while commonly used in europe, it is still not well established in the usa . Over the last 20 years the european medical community produced a large amount of high - quality evidence suggesting that slit is safer than scit [14, 15]. While no single case of mortality has ever been reported with slit [12, 16] this is not the case with scit [17, 18]. This study constitutes a retrospective, consecutive chart review of allergy patients treated by the author at his private office . Inclusion criteria were as follows: a patient of any age with nasal allergies with or without asthma that was treated with immunotherapy for at least for 6 months and had at least 2 complete evaluations . A complete evaluation implies symptom scoring, evaluation of medication use, and determination of the peak flow meter (pfm) value . These evaluations are done every 36 months as treatment progresses . Because evaluations depend on patient's cooperation not all the patients had the same number of evaluations, but any patient that was considered a candidate had to have 2 evaluations as a minimum . We compared the first evaluation (pretreatment) and the last evaluation the patient had just at the time of inclusion for the study . The symptoms in the pretreatment evaluation and the amount of medications the patient was taking at that time reflect how the patient was doing without immunotherapy treatment . Ethical considerations . Subjects' privacy was respected by collecting and recording data in such a way that the subjects could not be identified, directly or indirectly, through identifiers linked to the subject . In other words, a patient's confidentiality would be protected by entering data in a simple spreadsheet with nonspecific identifiers as patient no . 1, patient no . 2, and so forth with subsequent refiling of the patient's chart, according to usual procedure . The content of the spread sheet became anonymous and ready for statistical analysis . After discussing with patient about their allergies and advising about environmental modification maneuvers a discussion about treatment options including immunotherapy follows . In our office scit or slit is used to treat patients with inhalant allergies with or without bronchial involvement . The decision to use one or the other economical considerations, living far from the office, busy schedule, or needle phobia, are examples of when a patient may chose slit . Having severe asthma, being a very young patient or having medical problems that may render administration of scit risky are examples of why the treating physician will advise slit . All patients were tested using a fivefold intradermal dilution skin test (idt) as taught by the aaoa [20, 21]. The test includes several panels: dust, dander, epidermals, molds, and pollens for our geographic area (table 1). Standardized antigens were used for testing and treatment whenever these were available; otherwise weight / volume antigen extracts were used . After identifying the minimally reactive antigen concentration (meaning first reactive wheal) for each of the patient's reactive allergens, scit vials or slit bottles were formulated including all of the positive results (reactive allergens in the intradermal test) in the treatment mixture . Patients on slit were treated according to a previously published protocol where the dose is slowly advanced from 1 drop per day to 5 drops per day until attaining the most concentrated mixture in the slit bottle . The formulation was the same for both injectable and oral vaccines . While the concentration of antigens is exactly the same for both scit and slit but slit is administered daily, patients on slit will receive a larger amount of antigen each week than those treated with scit . The slit bottles are mixed with 7.5 ml . If we consider a single allergen, for example, dermatophagoides pteronyssinus (dp), standardized dust mite dp has a concentration of 10,000 au / ml containing 68 mcg / ml of der p 1 and 71 mcg / ml of der p 2 antigens . If the minimally reactive antigen concentration occurred at dilution no . 3 and dose was advanced until mixing a vial from manufacturer's concentrate, the cumulative dose this patient would receive weekly by scit would be 200 au per week, while a patient treated by slit would receive 464 au per week . As stated before, the initial allergen concentration in both scit and slit is the same: 80 au / ml as in both circumstances the extract (with 10.000 au / ml) will be diluted 125 times . After one year of treatment the patient on scit would receive 9680 au and the patient treated by slit would receive 21149 au or 2.18 times more allergen . A chi - square test was applied for the following allergens: dust mite, cat, roach, mold, tree - pollens, grass - pollens, and weed - pollens for both groups, scit and slit . Asthma diagnosis was based on the presence of recurrent cough, chest tightness, sob, or wheezing, having a spirometry consistent with airflow obstruction or having the symptoms respond to the administration of a short - acting broncho - agonist (saba). Recorded symptoms included runny nose, sneezing, nasal obstruction, itchy eyes, itchy ears, cough, shortness, and wheezing . These were scored according to fell's method with a numerical analog from 0 through 3 as follows: 0 = symptom not present, 2 = symptom is moderate, 3 = symptom is severe . Medication use was also evaluated on a similar numerical scale as follows: 0 = medication is not being used, 1 = medication is being used once a week or less, 2 = medication is being used 23 times per week, 3 = medication is being used 4 or more times per week . Medications were generically grouped as allergy pills, intranasal steroids (inss), and short - acting broncho - agonists (sabas) in the case of asthmatic patients . The value of the pfm determination was used as the parameter to be recorded at each patient's encounter . Ninety - three charts met the inclusion criteria, 50 on scit and 43 on slit . Among the 50 patient's on scit, 20 (40%) were male, 30 (60%) female ranging in age from 2.33 to 75 years (mean 45 17.8 sd). This compared to 43 patients on slit of whom 21 (49%) were male, 22 (51%) female ranging in age from 1.66 to 75 years (mean 35 20.8 sd). Analysis of covariance for the dependent variables for which a significant pre / posttreatment by treatment modality interaction effects was obtained did not reveal gender or age to account for significant dependent variable variance; in other words the results were not affected by age or gender so both groups can be considered homogeneous . A chi - square test was applied for the following allergens: dust mite, cat, roach, mold, tree - pollens, grass - pollens, and weed - pollens . Results indicate that there are no statistical differences between both groups (at the p <0.05 level); therefore in their reactivity to allergens both groups can also be considered homogeneous . There were 3 children <12 years on scit (mean 7.8 years) versus 11 on slit (mean 6.9 years). Ten (20%) scit patients had asthma versus 12 (28%) on slit . Thus a greater percentage of asthmatics (12/22 or 55%) and more children under 12 years of age (11/14 or 79%) were on slit . Length of treatment for the scit group was 12 to 86 (mean 31 18.7 sd) months and for the slit group was 10 to 32 (mean 19 6.3 sd) months . For all patients the pre- and posttreatment averages for each symptom, medication use, and pf value were statistically compared through the use of repeated measure analysis of variance (anova). The results for the two treatment modalities (scit versus slit) were also compared using the between - subjects factor of the anova (table 2). The same analyses were completed for medication use (table 3). For the pf evaluation the pre- and post - treatment values were compared (table 4). In table 2 the mean value for each symptom score before treatment and at the time of data collection the result of the test of significance is shown for each symptom within each treatment modality (paired t - test). Lastly, the result of the statistical analysis comparing symptom improvement with one or the other treatment modality is shown . Shortness of breath and wheezing had significant improvements at p <0.05 for both treatment modalities . The remaining symptoms had a significant improvement at p <0.001 for both treatment modalities . Wheezing and coughing were the only symptom scores which seemed to respond better to either scit (coughing slightly better, p = 0.037) or slit (wheezing slightly better, p = 0.024), though both symptoms significantly improved regardless of treatment modality . Both scit and slit provided equally significant reduction in use of medication (p <0.001) including allergy pills, ins, and, to a slightly lesser but still significant degree, saba (table 3) but without no significant difference between both treatment modalities . Pf value before treatment and at the time of the last patient evaluation is shown in table 4 . Both treatment modalities were equally effective in achieving a significant increase in pf values (p <0.001) but there was no significant difference between both treatment modalities . This paper is a retrospective chart review and as such lacks the rigor of a prospective randomized study with a placebo control group which is very difficult to do in a private office setting . While an analysis of covariance is useful, it is not a perfect solution . A future, larger - scale study should be planned to include the above design characteristics . We observed that patients usually come to the office already using one or more allergy medications . This study, like others, demonstrates that immunotherapy, whether scit or slit, will lead to the reduction of medication use for ar and/or asthma . It was not the purpose of this paper to evaluate the effect of medications on allergy symptoms but rather to compare the effects of scit versus slit on medication use . Both treatment modalities resulted in the reduction of antihistamines, inhaled nasal steroids, and sabas . The slight imbalances in demographic characteristics between the groups on scit versus slit were not statistically significant and did not affect the statistical results . The reason why there are more young patients and more asthmatic patients in the slit group can be explained by the fact that slit is safer and easier to administer therefore it is suggested more frequently for these difficult - to - manage patients . Indeed we would have expected a much more pronounced difference; yet fewer than expected chose slit because it is not covered by insurance . Patients on scit have been treated for a longer period of time because slit was added to our practice later than scit . The improvement of the asthmatic symptoms wheezing and sob and the decrease in saba use were significant at p <0.05 yet because of sample size this is not as strong as the improvement in other symptoms or medications that had an improvement at the level of p <0.001 . The advantage for scit in treating coughing is real, but the effect size (eta - squared) is only 0.025, meaning that it only accounts for 2.5% of the variance in pre- versus posttreatment differences, which is not much . The advantage of slit in treating wheezing may have been influenced by our own bias of suggesting slit use to asthmatic patients as a safer treating modality . It is therefore more likely that patients with higher symptom scores were present in the slit group . Our findings demonstrate that slit is not only effective in controlling symptoms in nasal allergy patients with or without asthma, in decreasing medication use in such patients, and in improving parameters of pulmonary function, but it also appears that slit is as effective as scit these findings are in agreement with those published in the european literature [26, 27] but certainly this presentation lacks the scientific validity of other reports that present a prospective, randomized, controlled study; therefore this presentation we hope will serve as a stimulus for centers with the capability to undertake such a study to continue with this line of research . This would help the fda to finally recognize slit as an effective and safe treatment modality . If slit became an fda - approved treatment modality (and hopefully) reimbursed by insurance companies many more patients might be receptive to immunotherapy which is a treatment capable of altering the immunological mechanisms responsible for the development of allergic conditions . Pf values for asthma control should be taken as a guideline only because the predicted lung function has a high degree of variability with significant differences in pf values according to presence or not of lung disease, smoking, age, sex, and even patient's social environment [2931]. Having the advantage of providing results quickly, and requiring little training (from the patient as well as from the technical staff), the pfm device is useful to monitor progress during immunotherapy . It is most useful when the changes in pf values are compared to the initial value of each patient, recorded at the time of treatment initiation . For the purpose of this study individual improvement with therapy is not reported, but rather an overall trend, thus the use of pfm provides a gross indicator of change . Some of our patients were children, and it is expected they grow during treatment . Certainly using a pfm as a tool to determine improvement in pulmonary function adds uncertainty as to whether the improvement in pf value is related to clinical improvement or to the growth of the patient during treatment . In this we have demonstrated that the pf value in patients treated by immunotherapy increases regardless of age or asthmatic condition . In our experience, the use of slit with multiple antigens has enabled us to treat patients that otherwise would have not received immunotherapy, or would have not continued to receive immunotherapy, like asthmatic patients with poorly controlled asthma, patients that had severe arm reactions, very young patients to whom it is difficult to administer shots or patients whose schedules prevent them from being compliant . These results suggest that scit and slit exhibit similar efficacy . Slit objectively improves symptom scores for asthma and ar while decreasing medication usage of allergy medications and sabas . Given the increased risk and difficulty in treating asthmatic and young patients, these results would suggest that slit should be considered as the main treatment modality for these patients, considering scit only for treatment failures . The results of this study are in agreement with the european literature and therefore would support the inclusion of slit in the routine management of the allergic disease.

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